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Mikrobicidno delovanje α in θ defenzinov na antibiotik-odporne Staphylococcus aureus in Pseudomonas aeruginosa

2015-04-07T19:53:51Z

Ana90:

<h3>UVOD</h3>

Bakterije odporne na antibiotike predstavljajo tako zdravstveni kot tudi ekonomski problem. Številni antibiotiki ciljajo na specifične bakterijske encime ali reakcije in lahko se zgodi, da geni odgovorni zanje mutirajo pod vplivom selekcijskega pritiska kar lahko pripelje do odpornosti na antibiotike. Velik potencial v boji proti patogenim bakterijam kažejo protimikrobne snovi, ki vplivajo na integriteto celične membrane. To so sesalski α in θ defenzini, kationski peptidi s specifičnmi tridisulfidnimi mrežami. Alfa defenzini se večinoma nahajajo v neutrofilnih azurofilnih granulah, najdemo jih tudi v naravnih celicah ubijalkah. θ defenzine pa so zaenkrat odkrili le v kostnem mozgu nekaterih primatov. Defenzini delujejo tako, da se selektivno vežejo na bakterijske membrane in povzročijo izhajanje kalijevih ionov in hranil, depolarizacijo membrane in posledično celično smrt. Da potrdijo hipoteze o njihovem baktericidnem delovanju, so preverili učinek α defenzinov (Crp-4, RMAD-4, HNP 1-3) in θ defenzinov (RTD 1) na meticilin odporno ''Staphylococcus aureus'' (MRSA), skupaj z vankomicin rezistentnimi sevi in na ciprofloksacin odporne ''Pseudomonas aeruginosa'' (PA).

<h3>MATERIALI IN METODE</h3>

Peptide so homogenizirali z reverzno-faznim HPLC in karakterizirali z MALDI-TOF MS in AU-PAGE. Rekombinantne Crp - 4 in RMAD -4 peptide so izrazili v bakteriji ''Escherichia coli'' kot N- terminalni fuzijski protein s His6 oznako preko pET28a ekspresijskega vektorja. Sledila je izolacija s His6 označenih fuzijskih Crp- 4 peptidov z afinitetno kromatografijo z uporabo nikelj-nitrilotriocetno kislino. Cianogen bromid je služil za odstranitev His6 fuzije, proteine pa so prečistili s sekvenčnim C18 Rp-HPLC in določili molekulsko maso z MALDI-TOF MS. Natančna določitev strukture je bila opravljena z metodo NMR. HNP so izolirali iz vzorcev obogatenih z nevtrofilnimi granulami pripravljenimi iz perifernih levkocitov. HNP 1-3 so pridobili z gelsko permeacijsko kromatografijo z uporabo BioGel P10, HNP 2 so očistili iz druženih HNP 1-3 z metodo C18 RP-HPLC. Θ defenzin RTD-1 pa so sintetizirali.
Klinične izolate so pridobili iz hospitaliziranih pacientov. MRSA izolate odporne na vankomicin so določili z uporabo Etest glikopeptid rezistentne detekcijske metode. PA dovzetnost na ciproflokasin pa so določili z mikrodilucijsko metodo. Ciprofloksacinska odpornost je bila definirana z minimalno inhibitorno koncentracijo 2μg/ml ali več. MRSA in PA seve, ki povzročajo pljučnico, bakteriemijo, infekcije ran in urinarnega trakta so izbrali kot reprezentativne klinične izolate z različnimi stopnjami odpornosti na vankomicin (MRSA) in ciprofloksacin (PA). Alfa in θ defenzine so testirali na baktericidno aktivnost pri kliničnih izolatih MRSA in PA v in vitro celičnih suspenizijskih testih. Preživetje bakterij glede na izpostavitev peptidom so določili z štetjem CFU. 1h po izpostavitvi peptidom so peptidne in bakterijske mešanice nagojili na agarne plošče.

<h3>REZULTATI</h3>

Crp-4, RMAD-4 in RTD–1, testirani s kliničnimi izolati MRSA z različnimi stopnjami odpornosti na vankomicin, na podoben način zmanjšajo viabilnost sevov, ne glede na vankomicinsko rezistenco izolatov. Enako so storili tudi s sevi PA odporne na ciprofloksacin, ki so jih testirali s Crp-4, RMAD-4, RTD-1 in HNP 1-3 ter določali učinkovitost glede na različne odpornosti na ciprofloksacin, mesta izolacije,TTSS efektorskega genotipa in citotoksični potencial. Ugotovili so, da je Crp-4 bolj učinkovit pri PA izolatih, RMAD-4 pa pri izolatih MRSA. Pri sevih pridobljenih iz različnih mest (opekline, urin, kri), ki se med seboj tudi fenotipsko razlikujejo, sta Crp-4 in RMAD-4 delovala z enako občutljivostjo. Sicer so pri RMAD-4 opazili večjo aktivnosti pri sevih izoliranih iz opeklin, kar nakazuje na morebitno povezavo med mestom izolacije PA in odpornostjo na α defenzine. Rezultati so pokazali,da HNP1-3 in HNP-2 nimajo velikega vpliva na preživetje PA, napredek niso opazili niti pri koncetracijah pri katerih sicer Crp-4 in RTD-1 delujeta močno baktericidno.
Tudi kationski naboj vpliva na baktericidno delovanje defenzinov. Tako je na primer Crp-4 veliko bolj učinkovit proti PA, RMAD pa proti MRSA izolatom. Verjetno gre tu za vpliv različnih porazdelitev površinskih nabojev ali hidrofobnosti.

<h3>ZAKLJUČKI</h3>

Protimikrobni peptidi predstavljajo dobro platformo za razvoj novih terapevtikov proti odpornim patogenom. Defenzini vplivajo na celično integriteto, vplivajo na delovanje membrane in se po mehanizmu razlikujejo od beta laktamskih inhibitorjev in vankomicina. Defenzine bi tako lahko združili z obstoječimi antibiotiki in tako izboljšali njihovo delovanje ter posledično povečali učinkovitost zdravljenja proti odpornim patogenom.

Mikrobicidno delovanje α in θ defenzinov na antibiotik-odporne Staphylococcus aureus in Pseudomonas aeruginosa

2015-04-07T19:46:22Z

Ana90:

<h3>UVOD</h3>

Bakterije odporne na antibiotike predstavljajo tako zdravstveni kot tudi ekonomski problem. Številni antibiotiki ciljajo na specifične bakterijske encime ali reakcije in lahko se zgodi, da geni odgovorni zanje mutirajo pod vplivom selekcijskega pritiska kar lahko pripelje do odpornosti na antibiotike. Velik potencial v boji proti patogenim bakterijam kažejo protimikrobne snovi, ki vplivajo na integriteto celične membrane . Gre za sesalske α in θ defenzine , ki so kationski peptidi s specifičnmi tridisulfidnimi mrežami. Alfa defenzini se večinoma akomulirajo v neutrofilnih azurofilnih granulah, najdemo jih tudi v naravnih celicah ubijalkah. θ defenzine pa so zaenkrat odkrili le v kostnem mozgu nekaterih primatov. Defenzini delujejo tako, da se selektivno vežejo na bakterijske membrane in povzročijo izhajanje kalijevih ionov in hranil, depolarizacijo membrane in posledično celično smrt. Da preverijo hipoteze o njihovem baktericidnem delovanju, so preverili učinek α defenzinov (Crp-4, RMAD-4, HNP 1-3) in θ defenzinov (RTD 1) na meticilin odporno ''Staphylococcus aureus'' (MRSA), skupaj z vankomicin rezistentnimi sevi in na ciprofloksacin odporne ''Pseudomonas aeruginosa'' (PA).

<h3>MATERIALI IN METODE</h3>

Peptide so homogenizirali z reverzno-faznim HPLC in karakterizirali z MALDI-TOF MS in AU-PAGE. Rekombinantne Crp - 4 in RMAD -4 peptide so izrazili v bakteriji ''Escherichia coli'' kot N- terminalni fuzijski protein s His6 oznako preko pET28a ekspresijskega vektorja. Sledila je izolacija s His6 označenih fuzijskih Crp- 4 peptidov z afinitetno kromatografijo z uporabo nikelj-nitrilotriocetno kislino. Cianogen bromid je služil za odstranitev His6 fuzije, proteine pa so prečistili s sekvenčnim C18 Rp-HPLC in določili molekulsko maso z MALDI-TOF MS. Natančna določitev strukture je bila opravljena z metodo NMR. HNP so izolirali iz vzorcev obogatenih z nevtrofilnimi granulami pripravljenimi iz perifernih levkocitov. HNP 1-3 so pridobili z gelsko permeacijsko kromatografijo z uporabo BioGel P10, HNP 2 so očistili iz druženih HNP 1-3 z metodo C18 RP-HPLC. Θ defenzin RTD-1 pa so sintetizirali.
Klinične izolate so pridobili iz hospitaliziranih pacientov. MRSA izolate odporne na vankomicin so določili z uporabo Etest glikopeptid rezistentne detekcijske metode. PA dovzetnost na ciproflokasin pa so določili z mikrodilucijsko metodo. Ciprofloksacinska odpornost je bila definirana z minimalno inhibitorno koncentracijo 2μg/ml ali več. MRSA in PA seve, ki povzročajo pljučnico, bakteriemijo, infekcije ran in urinarnega trakta so izbrali kot reprezentativne klinične izolate z različnimi stopnjami odpornosti na vankomicin (MRSA) in ciprofloksacin (PA). Alfa in θ defenzine so testirali na baktericidno aktivnost pri kliničnih izolatih MRSA in PA v in vitro celičnih suspenizijskih testih. Preživetje bakterij glede na izpostavitev peptidom so določili z štetjem CFU. 1h po izpostavitvi peptidom so peptidne in bakterijske mešanice nagojili na agarne plošče.

<h3>REZULTATI</h3>

Crp-4, RMAD-4 in RTD–1, testirani s kliničnimi izolati MRSA z različnimi stopnjami odpornosti na vankomicin, na podoben način zmanjšajo viabilnost sevov, ne glede na vankomicinsko rezistenco izolatov. Klinični izolati so bili občutljivi na vse testirane defenzine. Enako so storili tudi s sevi PA odporne na ciprofloksacin, ki so jih testirali s Crp-4, RMAD-4, RTD-1 in HNP 1-3 ter določali učinkovitost glede na različne odpornosti na ciproflokascin, mesta izolacije,TTSS efektorskega genotipa in citotoksični potencial. Ugotovili so, da je Crp-4 bolj učinkovit pri PA izolatih, RMAD-4 pa pri izolatih MRSA. Pri sevih pridobljenih iz različnih mest (opekline, urin, kri), ki se med sabo tudi fentipsko razlikujejo, sta Crp-4 in RMAD-4 delovala z enako občutljivostjo. Sicer so pri RMAD-4 opazili večjo aktivnosti pri sevih izoliranih iz opeklin, kar nakazuje morebitno povezavo med mestom izolacije PA in odpornostjo na α defenzine. Rezultati so pokazali,da HNP1-3 in HNP-2 nimajo velikega vpliva na preživetje PA, napredek niso opazili niti pri koncetracijah pri katerih sicer Crp-4 in RTD-1 delujeta močno baktericidno.
Tudi kationski naboj vpliva na baktericidno delovanje defenzinov. Tako je na primer Crp-4 veliko bolj učinkovit proti PA izolatom, RMAD pa proti MRSA. Verjetno gre tu za vpliv različnih porazdelitev površinkih nabojev ali hidrofobnosti.

<h3>ZAKLJUČKI</h3>

Antimikrobni peptidi predstavljajo dobro platformo za razvoj novih terapevtikov proti odpornim patogenom. Defenzini vplivajo na celično integriteto, vplivajo na delovanje membrane in se po mehanizmu razlikujejo od beta laktamskih inhibitorjev in vankomicina. Defenzine bi tako lahko združili z obstoječimi antibiotiki in tako izboljšali njihovo delovanje ter posledično povečali učinkovitost zdravljenja proti odpornim patogenom.

MBT seminarji 2015

2015-04-07T19:32:56Z

Ana90:

'''Seznam seminarjev iz Molekularne biotehnologije v študijskem letu 2014/15'''

Tabela za razpored po tednih bo objavljena v spletni učilnici, vanjo pa se vpišite tudi za kratke predstavitve novic (3 min, dvakrat v semestru). Na tej strani bo samo seznam odobrenih člankov za seminar in povezave do člankov in do povzetkov, ki jih morate objaviti najkasneje tri dni pred predstavitvijo (ponedeljek oz. torek). Angleški naslov prevedite tudi v slovenščino - to bo naslov povzetka, ki ga objavite na posebni strani, tako kot so to naredili kolegi pred vami (oz. lani).

Način vnosa:

# The importance of ''Arabidopsis'' glutathione peroxidase 8 for protecting ''Arabidopsis'' plant and ''E. coli'' cells against oxidative stress (A. Gaber; GM Crops & Food 5(1), 2014; http://dx.doi.org/10.4161/gmcr.26979) Pomen glutation peroksidaze 8 iz repnjakovca za zaščito rastline ''Arabidopsis thaliana'' in bakterije ''Escherichia coli'' pred oksidativnim stresom. Janez Novak, 15. marca 2014
(slovenski naslov povežite z novo stranjo, na kateri bo povzetek)

'''Naslovi odobrenih člankov po temah:'''

'''Gensko spremenjene rastline'''<br>
# Successful high-level accumulation of fish oil omega-3 long-chain polyunsaturated fatty acids in a transgenic oilseed crop (Ruiz-Lopez, N., et al; The plant journal 77, 198-208, 2014; http://www.ncbi.nlm.nih.gov/pubmed/24308505). [[Uspešna priprava gensko spremenjene oljne rastline z visoko vsebnostjo omega-3 polinenasičenih maščobnih kislin.]] Petra Malavašič, 20. marca 2015
#A simpliﬁed and accurate detection of the genetically modiﬁed wheat MON71800 with one calibrator plasmid (Jae Juan, S.,et al; Food Chemistry 176, 1-6, ;http://www.sciencedirect.com.nukweb.nuk.uni-lj.si/science/article/pii/S03088146140196572015 [[Poenostavljena in točna detekcija gensko spemenjene pšenice MON71800 z enim kalibratorskim plazmidom]]. Matej Lesar, 20. marca 2015

'''Gensko spremenjene živali'''<br>
# [[A novel adenoviral vector carrying an all-in-one Tet-On system with an autoregulatory loop for tight, inducible transgene expresion]] (H. Chen; et all.; BMC Biotechnology 2015, 15:4, doi:10.1186/s12896-015-0121-4; http://www.biomedcentral.com/1472-6750/15/4). Edvinas Grauželis, 27. marca 2015 (in English)
# Production of functional active human growth factors in insects used as living biofactories (B. Dudognon, et al; Journal of Biotechnology 184, 229–239, 2014; http://dx.doi.org/10.1016/j.jbiotec.2014.05.030). [[Proizvodnja funkcionalno aktivnih človeških rastnih faktorjev v insektih uporabljenih kot žive biotovarne]] Maxi Sagmeister, 27. marca 2015

'''Okolje'''<br>
# Bioremediation of pesticide contaminated water using an organophosphate degrading enzyme immobilized on nonwoven polyester textiles (Yuan Gao ''et al.'', Enzyme and Microbial Technology, vol. 54, pages 38-44, 10.1.2014, http://www.sciencedirect.com/science/article/pii/S0141022913002044). [[Bioremediacija s pesticidi okužene vode z uporabo encima, ki razgrajuje organofosfate in je vezan na netkan poliestrski tekstil]]. Mitja Crček, 3. aprila 2015
# Biodegradation of atrazine by three transgenic grasses and alfalfa expressing a modified bacterial atrazine chlorohydrolase gene (A. W. Vail ''et al.''; Transgenic Research, 29. 11. 2014; http://link.springer.com/article/10.1007/s11248-014-9851-7). [[Biorazgradnja atrazina s tremi transgenskimi travami in lucerno, ki izražajo gen za modificirano bakterijsko atrazin klorohidrolazo]]. Mirjam Kmetič, 3. aprila 2015

'''Terapevtiki'''<br>
# Mind-controlled transgene expression by a wireless-powered optogenetic designer cell implant (M. Folcher; Nature Communications 5, 1–11, 2014; http://www.nature.com/ncomms/2014/141111/ncomms6392/full/ncomms6392.html) Z EEG nadzorovano izražanje transgena preko brezžično napajanega optogenetskega celičnega vsadka. Luka Smole, 10. aprila 2015
# Glycosylated enfuvirtide: A long-lasting glycopeptide with potent anti-HIV activity; http://pubs.acs.org/doi/full/10.1021/jm5016582 Sebastian Pleško, 10. aprila
# Microbicidal effects of α- and θ-defensins against antibiotic-resistant Staphylococcus aureus and Pseudomonas aeruginosa; http://ini.sagepub.com/content/21/1/17.long. [[Mikrobicidno delovanje α in θ defenzinov na antibiotik-odporne Staphylococcus aureus in Pseudomonas aeruginosa]]. Ana Kapraljević, 10. aprila

'''Encimi'''<br>
# Immobilization and controlled release of β-galactosidase from chitosan-grafted hydrogels; http://www.sciencedirect.com/science/article/pii/S0308814615001028. Mojca Banič, 16. aprila 2015
# Construction of efficient xylose utilizing ''Pichia pastoris'' for industrial enzyme production (Li ''et al''; Microbial Cell Factories 14:22, 1-10, 2015; http://www.microbialcellfactories.com/content/14/1/22). Priprava ''Pichie pastoris'', ki učinkovito uporablja ksilozo, za industrijsko proizvodnjo encimov. Špela Tomaž, 17. aprila 2015
# Postharvest application of a novel chitinase cloned from Metschnikowia fructicola and overexpressed in Pichia pastoris to control brown rot of peaches; http://www.sciencedirect.com/science/article/pii/S0168160515000033. Špela Pohleven, 17. aprila 2015

'''Protitelesa'''<br>
# Optimization of heavy chain and light chain signal peptides for high level expression of therapeutic antibodies in CHO cells; http://dx.plos.org/10.1371/journal.pone.0116878. Tjaša Blatnik, 23. aprila 2015
# Ethanol precipitation for purification of recombinant antibodies (A. Tscheliessnig ''et al''; Journal of Biotechnology 188, 17-28, 2014; http://www.sciencedirect.com/science/article/pii/S0168165614007810). Čiščenje rekombinantnih protiteles z obarjanjem z etanolom. Urška Rauter, 24. aprila 2015
# Functional mutations in and characterization of VHH against Helicobacter pylori urease (R. Hoseinpoor ''et al''; Applied Biochemistry and Biotechnology 172, 3079-3091, 2014; http://link.springer.com/article/10.1007/s12010-014-0750-4). Funkcionalne mutacije in karakterizacija VHH proti ureazi ''Helicobacter pylori''. Marko Radojković, 7. maja 2015

'''Cepiva'''<br>
# Development of anti-E6 pegylated lipoplexes for mucosal application in the context of cervical preneoplastic lesions; http://www.sciencedirect.com/science/article/pii/S0378517315001507. Tanja Korpar, 7. maja 2015
# A novel “priming-boosting” strategy for immune interventions in cervical cancer (S. Liao et al.; Molecular Immunology 64, 295-305, 2015, http://www.sciencedirect.com/science/article/pii/S0161589014003460. Nova "priming-boosting" strategija za imunsko posredovanje pri raku materničnega vratu. Anita Kustec, 8. maja 2015
# Potentiation of anthrax vaccines using protective antigen-expressing viral replicon vectors (H.C. Wang et al.; Immunology letters 163, 206-213, 2015, http://www.ncbi.nlm.nih.gov/pubmed/25102364 ) Izboljšava cepiv proti antraksu z uporabo iz virusnih replikonov izvedenih vektorjev, ki omogočajo izražanje zaščitnega antigena. Daša Pavc, 8. maja 2015

'''Male molekule in polimeri'''<br>
# Methanol-induced chain termination in poly(3-hydroxybutyrate) biopolymers: Molecular weight control; http://www.sciencedirect.com/science/article/pii/S0141813014008307. Gašper Lavrenčič, 14. maja 2015
# Purification and characterization of gamma poly glutamic acid from newly Bacillus licheniformis NRC20; http://www.sciencedirect.com/science/article/pii/S0141813014008216. Uroš Stupar, 14. maja 2015
# Iza Ogris, 15. maja 2015
# Chromosomal integration of hyaluronic acid synthesis (''has'') genes enhances the molecular weight of hyaluronan produced in ''Lactococcus lactis'' (R. V. Hmar et al; Biotechnol. J. 9 (12), 2014; http://dx.doi.org/10.1002/biot.201400215) Integracija genov za sintezo hialuronske kisline v kromosom bakterije ''Lactococcus lactis'' izboljša sintezo visokomolekularne hialuronske kisline. Maja Grdadolnik, 15. maja 2015

'''Pretvorba biomase'''<br>
# Effect of pretreatment methods on the synergism of cellulase and xylanase during the hydrolysis of bagasse; http://www.sciencedirect.com/science/article/pii/S0960852415002114. Eva Lucija Kozak, 21. maja 2015
# Third generation biohydrogen production by Clostridium butyricum and adapted mixed cultures from Scenedesmus obliquus microalga biomass; http://www.sciencedirect.com/science/article/pii/S0016236115002550?np=y. Nives Naraglav, 22. maja 2015
# Bio-catalytic action of twin-screw extruder enzymatic hydrolysis on the deconstruction of annual plant material: Case of sweet corn co-products; http://www.sciencedirect.com/science/article/pii/S0926669015000436. Griša Prinčič, 22. maja 2015

'''Metabolično inženirstvo'''<br>
# Engineering lipid overproduction in the oleaginous yeast Yarrowia lipolytica;http://www.sciencedirect.com/science/article/pii/S1096717615000166. Andreja Bratovš, 28. maja 2015
# Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals (Weerawat Runguphana, Jay D. Keasling; Metabolic Engineering, vol 21, January 2014, Pages 103–113; http://www.sciencedirect.com/science/article/pii/S1096717613000670). Metabolično inženirstvo ''Saccharomyces cerevisiae'' za proizvodnjo derivatov maščobnih kislin, ki so primerni za biogorivo in kemikalije. Dominik Kert, 29. maja 2015
# Metabolic engineering of Klebsiella pneumoniae for the production of cis,cis-muconic acid (Jung,H.-M. Jung,M.-Y. Oh, M.-K.;Applied Microbiology and Biotechnology, Published online: 14 February 2015; http://link.springer.com/article/10.1007/s00253-015-6442-3). Metabolno inženirstvo Klebsiella pneumoniae za produkcijo cis,cis-mukonične kisline. Jure Zabret, 29. maja 2015

'''Biološki viri energije'''<br>
# Anodic and cathodic microbial communities in single chamber microbial fuel cells; http://www.sciencedirect.com/science/article/pii/S1871678414021694. Tamara Marić, 4. junija 2015
# Combination of dry dark fermentation and mechanical pretreatment for lignocellulosic deconstruction: An innovative strategy for biofuels and volatile fatty acids recovery; http://www.sciencedirect.com/science/article/pii/S0306261915002196. Jernej Pušnik, 4. junija 2015
# Potential use of feedlot cattle manure for bioethanol production; http://www.sciencedirect.com/science/article/pii/S0960852415001960. Nastja Pirman, 5. junija 2015
# Cellulolytic enzymes produced by a newly isolated soil fungus Penicillium sp. TG2 with potential for use in cellulosic ethanol production; http://www.sciencedirect.com/science/article/pii/S0960148114007022. Jana Verbančič, 5. junija 2015

'''Novi pristopi v molekularni biotehnologiji'''<br>
# Exploring the potential of algae/bacteria interactions; http://www.sciencedirect.com/science/article/pii/S0958166915000269. Matja Zalar, 11. junija
# How close we are to achieving commercially viable large-scale photobiological hydrogen production by cyanobacteria: A review of the biological aspects; http://www.mdpi.com/2075-1729/5/1/997/htm. Monika Škrjanc, 11. junija

MBT seminarji 2015

2015-04-07T19:32:12Z

Ana90:

'''Seznam seminarjev iz Molekularne biotehnologije v študijskem letu 2014/15'''

Tabela za razpored po tednih bo objavljena v spletni učilnici, vanjo pa se vpišite tudi za kratke predstavitve novic (3 min, dvakrat v semestru). Na tej strani bo samo seznam odobrenih člankov za seminar in povezave do člankov in do povzetkov, ki jih morate objaviti najkasneje tri dni pred predstavitvijo (ponedeljek oz. torek). Angleški naslov prevedite tudi v slovenščino - to bo naslov povzetka, ki ga objavite na posebni strani, tako kot so to naredili kolegi pred vami (oz. lani).

Način vnosa:

# The importance of ''Arabidopsis'' glutathione peroxidase 8 for protecting ''Arabidopsis'' plant and ''E. coli'' cells against oxidative stress (A. Gaber; GM Crops & Food 5(1), 2014; http://dx.doi.org/10.4161/gmcr.26979) Pomen glutation peroksidaze 8 iz repnjakovca za zaščito rastline ''Arabidopsis thaliana'' in bakterije ''Escherichia coli'' pred oksidativnim stresom. Janez Novak, 15. marca 2014
(slovenski naslov povežite z novo stranjo, na kateri bo povzetek)

'''Naslovi odobrenih člankov po temah:'''

'''Gensko spremenjene rastline'''<br>
# Successful high-level accumulation of fish oil omega-3 long-chain polyunsaturated fatty acids in a transgenic oilseed crop (Ruiz-Lopez, N., et al; The plant journal 77, 198-208, 2014; http://www.ncbi.nlm.nih.gov/pubmed/24308505). [[Uspešna priprava gensko spremenjene oljne rastline z visoko vsebnostjo omega-3 polinenasičenih maščobnih kislin.]] Petra Malavašič, 20. marca 2015
#A simpliﬁed and accurate detection of the genetically modiﬁed wheat MON71800 with one calibrator plasmid (Jae Juan, S.,et al; Food Chemistry 176, 1-6, ;http://www.sciencedirect.com.nukweb.nuk.uni-lj.si/science/article/pii/S03088146140196572015 [[Poenostavljena in točna detekcija gensko spemenjene pšenice MON71800 z enim kalibratorskim plazmidom]]. Matej Lesar, 20. marca 2015

'''Gensko spremenjene živali'''<br>
# [[A novel adenoviral vector carrying an all-in-one Tet-On system with an autoregulatory loop for tight, inducible transgene expresion]] (H. Chen; et all.; BMC Biotechnology 2015, 15:4, doi:10.1186/s12896-015-0121-4; http://www.biomedcentral.com/1472-6750/15/4). Edvinas Grauželis, 27. marca 2015 (in English)
# Production of functional active human growth factors in insects used as living biofactories (B. Dudognon, et al; Journal of Biotechnology 184, 229–239, 2014; http://dx.doi.org/10.1016/j.jbiotec.2014.05.030). [[Proizvodnja funkcionalno aktivnih človeških rastnih faktorjev v insektih uporabljenih kot žive biotovarne]] Maxi Sagmeister, 27. marca 2015

'''Okolje'''<br>
# Bioremediation of pesticide contaminated water using an organophosphate degrading enzyme immobilized on nonwoven polyester textiles (Yuan Gao ''et al.'', Enzyme and Microbial Technology, vol. 54, pages 38-44, 10.1.2014, http://www.sciencedirect.com/science/article/pii/S0141022913002044). [[Bioremediacija s pesticidi okužene vode z uporabo encima, ki razgrajuje organofosfate in je vezan na netkan poliestrski tekstil]]. Mitja Crček, 3. aprila 2015
# Biodegradation of atrazine by three transgenic grasses and alfalfa expressing a modified bacterial atrazine chlorohydrolase gene (A. W. Vail ''et al.''; Transgenic Research, 29. 11. 2014; http://link.springer.com/article/10.1007/s11248-014-9851-7). [[Biorazgradnja atrazina s tremi transgenskimi travami in lucerno, ki izražajo gen za modificirano bakterijsko atrazin klorohidrolazo]]. Mirjam Kmetič, 3. aprila 2015

'''Terapevtiki'''<br>
# Mind-controlled transgene expression by a wireless-powered optogenetic designer cell implant (M. Folcher; Nature Communications 5, 1–11, 2014; http://www.nature.com/ncomms/2014/141111/ncomms6392/full/ncomms6392.html) Z EEG nadzorovano izražanje transgena preko brezžično napajanega optogenetskega celičnega vsadka. Luka Smole, 10. aprila 2015
# Glycosylated enfuvirtide: A long-lasting glycopeptide with potent anti-HIV activity; http://pubs.acs.org/doi/full/10.1021/jm5016582 Sebastian Pleško, 10. aprila
# Microbicidal effects of α- and θ-defensins against antibiotic-resistant Staphylococcus aureus and Pseudomonas aeruginosa; http://ini.sagepub.com/content/21/1/17.long. [[Mikrobicidno delovanje α in θ defenzinov na antibiotik-odporne Staphylococcus aureus in Pseudomonas aeruginosa]] Ana Kapraljević, 10. aprila

'''Encimi'''<br>
# Immobilization and controlled release of β-galactosidase from chitosan-grafted hydrogels; http://www.sciencedirect.com/science/article/pii/S0308814615001028. Mojca Banič, 16. aprila 2015
# Construction of efficient xylose utilizing ''Pichia pastoris'' for industrial enzyme production (Li ''et al''; Microbial Cell Factories 14:22, 1-10, 2015; http://www.microbialcellfactories.com/content/14/1/22). Priprava ''Pichie pastoris'', ki učinkovito uporablja ksilozo, za industrijsko proizvodnjo encimov. Špela Tomaž, 17. aprila 2015
# Postharvest application of a novel chitinase cloned from Metschnikowia fructicola and overexpressed in Pichia pastoris to control brown rot of peaches; http://www.sciencedirect.com/science/article/pii/S0168160515000033. Špela Pohleven, 17. aprila 2015

'''Protitelesa'''<br>
# Optimization of heavy chain and light chain signal peptides for high level expression of therapeutic antibodies in CHO cells; http://dx.plos.org/10.1371/journal.pone.0116878. Tjaša Blatnik, 23. aprila 2015
# Ethanol precipitation for purification of recombinant antibodies (A. Tscheliessnig ''et al''; Journal of Biotechnology 188, 17-28, 2014; http://www.sciencedirect.com/science/article/pii/S0168165614007810). Čiščenje rekombinantnih protiteles z obarjanjem z etanolom. Urška Rauter, 24. aprila 2015
# Functional mutations in and characterization of VHH against Helicobacter pylori urease (R. Hoseinpoor ''et al''; Applied Biochemistry and Biotechnology 172, 3079-3091, 2014; http://link.springer.com/article/10.1007/s12010-014-0750-4). Funkcionalne mutacije in karakterizacija VHH proti ureazi ''Helicobacter pylori''. Marko Radojković, 7. maja 2015

'''Cepiva'''<br>
# Development of anti-E6 pegylated lipoplexes for mucosal application in the context of cervical preneoplastic lesions; http://www.sciencedirect.com/science/article/pii/S0378517315001507. Tanja Korpar, 7. maja 2015
# A novel “priming-boosting” strategy for immune interventions in cervical cancer (S. Liao et al.; Molecular Immunology 64, 295-305, 2015, http://www.sciencedirect.com/science/article/pii/S0161589014003460. Nova "priming-boosting" strategija za imunsko posredovanje pri raku materničnega vratu. Anita Kustec, 8. maja 2015
# Potentiation of anthrax vaccines using protective antigen-expressing viral replicon vectors (H.C. Wang et al.; Immunology letters 163, 206-213, 2015, http://www.ncbi.nlm.nih.gov/pubmed/25102364 ) Izboljšava cepiv proti antraksu z uporabo iz virusnih replikonov izvedenih vektorjev, ki omogočajo izražanje zaščitnega antigena. Daša Pavc, 8. maja 2015

'''Male molekule in polimeri'''<br>
# Methanol-induced chain termination in poly(3-hydroxybutyrate) biopolymers: Molecular weight control; http://www.sciencedirect.com/science/article/pii/S0141813014008307. Gašper Lavrenčič, 14. maja 2015
# Purification and characterization of gamma poly glutamic acid from newly Bacillus licheniformis NRC20; http://www.sciencedirect.com/science/article/pii/S0141813014008216. Uroš Stupar, 14. maja 2015
# Iza Ogris, 15. maja 2015
# Chromosomal integration of hyaluronic acid synthesis (''has'') genes enhances the molecular weight of hyaluronan produced in ''Lactococcus lactis'' (R. V. Hmar et al; Biotechnol. J. 9 (12), 2014; http://dx.doi.org/10.1002/biot.201400215) Integracija genov za sintezo hialuronske kisline v kromosom bakterije ''Lactococcus lactis'' izboljša sintezo visokomolekularne hialuronske kisline. Maja Grdadolnik, 15. maja 2015

'''Pretvorba biomase'''<br>
# Effect of pretreatment methods on the synergism of cellulase and xylanase during the hydrolysis of bagasse; http://www.sciencedirect.com/science/article/pii/S0960852415002114. Eva Lucija Kozak, 21. maja 2015
# Third generation biohydrogen production by Clostridium butyricum and adapted mixed cultures from Scenedesmus obliquus microalga biomass; http://www.sciencedirect.com/science/article/pii/S0016236115002550?np=y. Nives Naraglav, 22. maja 2015
# Bio-catalytic action of twin-screw extruder enzymatic hydrolysis on the deconstruction of annual plant material: Case of sweet corn co-products; http://www.sciencedirect.com/science/article/pii/S0926669015000436. Griša Prinčič, 22. maja 2015

'''Metabolično inženirstvo'''<br>
# Engineering lipid overproduction in the oleaginous yeast Yarrowia lipolytica;http://www.sciencedirect.com/science/article/pii/S1096717615000166. Andreja Bratovš, 28. maja 2015
# Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals (Weerawat Runguphana, Jay D. Keasling; Metabolic Engineering, vol 21, January 2014, Pages 103–113; http://www.sciencedirect.com/science/article/pii/S1096717613000670). Metabolično inženirstvo ''Saccharomyces cerevisiae'' za proizvodnjo derivatov maščobnih kislin, ki so primerni za biogorivo in kemikalije. Dominik Kert, 29. maja 2015
# Metabolic engineering of Klebsiella pneumoniae for the production of cis,cis-muconic acid (Jung,H.-M. Jung,M.-Y. Oh, M.-K.;Applied Microbiology and Biotechnology, Published online: 14 February 2015; http://link.springer.com/article/10.1007/s00253-015-6442-3). Metabolno inženirstvo Klebsiella pneumoniae za produkcijo cis,cis-mukonične kisline. Jure Zabret, 29. maja 2015

'''Biološki viri energije'''<br>
# Anodic and cathodic microbial communities in single chamber microbial fuel cells; http://www.sciencedirect.com/science/article/pii/S1871678414021694. Tamara Marić, 4. junija 2015
# Combination of dry dark fermentation and mechanical pretreatment for lignocellulosic deconstruction: An innovative strategy for biofuels and volatile fatty acids recovery; http://www.sciencedirect.com/science/article/pii/S0306261915002196. Jernej Pušnik, 4. junija 2015
# Potential use of feedlot cattle manure for bioethanol production; http://www.sciencedirect.com/science/article/pii/S0960852415001960. Nastja Pirman, 5. junija 2015
# Cellulolytic enzymes produced by a newly isolated soil fungus Penicillium sp. TG2 with potential for use in cellulosic ethanol production; http://www.sciencedirect.com/science/article/pii/S0960148114007022. Jana Verbančič, 5. junija 2015

'''Novi pristopi v molekularni biotehnologiji'''<br>
# Exploring the potential of algae/bacteria interactions; http://www.sciencedirect.com/science/article/pii/S0958166915000269. Matja Zalar, 11. junija
# How close we are to achieving commercially viable large-scale photobiological hydrogen production by cyanobacteria: A review of the biological aspects; http://www.mdpi.com/2075-1729/5/1/997/htm. Monika Škrjanc, 11. junija

MBT seminarji 2015

2015-04-07T19:31:00Z

Ana90:

'''Seznam seminarjev iz Molekularne biotehnologije v študijskem letu 2014/15'''

Tabela za razpored po tednih bo objavljena v spletni učilnici, vanjo pa se vpišite tudi za kratke predstavitve novic (3 min, dvakrat v semestru). Na tej strani bo samo seznam odobrenih člankov za seminar in povezave do člankov in do povzetkov, ki jih morate objaviti najkasneje tri dni pred predstavitvijo (ponedeljek oz. torek). Angleški naslov prevedite tudi v slovenščino - to bo naslov povzetka, ki ga objavite na posebni strani, tako kot so to naredili kolegi pred vami (oz. lani).

Način vnosa:

# The importance of ''Arabidopsis'' glutathione peroxidase 8 for protecting ''Arabidopsis'' plant and ''E. coli'' cells against oxidative stress (A. Gaber; GM Crops & Food 5(1), 2014; http://dx.doi.org/10.4161/gmcr.26979) Pomen glutation peroksidaze 8 iz repnjakovca za zaščito rastline ''Arabidopsis thaliana'' in bakterije ''Escherichia coli'' pred oksidativnim stresom. Janez Novak, 15. marca 2014
(slovenski naslov povežite z novo stranjo, na kateri bo povzetek)

'''Naslovi odobrenih člankov po temah:'''

'''Gensko spremenjene rastline'''<br>
# Successful high-level accumulation of fish oil omega-3 long-chain polyunsaturated fatty acids in a transgenic oilseed crop (Ruiz-Lopez, N., et al; The plant journal 77, 198-208, 2014; http://www.ncbi.nlm.nih.gov/pubmed/24308505). [[Uspešna priprava gensko spremenjene oljne rastline z visoko vsebnostjo omega-3 polinenasičenih maščobnih kislin.]] Petra Malavašič, 20. marca 2015
#A simpliﬁed and accurate detection of the genetically modiﬁed wheat MON71800 with one calibrator plasmid (Jae Juan, S.,et al; Food Chemistry 176, 1-6, ;http://www.sciencedirect.com.nukweb.nuk.uni-lj.si/science/article/pii/S03088146140196572015 [[Poenostavljena in točna detekcija gensko spemenjene pšenice MON71800 z enim kalibratorskim plazmidom]]. Matej Lesar, 20. marca 2015

'''Gensko spremenjene živali'''<br>
# [[A novel adenoviral vector carrying an all-in-one Tet-On system with an autoregulatory loop for tight, inducible transgene expresion]] (H. Chen; et all.; BMC Biotechnology 2015, 15:4, doi:10.1186/s12896-015-0121-4; http://www.biomedcentral.com/1472-6750/15/4). Edvinas Grauželis, 27. marca 2015 (in English)
# Production of functional active human growth factors in insects used as living biofactories (B. Dudognon, et al; Journal of Biotechnology 184, 229–239, 2014; http://dx.doi.org/10.1016/j.jbiotec.2014.05.030). [[Proizvodnja funkcionalno aktivnih človeških rastnih faktorjev v insektih uporabljenih kot žive biotovarne]] Maxi Sagmeister, 27. marca 2015

'''Okolje'''<br>
# Bioremediation of pesticide contaminated water using an organophosphate degrading enzyme immobilized on nonwoven polyester textiles (Yuan Gao ''et al.'', Enzyme and Microbial Technology, vol. 54, pages 38-44, 10.1.2014, http://www.sciencedirect.com/science/article/pii/S0141022913002044). [[Bioremediacija s pesticidi okužene vode z uporabo encima, ki razgrajuje organofosfate in je vezan na netkan poliestrski tekstil]]. Mitja Crček, 3. aprila 2015
# Biodegradation of atrazine by three transgenic grasses and alfalfa expressing a modified bacterial atrazine chlorohydrolase gene (A. W. Vail ''et al.''; Transgenic Research, 29. 11. 2014; http://link.springer.com/article/10.1007/s11248-014-9851-7). [[Biorazgradnja atrazina s tremi transgenskimi travami in lucerno, ki izražajo gen za modificirano bakterijsko atrazin klorohidrolazo]]. Mirjam Kmetič, 3. aprila 2015

'''Terapevtiki'''<br>
# Mind-controlled transgene expression by a wireless-powered optogenetic designer cell implant (M. Folcher; Nature Communications 5, 1–11, 2014; http://www.nature.com/ncomms/2014/141111/ncomms6392/full/ncomms6392.html) Z EEG nadzorovano izražanje transgena preko brezžično napajanega optogenetskega celičnega vsadka. Luka Smole, 10. aprila 2015
# Glycosylated enfuvirtide: A long-lasting glycopeptide with potent anti-HIV activity; http://pubs.acs.org/doi/full/10.1021/jm5016582 Sebastian Pleško, 10. aprila
# Microbicidal effects of α- and θ-defensins against antibiotic-resistant Staphylococcus aureus and Pseudomonas aeruginosa; http://ini.sagepub.com/content/21/1/17.long. [Mikrobicidno delovanje α in θ defenzinov na antibiotik-odporne Staphylococcus aureus in Pseudomonas aeruginosa] Ana Kapraljević, 10. aprila

'''Encimi'''<br>
# Immobilization and controlled release of β-galactosidase from chitosan-grafted hydrogels; http://www.sciencedirect.com/science/article/pii/S0308814615001028. Mojca Banič, 16. aprila 2015
# Construction of efficient xylose utilizing ''Pichia pastoris'' for industrial enzyme production (Li ''et al''; Microbial Cell Factories 14:22, 1-10, 2015; http://www.microbialcellfactories.com/content/14/1/22). Priprava ''Pichie pastoris'', ki učinkovito uporablja ksilozo, za industrijsko proizvodnjo encimov. Špela Tomaž, 17. aprila 2015
# Postharvest application of a novel chitinase cloned from Metschnikowia fructicola and overexpressed in Pichia pastoris to control brown rot of peaches; http://www.sciencedirect.com/science/article/pii/S0168160515000033. Špela Pohleven, 17. aprila 2015

'''Protitelesa'''<br>
# Optimization of heavy chain and light chain signal peptides for high level expression of therapeutic antibodies in CHO cells; http://dx.plos.org/10.1371/journal.pone.0116878. Tjaša Blatnik, 23. aprila 2015
# Ethanol precipitation for purification of recombinant antibodies (A. Tscheliessnig ''et al''; Journal of Biotechnology 188, 17-28, 2014; http://www.sciencedirect.com/science/article/pii/S0168165614007810). Čiščenje rekombinantnih protiteles z obarjanjem z etanolom. Urška Rauter, 24. aprila 2015
# Functional mutations in and characterization of VHH against Helicobacter pylori urease (R. Hoseinpoor ''et al''; Applied Biochemistry and Biotechnology 172, 3079-3091, 2014; http://link.springer.com/article/10.1007/s12010-014-0750-4). Funkcionalne mutacije in karakterizacija VHH proti ureazi ''Helicobacter pylori''. Marko Radojković, 7. maja 2015

'''Cepiva'''<br>
# Development of anti-E6 pegylated lipoplexes for mucosal application in the context of cervical preneoplastic lesions; http://www.sciencedirect.com/science/article/pii/S0378517315001507. Tanja Korpar, 7. maja 2015
# A novel “priming-boosting” strategy for immune interventions in cervical cancer (S. Liao et al.; Molecular Immunology 64, 295-305, 2015, http://www.sciencedirect.com/science/article/pii/S0161589014003460. Nova "priming-boosting" strategija za imunsko posredovanje pri raku materničnega vratu. Anita Kustec, 8. maja 2015
# Potentiation of anthrax vaccines using protective antigen-expressing viral replicon vectors (H.C. Wang et al.; Immunology letters 163, 206-213, 2015, http://www.ncbi.nlm.nih.gov/pubmed/25102364 ) Izboljšava cepiv proti antraksu z uporabo iz virusnih replikonov izvedenih vektorjev, ki omogočajo izražanje zaščitnega antigena. Daša Pavc, 8. maja 2015

'''Male molekule in polimeri'''<br>
# Methanol-induced chain termination in poly(3-hydroxybutyrate) biopolymers: Molecular weight control; http://www.sciencedirect.com/science/article/pii/S0141813014008307. Gašper Lavrenčič, 14. maja 2015
# Purification and characterization of gamma poly glutamic acid from newly Bacillus licheniformis NRC20; http://www.sciencedirect.com/science/article/pii/S0141813014008216. Uroš Stupar, 14. maja 2015
# Iza Ogris, 15. maja 2015
# Chromosomal integration of hyaluronic acid synthesis (''has'') genes enhances the molecular weight of hyaluronan produced in ''Lactococcus lactis'' (R. V. Hmar et al; Biotechnol. J. 9 (12), 2014; http://dx.doi.org/10.1002/biot.201400215) Integracija genov za sintezo hialuronske kisline v kromosom bakterije ''Lactococcus lactis'' izboljša sintezo visokomolekularne hialuronske kisline. Maja Grdadolnik, 15. maja 2015

'''Pretvorba biomase'''<br>
# Effect of pretreatment methods on the synergism of cellulase and xylanase during the hydrolysis of bagasse; http://www.sciencedirect.com/science/article/pii/S0960852415002114. Eva Lucija Kozak, 21. maja 2015
# Third generation biohydrogen production by Clostridium butyricum and adapted mixed cultures from Scenedesmus obliquus microalga biomass; http://www.sciencedirect.com/science/article/pii/S0016236115002550?np=y. Nives Naraglav, 22. maja 2015
# Bio-catalytic action of twin-screw extruder enzymatic hydrolysis on the deconstruction of annual plant material: Case of sweet corn co-products; http://www.sciencedirect.com/science/article/pii/S0926669015000436. Griša Prinčič, 22. maja 2015

'''Metabolično inženirstvo'''<br>
# Engineering lipid overproduction in the oleaginous yeast Yarrowia lipolytica;http://www.sciencedirect.com/science/article/pii/S1096717615000166. Andreja Bratovš, 28. maja 2015
# Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals (Weerawat Runguphana, Jay D. Keasling; Metabolic Engineering, vol 21, January 2014, Pages 103–113; http://www.sciencedirect.com/science/article/pii/S1096717613000670). Metabolično inženirstvo ''Saccharomyces cerevisiae'' za proizvodnjo derivatov maščobnih kislin, ki so primerni za biogorivo in kemikalije. Dominik Kert, 29. maja 2015
# Metabolic engineering of Klebsiella pneumoniae for the production of cis,cis-muconic acid (Jung,H.-M. Jung,M.-Y. Oh, M.-K.;Applied Microbiology and Biotechnology, Published online: 14 February 2015; http://link.springer.com/article/10.1007/s00253-015-6442-3). Metabolno inženirstvo Klebsiella pneumoniae za produkcijo cis,cis-mukonične kisline. Jure Zabret, 29. maja 2015

'''Biološki viri energije'''<br>
# Anodic and cathodic microbial communities in single chamber microbial fuel cells; http://www.sciencedirect.com/science/article/pii/S1871678414021694. Tamara Marić, 4. junija 2015
# Combination of dry dark fermentation and mechanical pretreatment for lignocellulosic deconstruction: An innovative strategy for biofuels and volatile fatty acids recovery; http://www.sciencedirect.com/science/article/pii/S0306261915002196. Jernej Pušnik, 4. junija 2015
# Potential use of feedlot cattle manure for bioethanol production; http://www.sciencedirect.com/science/article/pii/S0960852415001960. Nastja Pirman, 5. junija 2015
# Cellulolytic enzymes produced by a newly isolated soil fungus Penicillium sp. TG2 with potential for use in cellulosic ethanol production; http://www.sciencedirect.com/science/article/pii/S0960148114007022. Jana Verbančič, 5. junija 2015

'''Novi pristopi v molekularni biotehnologiji'''<br>
# Exploring the potential of algae/bacteria interactions; http://www.sciencedirect.com/science/article/pii/S0958166915000269. Matja Zalar, 11. junija
# How close we are to achieving commercially viable large-scale photobiological hydrogen production by cyanobacteria: A review of the biological aspects; http://www.mdpi.com/2075-1729/5/1/997/htm. Monika Škrjanc, 11. junija

MBT seminarji 2015

2015-04-07T19:29:41Z

Ana90:

'''Seznam seminarjev iz Molekularne biotehnologije v študijskem letu 2014/15'''

Tabela za razpored po tednih bo objavljena v spletni učilnici, vanjo pa se vpišite tudi za kratke predstavitve novic (3 min, dvakrat v semestru). Na tej strani bo samo seznam odobrenih člankov za seminar in povezave do člankov in do povzetkov, ki jih morate objaviti najkasneje tri dni pred predstavitvijo (ponedeljek oz. torek). Angleški naslov prevedite tudi v slovenščino - to bo naslov povzetka, ki ga objavite na posebni strani, tako kot so to naredili kolegi pred vami (oz. lani).

Način vnosa:

# The importance of ''Arabidopsis'' glutathione peroxidase 8 for protecting ''Arabidopsis'' plant and ''E. coli'' cells against oxidative stress (A. Gaber; GM Crops & Food 5(1), 2014; http://dx.doi.org/10.4161/gmcr.26979) Pomen glutation peroksidaze 8 iz repnjakovca za zaščito rastline ''Arabidopsis thaliana'' in bakterije ''Escherichia coli'' pred oksidativnim stresom. Janez Novak, 15. marca 2014
(slovenski naslov povežite z novo stranjo, na kateri bo povzetek)

'''Naslovi odobrenih člankov po temah:'''

'''Gensko spremenjene rastline'''<br>
# Successful high-level accumulation of fish oil omega-3 long-chain polyunsaturated fatty acids in a transgenic oilseed crop (Ruiz-Lopez, N., et al; The plant journal 77, 198-208, 2014; http://www.ncbi.nlm.nih.gov/pubmed/24308505). [[Uspešna priprava gensko spremenjene oljne rastline z visoko vsebnostjo omega-3 polinenasičenih maščobnih kislin.]] Petra Malavašič, 20. marca 2015
#A simpliﬁed and accurate detection of the genetically modiﬁed wheat MON71800 with one calibrator plasmid (Jae Juan, S.,et al; Food Chemistry 176, 1-6, ;http://www.sciencedirect.com.nukweb.nuk.uni-lj.si/science/article/pii/S03088146140196572015 [[Poenostavljena in točna detekcija gensko spemenjene pšenice MON71800 z enim kalibratorskim plazmidom]]. Matej Lesar, 20. marca 2015

'''Gensko spremenjene živali'''<br>
# [[A novel adenoviral vector carrying an all-in-one Tet-On system with an autoregulatory loop for tight, inducible transgene expresion]] (H. Chen; et all.; BMC Biotechnology 2015, 15:4, doi:10.1186/s12896-015-0121-4; http://www.biomedcentral.com/1472-6750/15/4). Edvinas Grauželis, 27. marca 2015 (in English)
# Production of functional active human growth factors in insects used as living biofactories (B. Dudognon, et al; Journal of Biotechnology 184, 229–239, 2014; http://dx.doi.org/10.1016/j.jbiotec.2014.05.030). [[Proizvodnja funkcionalno aktivnih človeških rastnih faktorjev v insektih uporabljenih kot žive biotovarne]] Maxi Sagmeister, 27. marca 2015

'''Okolje'''<br>
# Bioremediation of pesticide contaminated water using an organophosphate degrading enzyme immobilized on nonwoven polyester textiles (Yuan Gao ''et al.'', Enzyme and Microbial Technology, vol. 54, pages 38-44, 10.1.2014, http://www.sciencedirect.com/science/article/pii/S0141022913002044). [[Bioremediacija s pesticidi okužene vode z uporabo encima, ki razgrajuje organofosfate in je vezan na netkan poliestrski tekstil]]. Mitja Crček, 3. aprila 2015
# Biodegradation of atrazine by three transgenic grasses and alfalfa expressing a modified bacterial atrazine chlorohydrolase gene (A. W. Vail ''et al.''; Transgenic Research, 29. 11. 2014; http://link.springer.com/article/10.1007/s11248-014-9851-7). [[Biorazgradnja atrazina s tremi transgenskimi travami in lucerno, ki izražajo gen za modificirano bakterijsko atrazin klorohidrolazo]]. Mirjam Kmetič, 3. aprila 2015

'''Terapevtiki'''<br>
# Mind-controlled transgene expression by a wireless-powered optogenetic designer cell implant (M. Folcher; Nature Communications 5, 1–11, 2014; http://www.nature.com/ncomms/2014/141111/ncomms6392/full/ncomms6392.html) Z EEG nadzorovano izražanje transgena preko brezžično napajanega optogenetskega celičnega vsadka. Luka Smole, 10. aprila 2015
# Glycosylated enfuvirtide: A long-lasting glycopeptide with potent anti-HIV activity; http://pubs.acs.org/doi/full/10.1021/jm5016582 Sebastian Pleško, 10. aprila
# Microbicidal effects of α- and θ-defensins against antibiotic-resistant Staphylococcus aureus and Pseudomonas aeruginosa; http://ini.sagepub.com/content/21/1/17.long. [Mikrobicidno delovanje α in θ defenzinov na antibiotik-odporne ''Staphylococcus aureus in Pseudomonas aeruginosa''] Ana Kapraljević, 10. aprila

'''Encimi'''<br>
# Immobilization and controlled release of β-galactosidase from chitosan-grafted hydrogels; http://www.sciencedirect.com/science/article/pii/S0308814615001028. Mojca Banič, 16. aprila 2015
# Construction of efficient xylose utilizing ''Pichia pastoris'' for industrial enzyme production (Li ''et al''; Microbial Cell Factories 14:22, 1-10, 2015; http://www.microbialcellfactories.com/content/14/1/22). Priprava ''Pichie pastoris'', ki učinkovito uporablja ksilozo, za industrijsko proizvodnjo encimov. Špela Tomaž, 17. aprila 2015
# Postharvest application of a novel chitinase cloned from Metschnikowia fructicola and overexpressed in Pichia pastoris to control brown rot of peaches; http://www.sciencedirect.com/science/article/pii/S0168160515000033. Špela Pohleven, 17. aprila 2015

'''Protitelesa'''<br>
# Optimization of heavy chain and light chain signal peptides for high level expression of therapeutic antibodies in CHO cells; http://dx.plos.org/10.1371/journal.pone.0116878. Tjaša Blatnik, 23. aprila 2015
# Ethanol precipitation for purification of recombinant antibodies (A. Tscheliessnig ''et al''; Journal of Biotechnology 188, 17-28, 2014; http://www.sciencedirect.com/science/article/pii/S0168165614007810). Čiščenje rekombinantnih protiteles z obarjanjem z etanolom. Urška Rauter, 24. aprila 2015
# Functional mutations in and characterization of VHH against Helicobacter pylori urease (R. Hoseinpoor ''et al''; Applied Biochemistry and Biotechnology 172, 3079-3091, 2014; http://link.springer.com/article/10.1007/s12010-014-0750-4). Funkcionalne mutacije in karakterizacija VHH proti ureazi ''Helicobacter pylori''. Marko Radojković, 7. maja 2015

'''Cepiva'''<br>
# Development of anti-E6 pegylated lipoplexes for mucosal application in the context of cervical preneoplastic lesions; http://www.sciencedirect.com/science/article/pii/S0378517315001507. Tanja Korpar, 7. maja 2015
# A novel “priming-boosting” strategy for immune interventions in cervical cancer (S. Liao et al.; Molecular Immunology 64, 295-305, 2015, http://www.sciencedirect.com/science/article/pii/S0161589014003460. Nova "priming-boosting" strategija za imunsko posredovanje pri raku materničnega vratu. Anita Kustec, 8. maja 2015
# Potentiation of anthrax vaccines using protective antigen-expressing viral replicon vectors (H.C. Wang et al.; Immunology letters 163, 206-213, 2015, http://www.ncbi.nlm.nih.gov/pubmed/25102364 ) Izboljšava cepiv proti antraksu z uporabo iz virusnih replikonov izvedenih vektorjev, ki omogočajo izražanje zaščitnega antigena. Daša Pavc, 8. maja 2015

'''Male molekule in polimeri'''<br>
# Methanol-induced chain termination in poly(3-hydroxybutyrate) biopolymers: Molecular weight control; http://www.sciencedirect.com/science/article/pii/S0141813014008307. Gašper Lavrenčič, 14. maja 2015
# Purification and characterization of gamma poly glutamic acid from newly Bacillus licheniformis NRC20; http://www.sciencedirect.com/science/article/pii/S0141813014008216. Uroš Stupar, 14. maja 2015
# Iza Ogris, 15. maja 2015
# Chromosomal integration of hyaluronic acid synthesis (''has'') genes enhances the molecular weight of hyaluronan produced in ''Lactococcus lactis'' (R. V. Hmar et al; Biotechnol. J. 9 (12), 2014; http://dx.doi.org/10.1002/biot.201400215) Integracija genov za sintezo hialuronske kisline v kromosom bakterije ''Lactococcus lactis'' izboljša sintezo visokomolekularne hialuronske kisline. Maja Grdadolnik, 15. maja 2015

'''Pretvorba biomase'''<br>
# Effect of pretreatment methods on the synergism of cellulase and xylanase during the hydrolysis of bagasse; http://www.sciencedirect.com/science/article/pii/S0960852415002114. Eva Lucija Kozak, 21. maja 2015
# Third generation biohydrogen production by Clostridium butyricum and adapted mixed cultures from Scenedesmus obliquus microalga biomass; http://www.sciencedirect.com/science/article/pii/S0016236115002550?np=y. Nives Naraglav, 22. maja 2015
# Bio-catalytic action of twin-screw extruder enzymatic hydrolysis on the deconstruction of annual plant material: Case of sweet corn co-products; http://www.sciencedirect.com/science/article/pii/S0926669015000436. Griša Prinčič, 22. maja 2015

'''Metabolično inženirstvo'''<br>
# Engineering lipid overproduction in the oleaginous yeast Yarrowia lipolytica;http://www.sciencedirect.com/science/article/pii/S1096717615000166. Andreja Bratovš, 28. maja 2015
# Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals (Weerawat Runguphana, Jay D. Keasling; Metabolic Engineering, vol 21, January 2014, Pages 103–113; http://www.sciencedirect.com/science/article/pii/S1096717613000670). Metabolično inženirstvo ''Saccharomyces cerevisiae'' za proizvodnjo derivatov maščobnih kislin, ki so primerni za biogorivo in kemikalije. Dominik Kert, 29. maja 2015
# Metabolic engineering of Klebsiella pneumoniae for the production of cis,cis-muconic acid (Jung,H.-M. Jung,M.-Y. Oh, M.-K.;Applied Microbiology and Biotechnology, Published online: 14 February 2015; http://link.springer.com/article/10.1007/s00253-015-6442-3). Metabolno inženirstvo Klebsiella pneumoniae za produkcijo cis,cis-mukonične kisline. Jure Zabret, 29. maja 2015

'''Biološki viri energije'''<br>
# Anodic and cathodic microbial communities in single chamber microbial fuel cells; http://www.sciencedirect.com/science/article/pii/S1871678414021694. Tamara Marić, 4. junija 2015
# Combination of dry dark fermentation and mechanical pretreatment for lignocellulosic deconstruction: An innovative strategy for biofuels and volatile fatty acids recovery; http://www.sciencedirect.com/science/article/pii/S0306261915002196. Jernej Pušnik, 4. junija 2015
# Potential use of feedlot cattle manure for bioethanol production; http://www.sciencedirect.com/science/article/pii/S0960852415001960. Nastja Pirman, 5. junija 2015
# Cellulolytic enzymes produced by a newly isolated soil fungus Penicillium sp. TG2 with potential for use in cellulosic ethanol production; http://www.sciencedirect.com/science/article/pii/S0960148114007022. Jana Verbančič, 5. junija 2015

'''Novi pristopi v molekularni biotehnologiji'''<br>
# Exploring the potential of algae/bacteria interactions; http://www.sciencedirect.com/science/article/pii/S0958166915000269. Matja Zalar, 11. junija
# How close we are to achieving commercially viable large-scale photobiological hydrogen production by cyanobacteria: A review of the biological aspects; http://www.mdpi.com/2075-1729/5/1/997/htm. Monika Škrjanc, 11. junija

MBT seminarji 2015

2015-04-07T19:28:01Z

Ana90:

'''Seznam seminarjev iz Molekularne biotehnologije v študijskem letu 2014/15'''

Tabela za razpored po tednih bo objavljena v spletni učilnici, vanjo pa se vpišite tudi za kratke predstavitve novic (3 min, dvakrat v semestru). Na tej strani bo samo seznam odobrenih člankov za seminar in povezave do člankov in do povzetkov, ki jih morate objaviti najkasneje tri dni pred predstavitvijo (ponedeljek oz. torek). Angleški naslov prevedite tudi v slovenščino - to bo naslov povzetka, ki ga objavite na posebni strani, tako kot so to naredili kolegi pred vami (oz. lani).

Način vnosa:

# The importance of ''Arabidopsis'' glutathione peroxidase 8 for protecting ''Arabidopsis'' plant and ''E. coli'' cells against oxidative stress (A. Gaber; GM Crops & Food 5(1), 2014; http://dx.doi.org/10.4161/gmcr.26979) Pomen glutation peroksidaze 8 iz repnjakovca za zaščito rastline ''Arabidopsis thaliana'' in bakterije ''Escherichia coli'' pred oksidativnim stresom. Janez Novak, 15. marca 2014
(slovenski naslov povežite z novo stranjo, na kateri bo povzetek)

'''Naslovi odobrenih člankov po temah:'''

'''Gensko spremenjene rastline'''<br>
# Successful high-level accumulation of fish oil omega-3 long-chain polyunsaturated fatty acids in a transgenic oilseed crop (Ruiz-Lopez, N., et al; The plant journal 77, 198-208, 2014; http://www.ncbi.nlm.nih.gov/pubmed/24308505). [[Uspešna priprava gensko spremenjene oljne rastline z visoko vsebnostjo omega-3 polinenasičenih maščobnih kislin.]] Petra Malavašič, 20. marca 2015
#A simpliﬁed and accurate detection of the genetically modiﬁed wheat MON71800 with one calibrator plasmid (Jae Juan, S.,et al; Food Chemistry 176, 1-6, ;http://www.sciencedirect.com.nukweb.nuk.uni-lj.si/science/article/pii/S03088146140196572015 [[Poenostavljena in točna detekcija gensko spemenjene pšenice MON71800 z enim kalibratorskim plazmidom]]. Matej Lesar, 20. marca 2015

'''Gensko spremenjene živali'''<br>
# [[A novel adenoviral vector carrying an all-in-one Tet-On system with an autoregulatory loop for tight, inducible transgene expresion]] (H. Chen; et all.; BMC Biotechnology 2015, 15:4, doi:10.1186/s12896-015-0121-4; http://www.biomedcentral.com/1472-6750/15/4). Edvinas Grauželis, 27. marca 2015 (in English)
# Production of functional active human growth factors in insects used as living biofactories (B. Dudognon, et al; Journal of Biotechnology 184, 229–239, 2014; http://dx.doi.org/10.1016/j.jbiotec.2014.05.030). [[Proizvodnja funkcionalno aktivnih človeških rastnih faktorjev v insektih uporabljenih kot žive biotovarne]] Maxi Sagmeister, 27. marca 2015

'''Okolje'''<br>
# Bioremediation of pesticide contaminated water using an organophosphate degrading enzyme immobilized on nonwoven polyester textiles (Yuan Gao ''et al.'', Enzyme and Microbial Technology, vol. 54, pages 38-44, 10.1.2014, http://www.sciencedirect.com/science/article/pii/S0141022913002044). [[Bioremediacija s pesticidi okužene vode z uporabo encima, ki razgrajuje organofosfate in je vezan na netkan poliestrski tekstil]]. Mitja Crček, 3. aprila 2015
# Biodegradation of atrazine by three transgenic grasses and alfalfa expressing a modified bacterial atrazine chlorohydrolase gene (A. W. Vail ''et al.''; Transgenic Research, 29. 11. 2014; http://link.springer.com/article/10.1007/s11248-014-9851-7). [[Biorazgradnja atrazina s tremi transgenskimi travami in lucerno, ki izražajo gen za modificirano bakterijsko atrazin klorohidrolazo]]. Mirjam Kmetič, 3. aprila 2015

'''Terapevtiki'''<br>
# Mind-controlled transgene expression by a wireless-powered optogenetic designer cell implant (M. Folcher; Nature Communications 5, 1–11, 2014; http://www.nature.com/ncomms/2014/141111/ncomms6392/full/ncomms6392.html) Z EEG nadzorovano izražanje transgena preko brezžično napajanega optogenetskega celičnega vsadka. Luka Smole, 10. aprila 2015
# Glycosylated enfuvirtide: A long-lasting glycopeptide with potent anti-HIV activity; http://pubs.acs.org/doi/full/10.1021/jm5016582 Sebastian Pleško, 10. aprila
# Microbicidal effects of α- and θ-defensins against antibiotic-resistant Staphylococcus aureus and Pseudomonas aeruginosa; http://ini.sagepub.com/content/21/1/17.long. [[Mikrobicidno delovanje α in θ defenzinov na antibiotik-odporne ''Staphylococcus aureus in Pseudomonas aeruginosa'']] Ana Kapraljević, 10. aprila

'''Encimi'''<br>
# Immobilization and controlled release of β-galactosidase from chitosan-grafted hydrogels; http://www.sciencedirect.com/science/article/pii/S0308814615001028. Mojca Banič, 16. aprila 2015
# Construction of efficient xylose utilizing ''Pichia pastoris'' for industrial enzyme production (Li ''et al''; Microbial Cell Factories 14:22, 1-10, 2015; http://www.microbialcellfactories.com/content/14/1/22). Priprava ''Pichie pastoris'', ki učinkovito uporablja ksilozo, za industrijsko proizvodnjo encimov. Špela Tomaž, 17. aprila 2015
# Postharvest application of a novel chitinase cloned from Metschnikowia fructicola and overexpressed in Pichia pastoris to control brown rot of peaches; http://www.sciencedirect.com/science/article/pii/S0168160515000033. Špela Pohleven, 17. aprila 2015

'''Protitelesa'''<br>
# Optimization of heavy chain and light chain signal peptides for high level expression of therapeutic antibodies in CHO cells; http://dx.plos.org/10.1371/journal.pone.0116878. Tjaša Blatnik, 23. aprila 2015
# Ethanol precipitation for purification of recombinant antibodies (A. Tscheliessnig ''et al''; Journal of Biotechnology 188, 17-28, 2014; http://www.sciencedirect.com/science/article/pii/S0168165614007810). Čiščenje rekombinantnih protiteles z obarjanjem z etanolom. Urška Rauter, 24. aprila 2015
# Functional mutations in and characterization of VHH against Helicobacter pylori urease (R. Hoseinpoor ''et al''; Applied Biochemistry and Biotechnology 172, 3079-3091, 2014; http://link.springer.com/article/10.1007/s12010-014-0750-4). Funkcionalne mutacije in karakterizacija VHH proti ureazi ''Helicobacter pylori''. Marko Radojković, 7. maja 2015

'''Cepiva'''<br>
# Development of anti-E6 pegylated lipoplexes for mucosal application in the context of cervical preneoplastic lesions; http://www.sciencedirect.com/science/article/pii/S0378517315001507. Tanja Korpar, 7. maja 2015
# A novel “priming-boosting” strategy for immune interventions in cervical cancer (S. Liao et al.; Molecular Immunology 64, 295-305, 2015, http://www.sciencedirect.com/science/article/pii/S0161589014003460. Nova "priming-boosting" strategija za imunsko posredovanje pri raku materničnega vratu. Anita Kustec, 8. maja 2015
# Potentiation of anthrax vaccines using protective antigen-expressing viral replicon vectors (H.C. Wang et al.; Immunology letters 163, 206-213, 2015, http://www.ncbi.nlm.nih.gov/pubmed/25102364 ) Izboljšava cepiv proti antraksu z uporabo iz virusnih replikonov izvedenih vektorjev, ki omogočajo izražanje zaščitnega antigena. Daša Pavc, 8. maja 2015

'''Male molekule in polimeri'''<br>
# Methanol-induced chain termination in poly(3-hydroxybutyrate) biopolymers: Molecular weight control; http://www.sciencedirect.com/science/article/pii/S0141813014008307. Gašper Lavrenčič, 14. maja 2015
# Purification and characterization of gamma poly glutamic acid from newly Bacillus licheniformis NRC20; http://www.sciencedirect.com/science/article/pii/S0141813014008216. Uroš Stupar, 14. maja 2015
# Iza Ogris, 15. maja 2015
# Chromosomal integration of hyaluronic acid synthesis (''has'') genes enhances the molecular weight of hyaluronan produced in ''Lactococcus lactis'' (R. V. Hmar et al; Biotechnol. J. 9 (12), 2014; http://dx.doi.org/10.1002/biot.201400215) Integracija genov za sintezo hialuronske kisline v kromosom bakterije ''Lactococcus lactis'' izboljša sintezo visokomolekularne hialuronske kisline. Maja Grdadolnik, 15. maja 2015

'''Pretvorba biomase'''<br>
# Effect of pretreatment methods on the synergism of cellulase and xylanase during the hydrolysis of bagasse; http://www.sciencedirect.com/science/article/pii/S0960852415002114. Eva Lucija Kozak, 21. maja 2015
# Third generation biohydrogen production by Clostridium butyricum and adapted mixed cultures from Scenedesmus obliquus microalga biomass; http://www.sciencedirect.com/science/article/pii/S0016236115002550?np=y. Nives Naraglav, 22. maja 2015
# Bio-catalytic action of twin-screw extruder enzymatic hydrolysis on the deconstruction of annual plant material: Case of sweet corn co-products; http://www.sciencedirect.com/science/article/pii/S0926669015000436. Griša Prinčič, 22. maja 2015

'''Metabolično inženirstvo'''<br>
# Engineering lipid overproduction in the oleaginous yeast Yarrowia lipolytica;http://www.sciencedirect.com/science/article/pii/S1096717615000166. Andreja Bratovš, 28. maja 2015
# Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals (Weerawat Runguphana, Jay D. Keasling; Metabolic Engineering, vol 21, January 2014, Pages 103–113; http://www.sciencedirect.com/science/article/pii/S1096717613000670). Metabolično inženirstvo ''Saccharomyces cerevisiae'' za proizvodnjo derivatov maščobnih kislin, ki so primerni za biogorivo in kemikalije. Dominik Kert, 29. maja 2015
# Metabolic engineering of Klebsiella pneumoniae for the production of cis,cis-muconic acid (Jung,H.-M. Jung,M.-Y. Oh, M.-K.;Applied Microbiology and Biotechnology, Published online: 14 February 2015; http://link.springer.com/article/10.1007/s00253-015-6442-3). Metabolno inženirstvo Klebsiella pneumoniae za produkcijo cis,cis-mukonične kisline. Jure Zabret, 29. maja 2015

'''Biološki viri energije'''<br>
# Anodic and cathodic microbial communities in single chamber microbial fuel cells; http://www.sciencedirect.com/science/article/pii/S1871678414021694. Tamara Marić, 4. junija 2015
# Combination of dry dark fermentation and mechanical pretreatment for lignocellulosic deconstruction: An innovative strategy for biofuels and volatile fatty acids recovery; http://www.sciencedirect.com/science/article/pii/S0306261915002196. Jernej Pušnik, 4. junija 2015
# Potential use of feedlot cattle manure for bioethanol production; http://www.sciencedirect.com/science/article/pii/S0960852415001960. Nastja Pirman, 5. junija 2015
# Cellulolytic enzymes produced by a newly isolated soil fungus Penicillium sp. TG2 with potential for use in cellulosic ethanol production; http://www.sciencedirect.com/science/article/pii/S0960148114007022. Jana Verbančič, 5. junija 2015

'''Novi pristopi v molekularni biotehnologiji'''<br>
# Exploring the potential of algae/bacteria interactions; http://www.sciencedirect.com/science/article/pii/S0958166915000269. Matja Zalar, 11. junija
# How close we are to achieving commercially viable large-scale photobiological hydrogen production by cyanobacteria: A review of the biological aspects; http://www.mdpi.com/2075-1729/5/1/997/htm. Monika Škrjanc, 11. junija

Mikrobicidno delovanje α in θ defenzinov na antibiotik-odporne Staphylococcus aureus in Pseudomonas aeruginosa

2015-04-07T19:21:40Z

Ana90: New page: <h3>UVOD</h3> Bakterije odporne na antibiotike predstavljajo tako zdravstveni kot tudi ekonomski problem. Številni antibiotiki ciljajo na specifične bakterijske encime ali reakcije in ...

<h3>UVOD</h3>

Bakterije odporne na antibiotike predstavljajo tako zdravstveni kot tudi ekonomski problem. Številni antibiotiki ciljajo na specifične bakterijske encime ali reakcije in lahko se zgodi, da geni odgovorni zanje mutirajo pod vplivom selekcijskega pritiska kar lahko pripelje do odpornosti na antibiotike. Velik potencial v boji proti patogenim bakterijam kažejo protimikrobne snovi, ki vplivajo na integriteto celične membrane . Gre za sesalske α in θ defenzine , ki so kationski peptidi s specifičnmi tridisulfidnimi mrežami. Alfa defenzini se večinoma akomulirajo v neutrofilnih azurofilnih granulah, najdemo jih tudi v naravnih celicah ubijalkah. θ defenzine pa so zaenkrat odkrili le v kostnem mozgu nekaterih primatov. Defenzini delujejo tako, da se selektivno vežejo na bakterijske membrane in povzročijo izhajanje kalijevih ionov in hranil, depolarizacijo membrane in posledično celično smrt. Da preverijo hipoteze o njihovem baktericidnem delovanju, so preverili učinek α defenzinov (Crp-4, RMAD-4, HNP 1-3) in θ defenzinov (RTD 1) na meticilin odporno ''Staphylococcus aureus'' (MRSA), skupaj z vankomicin rezistentnimi sevi in na ciprofloksacin odporne ''Pseudomonas aeruginosa'' (PA).

<h3>MATERIALI IN METODE</h3>

Peptide so homogenizirali z reverzno-faznim HPLC in karakterizirali z MALDI-TOF MS in AU-PAGE. Rekombinantne Crp - 4 in RMAD -4 peptide so izrazili v bakteriji ''Escherichia coli'' kot N- terminalni fuzijski protein s His6 oznako preko pET28a ekspresijskega vektorja. Sledila je izolacija s His6 označenih fuzijskih Crp- 4 peptidov z afinitetno kromatografijo z uporabo nikelj-nitrilotriocetno kislino. Cianogen bromid je služil za odstranitev His6 fuzije, proteine pa so prečistili s sekvenčnim C18 Rp-HPLC in določili molekulsko maso z MALDI-TOF MS. Natančna določitev strukture je bila opravljena z metodo NMR. HNP so izolirali iz vzorcev obogatenih z nevtrofilnimi granulami pripravljenimi iz perifernih levkocitov. HNP 1-3 so pridobili z gelsko permeacijsko kromatografijo z uporabo BioGel P10, HNP 2 so očistili iz druženih HNP 1-3 z metodo C18 RP-HPLC. Θ defenzin RTD-1 pa so sintetizirali.
Klinične izolate so pridobili iz hospitaliziranih pacientov. MRSA izolate odporne na vankomicin so določili z uporabo Etest glikopeptid rezistentne detekcijske metode. PA dovzetnost na ciproflokasin pa so določili z mikrodilucijsko metodo. Ciprofloksacinska odpornost je bila definirana z minimalno inhibitorno koncentracijo 2μg/ml ali več. MRSA in PA seve, ki povzročajo pljučnico, bakteriemijo, infekcije ran in urinarnega trakta so izbrali kot reprezentativne klinične izolate z različnimi stopnjami odpornosti na vankomicin (MRSA) in ciprofloksacin (PA). Alfa in θ defenzine so testirali na baktericidno aktivnost pri kliničnih izolatih MRSA in PA v in vitro celičnih suspenizijskih testih. Preživetje bakterij glede na izpostavitev peptidom so določili z štetjem CFU. 1h po izpostavitvi peptidom so peptidne in bakterijske mešanice nagojili na agarne plošče.

<h3>REZULTATI</h3>

Crp- 4, RMAD -4 in RTD – 1, testirani s kliničnimi izolati MRSA z različnimi stopnjami odpornosti na vankomicin, podobno zmanjšajo viabilnost sevov, ne glede na vankomicinsko rezistenco. Klinični izolati so bili občutljivi na vse testirane defenzine. Enako so storili tudi s sevi PA odporne na ciprofloksacin, ki so jih testirali s Crp-4, RMAD-4, RTD-1 in HNP 1-3 ter določali učinkovitost glede na različne odpornosti na ciproflokascin, mesta izolacije,TTSS efektorskega genotipa in citotoksični potencial. Ugotovili so, da je Crp-4 bolj učinkovit pri PA izolatih, RMAD-4 pa pri izolatih MRSA. Pri sevih pridobljenih iz različnih mest (opekline, urin, kri), ki se med sabo tudi fentipsko razlikujejo, sta Crp-4 in RMAD-4 delovala z enako občutljivostjo. Sicer so pri RMAD-4 opazili večjo aktivnosti pri sevih izoliranih iz opeklin, kar nakazuje morebitno povezavo med mestom izolacije PA in odpornostjo na α defenzine. Rezultati so pokazali,da HNP1-3 in HNP-2 nimajo velikega vpliva na preživetje PA, napredek niso opazili niti pri koncetracijah pri katerih sicer Crp-4 in RTD-1 delujeta močno baktericidno.
Tudi kationski naboj vpliva na baktericidno delovanje defenzinov. Tako je na primer Crp-4 veliko bolj učinkovit proti PA izolatom, RMAD pa proti MRSA. Verjetno gre tu za vpliv različnih porazdelitev površinkih nabojev ali hidrofobnosti.

<h3>ZAKLJUČKI</h3>

Antimikrobni peptidi predstavljajo dobro platformo za razvoj novih terapevtikov proti odpornim patogenom. Defenzini vplivajo na celično integriteto, vplivajo na delovanje membrane in se po mehanizmu razlikujejo od beta laktamskih inhibitorjev in vankomicina. Defenzine bi tako lahko združili z obstoječimi antibiotiki in tako izboljšali njihovo delovanje ter posledično povečali učinkovitost zdravljenja proti odpornim patogenom.

Engineering a mevalonate pathway in Escherichia coli for production of terpenoids

2015-01-20T00:02:37Z

Ana90: /* Synthase gene asemby and amorphadiene production */

'''Ana Kapraljević'''

[http://www2.lbl.gov/ttd/publications/2837pub1.pdf Engineering a mevalonate pathway in Escherichia coli for production of terpenoids]
Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling ;Nature biotehnology; July 2003; vol 21; pages 796-802

== INTRODUCTION ==

Plants can synthesise and accumulate a wide range of small molecules or natural products involved in psyhological an ecological process. With the knowledge we know about their biosyntesis we used them as commercial flavors, fragnance compunds, colorants and terapeutical drugs. Their therapeutic potential is used for production of anticancer or antimalarial drugs, such as artemisnin. The most numerous and diverse class of natural products used in production for antimalarial drug are isoprenoids or more corectly their subclass, terpenoids. Malaria represent a major health problem in tropic and subtropic. Majority of death occur in children living in Africa. But the problem is that terpenoids are in plants produced in very small quantites and consequently not very economically feasible. For that reason the drugs for treatin malaria, artemisnin, is in short supply and is still unaffordable for most people living in malaria-endanger countries. Scientists are now focusing on alternative and cheaper way for terpenoid synthesis that can be enchanched and became economically acceptable. The alternative way is the expression of synthetic amorphadiene, precursor to artemisnin, and mevalonate isoprenoid way from yeas ''Saccharomyces cerevisiae'' in bacteria. In this paper I will summerize the project made in the production of amorphadiene by biotehnology and synthetic biology approach [1,2].

=== Artemisnin ===

Artemisnin is a substance that has antimalarial potential. Malaria is a infectious disease caused by parasitic protozan of the ''Plasmodium'' species. In the certain parts of the world malaria takes lives of more people worldwide than any other infecious disease, so it is a global problem. Death to malaria has been nowdays reduced thanks to the use of artemisnin-based combination therapy. Artemisnin is a natural compoud from plant ''Artemisia annua''. Chemically it is a sesquiterpene lactone, amorphadiene. The total sythesis of artemisnin is difficult and not very economical,so the new aproach is to put the hopes in sythetic biology and develop the new way, synthetic way, for its production [1,3].

=== Biology of terpenoids ===

For initiation of any synthetic biology process in metabolic engineering in that matter it is important to have some basic understanding of biosynthesis and regulation of terpenoids. Terpenoids, a class of isoprenoids, are natural products often sythesized by plants. They represent a large and diverse class and have many functions as primarly metabolites involved in growth and development of plants, or as secundary metabolites that optimize the interaction between the plant and the environment. Eventhought they are structural diverse, the share a common biosynthetic origin and follow similar synthesis tracks. All terpenoids are derived from repetitive fusion of isoprene units (C5 H8) [3]. The number of isoprene units determines their classification. For isoprenoid synthesis plants use mevalonate-dependent (MEV) pathway and mevalonte-independent pathway or alternative deoxyxylulose 5-phosphate pathway (DXP). In higher plants the biosynthesis begins with the generation of isopentenyl pyrophosphate (IPP). IPP is derived via the classic mevalonic acid pathway from acetly-coenzime A (acetyl-CoA). The IPP is isomerized to its allylic isomer dimethylallyl pyrophosphate (DMAPP) [3]. The continous condensation of IPP and DMAPP units leads to the formaton of prenylated pyrophosphates, the immediate precursors of the different terpenoid classes. So IPP and DMAPP are combined to form larger isoprenoids, such as farnesyl diphosphate (FPP). Condensation of IPP and DMAPP than units leads to the formation of precursors of the different terpenoid classes. Shematic production of terpenoids in ilustraded in this picture. Similary to plants, yeast ''Sacharomyces cerevisae'' uses the clasical mevalonate pathway for the synthesis of isoprenoids[3]. Prokariontes mostly use the DXP pathway for production IPP and DMAPP. This is an alternative pathway for IPP synthesis. This pathway uses ''Escherichia coli'' and some other bacteria for their terpenoid synthesis. IPP and DMAPP precurosr are essential to E. Coli for prenylation (addition of hydrophobic molecules to a protein or chemical compound) of tRNA and the synthesis of farnesyl pyrophosphate (FPP). FPP is then used for quinone (class of organic compounds) and cell wall synthesis. The DPX pathway begins with the glycolytic intermediates glyceraldehyde-3-phosphate (G3P) and pyruvate and continues through enzymatic steps for production of IPP and DMAPP. Therefore IPP and DMAPP are the end product in both way,MEV and DPX pathway [1,3].

== METABOLIC ENGINEERING FOR PRODUCTION OD TERPENOIDS ==

Metabolic engineering represents an improvement of production, construction, or cellular properties through the modification of specific biochemical reactions. It is described as the use of genetic engineering to reshape or modify the metabolism of organism. The aim is to optimize genetic and regulatory processes in cells to increase the production of a certain substance that we find in nature. It is based on optimisation of existing biochemical pathways or the introduction of natural pathway components in bacteria, yeast or plants. The main goal is the production of high-yield of specific metabolites that we can use in medicine or biotechnology. With progress in synthetic biology we can design a biologic funcion that do not exist in nature. Consequently, we can create and construct new biological components or redesign exisitng biological systems. In plants, most terpenoids are produced in a very small quantities in their natural sources. Extraction of this compouds from plants is expensive and therfore not economicaly accetable. So there is a big disagreement between the insistence for supply of terpenoids and therefore that leads to delay for their widespread application. With the understanding of terpenoid synthesis and progress in synthetic biology we are now able of redesign and modify the isoprenoid pathway to increase the terpenoid productivity [2,4]. ([http://www.disketa.si/media/uploads/2015-01/cd201ade6a167488767dfb1b118587bc.jpg See image here])

=== Microbal host ===

To elimante the need for plant extraction it is practical to produce terpenoid compouds at high yield in microbal host by introducing a heterlogous isoprenoid pathway into bacteria ''E.coli.'' Microorganisms, such as bacteria are attractive alternatives as hosts because they have rapid doubling time, they can achieve robussnes under process conditions and because of the simplicity of product putrification. And the costs are significantly lower than working with plants. With the engineering the DXP pathway we can increase the supply of isoprenoid precurosor needed for high level productio of isoprenoids in E.coli [1,3].

=== Operons ===

Operon is a a funcional unit of DNA that contains a cluster of genes under the control of a single promotor. The synthesis of natural or synthetic products involves the introduction of a large number of genes. In this case these genes were encoding the enzymes needed for metabolic pathway. So it is useful to put certain genes under the control of a single promotor. For that reason Plac promotor was used in this study [1,5].

== EXPERIMENT ==

=== Synthase gene asemby and amorphadiene production ===

For high level production and changing the difficulties in expressing teprene synthases they first synthesized and expressed a codon-optimized variants of ADS . ADS is a gene encoding amorphadiene synthase and is sequentially oxidised by citorchromeP450 CYP71AV1 and reduced by artemisinic aldehyde reductase (DBR2). Amorphadyene synthase is an enzyme and catalyse the first step in the anitmalarial drug artemisnin synthesis. It has an inportant role in catalysing the reaction of FPP to amorphadiene. Beacause of its importaint step towards artemisnin biosynthesis it was designed and improved for the purpuse to acheve high-level expression in E.coli. For ADS gene synthesis they used two step assemly and one step amplification PCR.(1) Assemby PCR is a useful technique for production novel gene sequences. With this method we can put together short fragments of DNA and it is also a good choice for gene construction. Reserches in this article analysed the sequence of three ADS genes from independet clones and identified mutations in each of the genes. Than, two site directed mutagenesis reaction was used for assembly and generation a functional ADS gene from two clones. Than they measure the amorphadiene production by the amorphadiene synthase in E.coli DH10B with natural (nonengineered DPX pathway) and in ''E.coli'' with engineered DPX pathway. ''E. coli'' with engineerd pathway was harboring the pSOE4 plasmid (DXP pathway operon with ''dxs,IppHp and ispA'' gene). The result sugested that FPP synthesis limited amorphadiene production in this engineered host [1,6]. ([http://www.disketa.si/media/uploads/2015-01/0bddf07f946e6f5be0638090dee56490.jpg See image here])

=== Egineering the mevalonate-dependet pathway in E.coli ===

The aim was to increase the intracellular concentration of FPP substrate. Genes encoding the MEV pathway from S.cerevisae was assembled into operons and expressed in E.coli. Because of the complexity of engineering numerous gene biosynthetic pathway, genes were divided into to operons, top and bottom.Top operon, MevT, consisted of three enzymes: acetoacetyl-CoA thiolase (''atoB''), HMG-CoA synthases (''HMGS'') and HMG-CoA reductase (''tHMGR''). This operon was responsible for transformation of acetyl-coA to mevalonate. Thre different bottom operons pMevB (''ERG12, ERG8,MVD1''), pMBI (''ERG12, ERG8, MVD1, idi'') and pMBIS (''ERG12, ERG8, MVD1, idi, ispA'') transformed mevalonate to IPP, DMAPP and consequently to FPP. The next step was to analyse and test the funcionality of this pathway. For this purpuse E.coli strand DYM1 with incoplemplete isoprenoid synthesis was transformed with plasmid expressing three bottom operon constuct- pMevB, pMBI and pMBI. E.coli strand DYM1 had a deletion in the ispC gene for encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase, and was not able to synthesise 2-C-methyl-D-erythritol 4-phosphate(MEP), which is important intermediate in isoprenoid biosynthesis[1]. Because of this deletion the synthesis of precursors IPP and DMAP is blocked. So then they tested what is going to happen if they grew the strain DYM1 in the presence of 2-C-methyl-D-ertihritol and in medium with mevalonate without methylerythritol since DYM1 strain was not capable to synthesisie MEP. As expected , all strains expressing operon from S.cerevisiae grew on the plates in the presence of 2-c-methyl-D-erythritol. But only strains with pMBI or pMBIS grew on plates with 1mM mevalonate in the absence of methylerythol. This was the conformation that synthetic MBI and MBIS operons were functional, because they were capable to support the growth of E.coli without ispC gene by supplying cells with IPP and DMAPP. Next they completed the mevalonate pathway by transforming the pMevT plasmid (expressing three genes mentioned before-atoB, HMGS and tHMGR) with pMBI or pMBIS. This coexpresion of two operons, pMevT and pMBI /pMBIS ,completed the mevlonate pathway and allowed the synthesis of sesquiterpene precursors from acetyl-CoA, IPP and DMAPP. Therefore, coexpression complemented the ''ispC'' deletion even in the absence of mevalonate, what was the proof, that MevT operon was functional [1,7].

=== Amorpadiene synthesis from mevalonate ===

The aim was to achieve high-level production of amorphadiene and to find out if the amount of FPP was limiting amoprhadiene output. As mentioned, ''E.coli'' was used as heterologous microorganism in which coupled mevalonate pathway with amorphadiene synthesis. Cells with ''ADS'' gene were coexpressed with MBIS operon and grown in medium with increasing amounts (0 to 40 mM) of exogenus mevalonate. The culture extract was analysed with gas chromatography-mass spectrometry, analytical method for identification different substances in a test sample. It combines the features of mass spectrometry and gas-liquid chromatography. Results showed that MBIS operon did not limit the amorphadiene production at the highest mevalonate concentration(40mM). ([http://www.disketa.si/media/uploads/2015-01/7711d592584a53282f2ecb0fd678ef40.jpg See image here]) Cultures with 40 mM mevalonate added produced much more amoprhadiene. With time, amorphadiene concentration was dropping beacause of the loss of the volatile terpene[1]. By adding more than 10 mM mevalonate in control cultures without ADS, the severe growth ihibition has been reported. ([http://www.disketa.si/media/uploads/2015-01/2a21f55f0fea31ffcbe89c866c9150f7.jpg See image here]) To discover the cause ofthis inhibition, the growth of ''E.coli'' DH10B from strains with empty, pMKPMK, pMevB, pMBI or pMBIS plasmid was measured in media with increasing concentrations of mevalonate. The addition of 5mM mevalonate to the medium inhibited the growth of cells with pMevB. But 5mM concentrations of mevalonate did not alter the growth of cells with pMKPMK, PMBI or pMBIS plasmid. This indicated that the accomulation of IPP is toxic and inhibits normal cell growth. The accmulation mostly occurs in cells with high flux through mevalonate pathway. The next step was comparison of intracellular prenyl pyrophosphate (FPP) in strains. This was done by feeding the cells harboring the different mevalonte operon construct with radiolabeled mevalonate. Than the labeled metabolites were tracked. For this experiment cell suspensions of E.coli harboring pMevB+pTrc99A, pMBI+ pTrc99A, pMBIS+ pTrc99A or pMBIS+pADS were used. pTRc99A is the bacterial expression vector with inducible to IPTG lacI promoter. The result showed that strains with MevB operon accumulated IPP but not FPP, when MBI and MBIS strains accumulated FPP. ([http://www.disketa.si/media/uploads/2015-01/31ad7a215f016dc26aceacf92eb46712.jpg See image here]) Because of the simultaneous expression of the amorphadiene synthase the waste of FPP that accumulated in the MBIS host was consumed. This was shown by a decrease in intracellular FPP. Cells expresing MBIS acumulated FPP and showed growth inhibition in the presence of 10 mM mevalonate. The prediction was that the coexpression of ADS would ease the growth inhibition by forwarding the prenyl pyrophosphate intermediates to volatile terpene olefin. For this test, the cell with pMBIS and empty expression vector pTrc99A (without ADS)and cells with pMBIS and pADS vectors were used. The medium was supplemented with mevalonate concentations ( 0mM, 5mM, 10mM, 20mM or 40mM). Two hours after addition of mevalonate and inducer IPTG, the growth inhibition was observed. ([http://www.disketa.si/media/uploads/2015-01/94b5f3d5636fe767316f770f3e2b0448.jpg See image here]) The result showed that the strains with pMBIS +ADS started to grow, but strain lacking the ADS still showed growth inhbition at higher concentrations of mevalonate. This supported the conclusion that converion of FPP to amophadiene has an important role in minimising growth inhibition. Briefly these result showed that engineered mevalonate patway produces high levels of prenyl pyrophosphate precursors. If the IPP isomerase, FPP synthase and terpen synthase is absent, IPP can acumulate and be toxic to the cells. The toxic levels of intracellular prenyl pyrphosphate can accumulate [1].

=== Amorphadiene synthesis from acetyl-Coa ===

The main goal was to achieve high amoprhaidene production from a simple and inexpensive carbon source. ''E.coli'' strains harboring pMevT,pMBIS and pADS plasmids were transformed with pMevT plasmid. Subsequently the strains were tested for ability to produce amoprhadiene in the absence of exogenous mevalonate. The comparison between E.coli expressing the native DPX pathway and engineered pathway as made. They tested native DXP pathway, engineered DPX pathway, mevalonate bottom pathway in the absence of mevalonate, mevlonate bottom pathway in medium suplemented with mevalonate, the complete mevalonate pathway and the complete mevalonate pathway supplemented with 0,8% glycerol. Glycerol was addded to investigate the effect of amorphadiene production of supplying as single carbon source and for prolongation of amorphadiene production. Breifly the resulut showed that expression of the mevalonate-dependent isoprenoid biosynthetic pathway delivers hight levels of isoprenoid precursor for the production of sesquiterpene [1]. ([http://www.disketa.si/media/uploads/2015-01/a31fe07a7c66854b1dc3ff36f8ed01ef.jpg See image here])

== DISSCUSION ==
The identification of genes encoding the enzymes involved in the artemisinin-biosynthesis pathway together with advances in metabolic engineering provides new options for engineering artemisinin biosynthesis. Heterologous production of artemisnin in microorganism such as ''E.coli''. is an alternative way for production of artmeinsin in high yields. But eventhougt micoorganisms has shown great potential in that matter, optimisation of these heterlogous pathways still require maximal titer. Inequal gene expression can lead to accumulation of toxic metabolic intermediates and to over or under production of enzymes involved in pathway. (4). Conclusion based on these study shows that poor expression of the amoprhaidene synthase genes (''ADS'') limits high terpene olfein yields from the host. To achieve better results , in this study they chose to synthesiyse an amophaiene synthase gene (ADS). The comparison between ''E.coli'' expressing native sesquiterpene synthase genes and'' E.coli'' expressing the synthetic ADS gene showed big improvement in terpene synthesis due to synthetic ADS gene. The native DPX pathway showed poor supply of the prenyl pyrophosphate precuror. And that limited terpenoid -carotenoid yields in ''E.coli.'' With the metabolic engineering of the DPX pathway the flux of carotenoid accumulation showed signifcant increase. By using the mevalonate-dependent isoprenoid pathway from yeast S. cerevisae and engineering that pathway in bacteria ''E.coli'', we avoid the native regulatory elements found in yeast while bypassing those of ''E.coli'' DXP pathway. In this study results showed growth inhibition in cells without ''ADS'' due to the vast excess of preny pyrophosphate. But the coexpression of a synthetic sesquiterpene synthase can contribute to reducing the excess pool of precursors and eliminate growth inhibition. In this study total biosynthesis of artemisnin was not completely achieved. But these results can help in further investigation for poduction of artemisnic acid. This acid can then be putrified and chemically converted into artemsinin. [1,7].

== CONCLUSIONS ==
To sum things up, artmeinsin, a sesquiterpene isolated from Artemisia annua,represent a novel drug for malaria treatment. The present artemisnin derivates are too expensive for general use and plants can not produce artmeinsin in big quantities. This fact inspired scientist to focus on developing the way for production of artemisnin in high quantities and to avoid big costs. The promising tool in alternative source of artmeinsin is the use of microoganisms. The aim is to transfer the genes encoding enzymes involved in biosynthetic pathway for artemisnin into a hight producing host organism. Several study have been reporting on engineering the metabolic pathway in S.cerevisae to produce the precursor for artmeisnic acid. With the effort in synthetic biology they used modified mevalonate pathway . With the help of syntehtic bilogy the yeast cells were engineerd to express ezymes needed for production of artmemisnic acid. But in this paper the main focus was on engineering a mevalonate pathway in bacteria ''E.coli''. By engineering a new metabolic pathway in ''E.coli'' we can easily synthesise a precursor nedeed for artemisnin production. The goal is to produce drugs in better and cheape ways. But, why focus on artemisnin? Well, acording on recent studies artemisnin shows to be very sucesfull for the treatmen all strain of malaria. More than a milion people affected with malaria have already been cured by arteminsin. As already mentioned , because of very hight costs and limited production of arteminin in plants most populations in Africa can not afford it. Progress made in synthetic biology and ability to produce amorphadiene in microorganisms in big quantites opens a new possibility to produce low cost artemisnin and contributes to the brighter future od antimalaria drug deveopment, and therefore saving milions from suffering from malaria. [1,2,3].

[1]Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nature biotehnology; vol 21; pages 796-802, July 2003

[2] T. Mosses, J. Pollier, J. M. Thevelein, A. Goossens. Bioengineering of plant (tri)terpenoids: from metabolic engineering of plants to synthetic biology in vivo and in vitro. New Pathyologist. 200: 27-43, 2013

[3] M.Farhi, M. Kozin, S. Duchin, A. Vainstein. Metabolic engineering of plants for artemisinin synthesis. Biotehnology and genetic engineering rewiews.Vol.29,No.2: 135-148, 2013

[4] Synthetic biology ; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Synthetic_biology http://en.wikipedia.org/wiki/Synthetic_biology]

[5]Operon; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Operon http://en.wikipedia.org/wiki/Operon]

[6] M. K. Julsing, G. van Pouderoyen, J. van der Veen,H. J. Woerdenbag, O. Kayser, W. J. Quax. Amorphadiene synthase: probing the active site model with directed mutagenesis. Directed mutagenesis of AMDS.Chapter 4: 47-56

[7]J.R. Anthony, L. C. Anthony, F. Nowroozi, G. Kwon, J.D. Newman, J.D. Keasling. Optimization of themevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene. Metabolic Engineering. 11: 13-19, 2009

Engineering a mevalonate pathway in Escherichia coli for production of terpenoids

2015-01-20T00:00:59Z

Ana90: /* CONCLUSIONS */

'''Ana Kapraljević'''

[http://www2.lbl.gov/ttd/publications/2837pub1.pdf Engineering a mevalonate pathway in Escherichia coli for production of terpenoids]
Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling ;Nature biotehnology; July 2003; vol 21; pages 796-802

== INTRODUCTION ==

Plants can synthesise and accumulate a wide range of small molecules or natural products involved in psyhological an ecological process. With the knowledge we know about their biosyntesis we used them as commercial flavors, fragnance compunds, colorants and terapeutical drugs. Their therapeutic potential is used for production of anticancer or antimalarial drugs, such as artemisnin. The most numerous and diverse class of natural products used in production for antimalarial drug are isoprenoids or more corectly their subclass, terpenoids. Malaria represent a major health problem in tropic and subtropic. Majority of death occur in children living in Africa. But the problem is that terpenoids are in plants produced in very small quantites and consequently not very economically feasible. For that reason the drugs for treatin malaria, artemisnin, is in short supply and is still unaffordable for most people living in malaria-endanger countries. Scientists are now focusing on alternative and cheaper way for terpenoid synthesis that can be enchanched and became economically acceptable. The alternative way is the expression of synthetic amorphadiene, precursor to artemisnin, and mevalonate isoprenoid way from yeas ''Saccharomyces cerevisiae'' in bacteria. In this paper I will summerize the project made in the production of amorphadiene by biotehnology and synthetic biology approach [1,2].

=== Artemisnin ===

Artemisnin is a substance that has antimalarial potential. Malaria is a infectious disease caused by parasitic protozan of the ''Plasmodium'' species. In the certain parts of the world malaria takes lives of more people worldwide than any other infecious disease, so it is a global problem. Death to malaria has been nowdays reduced thanks to the use of artemisnin-based combination therapy. Artemisnin is a natural compoud from plant ''Artemisia annua''. Chemically it is a sesquiterpene lactone, amorphadiene. The total sythesis of artemisnin is difficult and not very economical,so the new aproach is to put the hopes in sythetic biology and develop the new way, synthetic way, for its production [1,3].

=== Biology of terpenoids ===

For initiation of any synthetic biology process in metabolic engineering in that matter it is important to have some basic understanding of biosynthesis and regulation of terpenoids. Terpenoids, a class of isoprenoids, are natural products often sythesized by plants. They represent a large and diverse class and have many functions as primarly metabolites involved in growth and development of plants, or as secundary metabolites that optimize the interaction between the plant and the environment. Eventhought they are structural diverse, the share a common biosynthetic origin and follow similar synthesis tracks. All terpenoids are derived from repetitive fusion of isoprene units (C5 H8) [3]. The number of isoprene units determines their classification. For isoprenoid synthesis plants use mevalonate-dependent (MEV) pathway and mevalonte-independent pathway or alternative deoxyxylulose 5-phosphate pathway (DXP). In higher plants the biosynthesis begins with the generation of isopentenyl pyrophosphate (IPP). IPP is derived via the classic mevalonic acid pathway from acetly-coenzime A (acetyl-CoA). The IPP is isomerized to its allylic isomer dimethylallyl pyrophosphate (DMAPP) [3]. The continous condensation of IPP and DMAPP units leads to the formaton of prenylated pyrophosphates, the immediate precursors of the different terpenoid classes. So IPP and DMAPP are combined to form larger isoprenoids, such as farnesyl diphosphate (FPP). Condensation of IPP and DMAPP than units leads to the formation of precursors of the different terpenoid classes. Shematic production of terpenoids in ilustraded in this picture. Similary to plants, yeast ''Sacharomyces cerevisae'' uses the clasical mevalonate pathway for the synthesis of isoprenoids[3]. Prokariontes mostly use the DXP pathway for production IPP and DMAPP. This is an alternative pathway for IPP synthesis. This pathway uses ''Escherichia coli'' and some other bacteria for their terpenoid synthesis. IPP and DMAPP precurosr are essential to E. Coli for prenylation (addition of hydrophobic molecules to a protein or chemical compound) of tRNA and the synthesis of farnesyl pyrophosphate (FPP). FPP is then used for quinone (class of organic compounds) and cell wall synthesis. The DPX pathway begins with the glycolytic intermediates glyceraldehyde-3-phosphate (G3P) and pyruvate and continues through enzymatic steps for production of IPP and DMAPP. Therefore IPP and DMAPP are the end product in both way,MEV and DPX pathway [1,3].

== METABOLIC ENGINEERING FOR PRODUCTION OD TERPENOIDS ==

Metabolic engineering represents an improvement of production, construction, or cellular properties through the modification of specific biochemical reactions. It is described as the use of genetic engineering to reshape or modify the metabolism of organism. The aim is to optimize genetic and regulatory processes in cells to increase the production of a certain substance that we find in nature. It is based on optimisation of existing biochemical pathways or the introduction of natural pathway components in bacteria, yeast or plants. The main goal is the production of high-yield of specific metabolites that we can use in medicine or biotechnology. With progress in synthetic biology we can design a biologic funcion that do not exist in nature. Consequently, we can create and construct new biological components or redesign exisitng biological systems. In plants, most terpenoids are produced in a very small quantities in their natural sources. Extraction of this compouds from plants is expensive and therfore not economicaly accetable. So there is a big disagreement between the insistence for supply of terpenoids and therefore that leads to delay for their widespread application. With the understanding of terpenoid synthesis and progress in synthetic biology we are now able of redesign and modify the isoprenoid pathway to increase the terpenoid productivity [2,4]. ([http://www.disketa.si/media/uploads/2015-01/cd201ade6a167488767dfb1b118587bc.jpg See image here])

=== Microbal host ===

To elimante the need for plant extraction it is practical to produce terpenoid compouds at high yield in microbal host by introducing a heterlogous isoprenoid pathway into bacteria ''E.coli.'' Microorganisms, such as bacteria are attractive alternatives as hosts because they have rapid doubling time, they can achieve robussnes under process conditions and because of the simplicity of product putrification. And the costs are significantly lower than working with plants. With the engineering the DXP pathway we can increase the supply of isoprenoid precurosor needed for high level productio of isoprenoids in E.coli [1,3].

=== Operons ===

Operon is a a funcional unit of DNA that contains a cluster of genes under the control of a single promotor. The synthesis of natural or synthetic products involves the introduction of a large number of genes. In this case these genes were encoding the enzymes needed for metabolic pathway. So it is useful to put certain genes under the control of a single promotor. For that reason Plac promotor was used in this study [1,5].

== EXPERIMENT ==

=== Synthase gene asemby and amorphadiene production ===

For high level production and changing the difficulties in expressing teprene synthases they first synthesized and expressed a codon-optimized variants of ADS . ADS is a gene encoding amorphadiene synthase and is sequentially oxidised by citorchromeP450 CYP71AV1 and reduced by artemisinic aldehyde reductase (DBR2). Amorphadyene synthase is an enzyme and catalyse the first step in the anitmalarial drug artemisnin synthesis. It has an inportant role in catalysing the reaction of FPP to amorphadiene. Beacause of its importaint step towards artemisnin biosynthesis it was designed and improved for the purpuse to acheve high-level expression in E.coli. For ADS gene synthesis they used two step assemly and one step amplification PCR.(1) Assemby PCR is a useful technique for production novel gene sequences. With this method we can put together short fragments of DNA and it is also a good choice for gene construction. Reserches in this article analysed the sequence of three ADS genes from independet clones and identified mutations in each of the genes. Than, two site directed mutagenesis reaction was used for assembly and generation a functional ADS gene from two clones. Than they measure the amorphadiene production by the amorphadiene synthase in E.coli DH10B with natural (nonengineered DPX pathway) and in E.coli with engineered DPX pathway. E. coli with engineerd pathway was harboring the pSOE4 plasmid (DXP pathway operon with dxs,IppHp and ispA gene). The result sugested that FPP synthesis limited amorphadiene production in this engineered host [1,6]. ([http://www.disketa.si/media/uploads/2015-01/0bddf07f946e6f5be0638090dee56490.jpg See image here])

=== Egineering the mevalonate-dependet pathway in E.coli ===

The aim was to increase the intracellular concentration of FPP substrate. Genes encoding the MEV pathway from S.cerevisae was assembled into operons and expressed in E.coli. Because of the complexity of engineering numerous gene biosynthetic pathway, genes were divided into to operons, top and bottom.Top operon, MevT, consisted of three enzymes: acetoacetyl-CoA thiolase (''atoB''), HMG-CoA synthases (''HMGS'') and HMG-CoA reductase (''tHMGR''). This operon was responsible for transformation of acetyl-coA to mevalonate. Thre different bottom operons pMevB (''ERG12, ERG8,MVD1''), pMBI (''ERG12, ERG8, MVD1, idi'') and pMBIS (''ERG12, ERG8, MVD1, idi, ispA'') transformed mevalonate to IPP, DMAPP and consequently to FPP. The next step was to analyse and test the funcionality of this pathway. For this purpuse E.coli strand DYM1 with incoplemplete isoprenoid synthesis was transformed with plasmid expressing three bottom operon constuct- pMevB, pMBI and pMBI. E.coli strand DYM1 had a deletion in the ispC gene for encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase, and was not able to synthesise 2-C-methyl-D-erythritol 4-phosphate(MEP), which is important intermediate in isoprenoid biosynthesis[1]. Because of this deletion the synthesis of precursors IPP and DMAP is blocked. So then they tested what is going to happen if they grew the strain DYM1 in the presence of 2-C-methyl-D-ertihritol and in medium with mevalonate without methylerythritol since DYM1 strain was not capable to synthesisie MEP. As expected , all strains expressing operon from S.cerevisiae grew on the plates in the presence of 2-c-methyl-D-erythritol. But only strains with pMBI or pMBIS grew on plates with 1mM mevalonate in the absence of methylerythol. This was the conformation that synthetic MBI and MBIS operons were functional, because they were capable to support the growth of E.coli without ispC gene by supplying cells with IPP and DMAPP. Next they completed the mevalonate pathway by transforming the pMevT plasmid (expressing three genes mentioned before-atoB, HMGS and tHMGR) with pMBI or pMBIS. This coexpresion of two operons, pMevT and pMBI /pMBIS ,completed the mevlonate pathway and allowed the synthesis of sesquiterpene precursors from acetyl-CoA, IPP and DMAPP. Therefore, coexpression complemented the ''ispC'' deletion even in the absence of mevalonate, what was the proof, that MevT operon was functional [1,7].

=== Amorpadiene synthesis from mevalonate ===

The aim was to achieve high-level production of amorphadiene and to find out if the amount of FPP was limiting amoprhadiene output. As mentioned, ''E.coli'' was used as heterologous microorganism in which coupled mevalonate pathway with amorphadiene synthesis. Cells with ''ADS'' gene were coexpressed with MBIS operon and grown in medium with increasing amounts (0 to 40 mM) of exogenus mevalonate. The culture extract was analysed with gas chromatography-mass spectrometry, analytical method for identification different substances in a test sample. It combines the features of mass spectrometry and gas-liquid chromatography. Results showed that MBIS operon did not limit the amorphadiene production at the highest mevalonate concentration(40mM). ([http://www.disketa.si/media/uploads/2015-01/7711d592584a53282f2ecb0fd678ef40.jpg See image here]) Cultures with 40 mM mevalonate added produced much more amoprhadiene. With time, amorphadiene concentration was dropping beacause of the loss of the volatile terpene[1]. By adding more than 10 mM mevalonate in control cultures without ADS, the severe growth ihibition has been reported. ([http://www.disketa.si/media/uploads/2015-01/2a21f55f0fea31ffcbe89c866c9150f7.jpg See image here]) To discover the cause ofthis inhibition, the growth of ''E.coli'' DH10B from strains with empty, pMKPMK, pMevB, pMBI or pMBIS plasmid was measured in media with increasing concentrations of mevalonate. The addition of 5mM mevalonate to the medium inhibited the growth of cells with pMevB. But 5mM concentrations of mevalonate did not alter the growth of cells with pMKPMK, PMBI or pMBIS plasmid. This indicated that the accomulation of IPP is toxic and inhibits normal cell growth. The accmulation mostly occurs in cells with high flux through mevalonate pathway. The next step was comparison of intracellular prenyl pyrophosphate (FPP) in strains. This was done by feeding the cells harboring the different mevalonte operon construct with radiolabeled mevalonate. Than the labeled metabolites were tracked. For this experiment cell suspensions of E.coli harboring pMevB+pTrc99A, pMBI+ pTrc99A, pMBIS+ pTrc99A or pMBIS+pADS were used. pTRc99A is the bacterial expression vector with inducible to IPTG lacI promoter. The result showed that strains with MevB operon accumulated IPP but not FPP, when MBI and MBIS strains accumulated FPP. ([http://www.disketa.si/media/uploads/2015-01/31ad7a215f016dc26aceacf92eb46712.jpg See image here]) Because of the simultaneous expression of the amorphadiene synthase the waste of FPP that accumulated in the MBIS host was consumed. This was shown by a decrease in intracellular FPP. Cells expresing MBIS acumulated FPP and showed growth inhibition in the presence of 10 mM mevalonate. The prediction was that the coexpression of ADS would ease the growth inhibition by forwarding the prenyl pyrophosphate intermediates to volatile terpene olefin. For this test, the cell with pMBIS and empty expression vector pTrc99A (without ADS)and cells with pMBIS and pADS vectors were used. The medium was supplemented with mevalonate concentations ( 0mM, 5mM, 10mM, 20mM or 40mM). Two hours after addition of mevalonate and inducer IPTG, the growth inhibition was observed. ([http://www.disketa.si/media/uploads/2015-01/94b5f3d5636fe767316f770f3e2b0448.jpg See image here]) The result showed that the strains with pMBIS +ADS started to grow, but strain lacking the ADS still showed growth inhbition at higher concentrations of mevalonate. This supported the conclusion that converion of FPP to amophadiene has an important role in minimising growth inhibition. Briefly these result showed that engineered mevalonate patway produces high levels of prenyl pyrophosphate precursors. If the IPP isomerase, FPP synthase and terpen synthase is absent, IPP can acumulate and be toxic to the cells. The toxic levels of intracellular prenyl pyrphosphate can accumulate [1].

=== Amorphadiene synthesis from acetyl-Coa ===

The main goal was to achieve high amoprhaidene production from a simple and inexpensive carbon source. ''E.coli'' strains harboring pMevT,pMBIS and pADS plasmids were transformed with pMevT plasmid. Subsequently the strains were tested for ability to produce amoprhadiene in the absence of exogenous mevalonate. The comparison between E.coli expressing the native DPX pathway and engineered pathway as made. They tested native DXP pathway, engineered DPX pathway, mevalonate bottom pathway in the absence of mevalonate, mevlonate bottom pathway in medium suplemented with mevalonate, the complete mevalonate pathway and the complete mevalonate pathway supplemented with 0,8% glycerol. Glycerol was addded to investigate the effect of amorphadiene production of supplying as single carbon source and for prolongation of amorphadiene production. Breifly the resulut showed that expression of the mevalonate-dependent isoprenoid biosynthetic pathway delivers hight levels of isoprenoid precursor for the production of sesquiterpene [1]. ([http://www.disketa.si/media/uploads/2015-01/a31fe07a7c66854b1dc3ff36f8ed01ef.jpg See image here])

== DISSCUSION ==
The identification of genes encoding the enzymes involved in the artemisinin-biosynthesis pathway together with advances in metabolic engineering provides new options for engineering artemisinin biosynthesis. Heterologous production of artemisnin in microorganism such as ''E.coli''. is an alternative way for production of artmeinsin in high yields. But eventhougt micoorganisms has shown great potential in that matter, optimisation of these heterlogous pathways still require maximal titer. Inequal gene expression can lead to accumulation of toxic metabolic intermediates and to over or under production of enzymes involved in pathway. (4). Conclusion based on these study shows that poor expression of the amoprhaidene synthase genes (''ADS'') limits high terpene olfein yields from the host. To achieve better results , in this study they chose to synthesiyse an amophaiene synthase gene (ADS). The comparison between ''E.coli'' expressing native sesquiterpene synthase genes and'' E.coli'' expressing the synthetic ADS gene showed big improvement in terpene synthesis due to synthetic ADS gene. The native DPX pathway showed poor supply of the prenyl pyrophosphate precuror. And that limited terpenoid -carotenoid yields in ''E.coli.'' With the metabolic engineering of the DPX pathway the flux of carotenoid accumulation showed signifcant increase. By using the mevalonate-dependent isoprenoid pathway from yeast S. cerevisae and engineering that pathway in bacteria ''E.coli'', we avoid the native regulatory elements found in yeast while bypassing those of ''E.coli'' DXP pathway. In this study results showed growth inhibition in cells without ''ADS'' due to the vast excess of preny pyrophosphate. But the coexpression of a synthetic sesquiterpene synthase can contribute to reducing the excess pool of precursors and eliminate growth inhibition. In this study total biosynthesis of artemisnin was not completely achieved. But these results can help in further investigation for poduction of artemisnic acid. This acid can then be putrified and chemically converted into artemsinin. [1,7].

== CONCLUSIONS ==
To sum things up, artmeinsin, a sesquiterpene isolated from Artemisia annua,represent a novel drug for malaria treatment. The present artemisnin derivates are too expensive for general use and plants can not produce artmeinsin in big quantities. This fact inspired scientist to focus on developing the way for production of artemisnin in high quantities and to avoid big costs. The promising tool in alternative source of artmeinsin is the use of microoganisms. The aim is to transfer the genes encoding enzymes involved in biosynthetic pathway for artemisnin into a hight producing host organism. Several study have been reporting on engineering the metabolic pathway in S.cerevisae to produce the precursor for artmeisnic acid. With the effort in synthetic biology they used modified mevalonate pathway . With the help of syntehtic bilogy the yeast cells were engineerd to express ezymes needed for production of artmemisnic acid. But in this paper the main focus was on engineering a mevalonate pathway in bacteria ''E.coli''. By engineering a new metabolic pathway in ''E.coli'' we can easily synthesise a precursor nedeed for artemisnin production. The goal is to produce drugs in better and cheape ways. But, why focus on artemisnin? Well, acording on recent studies artemisnin shows to be very sucesfull for the treatmen all strain of malaria. More than a milion people affected with malaria have already been cured by arteminsin. As already mentioned , because of very hight costs and limited production of arteminin in plants most populations in Africa can not afford it. Progress made in synthetic biology and ability to produce amorphadiene in microorganisms in big quantites opens a new possibility to produce low cost artemisnin and contributes to the brighter future od antimalaria drug deveopment, and therefore saving milions from suffering from malaria. [1,2,3].

[1]Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nature biotehnology; vol 21; pages 796-802, July 2003

[2] T. Mosses, J. Pollier, J. M. Thevelein, A. Goossens. Bioengineering of plant (tri)terpenoids: from metabolic engineering of plants to synthetic biology in vivo and in vitro. New Pathyologist. 200: 27-43, 2013

[3] M.Farhi, M. Kozin, S. Duchin, A. Vainstein. Metabolic engineering of plants for artemisinin synthesis. Biotehnology and genetic engineering rewiews.Vol.29,No.2: 135-148, 2013

[4] Synthetic biology ; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Synthetic_biology http://en.wikipedia.org/wiki/Synthetic_biology]

[5]Operon; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Operon http://en.wikipedia.org/wiki/Operon]

[6] M. K. Julsing, G. van Pouderoyen, J. van der Veen,H. J. Woerdenbag, O. Kayser, W. J. Quax. Amorphadiene synthase: probing the active site model with directed mutagenesis. Directed mutagenesis of AMDS.Chapter 4: 47-56

[7]J.R. Anthony, L. C. Anthony, F. Nowroozi, G. Kwon, J.D. Newman, J.D. Keasling. Optimization of themevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene. Metabolic Engineering. 11: 13-19, 2009

Engineering a mevalonate pathway in Escherichia coli for production of terpenoids

2015-01-20T00:00:17Z

Ana90: /* DISSCUSION */

'''Ana Kapraljević'''

[http://www2.lbl.gov/ttd/publications/2837pub1.pdf Engineering a mevalonate pathway in Escherichia coli for production of terpenoids]
Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling ;Nature biotehnology; July 2003; vol 21; pages 796-802

== INTRODUCTION ==

Plants can synthesise and accumulate a wide range of small molecules or natural products involved in psyhological an ecological process. With the knowledge we know about their biosyntesis we used them as commercial flavors, fragnance compunds, colorants and terapeutical drugs. Their therapeutic potential is used for production of anticancer or antimalarial drugs, such as artemisnin. The most numerous and diverse class of natural products used in production for antimalarial drug are isoprenoids or more corectly their subclass, terpenoids. Malaria represent a major health problem in tropic and subtropic. Majority of death occur in children living in Africa. But the problem is that terpenoids are in plants produced in very small quantites and consequently not very economically feasible. For that reason the drugs for treatin malaria, artemisnin, is in short supply and is still unaffordable for most people living in malaria-endanger countries. Scientists are now focusing on alternative and cheaper way for terpenoid synthesis that can be enchanched and became economically acceptable. The alternative way is the expression of synthetic amorphadiene, precursor to artemisnin, and mevalonate isoprenoid way from yeas ''Saccharomyces cerevisiae'' in bacteria. In this paper I will summerize the project made in the production of amorphadiene by biotehnology and synthetic biology approach [1,2].

=== Artemisnin ===

Artemisnin is a substance that has antimalarial potential. Malaria is a infectious disease caused by parasitic protozan of the ''Plasmodium'' species. In the certain parts of the world malaria takes lives of more people worldwide than any other infecious disease, so it is a global problem. Death to malaria has been nowdays reduced thanks to the use of artemisnin-based combination therapy. Artemisnin is a natural compoud from plant ''Artemisia annua''. Chemically it is a sesquiterpene lactone, amorphadiene. The total sythesis of artemisnin is difficult and not very economical,so the new aproach is to put the hopes in sythetic biology and develop the new way, synthetic way, for its production [1,3].

=== Biology of terpenoids ===

For initiation of any synthetic biology process in metabolic engineering in that matter it is important to have some basic understanding of biosynthesis and regulation of terpenoids. Terpenoids, a class of isoprenoids, are natural products often sythesized by plants. They represent a large and diverse class and have many functions as primarly metabolites involved in growth and development of plants, or as secundary metabolites that optimize the interaction between the plant and the environment. Eventhought they are structural diverse, the share a common biosynthetic origin and follow similar synthesis tracks. All terpenoids are derived from repetitive fusion of isoprene units (C5 H8) [3]. The number of isoprene units determines their classification. For isoprenoid synthesis plants use mevalonate-dependent (MEV) pathway and mevalonte-independent pathway or alternative deoxyxylulose 5-phosphate pathway (DXP). In higher plants the biosynthesis begins with the generation of isopentenyl pyrophosphate (IPP). IPP is derived via the classic mevalonic acid pathway from acetly-coenzime A (acetyl-CoA). The IPP is isomerized to its allylic isomer dimethylallyl pyrophosphate (DMAPP) [3]. The continous condensation of IPP and DMAPP units leads to the formaton of prenylated pyrophosphates, the immediate precursors of the different terpenoid classes. So IPP and DMAPP are combined to form larger isoprenoids, such as farnesyl diphosphate (FPP). Condensation of IPP and DMAPP than units leads to the formation of precursors of the different terpenoid classes. Shematic production of terpenoids in ilustraded in this picture. Similary to plants, yeast ''Sacharomyces cerevisae'' uses the clasical mevalonate pathway for the synthesis of isoprenoids[3]. Prokariontes mostly use the DXP pathway for production IPP and DMAPP. This is an alternative pathway for IPP synthesis. This pathway uses ''Escherichia coli'' and some other bacteria for their terpenoid synthesis. IPP and DMAPP precurosr are essential to E. Coli for prenylation (addition of hydrophobic molecules to a protein or chemical compound) of tRNA and the synthesis of farnesyl pyrophosphate (FPP). FPP is then used for quinone (class of organic compounds) and cell wall synthesis. The DPX pathway begins with the glycolytic intermediates glyceraldehyde-3-phosphate (G3P) and pyruvate and continues through enzymatic steps for production of IPP and DMAPP. Therefore IPP and DMAPP are the end product in both way,MEV and DPX pathway [1,3].

== METABOLIC ENGINEERING FOR PRODUCTION OD TERPENOIDS ==

Metabolic engineering represents an improvement of production, construction, or cellular properties through the modification of specific biochemical reactions. It is described as the use of genetic engineering to reshape or modify the metabolism of organism. The aim is to optimize genetic and regulatory processes in cells to increase the production of a certain substance that we find in nature. It is based on optimisation of existing biochemical pathways or the introduction of natural pathway components in bacteria, yeast or plants. The main goal is the production of high-yield of specific metabolites that we can use in medicine or biotechnology. With progress in synthetic biology we can design a biologic funcion that do not exist in nature. Consequently, we can create and construct new biological components or redesign exisitng biological systems. In plants, most terpenoids are produced in a very small quantities in their natural sources. Extraction of this compouds from plants is expensive and therfore not economicaly accetable. So there is a big disagreement between the insistence for supply of terpenoids and therefore that leads to delay for their widespread application. With the understanding of terpenoid synthesis and progress in synthetic biology we are now able of redesign and modify the isoprenoid pathway to increase the terpenoid productivity [2,4]. ([http://www.disketa.si/media/uploads/2015-01/cd201ade6a167488767dfb1b118587bc.jpg See image here])

=== Microbal host ===

To elimante the need for plant extraction it is practical to produce terpenoid compouds at high yield in microbal host by introducing a heterlogous isoprenoid pathway into bacteria ''E.coli.'' Microorganisms, such as bacteria are attractive alternatives as hosts because they have rapid doubling time, they can achieve robussnes under process conditions and because of the simplicity of product putrification. And the costs are significantly lower than working with plants. With the engineering the DXP pathway we can increase the supply of isoprenoid precurosor needed for high level productio of isoprenoids in E.coli [1,3].

=== Operons ===

Operon is a a funcional unit of DNA that contains a cluster of genes under the control of a single promotor. The synthesis of natural or synthetic products involves the introduction of a large number of genes. In this case these genes were encoding the enzymes needed for metabolic pathway. So it is useful to put certain genes under the control of a single promotor. For that reason Plac promotor was used in this study [1,5].

== EXPERIMENT ==

=== Synthase gene asemby and amorphadiene production ===

For high level production and changing the difficulties in expressing teprene synthases they first synthesized and expressed a codon-optimized variants of ADS . ADS is a gene encoding amorphadiene synthase and is sequentially oxidised by citorchromeP450 CYP71AV1 and reduced by artemisinic aldehyde reductase (DBR2). Amorphadyene synthase is an enzyme and catalyse the first step in the anitmalarial drug artemisnin synthesis. It has an inportant role in catalysing the reaction of FPP to amorphadiene. Beacause of its importaint step towards artemisnin biosynthesis it was designed and improved for the purpuse to acheve high-level expression in E.coli. For ADS gene synthesis they used two step assemly and one step amplification PCR.(1) Assemby PCR is a useful technique for production novel gene sequences. With this method we can put together short fragments of DNA and it is also a good choice for gene construction. Reserches in this article analysed the sequence of three ADS genes from independet clones and identified mutations in each of the genes. Than, two site directed mutagenesis reaction was used for assembly and generation a functional ADS gene from two clones. Than they measure the amorphadiene production by the amorphadiene synthase in E.coli DH10B with natural (nonengineered DPX pathway) and in E.coli with engineered DPX pathway. E. coli with engineerd pathway was harboring the pSOE4 plasmid (DXP pathway operon with dxs,IppHp and ispA gene). The result sugested that FPP synthesis limited amorphadiene production in this engineered host [1,6]. ([http://www.disketa.si/media/uploads/2015-01/0bddf07f946e6f5be0638090dee56490.jpg See image here])

=== Egineering the mevalonate-dependet pathway in E.coli ===

The aim was to increase the intracellular concentration of FPP substrate. Genes encoding the MEV pathway from S.cerevisae was assembled into operons and expressed in E.coli. Because of the complexity of engineering numerous gene biosynthetic pathway, genes were divided into to operons, top and bottom.Top operon, MevT, consisted of three enzymes: acetoacetyl-CoA thiolase (''atoB''), HMG-CoA synthases (''HMGS'') and HMG-CoA reductase (''tHMGR''). This operon was responsible for transformation of acetyl-coA to mevalonate. Thre different bottom operons pMevB (''ERG12, ERG8,MVD1''), pMBI (''ERG12, ERG8, MVD1, idi'') and pMBIS (''ERG12, ERG8, MVD1, idi, ispA'') transformed mevalonate to IPP, DMAPP and consequently to FPP. The next step was to analyse and test the funcionality of this pathway. For this purpuse E.coli strand DYM1 with incoplemplete isoprenoid synthesis was transformed with plasmid expressing three bottom operon constuct- pMevB, pMBI and pMBI. E.coli strand DYM1 had a deletion in the ispC gene for encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase, and was not able to synthesise 2-C-methyl-D-erythritol 4-phosphate(MEP), which is important intermediate in isoprenoid biosynthesis[1]. Because of this deletion the synthesis of precursors IPP and DMAP is blocked. So then they tested what is going to happen if they grew the strain DYM1 in the presence of 2-C-methyl-D-ertihritol and in medium with mevalonate without methylerythritol since DYM1 strain was not capable to synthesisie MEP. As expected , all strains expressing operon from S.cerevisiae grew on the plates in the presence of 2-c-methyl-D-erythritol. But only strains with pMBI or pMBIS grew on plates with 1mM mevalonate in the absence of methylerythol. This was the conformation that synthetic MBI and MBIS operons were functional, because they were capable to support the growth of E.coli without ispC gene by supplying cells with IPP and DMAPP. Next they completed the mevalonate pathway by transforming the pMevT plasmid (expressing three genes mentioned before-atoB, HMGS and tHMGR) with pMBI or pMBIS. This coexpresion of two operons, pMevT and pMBI /pMBIS ,completed the mevlonate pathway and allowed the synthesis of sesquiterpene precursors from acetyl-CoA, IPP and DMAPP. Therefore, coexpression complemented the ''ispC'' deletion even in the absence of mevalonate, what was the proof, that MevT operon was functional [1,7].

=== Amorpadiene synthesis from mevalonate ===

The aim was to achieve high-level production of amorphadiene and to find out if the amount of FPP was limiting amoprhadiene output. As mentioned, ''E.coli'' was used as heterologous microorganism in which coupled mevalonate pathway with amorphadiene synthesis. Cells with ''ADS'' gene were coexpressed with MBIS operon and grown in medium with increasing amounts (0 to 40 mM) of exogenus mevalonate. The culture extract was analysed with gas chromatography-mass spectrometry, analytical method for identification different substances in a test sample. It combines the features of mass spectrometry and gas-liquid chromatography. Results showed that MBIS operon did not limit the amorphadiene production at the highest mevalonate concentration(40mM). ([http://www.disketa.si/media/uploads/2015-01/7711d592584a53282f2ecb0fd678ef40.jpg See image here]) Cultures with 40 mM mevalonate added produced much more amoprhadiene. With time, amorphadiene concentration was dropping beacause of the loss of the volatile terpene[1]. By adding more than 10 mM mevalonate in control cultures without ADS, the severe growth ihibition has been reported. ([http://www.disketa.si/media/uploads/2015-01/2a21f55f0fea31ffcbe89c866c9150f7.jpg See image here]) To discover the cause ofthis inhibition, the growth of ''E.coli'' DH10B from strains with empty, pMKPMK, pMevB, pMBI or pMBIS plasmid was measured in media with increasing concentrations of mevalonate. The addition of 5mM mevalonate to the medium inhibited the growth of cells with pMevB. But 5mM concentrations of mevalonate did not alter the growth of cells with pMKPMK, PMBI or pMBIS plasmid. This indicated that the accomulation of IPP is toxic and inhibits normal cell growth. The accmulation mostly occurs in cells with high flux through mevalonate pathway. The next step was comparison of intracellular prenyl pyrophosphate (FPP) in strains. This was done by feeding the cells harboring the different mevalonte operon construct with radiolabeled mevalonate. Than the labeled metabolites were tracked. For this experiment cell suspensions of E.coli harboring pMevB+pTrc99A, pMBI+ pTrc99A, pMBIS+ pTrc99A or pMBIS+pADS were used. pTRc99A is the bacterial expression vector with inducible to IPTG lacI promoter. The result showed that strains with MevB operon accumulated IPP but not FPP, when MBI and MBIS strains accumulated FPP. ([http://www.disketa.si/media/uploads/2015-01/31ad7a215f016dc26aceacf92eb46712.jpg See image here]) Because of the simultaneous expression of the amorphadiene synthase the waste of FPP that accumulated in the MBIS host was consumed. This was shown by a decrease in intracellular FPP. Cells expresing MBIS acumulated FPP and showed growth inhibition in the presence of 10 mM mevalonate. The prediction was that the coexpression of ADS would ease the growth inhibition by forwarding the prenyl pyrophosphate intermediates to volatile terpene olefin. For this test, the cell with pMBIS and empty expression vector pTrc99A (without ADS)and cells with pMBIS and pADS vectors were used. The medium was supplemented with mevalonate concentations ( 0mM, 5mM, 10mM, 20mM or 40mM). Two hours after addition of mevalonate and inducer IPTG, the growth inhibition was observed. ([http://www.disketa.si/media/uploads/2015-01/94b5f3d5636fe767316f770f3e2b0448.jpg See image here]) The result showed that the strains with pMBIS +ADS started to grow, but strain lacking the ADS still showed growth inhbition at higher concentrations of mevalonate. This supported the conclusion that converion of FPP to amophadiene has an important role in minimising growth inhibition. Briefly these result showed that engineered mevalonate patway produces high levels of prenyl pyrophosphate precursors. If the IPP isomerase, FPP synthase and terpen synthase is absent, IPP can acumulate and be toxic to the cells. The toxic levels of intracellular prenyl pyrphosphate can accumulate [1].

=== Amorphadiene synthesis from acetyl-Coa ===

The main goal was to achieve high amoprhaidene production from a simple and inexpensive carbon source. ''E.coli'' strains harboring pMevT,pMBIS and pADS plasmids were transformed with pMevT plasmid. Subsequently the strains were tested for ability to produce amoprhadiene in the absence of exogenous mevalonate. The comparison between E.coli expressing the native DPX pathway and engineered pathway as made. They tested native DXP pathway, engineered DPX pathway, mevalonate bottom pathway in the absence of mevalonate, mevlonate bottom pathway in medium suplemented with mevalonate, the complete mevalonate pathway and the complete mevalonate pathway supplemented with 0,8% glycerol. Glycerol was addded to investigate the effect of amorphadiene production of supplying as single carbon source and for prolongation of amorphadiene production. Breifly the resulut showed that expression of the mevalonate-dependent isoprenoid biosynthetic pathway delivers hight levels of isoprenoid precursor for the production of sesquiterpene [1]. ([http://www.disketa.si/media/uploads/2015-01/a31fe07a7c66854b1dc3ff36f8ed01ef.jpg See image here])

== DISSCUSION ==
The identification of genes encoding the enzymes involved in the artemisinin-biosynthesis pathway together with advances in metabolic engineering provides new options for engineering artemisinin biosynthesis. Heterologous production of artemisnin in microorganism such as ''E.coli''. is an alternative way for production of artmeinsin in high yields. But eventhougt micoorganisms has shown great potential in that matter, optimisation of these heterlogous pathways still require maximal titer. Inequal gene expression can lead to accumulation of toxic metabolic intermediates and to over or under production of enzymes involved in pathway. (4). Conclusion based on these study shows that poor expression of the amoprhaidene synthase genes (''ADS'') limits high terpene olfein yields from the host. To achieve better results , in this study they chose to synthesiyse an amophaiene synthase gene (ADS). The comparison between ''E.coli'' expressing native sesquiterpene synthase genes and'' E.coli'' expressing the synthetic ADS gene showed big improvement in terpene synthesis due to synthetic ADS gene. The native DPX pathway showed poor supply of the prenyl pyrophosphate precuror. And that limited terpenoid -carotenoid yields in ''E.coli.'' With the metabolic engineering of the DPX pathway the flux of carotenoid accumulation showed signifcant increase. By using the mevalonate-dependent isoprenoid pathway from yeast S. cerevisae and engineering that pathway in bacteria ''E.coli'', we avoid the native regulatory elements found in yeast while bypassing those of ''E.coli'' DXP pathway. In this study results showed growth inhibition in cells without ''ADS'' due to the vast excess of preny pyrophosphate. But the coexpression of a synthetic sesquiterpene synthase can contribute to reducing the excess pool of precursors and eliminate growth inhibition. In this study total biosynthesis of artemisnin was not completely achieved. But these results can help in further investigation for poduction of artemisnic acid. This acid can then be putrified and chemically converted into artemsinin. [1,7].

== CONCLUSIONS ==
To sum things up, artmeinsin, a sesquiterpene isolated from Artemisia annua,represent a novel drug for malaria treatment. The present artemisnin derivates are too expensive for general use and plants can not produce artmeinsin in big quantities. This fact inspired scientist to focus on developing the way for production of artemisnin in high quantities and to avoid big costs. The promising tool in alternative source of artmeinsin is the use of microoganisms. The aim is to transfer the genes encoding enzymes involved in biosynthetic pathway for artemisnin into a hight producing host organism. Several study have been reporting on engineering the metabolic pathway in S.cerevisae to produce the precursor for artmeisnic acid. With the effort in synthetic biology they used modified mevalonate pathway . With the help of syntehtic bilogy the yeast cells were engineerd to express ezymes needed for production of artmemisnic acid. But in this paper the main focus was on engineering a mevalonate pathway in bacteria E.coli. By engineering a new metabolic pathway in E.coli we can easily synthesise a precursor nedeed for artemisnin production. The goal is to produce drugs in better and cheape ways. But, why focus on artemisnin? Well, acording on recent studies artemisnin shows to be very sucesfull for the treatmen all strain of malaria. More than a milion people affected with malaria have already been cured by arteminsin. As already mentioned , because of very hight costs and limited production of arteminin in plants most populations in Africa can not afford it. Progress made in synthetic biology and ability to produce amorphadiene in microorganisms in big quantites opens a new possibility to produce low cost artemisnin and contributes to the brighter future od antimalaria drug deveopment, and therefore saving milions from suffering from malaria. [1,2,3].

[1]Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nature biotehnology; vol 21; pages 796-802, July 2003

[2] T. Mosses, J. Pollier, J. M. Thevelein, A. Goossens. Bioengineering of plant (tri)terpenoids: from metabolic engineering of plants to synthetic biology in vivo and in vitro. New Pathyologist. 200: 27-43, 2013

[3] M.Farhi, M. Kozin, S. Duchin, A. Vainstein. Metabolic engineering of plants for artemisinin synthesis. Biotehnology and genetic engineering rewiews.Vol.29,No.2: 135-148, 2013

[4] Synthetic biology ; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Synthetic_biology http://en.wikipedia.org/wiki/Synthetic_biology]

[5]Operon; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Operon http://en.wikipedia.org/wiki/Operon]

[6] M. K. Julsing, G. van Pouderoyen, J. van der Veen,H. J. Woerdenbag, O. Kayser, W. J. Quax. Amorphadiene synthase: probing the active site model with directed mutagenesis. Directed mutagenesis of AMDS.Chapter 4: 47-56

[7]J.R. Anthony, L. C. Anthony, F. Nowroozi, G. Kwon, J.D. Newman, J.D. Keasling. Optimization of themevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene. Metabolic Engineering. 11: 13-19, 2009

Engineering a mevalonate pathway in Escherichia coli for production of terpenoids

2015-01-19T23:58:54Z

Ana90: /* Amorphadiene synthesis from acetyl-Coa */

'''Ana Kapraljević'''

[http://www2.lbl.gov/ttd/publications/2837pub1.pdf Engineering a mevalonate pathway in Escherichia coli for production of terpenoids]
Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling ;Nature biotehnology; July 2003; vol 21; pages 796-802

== INTRODUCTION ==

Plants can synthesise and accumulate a wide range of small molecules or natural products involved in psyhological an ecological process. With the knowledge we know about their biosyntesis we used them as commercial flavors, fragnance compunds, colorants and terapeutical drugs. Their therapeutic potential is used for production of anticancer or antimalarial drugs, such as artemisnin. The most numerous and diverse class of natural products used in production for antimalarial drug are isoprenoids or more corectly their subclass, terpenoids. Malaria represent a major health problem in tropic and subtropic. Majority of death occur in children living in Africa. But the problem is that terpenoids are in plants produced in very small quantites and consequently not very economically feasible. For that reason the drugs for treatin malaria, artemisnin, is in short supply and is still unaffordable for most people living in malaria-endanger countries. Scientists are now focusing on alternative and cheaper way for terpenoid synthesis that can be enchanched and became economically acceptable. The alternative way is the expression of synthetic amorphadiene, precursor to artemisnin, and mevalonate isoprenoid way from yeas ''Saccharomyces cerevisiae'' in bacteria. In this paper I will summerize the project made in the production of amorphadiene by biotehnology and synthetic biology approach [1,2].

=== Artemisnin ===

Artemisnin is a substance that has antimalarial potential. Malaria is a infectious disease caused by parasitic protozan of the ''Plasmodium'' species. In the certain parts of the world malaria takes lives of more people worldwide than any other infecious disease, so it is a global problem. Death to malaria has been nowdays reduced thanks to the use of artemisnin-based combination therapy. Artemisnin is a natural compoud from plant ''Artemisia annua''. Chemically it is a sesquiterpene lactone, amorphadiene. The total sythesis of artemisnin is difficult and not very economical,so the new aproach is to put the hopes in sythetic biology and develop the new way, synthetic way, for its production [1,3].

=== Biology of terpenoids ===

For initiation of any synthetic biology process in metabolic engineering in that matter it is important to have some basic understanding of biosynthesis and regulation of terpenoids. Terpenoids, a class of isoprenoids, are natural products often sythesized by plants. They represent a large and diverse class and have many functions as primarly metabolites involved in growth and development of plants, or as secundary metabolites that optimize the interaction between the plant and the environment. Eventhought they are structural diverse, the share a common biosynthetic origin and follow similar synthesis tracks. All terpenoids are derived from repetitive fusion of isoprene units (C5 H8) [3]. The number of isoprene units determines their classification. For isoprenoid synthesis plants use mevalonate-dependent (MEV) pathway and mevalonte-independent pathway or alternative deoxyxylulose 5-phosphate pathway (DXP). In higher plants the biosynthesis begins with the generation of isopentenyl pyrophosphate (IPP). IPP is derived via the classic mevalonic acid pathway from acetly-coenzime A (acetyl-CoA). The IPP is isomerized to its allylic isomer dimethylallyl pyrophosphate (DMAPP) [3]. The continous condensation of IPP and DMAPP units leads to the formaton of prenylated pyrophosphates, the immediate precursors of the different terpenoid classes. So IPP and DMAPP are combined to form larger isoprenoids, such as farnesyl diphosphate (FPP). Condensation of IPP and DMAPP than units leads to the formation of precursors of the different terpenoid classes. Shematic production of terpenoids in ilustraded in this picture. Similary to plants, yeast ''Sacharomyces cerevisae'' uses the clasical mevalonate pathway for the synthesis of isoprenoids[3]. Prokariontes mostly use the DXP pathway for production IPP and DMAPP. This is an alternative pathway for IPP synthesis. This pathway uses ''Escherichia coli'' and some other bacteria for their terpenoid synthesis. IPP and DMAPP precurosr are essential to E. Coli for prenylation (addition of hydrophobic molecules to a protein or chemical compound) of tRNA and the synthesis of farnesyl pyrophosphate (FPP). FPP is then used for quinone (class of organic compounds) and cell wall synthesis. The DPX pathway begins with the glycolytic intermediates glyceraldehyde-3-phosphate (G3P) and pyruvate and continues through enzymatic steps for production of IPP and DMAPP. Therefore IPP and DMAPP are the end product in both way,MEV and DPX pathway [1,3].

== METABOLIC ENGINEERING FOR PRODUCTION OD TERPENOIDS ==

Metabolic engineering represents an improvement of production, construction, or cellular properties through the modification of specific biochemical reactions. It is described as the use of genetic engineering to reshape or modify the metabolism of organism. The aim is to optimize genetic and regulatory processes in cells to increase the production of a certain substance that we find in nature. It is based on optimisation of existing biochemical pathways or the introduction of natural pathway components in bacteria, yeast or plants. The main goal is the production of high-yield of specific metabolites that we can use in medicine or biotechnology. With progress in synthetic biology we can design a biologic funcion that do not exist in nature. Consequently, we can create and construct new biological components or redesign exisitng biological systems. In plants, most terpenoids are produced in a very small quantities in their natural sources. Extraction of this compouds from plants is expensive and therfore not economicaly accetable. So there is a big disagreement between the insistence for supply of terpenoids and therefore that leads to delay for their widespread application. With the understanding of terpenoid synthesis and progress in synthetic biology we are now able of redesign and modify the isoprenoid pathway to increase the terpenoid productivity [2,4]. ([http://www.disketa.si/media/uploads/2015-01/cd201ade6a167488767dfb1b118587bc.jpg See image here])

=== Microbal host ===

To elimante the need for plant extraction it is practical to produce terpenoid compouds at high yield in microbal host by introducing a heterlogous isoprenoid pathway into bacteria ''E.coli.'' Microorganisms, such as bacteria are attractive alternatives as hosts because they have rapid doubling time, they can achieve robussnes under process conditions and because of the simplicity of product putrification. And the costs are significantly lower than working with plants. With the engineering the DXP pathway we can increase the supply of isoprenoid precurosor needed for high level productio of isoprenoids in E.coli [1,3].

=== Operons ===

Operon is a a funcional unit of DNA that contains a cluster of genes under the control of a single promotor. The synthesis of natural or synthetic products involves the introduction of a large number of genes. In this case these genes were encoding the enzymes needed for metabolic pathway. So it is useful to put certain genes under the control of a single promotor. For that reason Plac promotor was used in this study [1,5].

== EXPERIMENT ==

=== Synthase gene asemby and amorphadiene production ===

For high level production and changing the difficulties in expressing teprene synthases they first synthesized and expressed a codon-optimized variants of ADS . ADS is a gene encoding amorphadiene synthase and is sequentially oxidised by citorchromeP450 CYP71AV1 and reduced by artemisinic aldehyde reductase (DBR2). Amorphadyene synthase is an enzyme and catalyse the first step in the anitmalarial drug artemisnin synthesis. It has an inportant role in catalysing the reaction of FPP to amorphadiene. Beacause of its importaint step towards artemisnin biosynthesis it was designed and improved for the purpuse to acheve high-level expression in E.coli. For ADS gene synthesis they used two step assemly and one step amplification PCR.(1) Assemby PCR is a useful technique for production novel gene sequences. With this method we can put together short fragments of DNA and it is also a good choice for gene construction. Reserches in this article analysed the sequence of three ADS genes from independet clones and identified mutations in each of the genes. Than, two site directed mutagenesis reaction was used for assembly and generation a functional ADS gene from two clones. Than they measure the amorphadiene production by the amorphadiene synthase in E.coli DH10B with natural (nonengineered DPX pathway) and in E.coli with engineered DPX pathway. E. coli with engineerd pathway was harboring the pSOE4 plasmid (DXP pathway operon with dxs,IppHp and ispA gene). The result sugested that FPP synthesis limited amorphadiene production in this engineered host [1,6]. ([http://www.disketa.si/media/uploads/2015-01/0bddf07f946e6f5be0638090dee56490.jpg See image here])

=== Egineering the mevalonate-dependet pathway in E.coli ===

The aim was to increase the intracellular concentration of FPP substrate. Genes encoding the MEV pathway from S.cerevisae was assembled into operons and expressed in E.coli. Because of the complexity of engineering numerous gene biosynthetic pathway, genes were divided into to operons, top and bottom.Top operon, MevT, consisted of three enzymes: acetoacetyl-CoA thiolase (''atoB''), HMG-CoA synthases (''HMGS'') and HMG-CoA reductase (''tHMGR''). This operon was responsible for transformation of acetyl-coA to mevalonate. Thre different bottom operons pMevB (''ERG12, ERG8,MVD1''), pMBI (''ERG12, ERG8, MVD1, idi'') and pMBIS (''ERG12, ERG8, MVD1, idi, ispA'') transformed mevalonate to IPP, DMAPP and consequently to FPP. The next step was to analyse and test the funcionality of this pathway. For this purpuse E.coli strand DYM1 with incoplemplete isoprenoid synthesis was transformed with plasmid expressing three bottom operon constuct- pMevB, pMBI and pMBI. E.coli strand DYM1 had a deletion in the ispC gene for encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase, and was not able to synthesise 2-C-methyl-D-erythritol 4-phosphate(MEP), which is important intermediate in isoprenoid biosynthesis[1]. Because of this deletion the synthesis of precursors IPP and DMAP is blocked. So then they tested what is going to happen if they grew the strain DYM1 in the presence of 2-C-methyl-D-ertihritol and in medium with mevalonate without methylerythritol since DYM1 strain was not capable to synthesisie MEP. As expected , all strains expressing operon from S.cerevisiae grew on the plates in the presence of 2-c-methyl-D-erythritol. But only strains with pMBI or pMBIS grew on plates with 1mM mevalonate in the absence of methylerythol. This was the conformation that synthetic MBI and MBIS operons were functional, because they were capable to support the growth of E.coli without ispC gene by supplying cells with IPP and DMAPP. Next they completed the mevalonate pathway by transforming the pMevT plasmid (expressing three genes mentioned before-atoB, HMGS and tHMGR) with pMBI or pMBIS. This coexpresion of two operons, pMevT and pMBI /pMBIS ,completed the mevlonate pathway and allowed the synthesis of sesquiterpene precursors from acetyl-CoA, IPP and DMAPP. Therefore, coexpression complemented the ''ispC'' deletion even in the absence of mevalonate, what was the proof, that MevT operon was functional [1,7].

=== Amorpadiene synthesis from mevalonate ===

The aim was to achieve high-level production of amorphadiene and to find out if the amount of FPP was limiting amoprhadiene output. As mentioned, ''E.coli'' was used as heterologous microorganism in which coupled mevalonate pathway with amorphadiene synthesis. Cells with ''ADS'' gene were coexpressed with MBIS operon and grown in medium with increasing amounts (0 to 40 mM) of exogenus mevalonate. The culture extract was analysed with gas chromatography-mass spectrometry, analytical method for identification different substances in a test sample. It combines the features of mass spectrometry and gas-liquid chromatography. Results showed that MBIS operon did not limit the amorphadiene production at the highest mevalonate concentration(40mM). ([http://www.disketa.si/media/uploads/2015-01/7711d592584a53282f2ecb0fd678ef40.jpg See image here]) Cultures with 40 mM mevalonate added produced much more amoprhadiene. With time, amorphadiene concentration was dropping beacause of the loss of the volatile terpene[1]. By adding more than 10 mM mevalonate in control cultures without ADS, the severe growth ihibition has been reported. ([http://www.disketa.si/media/uploads/2015-01/2a21f55f0fea31ffcbe89c866c9150f7.jpg See image here]) To discover the cause ofthis inhibition, the growth of ''E.coli'' DH10B from strains with empty, pMKPMK, pMevB, pMBI or pMBIS plasmid was measured in media with increasing concentrations of mevalonate. The addition of 5mM mevalonate to the medium inhibited the growth of cells with pMevB. But 5mM concentrations of mevalonate did not alter the growth of cells with pMKPMK, PMBI or pMBIS plasmid. This indicated that the accomulation of IPP is toxic and inhibits normal cell growth. The accmulation mostly occurs in cells with high flux through mevalonate pathway. The next step was comparison of intracellular prenyl pyrophosphate (FPP) in strains. This was done by feeding the cells harboring the different mevalonte operon construct with radiolabeled mevalonate. Than the labeled metabolites were tracked. For this experiment cell suspensions of E.coli harboring pMevB+pTrc99A, pMBI+ pTrc99A, pMBIS+ pTrc99A or pMBIS+pADS were used. pTRc99A is the bacterial expression vector with inducible to IPTG lacI promoter. The result showed that strains with MevB operon accumulated IPP but not FPP, when MBI and MBIS strains accumulated FPP. ([http://www.disketa.si/media/uploads/2015-01/31ad7a215f016dc26aceacf92eb46712.jpg See image here]) Because of the simultaneous expression of the amorphadiene synthase the waste of FPP that accumulated in the MBIS host was consumed. This was shown by a decrease in intracellular FPP. Cells expresing MBIS acumulated FPP and showed growth inhibition in the presence of 10 mM mevalonate. The prediction was that the coexpression of ADS would ease the growth inhibition by forwarding the prenyl pyrophosphate intermediates to volatile terpene olefin. For this test, the cell with pMBIS and empty expression vector pTrc99A (without ADS)and cells with pMBIS and pADS vectors were used. The medium was supplemented with mevalonate concentations ( 0mM, 5mM, 10mM, 20mM or 40mM). Two hours after addition of mevalonate and inducer IPTG, the growth inhibition was observed. ([http://www.disketa.si/media/uploads/2015-01/94b5f3d5636fe767316f770f3e2b0448.jpg See image here]) The result showed that the strains with pMBIS +ADS started to grow, but strain lacking the ADS still showed growth inhbition at higher concentrations of mevalonate. This supported the conclusion that converion of FPP to amophadiene has an important role in minimising growth inhibition. Briefly these result showed that engineered mevalonate patway produces high levels of prenyl pyrophosphate precursors. If the IPP isomerase, FPP synthase and terpen synthase is absent, IPP can acumulate and be toxic to the cells. The toxic levels of intracellular prenyl pyrphosphate can accumulate [1].

=== Amorphadiene synthesis from acetyl-Coa ===

The main goal was to achieve high amoprhaidene production from a simple and inexpensive carbon source. ''E.coli'' strains harboring pMevT,pMBIS and pADS plasmids were transformed with pMevT plasmid. Subsequently the strains were tested for ability to produce amoprhadiene in the absence of exogenous mevalonate. The comparison between E.coli expressing the native DPX pathway and engineered pathway as made. They tested native DXP pathway, engineered DPX pathway, mevalonate bottom pathway in the absence of mevalonate, mevlonate bottom pathway in medium suplemented with mevalonate, the complete mevalonate pathway and the complete mevalonate pathway supplemented with 0,8% glycerol. Glycerol was addded to investigate the effect of amorphadiene production of supplying as single carbon source and for prolongation of amorphadiene production. Breifly the resulut showed that expression of the mevalonate-dependent isoprenoid biosynthetic pathway delivers hight levels of isoprenoid precursor for the production of sesquiterpene [1]. ([http://www.disketa.si/media/uploads/2015-01/a31fe07a7c66854b1dc3ff36f8ed01ef.jpg See image here])

== DISSCUSION ==
The identification of genes encoding the enzymes involved in the artemisinin-biosynthesis pathway together with advances in metabolic engineering provides new options for engineering artemisinin biosynthesis. Heterologous production of artemisnin in microorganism such as E.coli. is an alternative way for production of artmeinsin in high yields. But eventhougt micoorganisms has shown great potential in that matter, optimisation of these heterlogous pathways still require maximal titer. Inequal gene expression can lead to accumulation of toxic metabolic intermediates and to over or under production of enzymes involved in pathway. (4). Conclusion based on these study shows that poor expression of the amoprhaidene synthase genes (ADS) limits high terpene olfein yields from the host. To achieve better results , in this study they chose to synthesiyse an amophaiene synthase gene (ADS). The comparison between E.coli expressing native sesquiterpene synthase genes and E.coli expressing the synthetic ADS gene showed big improvement in terpene synthesis due to synthetic ADS gene. The native DPX pathway showed poor supply of the prenyl pyrophosphate precuror. And that limited terpenoid -carotenoid yields in E.coli. With the metabolic engineering of the DPX pathway the flux of carotenoid accumulation showed signifcant increase. By using the mevalonate-dependent isoprenoid pathway from yeast S. cerevisae and engineering that pathway in bacteria E.coli, we avoid the native regulatory elements found in yeast while bypassing those of E.coli DXP pathway. In this study results showed growth inhibition in cells without ADS due to the vast excess of preny pyrophosphate. But the coexpression of a synthetic sesquiterpene synthase can contribute to reducing the excess pool of precursors and eliminate growth inhibition. In this study total biosynthesis of artemisnin was not completely achieved. But these results can help in further investigation for poduction of artemisnic acid. This acid can then be putrified and chemically converted into artemsinin. [1,7].

== CONCLUSIONS ==
To sum things up, artmeinsin, a sesquiterpene isolated from Artemisia annua,represent a novel drug for malaria treatment. The present artemisnin derivates are too expensive for general use and plants can not produce artmeinsin in big quantities. This fact inspired scientist to focus on developing the way for production of artemisnin in high quantities and to avoid big costs. The promising tool in alternative source of artmeinsin is the use of microoganisms. The aim is to transfer the genes encoding enzymes involved in biosynthetic pathway for artemisnin into a hight producing host organism. Several study have been reporting on engineering the metabolic pathway in S.cerevisae to produce the precursor for artmeisnic acid. With the effort in synthetic biology they used modified mevalonate pathway . With the help of syntehtic bilogy the yeast cells were engineerd to express ezymes needed for production of artmemisnic acid. But in this paper the main focus was on engineering a mevalonate pathway in bacteria E.coli. By engineering a new metabolic pathway in E.coli we can easily synthesise a precursor nedeed for artemisnin production. The goal is to produce drugs in better and cheape ways. But, why focus on artemisnin? Well, acording on recent studies artemisnin shows to be very sucesfull for the treatmen all strain of malaria. More than a milion people affected with malaria have already been cured by arteminsin. As already mentioned , because of very hight costs and limited production of arteminin in plants most populations in Africa can not afford it. Progress made in synthetic biology and ability to produce amorphadiene in microorganisms in big quantites opens a new possibility to produce low cost artemisnin and contributes to the brighter future od antimalaria drug deveopment, and therefore saving milions from suffering from malaria. [1,2,3].

[1]Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nature biotehnology; vol 21; pages 796-802, July 2003

[2] T. Mosses, J. Pollier, J. M. Thevelein, A. Goossens. Bioengineering of plant (tri)terpenoids: from metabolic engineering of plants to synthetic biology in vivo and in vitro. New Pathyologist. 200: 27-43, 2013

[3] M.Farhi, M. Kozin, S. Duchin, A. Vainstein. Metabolic engineering of plants for artemisinin synthesis. Biotehnology and genetic engineering rewiews.Vol.29,No.2: 135-148, 2013

[4] Synthetic biology ; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Synthetic_biology http://en.wikipedia.org/wiki/Synthetic_biology]

[5]Operon; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Operon http://en.wikipedia.org/wiki/Operon]

[6] M. K. Julsing, G. van Pouderoyen, J. van der Veen,H. J. Woerdenbag, O. Kayser, W. J. Quax. Amorphadiene synthase: probing the active site model with directed mutagenesis. Directed mutagenesis of AMDS.Chapter 4: 47-56

[7]J.R. Anthony, L. C. Anthony, F. Nowroozi, G. Kwon, J.D. Newman, J.D. Keasling. Optimization of themevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene. Metabolic Engineering. 11: 13-19, 2009

Engineering a mevalonate pathway in Escherichia coli for production of terpenoids

2015-01-19T23:58:09Z

Ana90: /* Amorpadiene synthesis from mevalonate */

'''Ana Kapraljević'''

[http://www2.lbl.gov/ttd/publications/2837pub1.pdf Engineering a mevalonate pathway in Escherichia coli for production of terpenoids]
Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling ;Nature biotehnology; July 2003; vol 21; pages 796-802

== INTRODUCTION ==

Plants can synthesise and accumulate a wide range of small molecules or natural products involved in psyhological an ecological process. With the knowledge we know about their biosyntesis we used them as commercial flavors, fragnance compunds, colorants and terapeutical drugs. Their therapeutic potential is used for production of anticancer or antimalarial drugs, such as artemisnin. The most numerous and diverse class of natural products used in production for antimalarial drug are isoprenoids or more corectly their subclass, terpenoids. Malaria represent a major health problem in tropic and subtropic. Majority of death occur in children living in Africa. But the problem is that terpenoids are in plants produced in very small quantites and consequently not very economically feasible. For that reason the drugs for treatin malaria, artemisnin, is in short supply and is still unaffordable for most people living in malaria-endanger countries. Scientists are now focusing on alternative and cheaper way for terpenoid synthesis that can be enchanched and became economically acceptable. The alternative way is the expression of synthetic amorphadiene, precursor to artemisnin, and mevalonate isoprenoid way from yeas ''Saccharomyces cerevisiae'' in bacteria. In this paper I will summerize the project made in the production of amorphadiene by biotehnology and synthetic biology approach [1,2].

=== Artemisnin ===

Artemisnin is a substance that has antimalarial potential. Malaria is a infectious disease caused by parasitic protozan of the ''Plasmodium'' species. In the certain parts of the world malaria takes lives of more people worldwide than any other infecious disease, so it is a global problem. Death to malaria has been nowdays reduced thanks to the use of artemisnin-based combination therapy. Artemisnin is a natural compoud from plant ''Artemisia annua''. Chemically it is a sesquiterpene lactone, amorphadiene. The total sythesis of artemisnin is difficult and not very economical,so the new aproach is to put the hopes in sythetic biology and develop the new way, synthetic way, for its production [1,3].

=== Biology of terpenoids ===

For initiation of any synthetic biology process in metabolic engineering in that matter it is important to have some basic understanding of biosynthesis and regulation of terpenoids. Terpenoids, a class of isoprenoids, are natural products often sythesized by plants. They represent a large and diverse class and have many functions as primarly metabolites involved in growth and development of plants, or as secundary metabolites that optimize the interaction between the plant and the environment. Eventhought they are structural diverse, the share a common biosynthetic origin and follow similar synthesis tracks. All terpenoids are derived from repetitive fusion of isoprene units (C5 H8) [3]. The number of isoprene units determines their classification. For isoprenoid synthesis plants use mevalonate-dependent (MEV) pathway and mevalonte-independent pathway or alternative deoxyxylulose 5-phosphate pathway (DXP). In higher plants the biosynthesis begins with the generation of isopentenyl pyrophosphate (IPP). IPP is derived via the classic mevalonic acid pathway from acetly-coenzime A (acetyl-CoA). The IPP is isomerized to its allylic isomer dimethylallyl pyrophosphate (DMAPP) [3]. The continous condensation of IPP and DMAPP units leads to the formaton of prenylated pyrophosphates, the immediate precursors of the different terpenoid classes. So IPP and DMAPP are combined to form larger isoprenoids, such as farnesyl diphosphate (FPP). Condensation of IPP and DMAPP than units leads to the formation of precursors of the different terpenoid classes. Shematic production of terpenoids in ilustraded in this picture. Similary to plants, yeast ''Sacharomyces cerevisae'' uses the clasical mevalonate pathway for the synthesis of isoprenoids[3]. Prokariontes mostly use the DXP pathway for production IPP and DMAPP. This is an alternative pathway for IPP synthesis. This pathway uses ''Escherichia coli'' and some other bacteria for their terpenoid synthesis. IPP and DMAPP precurosr are essential to E. Coli for prenylation (addition of hydrophobic molecules to a protein or chemical compound) of tRNA and the synthesis of farnesyl pyrophosphate (FPP). FPP is then used for quinone (class of organic compounds) and cell wall synthesis. The DPX pathway begins with the glycolytic intermediates glyceraldehyde-3-phosphate (G3P) and pyruvate and continues through enzymatic steps for production of IPP and DMAPP. Therefore IPP and DMAPP are the end product in both way,MEV and DPX pathway [1,3].

== METABOLIC ENGINEERING FOR PRODUCTION OD TERPENOIDS ==

Metabolic engineering represents an improvement of production, construction, or cellular properties through the modification of specific biochemical reactions. It is described as the use of genetic engineering to reshape or modify the metabolism of organism. The aim is to optimize genetic and regulatory processes in cells to increase the production of a certain substance that we find in nature. It is based on optimisation of existing biochemical pathways or the introduction of natural pathway components in bacteria, yeast or plants. The main goal is the production of high-yield of specific metabolites that we can use in medicine or biotechnology. With progress in synthetic biology we can design a biologic funcion that do not exist in nature. Consequently, we can create and construct new biological components or redesign exisitng biological systems. In plants, most terpenoids are produced in a very small quantities in their natural sources. Extraction of this compouds from plants is expensive and therfore not economicaly accetable. So there is a big disagreement between the insistence for supply of terpenoids and therefore that leads to delay for their widespread application. With the understanding of terpenoid synthesis and progress in synthetic biology we are now able of redesign and modify the isoprenoid pathway to increase the terpenoid productivity [2,4]. ([http://www.disketa.si/media/uploads/2015-01/cd201ade6a167488767dfb1b118587bc.jpg See image here])

=== Microbal host ===

To elimante the need for plant extraction it is practical to produce terpenoid compouds at high yield in microbal host by introducing a heterlogous isoprenoid pathway into bacteria ''E.coli.'' Microorganisms, such as bacteria are attractive alternatives as hosts because they have rapid doubling time, they can achieve robussnes under process conditions and because of the simplicity of product putrification. And the costs are significantly lower than working with plants. With the engineering the DXP pathway we can increase the supply of isoprenoid precurosor needed for high level productio of isoprenoids in E.coli [1,3].

=== Operons ===

Operon is a a funcional unit of DNA that contains a cluster of genes under the control of a single promotor. The synthesis of natural or synthetic products involves the introduction of a large number of genes. In this case these genes were encoding the enzymes needed for metabolic pathway. So it is useful to put certain genes under the control of a single promotor. For that reason Plac promotor was used in this study [1,5].

== EXPERIMENT ==

=== Synthase gene asemby and amorphadiene production ===

For high level production and changing the difficulties in expressing teprene synthases they first synthesized and expressed a codon-optimized variants of ADS . ADS is a gene encoding amorphadiene synthase and is sequentially oxidised by citorchromeP450 CYP71AV1 and reduced by artemisinic aldehyde reductase (DBR2). Amorphadyene synthase is an enzyme and catalyse the first step in the anitmalarial drug artemisnin synthesis. It has an inportant role in catalysing the reaction of FPP to amorphadiene. Beacause of its importaint step towards artemisnin biosynthesis it was designed and improved for the purpuse to acheve high-level expression in E.coli. For ADS gene synthesis they used two step assemly and one step amplification PCR.(1) Assemby PCR is a useful technique for production novel gene sequences. With this method we can put together short fragments of DNA and it is also a good choice for gene construction. Reserches in this article analysed the sequence of three ADS genes from independet clones and identified mutations in each of the genes. Than, two site directed mutagenesis reaction was used for assembly and generation a functional ADS gene from two clones. Than they measure the amorphadiene production by the amorphadiene synthase in E.coli DH10B with natural (nonengineered DPX pathway) and in E.coli with engineered DPX pathway. E. coli with engineerd pathway was harboring the pSOE4 plasmid (DXP pathway operon with dxs,IppHp and ispA gene). The result sugested that FPP synthesis limited amorphadiene production in this engineered host [1,6]. ([http://www.disketa.si/media/uploads/2015-01/0bddf07f946e6f5be0638090dee56490.jpg See image here])

=== Egineering the mevalonate-dependet pathway in E.coli ===

The aim was to increase the intracellular concentration of FPP substrate. Genes encoding the MEV pathway from S.cerevisae was assembled into operons and expressed in E.coli. Because of the complexity of engineering numerous gene biosynthetic pathway, genes were divided into to operons, top and bottom.Top operon, MevT, consisted of three enzymes: acetoacetyl-CoA thiolase (''atoB''), HMG-CoA synthases (''HMGS'') and HMG-CoA reductase (''tHMGR''). This operon was responsible for transformation of acetyl-coA to mevalonate. Thre different bottom operons pMevB (''ERG12, ERG8,MVD1''), pMBI (''ERG12, ERG8, MVD1, idi'') and pMBIS (''ERG12, ERG8, MVD1, idi, ispA'') transformed mevalonate to IPP, DMAPP and consequently to FPP. The next step was to analyse and test the funcionality of this pathway. For this purpuse E.coli strand DYM1 with incoplemplete isoprenoid synthesis was transformed with plasmid expressing three bottom operon constuct- pMevB, pMBI and pMBI. E.coli strand DYM1 had a deletion in the ispC gene for encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase, and was not able to synthesise 2-C-methyl-D-erythritol 4-phosphate(MEP), which is important intermediate in isoprenoid biosynthesis[1]. Because of this deletion the synthesis of precursors IPP and DMAP is blocked. So then they tested what is going to happen if they grew the strain DYM1 in the presence of 2-C-methyl-D-ertihritol and in medium with mevalonate without methylerythritol since DYM1 strain was not capable to synthesisie MEP. As expected , all strains expressing operon from S.cerevisiae grew on the plates in the presence of 2-c-methyl-D-erythritol. But only strains with pMBI or pMBIS grew on plates with 1mM mevalonate in the absence of methylerythol. This was the conformation that synthetic MBI and MBIS operons were functional, because they were capable to support the growth of E.coli without ispC gene by supplying cells with IPP and DMAPP. Next they completed the mevalonate pathway by transforming the pMevT plasmid (expressing three genes mentioned before-atoB, HMGS and tHMGR) with pMBI or pMBIS. This coexpresion of two operons, pMevT and pMBI /pMBIS ,completed the mevlonate pathway and allowed the synthesis of sesquiterpene precursors from acetyl-CoA, IPP and DMAPP. Therefore, coexpression complemented the ''ispC'' deletion even in the absence of mevalonate, what was the proof, that MevT operon was functional [1,7].

=== Amorpadiene synthesis from mevalonate ===

The aim was to achieve high-level production of amorphadiene and to find out if the amount of FPP was limiting amoprhadiene output. As mentioned, ''E.coli'' was used as heterologous microorganism in which coupled mevalonate pathway with amorphadiene synthesis. Cells with ''ADS'' gene were coexpressed with MBIS operon and grown in medium with increasing amounts (0 to 40 mM) of exogenus mevalonate. The culture extract was analysed with gas chromatography-mass spectrometry, analytical method for identification different substances in a test sample. It combines the features of mass spectrometry and gas-liquid chromatography. Results showed that MBIS operon did not limit the amorphadiene production at the highest mevalonate concentration(40mM). ([http://www.disketa.si/media/uploads/2015-01/7711d592584a53282f2ecb0fd678ef40.jpg See image here]) Cultures with 40 mM mevalonate added produced much more amoprhadiene. With time, amorphadiene concentration was dropping beacause of the loss of the volatile terpene[1]. By adding more than 10 mM mevalonate in control cultures without ADS, the severe growth ihibition has been reported. ([http://www.disketa.si/media/uploads/2015-01/2a21f55f0fea31ffcbe89c866c9150f7.jpg See image here]) To discover the cause ofthis inhibition, the growth of ''E.coli'' DH10B from strains with empty, pMKPMK, pMevB, pMBI or pMBIS plasmid was measured in media with increasing concentrations of mevalonate. The addition of 5mM mevalonate to the medium inhibited the growth of cells with pMevB. But 5mM concentrations of mevalonate did not alter the growth of cells with pMKPMK, PMBI or pMBIS plasmid. This indicated that the accomulation of IPP is toxic and inhibits normal cell growth. The accmulation mostly occurs in cells with high flux through mevalonate pathway. The next step was comparison of intracellular prenyl pyrophosphate (FPP) in strains. This was done by feeding the cells harboring the different mevalonte operon construct with radiolabeled mevalonate. Than the labeled metabolites were tracked. For this experiment cell suspensions of E.coli harboring pMevB+pTrc99A, pMBI+ pTrc99A, pMBIS+ pTrc99A or pMBIS+pADS were used. pTRc99A is the bacterial expression vector with inducible to IPTG lacI promoter. The result showed that strains with MevB operon accumulated IPP but not FPP, when MBI and MBIS strains accumulated FPP. ([http://www.disketa.si/media/uploads/2015-01/31ad7a215f016dc26aceacf92eb46712.jpg See image here]) Because of the simultaneous expression of the amorphadiene synthase the waste of FPP that accumulated in the MBIS host was consumed. This was shown by a decrease in intracellular FPP. Cells expresing MBIS acumulated FPP and showed growth inhibition in the presence of 10 mM mevalonate. The prediction was that the coexpression of ADS would ease the growth inhibition by forwarding the prenyl pyrophosphate intermediates to volatile terpene olefin. For this test, the cell with pMBIS and empty expression vector pTrc99A (without ADS)and cells with pMBIS and pADS vectors were used. The medium was supplemented with mevalonate concentations ( 0mM, 5mM, 10mM, 20mM or 40mM). Two hours after addition of mevalonate and inducer IPTG, the growth inhibition was observed. ([http://www.disketa.si/media/uploads/2015-01/94b5f3d5636fe767316f770f3e2b0448.jpg See image here]) The result showed that the strains with pMBIS +ADS started to grow, but strain lacking the ADS still showed growth inhbition at higher concentrations of mevalonate. This supported the conclusion that converion of FPP to amophadiene has an important role in minimising growth inhibition. Briefly these result showed that engineered mevalonate patway produces high levels of prenyl pyrophosphate precursors. If the IPP isomerase, FPP synthase and terpen synthase is absent, IPP can acumulate and be toxic to the cells. The toxic levels of intracellular prenyl pyrphosphate can accumulate [1].

=== Amorphadiene synthesis from acetyl-Coa ===

The main goal was to achieve high amoprhaidene production from a simple and inexpensive carbon source. E.coli strains harboring pMevT,pMBIS and pADS plasmids were transformed with pMevT plasmid. Subsequently the strains were tested for ability to produce amoprhadiene in the absence of exogenous mevalonate. The comparison between E.coli expressing the native DPX pathway and engineered pathway as made. They tested native DXP pathway, engineered DPX pathway, mevalonate bottom pathway in the absence of mevalonate, mevlonate bottom pathway in medium suplemented with mevalonate, the complete mevalonate pathway and the complete mevalonate pathway supplemented with 0,8% glycerol. Glycerol was addded to investigate the effect of amorphadiene production of supplying as single carbon source and for prolongation of amorphadiene production. Breifly the resulut showed that expression of the mevalonate-dependent isoprenoid biosynthetic pathway delivers hight levels of isoprenoid precursor for the production of sesquiterpene [1]. ([http://www.disketa.si/media/uploads/2015-01/a31fe07a7c66854b1dc3ff36f8ed01ef.jpg See image here])

== DISSCUSION ==
The identification of genes encoding the enzymes involved in the artemisinin-biosynthesis pathway together with advances in metabolic engineering provides new options for engineering artemisinin biosynthesis. Heterologous production of artemisnin in microorganism such as E.coli. is an alternative way for production of artmeinsin in high yields. But eventhougt micoorganisms has shown great potential in that matter, optimisation of these heterlogous pathways still require maximal titer. Inequal gene expression can lead to accumulation of toxic metabolic intermediates and to over or under production of enzymes involved in pathway. (4). Conclusion based on these study shows that poor expression of the amoprhaidene synthase genes (ADS) limits high terpene olfein yields from the host. To achieve better results , in this study they chose to synthesiyse an amophaiene synthase gene (ADS). The comparison between E.coli expressing native sesquiterpene synthase genes and E.coli expressing the synthetic ADS gene showed big improvement in terpene synthesis due to synthetic ADS gene. The native DPX pathway showed poor supply of the prenyl pyrophosphate precuror. And that limited terpenoid -carotenoid yields in E.coli. With the metabolic engineering of the DPX pathway the flux of carotenoid accumulation showed signifcant increase. By using the mevalonate-dependent isoprenoid pathway from yeast S. cerevisae and engineering that pathway in bacteria E.coli, we avoid the native regulatory elements found in yeast while bypassing those of E.coli DXP pathway. In this study results showed growth inhibition in cells without ADS due to the vast excess of preny pyrophosphate. But the coexpression of a synthetic sesquiterpene synthase can contribute to reducing the excess pool of precursors and eliminate growth inhibition. In this study total biosynthesis of artemisnin was not completely achieved. But these results can help in further investigation for poduction of artemisnic acid. This acid can then be putrified and chemically converted into artemsinin. [1,7].

== CONCLUSIONS ==
To sum things up, artmeinsin, a sesquiterpene isolated from Artemisia annua,represent a novel drug for malaria treatment. The present artemisnin derivates are too expensive for general use and plants can not produce artmeinsin in big quantities. This fact inspired scientist to focus on developing the way for production of artemisnin in high quantities and to avoid big costs. The promising tool in alternative source of artmeinsin is the use of microoganisms. The aim is to transfer the genes encoding enzymes involved in biosynthetic pathway for artemisnin into a hight producing host organism. Several study have been reporting on engineering the metabolic pathway in S.cerevisae to produce the precursor for artmeisnic acid. With the effort in synthetic biology they used modified mevalonate pathway . With the help of syntehtic bilogy the yeast cells were engineerd to express ezymes needed for production of artmemisnic acid. But in this paper the main focus was on engineering a mevalonate pathway in bacteria E.coli. By engineering a new metabolic pathway in E.coli we can easily synthesise a precursor nedeed for artemisnin production. The goal is to produce drugs in better and cheape ways. But, why focus on artemisnin? Well, acording on recent studies artemisnin shows to be very sucesfull for the treatmen all strain of malaria. More than a milion people affected with malaria have already been cured by arteminsin. As already mentioned , because of very hight costs and limited production of arteminin in plants most populations in Africa can not afford it. Progress made in synthetic biology and ability to produce amorphadiene in microorganisms in big quantites opens a new possibility to produce low cost artemisnin and contributes to the brighter future od antimalaria drug deveopment, and therefore saving milions from suffering from malaria. [1,2,3].

[1]Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nature biotehnology; vol 21; pages 796-802, July 2003

[2] T. Mosses, J. Pollier, J. M. Thevelein, A. Goossens. Bioengineering of plant (tri)terpenoids: from metabolic engineering of plants to synthetic biology in vivo and in vitro. New Pathyologist. 200: 27-43, 2013

[3] M.Farhi, M. Kozin, S. Duchin, A. Vainstein. Metabolic engineering of plants for artemisinin synthesis. Biotehnology and genetic engineering rewiews.Vol.29,No.2: 135-148, 2013

[4] Synthetic biology ; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Synthetic_biology http://en.wikipedia.org/wiki/Synthetic_biology]

[5]Operon; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Operon http://en.wikipedia.org/wiki/Operon]

[6] M. K. Julsing, G. van Pouderoyen, J. van der Veen,H. J. Woerdenbag, O. Kayser, W. J. Quax. Amorphadiene synthase: probing the active site model with directed mutagenesis. Directed mutagenesis of AMDS.Chapter 4: 47-56

[7]J.R. Anthony, L. C. Anthony, F. Nowroozi, G. Kwon, J.D. Newman, J.D. Keasling. Optimization of themevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene. Metabolic Engineering. 11: 13-19, 2009

Engineering a mevalonate pathway in Escherichia coli for production of terpenoids

2015-01-19T23:56:42Z

Ana90: /* Microbal host */

'''Ana Kapraljević'''

[http://www2.lbl.gov/ttd/publications/2837pub1.pdf Engineering a mevalonate pathway in Escherichia coli for production of terpenoids]
Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling ;Nature biotehnology; July 2003; vol 21; pages 796-802

== INTRODUCTION ==

Plants can synthesise and accumulate a wide range of small molecules or natural products involved in psyhological an ecological process. With the knowledge we know about their biosyntesis we used them as commercial flavors, fragnance compunds, colorants and terapeutical drugs. Their therapeutic potential is used for production of anticancer or antimalarial drugs, such as artemisnin. The most numerous and diverse class of natural products used in production for antimalarial drug are isoprenoids or more corectly their subclass, terpenoids. Malaria represent a major health problem in tropic and subtropic. Majority of death occur in children living in Africa. But the problem is that terpenoids are in plants produced in very small quantites and consequently not very economically feasible. For that reason the drugs for treatin malaria, artemisnin, is in short supply and is still unaffordable for most people living in malaria-endanger countries. Scientists are now focusing on alternative and cheaper way for terpenoid synthesis that can be enchanched and became economically acceptable. The alternative way is the expression of synthetic amorphadiene, precursor to artemisnin, and mevalonate isoprenoid way from yeas ''Saccharomyces cerevisiae'' in bacteria. In this paper I will summerize the project made in the production of amorphadiene by biotehnology and synthetic biology approach [1,2].

=== Artemisnin ===

Artemisnin is a substance that has antimalarial potential. Malaria is a infectious disease caused by parasitic protozan of the ''Plasmodium'' species. In the certain parts of the world malaria takes lives of more people worldwide than any other infecious disease, so it is a global problem. Death to malaria has been nowdays reduced thanks to the use of artemisnin-based combination therapy. Artemisnin is a natural compoud from plant ''Artemisia annua''. Chemically it is a sesquiterpene lactone, amorphadiene. The total sythesis of artemisnin is difficult and not very economical,so the new aproach is to put the hopes in sythetic biology and develop the new way, synthetic way, for its production [1,3].

=== Biology of terpenoids ===

For initiation of any synthetic biology process in metabolic engineering in that matter it is important to have some basic understanding of biosynthesis and regulation of terpenoids. Terpenoids, a class of isoprenoids, are natural products often sythesized by plants. They represent a large and diverse class and have many functions as primarly metabolites involved in growth and development of plants, or as secundary metabolites that optimize the interaction between the plant and the environment. Eventhought they are structural diverse, the share a common biosynthetic origin and follow similar synthesis tracks. All terpenoids are derived from repetitive fusion of isoprene units (C5 H8) [3]. The number of isoprene units determines their classification. For isoprenoid synthesis plants use mevalonate-dependent (MEV) pathway and mevalonte-independent pathway or alternative deoxyxylulose 5-phosphate pathway (DXP). In higher plants the biosynthesis begins with the generation of isopentenyl pyrophosphate (IPP). IPP is derived via the classic mevalonic acid pathway from acetly-coenzime A (acetyl-CoA). The IPP is isomerized to its allylic isomer dimethylallyl pyrophosphate (DMAPP) [3]. The continous condensation of IPP and DMAPP units leads to the formaton of prenylated pyrophosphates, the immediate precursors of the different terpenoid classes. So IPP and DMAPP are combined to form larger isoprenoids, such as farnesyl diphosphate (FPP). Condensation of IPP and DMAPP than units leads to the formation of precursors of the different terpenoid classes. Shematic production of terpenoids in ilustraded in this picture. Similary to plants, yeast ''Sacharomyces cerevisae'' uses the clasical mevalonate pathway for the synthesis of isoprenoids[3]. Prokariontes mostly use the DXP pathway for production IPP and DMAPP. This is an alternative pathway for IPP synthesis. This pathway uses ''Escherichia coli'' and some other bacteria for their terpenoid synthesis. IPP and DMAPP precurosr are essential to E. Coli for prenylation (addition of hydrophobic molecules to a protein or chemical compound) of tRNA and the synthesis of farnesyl pyrophosphate (FPP). FPP is then used for quinone (class of organic compounds) and cell wall synthesis. The DPX pathway begins with the glycolytic intermediates glyceraldehyde-3-phosphate (G3P) and pyruvate and continues through enzymatic steps for production of IPP and DMAPP. Therefore IPP and DMAPP are the end product in both way,MEV and DPX pathway [1,3].

== METABOLIC ENGINEERING FOR PRODUCTION OD TERPENOIDS ==

Metabolic engineering represents an improvement of production, construction, or cellular properties through the modification of specific biochemical reactions. It is described as the use of genetic engineering to reshape or modify the metabolism of organism. The aim is to optimize genetic and regulatory processes in cells to increase the production of a certain substance that we find in nature. It is based on optimisation of existing biochemical pathways or the introduction of natural pathway components in bacteria, yeast or plants. The main goal is the production of high-yield of specific metabolites that we can use in medicine or biotechnology. With progress in synthetic biology we can design a biologic funcion that do not exist in nature. Consequently, we can create and construct new biological components or redesign exisitng biological systems. In plants, most terpenoids are produced in a very small quantities in their natural sources. Extraction of this compouds from plants is expensive and therfore not economicaly accetable. So there is a big disagreement between the insistence for supply of terpenoids and therefore that leads to delay for their widespread application. With the understanding of terpenoid synthesis and progress in synthetic biology we are now able of redesign and modify the isoprenoid pathway to increase the terpenoid productivity [2,4]. ([http://www.disketa.si/media/uploads/2015-01/cd201ade6a167488767dfb1b118587bc.jpg See image here])

=== Microbal host ===

To elimante the need for plant extraction it is practical to produce terpenoid compouds at high yield in microbal host by introducing a heterlogous isoprenoid pathway into bacteria ''E.coli.'' Microorganisms, such as bacteria are attractive alternatives as hosts because they have rapid doubling time, they can achieve robussnes under process conditions and because of the simplicity of product putrification. And the costs are significantly lower than working with plants. With the engineering the DXP pathway we can increase the supply of isoprenoid precurosor needed for high level productio of isoprenoids in E.coli [1,3].

=== Operons ===

Operon is a a funcional unit of DNA that contains a cluster of genes under the control of a single promotor. The synthesis of natural or synthetic products involves the introduction of a large number of genes. In this case these genes were encoding the enzymes needed for metabolic pathway. So it is useful to put certain genes under the control of a single promotor. For that reason Plac promotor was used in this study [1,5].

== EXPERIMENT ==

=== Synthase gene asemby and amorphadiene production ===

For high level production and changing the difficulties in expressing teprene synthases they first synthesized and expressed a codon-optimized variants of ADS . ADS is a gene encoding amorphadiene synthase and is sequentially oxidised by citorchromeP450 CYP71AV1 and reduced by artemisinic aldehyde reductase (DBR2). Amorphadyene synthase is an enzyme and catalyse the first step in the anitmalarial drug artemisnin synthesis. It has an inportant role in catalysing the reaction of FPP to amorphadiene. Beacause of its importaint step towards artemisnin biosynthesis it was designed and improved for the purpuse to acheve high-level expression in E.coli. For ADS gene synthesis they used two step assemly and one step amplification PCR.(1) Assemby PCR is a useful technique for production novel gene sequences. With this method we can put together short fragments of DNA and it is also a good choice for gene construction. Reserches in this article analysed the sequence of three ADS genes from independet clones and identified mutations in each of the genes. Than, two site directed mutagenesis reaction was used for assembly and generation a functional ADS gene from two clones. Than they measure the amorphadiene production by the amorphadiene synthase in E.coli DH10B with natural (nonengineered DPX pathway) and in E.coli with engineered DPX pathway. E. coli with engineerd pathway was harboring the pSOE4 plasmid (DXP pathway operon with dxs,IppHp and ispA gene). The result sugested that FPP synthesis limited amorphadiene production in this engineered host [1,6]. ([http://www.disketa.si/media/uploads/2015-01/0bddf07f946e6f5be0638090dee56490.jpg See image here])

=== Egineering the mevalonate-dependet pathway in E.coli ===

The aim was to increase the intracellular concentration of FPP substrate. Genes encoding the MEV pathway from S.cerevisae was assembled into operons and expressed in E.coli. Because of the complexity of engineering numerous gene biosynthetic pathway, genes were divided into to operons, top and bottom.Top operon, MevT, consisted of three enzymes: acetoacetyl-CoA thiolase (''atoB''), HMG-CoA synthases (''HMGS'') and HMG-CoA reductase (''tHMGR''). This operon was responsible for transformation of acetyl-coA to mevalonate. Thre different bottom operons pMevB (''ERG12, ERG8,MVD1''), pMBI (''ERG12, ERG8, MVD1, idi'') and pMBIS (''ERG12, ERG8, MVD1, idi, ispA'') transformed mevalonate to IPP, DMAPP and consequently to FPP. The next step was to analyse and test the funcionality of this pathway. For this purpuse E.coli strand DYM1 with incoplemplete isoprenoid synthesis was transformed with plasmid expressing three bottom operon constuct- pMevB, pMBI and pMBI. E.coli strand DYM1 had a deletion in the ispC gene for encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase, and was not able to synthesise 2-C-methyl-D-erythritol 4-phosphate(MEP), which is important intermediate in isoprenoid biosynthesis[1]. Because of this deletion the synthesis of precursors IPP and DMAP is blocked. So then they tested what is going to happen if they grew the strain DYM1 in the presence of 2-C-methyl-D-ertihritol and in medium with mevalonate without methylerythritol since DYM1 strain was not capable to synthesisie MEP. As expected , all strains expressing operon from S.cerevisiae grew on the plates in the presence of 2-c-methyl-D-erythritol. But only strains with pMBI or pMBIS grew on plates with 1mM mevalonate in the absence of methylerythol. This was the conformation that synthetic MBI and MBIS operons were functional, because they were capable to support the growth of E.coli without ispC gene by supplying cells with IPP and DMAPP. Next they completed the mevalonate pathway by transforming the pMevT plasmid (expressing three genes mentioned before-atoB, HMGS and tHMGR) with pMBI or pMBIS. This coexpresion of two operons, pMevT and pMBI /pMBIS ,completed the mevlonate pathway and allowed the synthesis of sesquiterpene precursors from acetyl-CoA, IPP and DMAPP. Therefore, coexpression complemented the ''ispC'' deletion even in the absence of mevalonate, what was the proof, that MevT operon was functional [1,7].

=== Amorpadiene synthesis from mevalonate ===

The aim was to achieve high-level production of amorphadiene and to find out if the amount of FPP was limiting amoprhadiene output. As mentioned, E.coli was used as heterologous microorganism in which coupled mevalonate pathway with amorphadiene synthesis. Cells with ADS gene were coexpressed with MBIS operon and grown in medium with increasing amounts (0 to 40 mM) of exogenus mevalonate. The culture extract was analysed with gas chromatography-mass spectrometry, analytical method for identification different substances in a test sample. It combines the features of mass spectrometry and gas-liquid chromatography. Results showed that MBIS operon did not limit the amorphadiene production at the highest mevalonate concentration(40mM). ([http://www.disketa.si/media/uploads/2015-01/7711d592584a53282f2ecb0fd678ef40.jpg See image here]) Cultures with 40 mM mevalonate added produced much more amoprhadiene. With time, amorphadiene concentration was dropping beacause of the loss of the volatile terpene[1]. By adding more than 10 mM mevalonate in control cultures without ADS, the severe growth ihibition has been reported. ([http://www.disketa.si/media/uploads/2015-01/2a21f55f0fea31ffcbe89c866c9150f7.jpg See image here]) To discover the cause ofthis inhibition, the growth of E.coli DH10B from strains with empty, pMKPMK, pMevB, pMBI or pMBIS plasmid was measured in media with increasing concentrations of mevalonate. The addition of 5mM mevalonate to the medium inhibited the growth of cells with pMevB. But 5mM concentrations of mevalonate did not alter the growth of cells with pMKPMK, PMBI or pMBIS plasmid. This indicated that the accomulation of IPP is toxic and inhibits normal cell growth. The accmulation mostly occurs in cells with high flux through mevalonate pathway. The next step was comparison of intracellular prenyl pyrophosphate (FPP) in strains. This was done by feeding the cells harboring the different mevalonte operon construct with radiolabeled mevalonate. Than the labeled metabolites were tracked. For this experiment cell suspensions of E.coli harboring pMevB+pTrc99A, pMBI+ pTrc99A, pMBIS+ pTrc99A or pMBIS+pADS were used. pTRc99A is the bacterial expression vector with inducible to IPTG lacI promoter. The result showed that strains with MevB operon accumulated IPP but not FPP, when MBI and MBIS strains accumulated FPP. ([http://www.disketa.si/media/uploads/2015-01/31ad7a215f016dc26aceacf92eb46712.jpg See image here]) Because of the simultaneous expression of the amorphadiene synthase the waste of FPP that accumulated in the MBIS host was consumed. This was shown by a decrease in intracellular FPP. Cells expresing MBIS acumulated FPP and showed growth inhibition in the presence of 10 mM mevalonate. The prediction was that the coexpression of ADS would ease the growth inhibition by forwarding the prenyl pyrophosphate intermediates to volatile terpene olefin. For this test, the cell with pMBIS and empty expression vector pTrc99A (without ADS)and cells with pMBIS and pADS vectors were used. The medium was supplemented with mevalonate concentations ( 0mM, 5mM, 10mM, 20mM or 40mM). Two hours after addition of mevalonate and inducer IPTG, the growth inhibition was observed. ([http://www.disketa.si/media/uploads/2015-01/94b5f3d5636fe767316f770f3e2b0448.jpg See image here]) The result showed that the strains with pMBIS +ADS started to grow, but strain lacking the ADS still showed growth inhbition at higher concentrations of mevalonate. This supported the conclusion that converion of FPP to amophadiene has an important role in minimising growth inhibition. Briefly these result showed that engineered mevalonate patway produces high levels of prenyl pyrophosphate precursors. If the IPP isomerase, FPP synthase and terpen synthase is absent, IPP can acumulate and be toxic to the cells. The toxic levels of intracellular prenyl pyrphosphate can accumulate [1].

=== Amorphadiene synthesis from acetyl-Coa ===

The main goal was to achieve high amoprhaidene production from a simple and inexpensive carbon source. E.coli strains harboring pMevT,pMBIS and pADS plasmids were transformed with pMevT plasmid. Subsequently the strains were tested for ability to produce amoprhadiene in the absence of exogenous mevalonate. The comparison between E.coli expressing the native DPX pathway and engineered pathway as made. They tested native DXP pathway, engineered DPX pathway, mevalonate bottom pathway in the absence of mevalonate, mevlonate bottom pathway in medium suplemented with mevalonate, the complete mevalonate pathway and the complete mevalonate pathway supplemented with 0,8% glycerol. Glycerol was addded to investigate the effect of amorphadiene production of supplying as single carbon source and for prolongation of amorphadiene production. Breifly the resulut showed that expression of the mevalonate-dependent isoprenoid biosynthetic pathway delivers hight levels of isoprenoid precursor for the production of sesquiterpene [1]. ([http://www.disketa.si/media/uploads/2015-01/a31fe07a7c66854b1dc3ff36f8ed01ef.jpg See image here])

== DISSCUSION ==
The identification of genes encoding the enzymes involved in the artemisinin-biosynthesis pathway together with advances in metabolic engineering provides new options for engineering artemisinin biosynthesis. Heterologous production of artemisnin in microorganism such as E.coli. is an alternative way for production of artmeinsin in high yields. But eventhougt micoorganisms has shown great potential in that matter, optimisation of these heterlogous pathways still require maximal titer. Inequal gene expression can lead to accumulation of toxic metabolic intermediates and to over or under production of enzymes involved in pathway. (4). Conclusion based on these study shows that poor expression of the amoprhaidene synthase genes (ADS) limits high terpene olfein yields from the host. To achieve better results , in this study they chose to synthesiyse an amophaiene synthase gene (ADS). The comparison between E.coli expressing native sesquiterpene synthase genes and E.coli expressing the synthetic ADS gene showed big improvement in terpene synthesis due to synthetic ADS gene. The native DPX pathway showed poor supply of the prenyl pyrophosphate precuror. And that limited terpenoid -carotenoid yields in E.coli. With the metabolic engineering of the DPX pathway the flux of carotenoid accumulation showed signifcant increase. By using the mevalonate-dependent isoprenoid pathway from yeast S. cerevisae and engineering that pathway in bacteria E.coli, we avoid the native regulatory elements found in yeast while bypassing those of E.coli DXP pathway. In this study results showed growth inhibition in cells without ADS due to the vast excess of preny pyrophosphate. But the coexpression of a synthetic sesquiterpene synthase can contribute to reducing the excess pool of precursors and eliminate growth inhibition. In this study total biosynthesis of artemisnin was not completely achieved. But these results can help in further investigation for poduction of artemisnic acid. This acid can then be putrified and chemically converted into artemsinin. [1,7].

== CONCLUSIONS ==
To sum things up, artmeinsin, a sesquiterpene isolated from Artemisia annua,represent a novel drug for malaria treatment. The present artemisnin derivates are too expensive for general use and plants can not produce artmeinsin in big quantities. This fact inspired scientist to focus on developing the way for production of artemisnin in high quantities and to avoid big costs. The promising tool in alternative source of artmeinsin is the use of microoganisms. The aim is to transfer the genes encoding enzymes involved in biosynthetic pathway for artemisnin into a hight producing host organism. Several study have been reporting on engineering the metabolic pathway in S.cerevisae to produce the precursor for artmeisnic acid. With the effort in synthetic biology they used modified mevalonate pathway . With the help of syntehtic bilogy the yeast cells were engineerd to express ezymes needed for production of artmemisnic acid. But in this paper the main focus was on engineering a mevalonate pathway in bacteria E.coli. By engineering a new metabolic pathway in E.coli we can easily synthesise a precursor nedeed for artemisnin production. The goal is to produce drugs in better and cheape ways. But, why focus on artemisnin? Well, acording on recent studies artemisnin shows to be very sucesfull for the treatmen all strain of malaria. More than a milion people affected with malaria have already been cured by arteminsin. As already mentioned , because of very hight costs and limited production of arteminin in plants most populations in Africa can not afford it. Progress made in synthetic biology and ability to produce amorphadiene in microorganisms in big quantites opens a new possibility to produce low cost artemisnin and contributes to the brighter future od antimalaria drug deveopment, and therefore saving milions from suffering from malaria. [1,2,3].

[1]Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nature biotehnology; vol 21; pages 796-802, July 2003

[2] T. Mosses, J. Pollier, J. M. Thevelein, A. Goossens. Bioengineering of plant (tri)terpenoids: from metabolic engineering of plants to synthetic biology in vivo and in vitro. New Pathyologist. 200: 27-43, 2013

[3] M.Farhi, M. Kozin, S. Duchin, A. Vainstein. Metabolic engineering of plants for artemisinin synthesis. Biotehnology and genetic engineering rewiews.Vol.29,No.2: 135-148, 2013

[4] Synthetic biology ; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Synthetic_biology http://en.wikipedia.org/wiki/Synthetic_biology]

[5]Operon; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Operon http://en.wikipedia.org/wiki/Operon]

[6] M. K. Julsing, G. van Pouderoyen, J. van der Veen,H. J. Woerdenbag, O. Kayser, W. J. Quax. Amorphadiene synthase: probing the active site model with directed mutagenesis. Directed mutagenesis of AMDS.Chapter 4: 47-56

[7]J.R. Anthony, L. C. Anthony, F. Nowroozi, G. Kwon, J.D. Newman, J.D. Keasling. Optimization of themevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene. Metabolic Engineering. 11: 13-19, 2009

Engineering a mevalonate pathway in Escherichia coli for production of terpenoids

2015-01-19T23:56:20Z

Ana90: /* Biology of terpenoids */

'''Ana Kapraljević'''

[http://www2.lbl.gov/ttd/publications/2837pub1.pdf Engineering a mevalonate pathway in Escherichia coli for production of terpenoids]
Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling ;Nature biotehnology; July 2003; vol 21; pages 796-802

== INTRODUCTION ==

Plants can synthesise and accumulate a wide range of small molecules or natural products involved in psyhological an ecological process. With the knowledge we know about their biosyntesis we used them as commercial flavors, fragnance compunds, colorants and terapeutical drugs. Their therapeutic potential is used for production of anticancer or antimalarial drugs, such as artemisnin. The most numerous and diverse class of natural products used in production for antimalarial drug are isoprenoids or more corectly their subclass, terpenoids. Malaria represent a major health problem in tropic and subtropic. Majority of death occur in children living in Africa. But the problem is that terpenoids are in plants produced in very small quantites and consequently not very economically feasible. For that reason the drugs for treatin malaria, artemisnin, is in short supply and is still unaffordable for most people living in malaria-endanger countries. Scientists are now focusing on alternative and cheaper way for terpenoid synthesis that can be enchanched and became economically acceptable. The alternative way is the expression of synthetic amorphadiene, precursor to artemisnin, and mevalonate isoprenoid way from yeas ''Saccharomyces cerevisiae'' in bacteria. In this paper I will summerize the project made in the production of amorphadiene by biotehnology and synthetic biology approach [1,2].

=== Artemisnin ===

Artemisnin is a substance that has antimalarial potential. Malaria is a infectious disease caused by parasitic protozan of the ''Plasmodium'' species. In the certain parts of the world malaria takes lives of more people worldwide than any other infecious disease, so it is a global problem. Death to malaria has been nowdays reduced thanks to the use of artemisnin-based combination therapy. Artemisnin is a natural compoud from plant ''Artemisia annua''. Chemically it is a sesquiterpene lactone, amorphadiene. The total sythesis of artemisnin is difficult and not very economical,so the new aproach is to put the hopes in sythetic biology and develop the new way, synthetic way, for its production [1,3].

=== Biology of terpenoids ===

For initiation of any synthetic biology process in metabolic engineering in that matter it is important to have some basic understanding of biosynthesis and regulation of terpenoids. Terpenoids, a class of isoprenoids, are natural products often sythesized by plants. They represent a large and diverse class and have many functions as primarly metabolites involved in growth and development of plants, or as secundary metabolites that optimize the interaction between the plant and the environment. Eventhought they are structural diverse, the share a common biosynthetic origin and follow similar synthesis tracks. All terpenoids are derived from repetitive fusion of isoprene units (C5 H8) [3]. The number of isoprene units determines their classification. For isoprenoid synthesis plants use mevalonate-dependent (MEV) pathway and mevalonte-independent pathway or alternative deoxyxylulose 5-phosphate pathway (DXP). In higher plants the biosynthesis begins with the generation of isopentenyl pyrophosphate (IPP). IPP is derived via the classic mevalonic acid pathway from acetly-coenzime A (acetyl-CoA). The IPP is isomerized to its allylic isomer dimethylallyl pyrophosphate (DMAPP) [3]. The continous condensation of IPP and DMAPP units leads to the formaton of prenylated pyrophosphates, the immediate precursors of the different terpenoid classes. So IPP and DMAPP are combined to form larger isoprenoids, such as farnesyl diphosphate (FPP). Condensation of IPP and DMAPP than units leads to the formation of precursors of the different terpenoid classes. Shematic production of terpenoids in ilustraded in this picture. Similary to plants, yeast ''Sacharomyces cerevisae'' uses the clasical mevalonate pathway for the synthesis of isoprenoids[3]. Prokariontes mostly use the DXP pathway for production IPP and DMAPP. This is an alternative pathway for IPP synthesis. This pathway uses ''Escherichia coli'' and some other bacteria for their terpenoid synthesis. IPP and DMAPP precurosr are essential to E. Coli for prenylation (addition of hydrophobic molecules to a protein or chemical compound) of tRNA and the synthesis of farnesyl pyrophosphate (FPP). FPP is then used for quinone (class of organic compounds) and cell wall synthesis. The DPX pathway begins with the glycolytic intermediates glyceraldehyde-3-phosphate (G3P) and pyruvate and continues through enzymatic steps for production of IPP and DMAPP. Therefore IPP and DMAPP are the end product in both way,MEV and DPX pathway [1,3].

== METABOLIC ENGINEERING FOR PRODUCTION OD TERPENOIDS ==

Metabolic engineering represents an improvement of production, construction, or cellular properties through the modification of specific biochemical reactions. It is described as the use of genetic engineering to reshape or modify the metabolism of organism. The aim is to optimize genetic and regulatory processes in cells to increase the production of a certain substance that we find in nature. It is based on optimisation of existing biochemical pathways or the introduction of natural pathway components in bacteria, yeast or plants. The main goal is the production of high-yield of specific metabolites that we can use in medicine or biotechnology. With progress in synthetic biology we can design a biologic funcion that do not exist in nature. Consequently, we can create and construct new biological components or redesign exisitng biological systems. In plants, most terpenoids are produced in a very small quantities in their natural sources. Extraction of this compouds from plants is expensive and therfore not economicaly accetable. So there is a big disagreement between the insistence for supply of terpenoids and therefore that leads to delay for their widespread application. With the understanding of terpenoid synthesis and progress in synthetic biology we are now able of redesign and modify the isoprenoid pathway to increase the terpenoid productivity [2,4]. ([http://www.disketa.si/media/uploads/2015-01/cd201ade6a167488767dfb1b118587bc.jpg See image here])

=== Microbal host ===

To elimante the need for plant extraction it is practical to produce terpenoid compouds at high yield in microbal host by introducing a heterlogous isoprenoid pathway into bacteria E.coli. Microorganisms, such as bacteria are attractive alternatives as hosts because they have rapid doubling time, they can achieve robussnes under process conditions and because of the simplicity of product putrification. And the costs are significantly lower than working with plants. With the engineering the DXP pathway we can increase the supply of isoprenoid precurosor needed for high level productio of isoprenoids in E.coli [1,3].

=== Operons ===

Operon is a a funcional unit of DNA that contains a cluster of genes under the control of a single promotor. The synthesis of natural or synthetic products involves the introduction of a large number of genes. In this case these genes were encoding the enzymes needed for metabolic pathway. So it is useful to put certain genes under the control of a single promotor. For that reason Plac promotor was used in this study [1,5].

== EXPERIMENT ==

=== Synthase gene asemby and amorphadiene production ===

For high level production and changing the difficulties in expressing teprene synthases they first synthesized and expressed a codon-optimized variants of ADS . ADS is a gene encoding amorphadiene synthase and is sequentially oxidised by citorchromeP450 CYP71AV1 and reduced by artemisinic aldehyde reductase (DBR2). Amorphadyene synthase is an enzyme and catalyse the first step in the anitmalarial drug artemisnin synthesis. It has an inportant role in catalysing the reaction of FPP to amorphadiene. Beacause of its importaint step towards artemisnin biosynthesis it was designed and improved for the purpuse to acheve high-level expression in E.coli. For ADS gene synthesis they used two step assemly and one step amplification PCR.(1) Assemby PCR is a useful technique for production novel gene sequences. With this method we can put together short fragments of DNA and it is also a good choice for gene construction. Reserches in this article analysed the sequence of three ADS genes from independet clones and identified mutations in each of the genes. Than, two site directed mutagenesis reaction was used for assembly and generation a functional ADS gene from two clones. Than they measure the amorphadiene production by the amorphadiene synthase in E.coli DH10B with natural (nonengineered DPX pathway) and in E.coli with engineered DPX pathway. E. coli with engineerd pathway was harboring the pSOE4 plasmid (DXP pathway operon with dxs,IppHp and ispA gene). The result sugested that FPP synthesis limited amorphadiene production in this engineered host [1,6]. ([http://www.disketa.si/media/uploads/2015-01/0bddf07f946e6f5be0638090dee56490.jpg See image here])

=== Egineering the mevalonate-dependet pathway in E.coli ===

The aim was to increase the intracellular concentration of FPP substrate. Genes encoding the MEV pathway from S.cerevisae was assembled into operons and expressed in E.coli. Because of the complexity of engineering numerous gene biosynthetic pathway, genes were divided into to operons, top and bottom.Top operon, MevT, consisted of three enzymes: acetoacetyl-CoA thiolase (''atoB''), HMG-CoA synthases (''HMGS'') and HMG-CoA reductase (''tHMGR''). This operon was responsible for transformation of acetyl-coA to mevalonate. Thre different bottom operons pMevB (''ERG12, ERG8,MVD1''), pMBI (''ERG12, ERG8, MVD1, idi'') and pMBIS (''ERG12, ERG8, MVD1, idi, ispA'') transformed mevalonate to IPP, DMAPP and consequently to FPP. The next step was to analyse and test the funcionality of this pathway. For this purpuse E.coli strand DYM1 with incoplemplete isoprenoid synthesis was transformed with plasmid expressing three bottom operon constuct- pMevB, pMBI and pMBI. E.coli strand DYM1 had a deletion in the ispC gene for encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase, and was not able to synthesise 2-C-methyl-D-erythritol 4-phosphate(MEP), which is important intermediate in isoprenoid biosynthesis[1]. Because of this deletion the synthesis of precursors IPP and DMAP is blocked. So then they tested what is going to happen if they grew the strain DYM1 in the presence of 2-C-methyl-D-ertihritol and in medium with mevalonate without methylerythritol since DYM1 strain was not capable to synthesisie MEP. As expected , all strains expressing operon from S.cerevisiae grew on the plates in the presence of 2-c-methyl-D-erythritol. But only strains with pMBI or pMBIS grew on plates with 1mM mevalonate in the absence of methylerythol. This was the conformation that synthetic MBI and MBIS operons were functional, because they were capable to support the growth of E.coli without ispC gene by supplying cells with IPP and DMAPP. Next they completed the mevalonate pathway by transforming the pMevT plasmid (expressing three genes mentioned before-atoB, HMGS and tHMGR) with pMBI or pMBIS. This coexpresion of two operons, pMevT and pMBI /pMBIS ,completed the mevlonate pathway and allowed the synthesis of sesquiterpene precursors from acetyl-CoA, IPP and DMAPP. Therefore, coexpression complemented the ''ispC'' deletion even in the absence of mevalonate, what was the proof, that MevT operon was functional [1,7].

=== Amorpadiene synthesis from mevalonate ===

The aim was to achieve high-level production of amorphadiene and to find out if the amount of FPP was limiting amoprhadiene output. As mentioned, E.coli was used as heterologous microorganism in which coupled mevalonate pathway with amorphadiene synthesis. Cells with ADS gene were coexpressed with MBIS operon and grown in medium with increasing amounts (0 to 40 mM) of exogenus mevalonate. The culture extract was analysed with gas chromatography-mass spectrometry, analytical method for identification different substances in a test sample. It combines the features of mass spectrometry and gas-liquid chromatography. Results showed that MBIS operon did not limit the amorphadiene production at the highest mevalonate concentration(40mM). ([http://www.disketa.si/media/uploads/2015-01/7711d592584a53282f2ecb0fd678ef40.jpg See image here]) Cultures with 40 mM mevalonate added produced much more amoprhadiene. With time, amorphadiene concentration was dropping beacause of the loss of the volatile terpene[1]. By adding more than 10 mM mevalonate in control cultures without ADS, the severe growth ihibition has been reported. ([http://www.disketa.si/media/uploads/2015-01/2a21f55f0fea31ffcbe89c866c9150f7.jpg See image here]) To discover the cause ofthis inhibition, the growth of E.coli DH10B from strains with empty, pMKPMK, pMevB, pMBI or pMBIS plasmid was measured in media with increasing concentrations of mevalonate. The addition of 5mM mevalonate to the medium inhibited the growth of cells with pMevB. But 5mM concentrations of mevalonate did not alter the growth of cells with pMKPMK, PMBI or pMBIS plasmid. This indicated that the accomulation of IPP is toxic and inhibits normal cell growth. The accmulation mostly occurs in cells with high flux through mevalonate pathway. The next step was comparison of intracellular prenyl pyrophosphate (FPP) in strains. This was done by feeding the cells harboring the different mevalonte operon construct with radiolabeled mevalonate. Than the labeled metabolites were tracked. For this experiment cell suspensions of E.coli harboring pMevB+pTrc99A, pMBI+ pTrc99A, pMBIS+ pTrc99A or pMBIS+pADS were used. pTRc99A is the bacterial expression vector with inducible to IPTG lacI promoter. The result showed that strains with MevB operon accumulated IPP but not FPP, when MBI and MBIS strains accumulated FPP. ([http://www.disketa.si/media/uploads/2015-01/31ad7a215f016dc26aceacf92eb46712.jpg See image here]) Because of the simultaneous expression of the amorphadiene synthase the waste of FPP that accumulated in the MBIS host was consumed. This was shown by a decrease in intracellular FPP. Cells expresing MBIS acumulated FPP and showed growth inhibition in the presence of 10 mM mevalonate. The prediction was that the coexpression of ADS would ease the growth inhibition by forwarding the prenyl pyrophosphate intermediates to volatile terpene olefin. For this test, the cell with pMBIS and empty expression vector pTrc99A (without ADS)and cells with pMBIS and pADS vectors were used. The medium was supplemented with mevalonate concentations ( 0mM, 5mM, 10mM, 20mM or 40mM). Two hours after addition of mevalonate and inducer IPTG, the growth inhibition was observed. ([http://www.disketa.si/media/uploads/2015-01/94b5f3d5636fe767316f770f3e2b0448.jpg See image here]) The result showed that the strains with pMBIS +ADS started to grow, but strain lacking the ADS still showed growth inhbition at higher concentrations of mevalonate. This supported the conclusion that converion of FPP to amophadiene has an important role in minimising growth inhibition. Briefly these result showed that engineered mevalonate patway produces high levels of prenyl pyrophosphate precursors. If the IPP isomerase, FPP synthase and terpen synthase is absent, IPP can acumulate and be toxic to the cells. The toxic levels of intracellular prenyl pyrphosphate can accumulate [1].

=== Amorphadiene synthesis from acetyl-Coa ===

The main goal was to achieve high amoprhaidene production from a simple and inexpensive carbon source. E.coli strains harboring pMevT,pMBIS and pADS plasmids were transformed with pMevT plasmid. Subsequently the strains were tested for ability to produce amoprhadiene in the absence of exogenous mevalonate. The comparison between E.coli expressing the native DPX pathway and engineered pathway as made. They tested native DXP pathway, engineered DPX pathway, mevalonate bottom pathway in the absence of mevalonate, mevlonate bottom pathway in medium suplemented with mevalonate, the complete mevalonate pathway and the complete mevalonate pathway supplemented with 0,8% glycerol. Glycerol was addded to investigate the effect of amorphadiene production of supplying as single carbon source and for prolongation of amorphadiene production. Breifly the resulut showed that expression of the mevalonate-dependent isoprenoid biosynthetic pathway delivers hight levels of isoprenoid precursor for the production of sesquiterpene [1]. ([http://www.disketa.si/media/uploads/2015-01/a31fe07a7c66854b1dc3ff36f8ed01ef.jpg See image here])

== DISSCUSION ==
The identification of genes encoding the enzymes involved in the artemisinin-biosynthesis pathway together with advances in metabolic engineering provides new options for engineering artemisinin biosynthesis. Heterologous production of artemisnin in microorganism such as E.coli. is an alternative way for production of artmeinsin in high yields. But eventhougt micoorganisms has shown great potential in that matter, optimisation of these heterlogous pathways still require maximal titer. Inequal gene expression can lead to accumulation of toxic metabolic intermediates and to over or under production of enzymes involved in pathway. (4). Conclusion based on these study shows that poor expression of the amoprhaidene synthase genes (ADS) limits high terpene olfein yields from the host. To achieve better results , in this study they chose to synthesiyse an amophaiene synthase gene (ADS). The comparison between E.coli expressing native sesquiterpene synthase genes and E.coli expressing the synthetic ADS gene showed big improvement in terpene synthesis due to synthetic ADS gene. The native DPX pathway showed poor supply of the prenyl pyrophosphate precuror. And that limited terpenoid -carotenoid yields in E.coli. With the metabolic engineering of the DPX pathway the flux of carotenoid accumulation showed signifcant increase. By using the mevalonate-dependent isoprenoid pathway from yeast S. cerevisae and engineering that pathway in bacteria E.coli, we avoid the native regulatory elements found in yeast while bypassing those of E.coli DXP pathway. In this study results showed growth inhibition in cells without ADS due to the vast excess of preny pyrophosphate. But the coexpression of a synthetic sesquiterpene synthase can contribute to reducing the excess pool of precursors and eliminate growth inhibition. In this study total biosynthesis of artemisnin was not completely achieved. But these results can help in further investigation for poduction of artemisnic acid. This acid can then be putrified and chemically converted into artemsinin. [1,7].

== CONCLUSIONS ==
To sum things up, artmeinsin, a sesquiterpene isolated from Artemisia annua,represent a novel drug for malaria treatment. The present artemisnin derivates are too expensive for general use and plants can not produce artmeinsin in big quantities. This fact inspired scientist to focus on developing the way for production of artemisnin in high quantities and to avoid big costs. The promising tool in alternative source of artmeinsin is the use of microoganisms. The aim is to transfer the genes encoding enzymes involved in biosynthetic pathway for artemisnin into a hight producing host organism. Several study have been reporting on engineering the metabolic pathway in S.cerevisae to produce the precursor for artmeisnic acid. With the effort in synthetic biology they used modified mevalonate pathway . With the help of syntehtic bilogy the yeast cells were engineerd to express ezymes needed for production of artmemisnic acid. But in this paper the main focus was on engineering a mevalonate pathway in bacteria E.coli. By engineering a new metabolic pathway in E.coli we can easily synthesise a precursor nedeed for artemisnin production. The goal is to produce drugs in better and cheape ways. But, why focus on artemisnin? Well, acording on recent studies artemisnin shows to be very sucesfull for the treatmen all strain of malaria. More than a milion people affected with malaria have already been cured by arteminsin. As already mentioned , because of very hight costs and limited production of arteminin in plants most populations in Africa can not afford it. Progress made in synthetic biology and ability to produce amorphadiene in microorganisms in big quantites opens a new possibility to produce low cost artemisnin and contributes to the brighter future od antimalaria drug deveopment, and therefore saving milions from suffering from malaria. [1,2,3].

[1]Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nature biotehnology; vol 21; pages 796-802, July 2003

[2] T. Mosses, J. Pollier, J. M. Thevelein, A. Goossens. Bioengineering of plant (tri)terpenoids: from metabolic engineering of plants to synthetic biology in vivo and in vitro. New Pathyologist. 200: 27-43, 2013

[3] M.Farhi, M. Kozin, S. Duchin, A. Vainstein. Metabolic engineering of plants for artemisinin synthesis. Biotehnology and genetic engineering rewiews.Vol.29,No.2: 135-148, 2013

[4] Synthetic biology ; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Synthetic_biology http://en.wikipedia.org/wiki/Synthetic_biology]

[5]Operon; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Operon http://en.wikipedia.org/wiki/Operon]

[6] M. K. Julsing, G. van Pouderoyen, J. van der Veen,H. J. Woerdenbag, O. Kayser, W. J. Quax. Amorphadiene synthase: probing the active site model with directed mutagenesis. Directed mutagenesis of AMDS.Chapter 4: 47-56

[7]J.R. Anthony, L. C. Anthony, F. Nowroozi, G. Kwon, J.D. Newman, J.D. Keasling. Optimization of themevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene. Metabolic Engineering. 11: 13-19, 2009

Engineering a mevalonate pathway in Escherichia coli for production of terpenoids

2015-01-19T23:55:39Z

Ana90: /* INTRODUCTION */

'''Ana Kapraljević'''

[http://www2.lbl.gov/ttd/publications/2837pub1.pdf Engineering a mevalonate pathway in Escherichia coli for production of terpenoids]
Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling ;Nature biotehnology; July 2003; vol 21; pages 796-802

== INTRODUCTION ==

Plants can synthesise and accumulate a wide range of small molecules or natural products involved in psyhological an ecological process. With the knowledge we know about their biosyntesis we used them as commercial flavors, fragnance compunds, colorants and terapeutical drugs. Their therapeutic potential is used for production of anticancer or antimalarial drugs, such as artemisnin. The most numerous and diverse class of natural products used in production for antimalarial drug are isoprenoids or more corectly their subclass, terpenoids. Malaria represent a major health problem in tropic and subtropic. Majority of death occur in children living in Africa. But the problem is that terpenoids are in plants produced in very small quantites and consequently not very economically feasible. For that reason the drugs for treatin malaria, artemisnin, is in short supply and is still unaffordable for most people living in malaria-endanger countries. Scientists are now focusing on alternative and cheaper way for terpenoid synthesis that can be enchanched and became economically acceptable. The alternative way is the expression of synthetic amorphadiene, precursor to artemisnin, and mevalonate isoprenoid way from yeas ''Saccharomyces cerevisiae'' in bacteria. In this paper I will summerize the project made in the production of amorphadiene by biotehnology and synthetic biology approach [1,2].

=== Artemisnin ===

Artemisnin is a substance that has antimalarial potential. Malaria is a infectious disease caused by parasitic protozan of the ''Plasmodium'' species. In the certain parts of the world malaria takes lives of more people worldwide than any other infecious disease, so it is a global problem. Death to malaria has been nowdays reduced thanks to the use of artemisnin-based combination therapy. Artemisnin is a natural compoud from plant ''Artemisia annua''. Chemically it is a sesquiterpene lactone, amorphadiene. The total sythesis of artemisnin is difficult and not very economical,so the new aproach is to put the hopes in sythetic biology and develop the new way, synthetic way, for its production [1,3].

=== Biology of terpenoids ===

For initiation of any synthetic biology process in metabolic engineering in that matter it is important to have some basic understanding of biosynthesis and regulation of terpenoids. Terpenoids, a class of isoprenoids, are natural products often sythesized by plants. They represent a large and diverse class and have many functions as primarly metabolites involved in growth and development of plants, or as secundary metabolites that optimize the interaction between the plant and the environment. Eventhought they are structural diverse, the share a common biosynthetic origin and follow similar synthesis tracks. All terpenoids are derived from repetitive fusion of isoprene units (C5 H8) [3]. The number of isoprene units determines their classification. For isoprenoid synthesis plants use mevalonate-dependent (MEV) pathway and mevalonte-independent pathway or alternative deoxyxylulose 5-phosphate pathway (DXP). In higher plants the biosynthesis begins with the generation of isopentenyl pyrophosphate (IPP). IPP is derived via the classic mevalonic acid pathway from acetly-coenzime A (acetyl-CoA). The IPP is isomerized to its allylic isomer dimethylallyl pyrophosphate (DMAPP) [3]. The continous condensation of IPP and DMAPP units leads to the formaton of prenylated pyrophosphates, the immediate precursors of the different terpenoid classes. So IPP and DMAPP are combined to form larger isoprenoids, such as farnesyl diphosphate (FPP). Condensation of IPP and DMAPP than units leads to the formation of precursors of the different terpenoid classes. Shematic production of terpenoids in ilustraded in this picture. Similary to plants, yeast Sacharomyces cerevisae uses the clasical mevalonate pathway for the synthesis of isoprenoids[3]. Prokariontes mostly use the DXP pathway for production IPP and DMAPP. This is an alternative pathway for IPP synthesis. This pathway uses Escherichia coli and some other bacteria for their terpenoid synthesis. IPP and DMAPP precurosr are essential to E. Coli for prenylation (addition of hydrophobic molecules to a protein or chemical compound) of tRNA and the synthesis of farnesyl pyrophosphate (FPP). FPP is then used for quinone (class of organic compounds) and cell wall synthesis. The DPX pathway begins with the glycolytic intermediates glyceraldehyde-3-phosphate (G3P) and pyruvate and continues through enzymatic steps for production of IPP and DMAPP. Therefore IPP and DMAPP are the end product in both way,MEV and DPX pathway [1,3].

== METABOLIC ENGINEERING FOR PRODUCTION OD TERPENOIDS ==

Metabolic engineering represents an improvement of production, construction, or cellular properties through the modification of specific biochemical reactions. It is described as the use of genetic engineering to reshape or modify the metabolism of organism. The aim is to optimize genetic and regulatory processes in cells to increase the production of a certain substance that we find in nature. It is based on optimisation of existing biochemical pathways or the introduction of natural pathway components in bacteria, yeast or plants. The main goal is the production of high-yield of specific metabolites that we can use in medicine or biotechnology. With progress in synthetic biology we can design a biologic funcion that do not exist in nature. Consequently, we can create and construct new biological components or redesign exisitng biological systems. In plants, most terpenoids are produced in a very small quantities in their natural sources. Extraction of this compouds from plants is expensive and therfore not economicaly accetable. So there is a big disagreement between the insistence for supply of terpenoids and therefore that leads to delay for their widespread application. With the understanding of terpenoid synthesis and progress in synthetic biology we are now able of redesign and modify the isoprenoid pathway to increase the terpenoid productivity [2,4]. ([http://www.disketa.si/media/uploads/2015-01/cd201ade6a167488767dfb1b118587bc.jpg See image here])

=== Microbal host ===

To elimante the need for plant extraction it is practical to produce terpenoid compouds at high yield in microbal host by introducing a heterlogous isoprenoid pathway into bacteria E.coli. Microorganisms, such as bacteria are attractive alternatives as hosts because they have rapid doubling time, they can achieve robussnes under process conditions and because of the simplicity of product putrification. And the costs are significantly lower than working with plants. With the engineering the DXP pathway we can increase the supply of isoprenoid precurosor needed for high level productio of isoprenoids in E.coli [1,3].

=== Operons ===

Operon is a a funcional unit of DNA that contains a cluster of genes under the control of a single promotor. The synthesis of natural or synthetic products involves the introduction of a large number of genes. In this case these genes were encoding the enzymes needed for metabolic pathway. So it is useful to put certain genes under the control of a single promotor. For that reason Plac promotor was used in this study [1,5].

== EXPERIMENT ==

=== Synthase gene asemby and amorphadiene production ===

For high level production and changing the difficulties in expressing teprene synthases they first synthesized and expressed a codon-optimized variants of ADS . ADS is a gene encoding amorphadiene synthase and is sequentially oxidised by citorchromeP450 CYP71AV1 and reduced by artemisinic aldehyde reductase (DBR2). Amorphadyene synthase is an enzyme and catalyse the first step in the anitmalarial drug artemisnin synthesis. It has an inportant role in catalysing the reaction of FPP to amorphadiene. Beacause of its importaint step towards artemisnin biosynthesis it was designed and improved for the purpuse to acheve high-level expression in E.coli. For ADS gene synthesis they used two step assemly and one step amplification PCR.(1) Assemby PCR is a useful technique for production novel gene sequences. With this method we can put together short fragments of DNA and it is also a good choice for gene construction. Reserches in this article analysed the sequence of three ADS genes from independet clones and identified mutations in each of the genes. Than, two site directed mutagenesis reaction was used for assembly and generation a functional ADS gene from two clones. Than they measure the amorphadiene production by the amorphadiene synthase in E.coli DH10B with natural (nonengineered DPX pathway) and in E.coli with engineered DPX pathway. E. coli with engineerd pathway was harboring the pSOE4 plasmid (DXP pathway operon with dxs,IppHp and ispA gene). The result sugested that FPP synthesis limited amorphadiene production in this engineered host [1,6]. ([http://www.disketa.si/media/uploads/2015-01/0bddf07f946e6f5be0638090dee56490.jpg See image here])

=== Egineering the mevalonate-dependet pathway in E.coli ===

The aim was to increase the intracellular concentration of FPP substrate. Genes encoding the MEV pathway from S.cerevisae was assembled into operons and expressed in E.coli. Because of the complexity of engineering numerous gene biosynthetic pathway, genes were divided into to operons, top and bottom.Top operon, MevT, consisted of three enzymes: acetoacetyl-CoA thiolase (''atoB''), HMG-CoA synthases (''HMGS'') and HMG-CoA reductase (''tHMGR''). This operon was responsible for transformation of acetyl-coA to mevalonate. Thre different bottom operons pMevB (''ERG12, ERG8,MVD1''), pMBI (''ERG12, ERG8, MVD1, idi'') and pMBIS (''ERG12, ERG8, MVD1, idi, ispA'') transformed mevalonate to IPP, DMAPP and consequently to FPP. The next step was to analyse and test the funcionality of this pathway. For this purpuse E.coli strand DYM1 with incoplemplete isoprenoid synthesis was transformed with plasmid expressing three bottom operon constuct- pMevB, pMBI and pMBI. E.coli strand DYM1 had a deletion in the ispC gene for encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase, and was not able to synthesise 2-C-methyl-D-erythritol 4-phosphate(MEP), which is important intermediate in isoprenoid biosynthesis[1]. Because of this deletion the synthesis of precursors IPP and DMAP is blocked. So then they tested what is going to happen if they grew the strain DYM1 in the presence of 2-C-methyl-D-ertihritol and in medium with mevalonate without methylerythritol since DYM1 strain was not capable to synthesisie MEP. As expected , all strains expressing operon from S.cerevisiae grew on the plates in the presence of 2-c-methyl-D-erythritol. But only strains with pMBI or pMBIS grew on plates with 1mM mevalonate in the absence of methylerythol. This was the conformation that synthetic MBI and MBIS operons were functional, because they were capable to support the growth of E.coli without ispC gene by supplying cells with IPP and DMAPP. Next they completed the mevalonate pathway by transforming the pMevT plasmid (expressing three genes mentioned before-atoB, HMGS and tHMGR) with pMBI or pMBIS. This coexpresion of two operons, pMevT and pMBI /pMBIS ,completed the mevlonate pathway and allowed the synthesis of sesquiterpene precursors from acetyl-CoA, IPP and DMAPP. Therefore, coexpression complemented the ''ispC'' deletion even in the absence of mevalonate, what was the proof, that MevT operon was functional [1,7].

=== Amorpadiene synthesis from mevalonate ===

The aim was to achieve high-level production of amorphadiene and to find out if the amount of FPP was limiting amoprhadiene output. As mentioned, E.coli was used as heterologous microorganism in which coupled mevalonate pathway with amorphadiene synthesis. Cells with ADS gene were coexpressed with MBIS operon and grown in medium with increasing amounts (0 to 40 mM) of exogenus mevalonate. The culture extract was analysed with gas chromatography-mass spectrometry, analytical method for identification different substances in a test sample. It combines the features of mass spectrometry and gas-liquid chromatography. Results showed that MBIS operon did not limit the amorphadiene production at the highest mevalonate concentration(40mM). ([http://www.disketa.si/media/uploads/2015-01/7711d592584a53282f2ecb0fd678ef40.jpg See image here]) Cultures with 40 mM mevalonate added produced much more amoprhadiene. With time, amorphadiene concentration was dropping beacause of the loss of the volatile terpene[1]. By adding more than 10 mM mevalonate in control cultures without ADS, the severe growth ihibition has been reported. ([http://www.disketa.si/media/uploads/2015-01/2a21f55f0fea31ffcbe89c866c9150f7.jpg See image here]) To discover the cause ofthis inhibition, the growth of E.coli DH10B from strains with empty, pMKPMK, pMevB, pMBI or pMBIS plasmid was measured in media with increasing concentrations of mevalonate. The addition of 5mM mevalonate to the medium inhibited the growth of cells with pMevB. But 5mM concentrations of mevalonate did not alter the growth of cells with pMKPMK, PMBI or pMBIS plasmid. This indicated that the accomulation of IPP is toxic and inhibits normal cell growth. The accmulation mostly occurs in cells with high flux through mevalonate pathway. The next step was comparison of intracellular prenyl pyrophosphate (FPP) in strains. This was done by feeding the cells harboring the different mevalonte operon construct with radiolabeled mevalonate. Than the labeled metabolites were tracked. For this experiment cell suspensions of E.coli harboring pMevB+pTrc99A, pMBI+ pTrc99A, pMBIS+ pTrc99A or pMBIS+pADS were used. pTRc99A is the bacterial expression vector with inducible to IPTG lacI promoter. The result showed that strains with MevB operon accumulated IPP but not FPP, when MBI and MBIS strains accumulated FPP. ([http://www.disketa.si/media/uploads/2015-01/31ad7a215f016dc26aceacf92eb46712.jpg See image here]) Because of the simultaneous expression of the amorphadiene synthase the waste of FPP that accumulated in the MBIS host was consumed. This was shown by a decrease in intracellular FPP. Cells expresing MBIS acumulated FPP and showed growth inhibition in the presence of 10 mM mevalonate. The prediction was that the coexpression of ADS would ease the growth inhibition by forwarding the prenyl pyrophosphate intermediates to volatile terpene olefin. For this test, the cell with pMBIS and empty expression vector pTrc99A (without ADS)and cells with pMBIS and pADS vectors were used. The medium was supplemented with mevalonate concentations ( 0mM, 5mM, 10mM, 20mM or 40mM). Two hours after addition of mevalonate and inducer IPTG, the growth inhibition was observed. ([http://www.disketa.si/media/uploads/2015-01/94b5f3d5636fe767316f770f3e2b0448.jpg See image here]) The result showed that the strains with pMBIS +ADS started to grow, but strain lacking the ADS still showed growth inhbition at higher concentrations of mevalonate. This supported the conclusion that converion of FPP to amophadiene has an important role in minimising growth inhibition. Briefly these result showed that engineered mevalonate patway produces high levels of prenyl pyrophosphate precursors. If the IPP isomerase, FPP synthase and terpen synthase is absent, IPP can acumulate and be toxic to the cells. The toxic levels of intracellular prenyl pyrphosphate can accumulate [1].

=== Amorphadiene synthesis from acetyl-Coa ===

The main goal was to achieve high amoprhaidene production from a simple and inexpensive carbon source. E.coli strains harboring pMevT,pMBIS and pADS plasmids were transformed with pMevT plasmid. Subsequently the strains were tested for ability to produce amoprhadiene in the absence of exogenous mevalonate. The comparison between E.coli expressing the native DPX pathway and engineered pathway as made. They tested native DXP pathway, engineered DPX pathway, mevalonate bottom pathway in the absence of mevalonate, mevlonate bottom pathway in medium suplemented with mevalonate, the complete mevalonate pathway and the complete mevalonate pathway supplemented with 0,8% glycerol. Glycerol was addded to investigate the effect of amorphadiene production of supplying as single carbon source and for prolongation of amorphadiene production. Breifly the resulut showed that expression of the mevalonate-dependent isoprenoid biosynthetic pathway delivers hight levels of isoprenoid precursor for the production of sesquiterpene [1]. ([http://www.disketa.si/media/uploads/2015-01/a31fe07a7c66854b1dc3ff36f8ed01ef.jpg See image here])

== DISSCUSION ==
The identification of genes encoding the enzymes involved in the artemisinin-biosynthesis pathway together with advances in metabolic engineering provides new options for engineering artemisinin biosynthesis. Heterologous production of artemisnin in microorganism such as E.coli. is an alternative way for production of artmeinsin in high yields. But eventhougt micoorganisms has shown great potential in that matter, optimisation of these heterlogous pathways still require maximal titer. Inequal gene expression can lead to accumulation of toxic metabolic intermediates and to over or under production of enzymes involved in pathway. (4). Conclusion based on these study shows that poor expression of the amoprhaidene synthase genes (ADS) limits high terpene olfein yields from the host. To achieve better results , in this study they chose to synthesiyse an amophaiene synthase gene (ADS). The comparison between E.coli expressing native sesquiterpene synthase genes and E.coli expressing the synthetic ADS gene showed big improvement in terpene synthesis due to synthetic ADS gene. The native DPX pathway showed poor supply of the prenyl pyrophosphate precuror. And that limited terpenoid -carotenoid yields in E.coli. With the metabolic engineering of the DPX pathway the flux of carotenoid accumulation showed signifcant increase. By using the mevalonate-dependent isoprenoid pathway from yeast S. cerevisae and engineering that pathway in bacteria E.coli, we avoid the native regulatory elements found in yeast while bypassing those of E.coli DXP pathway. In this study results showed growth inhibition in cells without ADS due to the vast excess of preny pyrophosphate. But the coexpression of a synthetic sesquiterpene synthase can contribute to reducing the excess pool of precursors and eliminate growth inhibition. In this study total biosynthesis of artemisnin was not completely achieved. But these results can help in further investigation for poduction of artemisnic acid. This acid can then be putrified and chemically converted into artemsinin. [1,7].

== CONCLUSIONS ==
To sum things up, artmeinsin, a sesquiterpene isolated from Artemisia annua,represent a novel drug for malaria treatment. The present artemisnin derivates are too expensive for general use and plants can not produce artmeinsin in big quantities. This fact inspired scientist to focus on developing the way for production of artemisnin in high quantities and to avoid big costs. The promising tool in alternative source of artmeinsin is the use of microoganisms. The aim is to transfer the genes encoding enzymes involved in biosynthetic pathway for artemisnin into a hight producing host organism. Several study have been reporting on engineering the metabolic pathway in S.cerevisae to produce the precursor for artmeisnic acid. With the effort in synthetic biology they used modified mevalonate pathway . With the help of syntehtic bilogy the yeast cells were engineerd to express ezymes needed for production of artmemisnic acid. But in this paper the main focus was on engineering a mevalonate pathway in bacteria E.coli. By engineering a new metabolic pathway in E.coli we can easily synthesise a precursor nedeed for artemisnin production. The goal is to produce drugs in better and cheape ways. But, why focus on artemisnin? Well, acording on recent studies artemisnin shows to be very sucesfull for the treatmen all strain of malaria. More than a milion people affected with malaria have already been cured by arteminsin. As already mentioned , because of very hight costs and limited production of arteminin in plants most populations in Africa can not afford it. Progress made in synthetic biology and ability to produce amorphadiene in microorganisms in big quantites opens a new possibility to produce low cost artemisnin and contributes to the brighter future od antimalaria drug deveopment, and therefore saving milions from suffering from malaria. [1,2,3].

[1]Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nature biotehnology; vol 21; pages 796-802, July 2003

[2] T. Mosses, J. Pollier, J. M. Thevelein, A. Goossens. Bioengineering of plant (tri)terpenoids: from metabolic engineering of plants to synthetic biology in vivo and in vitro. New Pathyologist. 200: 27-43, 2013

[3] M.Farhi, M. Kozin, S. Duchin, A. Vainstein. Metabolic engineering of plants for artemisinin synthesis. Biotehnology and genetic engineering rewiews.Vol.29,No.2: 135-148, 2013

[4] Synthetic biology ; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Synthetic_biology http://en.wikipedia.org/wiki/Synthetic_biology]

[5]Operon; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Operon http://en.wikipedia.org/wiki/Operon]

[6] M. K. Julsing, G. van Pouderoyen, J. van der Veen,H. J. Woerdenbag, O. Kayser, W. J. Quax. Amorphadiene synthase: probing the active site model with directed mutagenesis. Directed mutagenesis of AMDS.Chapter 4: 47-56

[7]J.R. Anthony, L. C. Anthony, F. Nowroozi, G. Kwon, J.D. Newman, J.D. Keasling. Optimization of themevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene. Metabolic Engineering. 11: 13-19, 2009

Engineering a mevalonate pathway in Escherichia coli for production of terpenoids

2015-01-19T23:54:35Z

Ana90: /* Egineering the mevalonate-dependet pathway in E.coli */

'''Ana Kapraljević'''

[http://www2.lbl.gov/ttd/publications/2837pub1.pdf Engineering a mevalonate pathway in Escherichia coli for production of terpenoids]
Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling ;Nature biotehnology; July 2003; vol 21; pages 796-802

== INTRODUCTION ==

Plants can synthesise and accumulate a wide range of small molecules or natural products involved in psyhological an ecological process. With the knowledge we know about their biosyntesis we used them as commercial flavors, fragnance compunds, colorants and terapeutical drugs. Their therapeutic potential is used for production of anticancer or antimalarial drugs, such as artemisnin. The most numerous and diverse class of natural products used in production for antimalarial drug are isoprenoids or more corectly their subclass, terpenoids. Malaria represent a major health problem in tropic and subtropic. Majority of death occur in children living in Africa. But the problem is that terpenoids are in plants produced in very small quantites and consequently not very economically feasible. For that reason the drugs for treatin malaria, artemisnin, is in short supply and is still unaffordable for most people living in malaria-endanger countries. Scientists are now focusing on alternative and cheaper way for terpenoid synthesis that can be enchanched and became economically acceptable. The alternative way is the expression of synthetic amorphadiene, precursor to artemisnin, and mevalonate isoprenoid way from yeas Saccharomyces cerevisiae in bacteria. In this paper I will summerize the project made in the production of amorphadiene by biotehnology and synthetic biology approach [1,2].

=== Artemisnin ===

Artemisnin is a substance that has antimalarial potential. Malaria is a infectious disease caused by parasitic protozan of the Plasmodium species. In the certain parts of the world malaria takes lives of more people worldwide than any other infecious disease, so it is a global problem. Death to malaria has been nowdays reduced thanks to the use of artemisnin-based combination therapy. Artemisnin is a natural compoud from plant ''Artemisia annua''. Chemically it is a sesquiterpene lactone, amorphadiene. The total sythesis of artemisnin is difficult and not very economical,so the new aproach is to put the hopes in sythetic biology and develop the new way, synthetic way, for its production [1,3].

=== Biology of terpenoids ===

For initiation of any synthetic biology process in metabolic engineering in that matter it is important to have some basic understanding of biosynthesis and regulation of terpenoids. Terpenoids, a class of isoprenoids, are natural products often sythesized by plants. They represent a large and diverse class and have many functions as primarly metabolites involved in growth and development of plants, or as secundary metabolites that optimize the interaction between the plant and the environment. Eventhought they are structural diverse, the share a common biosynthetic origin and follow similar synthesis tracks. All terpenoids are derived from repetitive fusion of isoprene units (C5 H8) [3]. The number of isoprene units determines their classification. For isoprenoid synthesis plants use mevalonate-dependent (MEV) pathway and mevalonte-independent pathway or alternative deoxyxylulose 5-phosphate pathway (DXP). In higher plants the biosynthesis begins with the generation of isopentenyl pyrophosphate (IPP). IPP is derived via the classic mevalonic acid pathway from acetly-coenzime A (acetyl-CoA). The IPP is isomerized to its allylic isomer dimethylallyl pyrophosphate (DMAPP) [3]. The continous condensation of IPP and DMAPP units leads to the formaton of prenylated pyrophosphates, the immediate precursors of the different terpenoid classes. So IPP and DMAPP are combined to form larger isoprenoids, such as farnesyl diphosphate (FPP). Condensation of IPP and DMAPP than units leads to the formation of precursors of the different terpenoid classes. Shematic production of terpenoids in ilustraded in this picture. Similary to plants, yeast Sacharomyces cerevisae uses the clasical mevalonate pathway for the synthesis of isoprenoids[3]. Prokariontes mostly use the DXP pathway for production IPP and DMAPP. This is an alternative pathway for IPP synthesis. This pathway uses Escherichia coli and some other bacteria for their terpenoid synthesis. IPP and DMAPP precurosr are essential to E. Coli for prenylation (addition of hydrophobic molecules to a protein or chemical compound) of tRNA and the synthesis of farnesyl pyrophosphate (FPP). FPP is then used for quinone (class of organic compounds) and cell wall synthesis. The DPX pathway begins with the glycolytic intermediates glyceraldehyde-3-phosphate (G3P) and pyruvate and continues through enzymatic steps for production of IPP and DMAPP. Therefore IPP and DMAPP are the end product in both way,MEV and DPX pathway [1,3].

== METABOLIC ENGINEERING FOR PRODUCTION OD TERPENOIDS ==

Metabolic engineering represents an improvement of production, construction, or cellular properties through the modification of specific biochemical reactions. It is described as the use of genetic engineering to reshape or modify the metabolism of organism. The aim is to optimize genetic and regulatory processes in cells to increase the production of a certain substance that we find in nature. It is based on optimisation of existing biochemical pathways or the introduction of natural pathway components in bacteria, yeast or plants. The main goal is the production of high-yield of specific metabolites that we can use in medicine or biotechnology. With progress in synthetic biology we can design a biologic funcion that do not exist in nature. Consequently, we can create and construct new biological components or redesign exisitng biological systems. In plants, most terpenoids are produced in a very small quantities in their natural sources. Extraction of this compouds from plants is expensive and therfore not economicaly accetable. So there is a big disagreement between the insistence for supply of terpenoids and therefore that leads to delay for their widespread application. With the understanding of terpenoid synthesis and progress in synthetic biology we are now able of redesign and modify the isoprenoid pathway to increase the terpenoid productivity [2,4]. ([http://www.disketa.si/media/uploads/2015-01/cd201ade6a167488767dfb1b118587bc.jpg See image here])

=== Microbal host ===

To elimante the need for plant extraction it is practical to produce terpenoid compouds at high yield in microbal host by introducing a heterlogous isoprenoid pathway into bacteria E.coli. Microorganisms, such as bacteria are attractive alternatives as hosts because they have rapid doubling time, they can achieve robussnes under process conditions and because of the simplicity of product putrification. And the costs are significantly lower than working with plants. With the engineering the DXP pathway we can increase the supply of isoprenoid precurosor needed for high level productio of isoprenoids in E.coli [1,3].

=== Operons ===

Operon is a a funcional unit of DNA that contains a cluster of genes under the control of a single promotor. The synthesis of natural or synthetic products involves the introduction of a large number of genes. In this case these genes were encoding the enzymes needed for metabolic pathway. So it is useful to put certain genes under the control of a single promotor. For that reason Plac promotor was used in this study [1,5].

== EXPERIMENT ==

=== Synthase gene asemby and amorphadiene production ===

For high level production and changing the difficulties in expressing teprene synthases they first synthesized and expressed a codon-optimized variants of ADS . ADS is a gene encoding amorphadiene synthase and is sequentially oxidised by citorchromeP450 CYP71AV1 and reduced by artemisinic aldehyde reductase (DBR2). Amorphadyene synthase is an enzyme and catalyse the first step in the anitmalarial drug artemisnin synthesis. It has an inportant role in catalysing the reaction of FPP to amorphadiene. Beacause of its importaint step towards artemisnin biosynthesis it was designed and improved for the purpuse to acheve high-level expression in E.coli. For ADS gene synthesis they used two step assemly and one step amplification PCR.(1) Assemby PCR is a useful technique for production novel gene sequences. With this method we can put together short fragments of DNA and it is also a good choice for gene construction. Reserches in this article analysed the sequence of three ADS genes from independet clones and identified mutations in each of the genes. Than, two site directed mutagenesis reaction was used for assembly and generation a functional ADS gene from two clones. Than they measure the amorphadiene production by the amorphadiene synthase in E.coli DH10B with natural (nonengineered DPX pathway) and in E.coli with engineered DPX pathway. E. coli with engineerd pathway was harboring the pSOE4 plasmid (DXP pathway operon with dxs,IppHp and ispA gene). The result sugested that FPP synthesis limited amorphadiene production in this engineered host [1,6]. ([http://www.disketa.si/media/uploads/2015-01/0bddf07f946e6f5be0638090dee56490.jpg See image here])

=== Egineering the mevalonate-dependet pathway in E.coli ===

The aim was to increase the intracellular concentration of FPP substrate. Genes encoding the MEV pathway from S.cerevisae was assembled into operons and expressed in E.coli. Because of the complexity of engineering numerous gene biosynthetic pathway, genes were divided into to operons, top and bottom.Top operon, MevT, consisted of three enzymes: acetoacetyl-CoA thiolase (''atoB''), HMG-CoA synthases (''HMGS'') and HMG-CoA reductase (''tHMGR''). This operon was responsible for transformation of acetyl-coA to mevalonate. Thre different bottom operons pMevB (''ERG12, ERG8,MVD1''), pMBI (''ERG12, ERG8, MVD1, idi'') and pMBIS (''ERG12, ERG8, MVD1, idi, ispA'') transformed mevalonate to IPP, DMAPP and consequently to FPP. The next step was to analyse and test the funcionality of this pathway. For this purpuse E.coli strand DYM1 with incoplemplete isoprenoid synthesis was transformed with plasmid expressing three bottom operon constuct- pMevB, pMBI and pMBI. E.coli strand DYM1 had a deletion in the ispC gene for encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase, and was not able to synthesise 2-C-methyl-D-erythritol 4-phosphate(MEP), which is important intermediate in isoprenoid biosynthesis[1]. Because of this deletion the synthesis of precursors IPP and DMAP is blocked. So then they tested what is going to happen if they grew the strain DYM1 in the presence of 2-C-methyl-D-ertihritol and in medium with mevalonate without methylerythritol since DYM1 strain was not capable to synthesisie MEP. As expected , all strains expressing operon from S.cerevisiae grew on the plates in the presence of 2-c-methyl-D-erythritol. But only strains with pMBI or pMBIS grew on plates with 1mM mevalonate in the absence of methylerythol. This was the conformation that synthetic MBI and MBIS operons were functional, because they were capable to support the growth of E.coli without ispC gene by supplying cells with IPP and DMAPP. Next they completed the mevalonate pathway by transforming the pMevT plasmid (expressing three genes mentioned before-atoB, HMGS and tHMGR) with pMBI or pMBIS. This coexpresion of two operons, pMevT and pMBI /pMBIS ,completed the mevlonate pathway and allowed the synthesis of sesquiterpene precursors from acetyl-CoA, IPP and DMAPP. Therefore, coexpression complemented the ''ispC'' deletion even in the absence of mevalonate, what was the proof, that MevT operon was functional [1,7].

=== Amorpadiene synthesis from mevalonate ===

The aim was to achieve high-level production of amorphadiene and to find out if the amount of FPP was limiting amoprhadiene output. As mentioned, E.coli was used as heterologous microorganism in which coupled mevalonate pathway with amorphadiene synthesis. Cells with ADS gene were coexpressed with MBIS operon and grown in medium with increasing amounts (0 to 40 mM) of exogenus mevalonate. The culture extract was analysed with gas chromatography-mass spectrometry, analytical method for identification different substances in a test sample. It combines the features of mass spectrometry and gas-liquid chromatography. Results showed that MBIS operon did not limit the amorphadiene production at the highest mevalonate concentration(40mM). ([http://www.disketa.si/media/uploads/2015-01/7711d592584a53282f2ecb0fd678ef40.jpg See image here]) Cultures with 40 mM mevalonate added produced much more amoprhadiene. With time, amorphadiene concentration was dropping beacause of the loss of the volatile terpene[1]. By adding more than 10 mM mevalonate in control cultures without ADS, the severe growth ihibition has been reported. ([http://www.disketa.si/media/uploads/2015-01/2a21f55f0fea31ffcbe89c866c9150f7.jpg See image here]) To discover the cause ofthis inhibition, the growth of E.coli DH10B from strains with empty, pMKPMK, pMevB, pMBI or pMBIS plasmid was measured in media with increasing concentrations of mevalonate. The addition of 5mM mevalonate to the medium inhibited the growth of cells with pMevB. But 5mM concentrations of mevalonate did not alter the growth of cells with pMKPMK, PMBI or pMBIS plasmid. This indicated that the accomulation of IPP is toxic and inhibits normal cell growth. The accmulation mostly occurs in cells with high flux through mevalonate pathway. The next step was comparison of intracellular prenyl pyrophosphate (FPP) in strains. This was done by feeding the cells harboring the different mevalonte operon construct with radiolabeled mevalonate. Than the labeled metabolites were tracked. For this experiment cell suspensions of E.coli harboring pMevB+pTrc99A, pMBI+ pTrc99A, pMBIS+ pTrc99A or pMBIS+pADS were used. pTRc99A is the bacterial expression vector with inducible to IPTG lacI promoter. The result showed that strains with MevB operon accumulated IPP but not FPP, when MBI and MBIS strains accumulated FPP. ([http://www.disketa.si/media/uploads/2015-01/31ad7a215f016dc26aceacf92eb46712.jpg See image here]) Because of the simultaneous expression of the amorphadiene synthase the waste of FPP that accumulated in the MBIS host was consumed. This was shown by a decrease in intracellular FPP. Cells expresing MBIS acumulated FPP and showed growth inhibition in the presence of 10 mM mevalonate. The prediction was that the coexpression of ADS would ease the growth inhibition by forwarding the prenyl pyrophosphate intermediates to volatile terpene olefin. For this test, the cell with pMBIS and empty expression vector pTrc99A (without ADS)and cells with pMBIS and pADS vectors were used. The medium was supplemented with mevalonate concentations ( 0mM, 5mM, 10mM, 20mM or 40mM). Two hours after addition of mevalonate and inducer IPTG, the growth inhibition was observed. ([http://www.disketa.si/media/uploads/2015-01/94b5f3d5636fe767316f770f3e2b0448.jpg See image here]) The result showed that the strains with pMBIS +ADS started to grow, but strain lacking the ADS still showed growth inhbition at higher concentrations of mevalonate. This supported the conclusion that converion of FPP to amophadiene has an important role in minimising growth inhibition. Briefly these result showed that engineered mevalonate patway produces high levels of prenyl pyrophosphate precursors. If the IPP isomerase, FPP synthase and terpen synthase is absent, IPP can acumulate and be toxic to the cells. The toxic levels of intracellular prenyl pyrphosphate can accumulate [1].

=== Amorphadiene synthesis from acetyl-Coa ===

The main goal was to achieve high amoprhaidene production from a simple and inexpensive carbon source. E.coli strains harboring pMevT,pMBIS and pADS plasmids were transformed with pMevT plasmid. Subsequently the strains were tested for ability to produce amoprhadiene in the absence of exogenous mevalonate. The comparison between E.coli expressing the native DPX pathway and engineered pathway as made. They tested native DXP pathway, engineered DPX pathway, mevalonate bottom pathway in the absence of mevalonate, mevlonate bottom pathway in medium suplemented with mevalonate, the complete mevalonate pathway and the complete mevalonate pathway supplemented with 0,8% glycerol. Glycerol was addded to investigate the effect of amorphadiene production of supplying as single carbon source and for prolongation of amorphadiene production. Breifly the resulut showed that expression of the mevalonate-dependent isoprenoid biosynthetic pathway delivers hight levels of isoprenoid precursor for the production of sesquiterpene [1]. ([http://www.disketa.si/media/uploads/2015-01/a31fe07a7c66854b1dc3ff36f8ed01ef.jpg See image here])

== DISSCUSION ==
The identification of genes encoding the enzymes involved in the artemisinin-biosynthesis pathway together with advances in metabolic engineering provides new options for engineering artemisinin biosynthesis. Heterologous production of artemisnin in microorganism such as E.coli. is an alternative way for production of artmeinsin in high yields. But eventhougt micoorganisms has shown great potential in that matter, optimisation of these heterlogous pathways still require maximal titer. Inequal gene expression can lead to accumulation of toxic metabolic intermediates and to over or under production of enzymes involved in pathway. (4). Conclusion based on these study shows that poor expression of the amoprhaidene synthase genes (ADS) limits high terpene olfein yields from the host. To achieve better results , in this study they chose to synthesiyse an amophaiene synthase gene (ADS). The comparison between E.coli expressing native sesquiterpene synthase genes and E.coli expressing the synthetic ADS gene showed big improvement in terpene synthesis due to synthetic ADS gene. The native DPX pathway showed poor supply of the prenyl pyrophosphate precuror. And that limited terpenoid -carotenoid yields in E.coli. With the metabolic engineering of the DPX pathway the flux of carotenoid accumulation showed signifcant increase. By using the mevalonate-dependent isoprenoid pathway from yeast S. cerevisae and engineering that pathway in bacteria E.coli, we avoid the native regulatory elements found in yeast while bypassing those of E.coli DXP pathway. In this study results showed growth inhibition in cells without ADS due to the vast excess of preny pyrophosphate. But the coexpression of a synthetic sesquiterpene synthase can contribute to reducing the excess pool of precursors and eliminate growth inhibition. In this study total biosynthesis of artemisnin was not completely achieved. But these results can help in further investigation for poduction of artemisnic acid. This acid can then be putrified and chemically converted into artemsinin. [1,7].

== CONCLUSIONS ==
To sum things up, artmeinsin, a sesquiterpene isolated from Artemisia annua,represent a novel drug for malaria treatment. The present artemisnin derivates are too expensive for general use and plants can not produce artmeinsin in big quantities. This fact inspired scientist to focus on developing the way for production of artemisnin in high quantities and to avoid big costs. The promising tool in alternative source of artmeinsin is the use of microoganisms. The aim is to transfer the genes encoding enzymes involved in biosynthetic pathway for artemisnin into a hight producing host organism. Several study have been reporting on engineering the metabolic pathway in S.cerevisae to produce the precursor for artmeisnic acid. With the effort in synthetic biology they used modified mevalonate pathway . With the help of syntehtic bilogy the yeast cells were engineerd to express ezymes needed for production of artmemisnic acid. But in this paper the main focus was on engineering a mevalonate pathway in bacteria E.coli. By engineering a new metabolic pathway in E.coli we can easily synthesise a precursor nedeed for artemisnin production. The goal is to produce drugs in better and cheape ways. But, why focus on artemisnin? Well, acording on recent studies artemisnin shows to be very sucesfull for the treatmen all strain of malaria. More than a milion people affected with malaria have already been cured by arteminsin. As already mentioned , because of very hight costs and limited production of arteminin in plants most populations in Africa can not afford it. Progress made in synthetic biology and ability to produce amorphadiene in microorganisms in big quantites opens a new possibility to produce low cost artemisnin and contributes to the brighter future od antimalaria drug deveopment, and therefore saving milions from suffering from malaria. [1,2,3].

[1]Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nature biotehnology; vol 21; pages 796-802, July 2003

[2] T. Mosses, J. Pollier, J. M. Thevelein, A. Goossens. Bioengineering of plant (tri)terpenoids: from metabolic engineering of plants to synthetic biology in vivo and in vitro. New Pathyologist. 200: 27-43, 2013

[3] M.Farhi, M. Kozin, S. Duchin, A. Vainstein. Metabolic engineering of plants for artemisinin synthesis. Biotehnology and genetic engineering rewiews.Vol.29,No.2: 135-148, 2013

[4] Synthetic biology ; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Synthetic_biology http://en.wikipedia.org/wiki/Synthetic_biology]

[5]Operon; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Operon http://en.wikipedia.org/wiki/Operon]

[6] M. K. Julsing, G. van Pouderoyen, J. van der Veen,H. J. Woerdenbag, O. Kayser, W. J. Quax. Amorphadiene synthase: probing the active site model with directed mutagenesis. Directed mutagenesis of AMDS.Chapter 4: 47-56

[7]J.R. Anthony, L. C. Anthony, F. Nowroozi, G. Kwon, J.D. Newman, J.D. Keasling. Optimization of themevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene. Metabolic Engineering. 11: 13-19, 2009

Engineering a mevalonate pathway in Escherichia coli for production of terpenoids

2015-01-19T23:54:02Z

Ana90: /* Egineering the mevalonate-dependet pathway in E.coli */

'''Ana Kapraljević'''

[http://www2.lbl.gov/ttd/publications/2837pub1.pdf Engineering a mevalonate pathway in Escherichia coli for production of terpenoids]
Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling ;Nature biotehnology; July 2003; vol 21; pages 796-802

== INTRODUCTION ==

Plants can synthesise and accumulate a wide range of small molecules or natural products involved in psyhological an ecological process. With the knowledge we know about their biosyntesis we used them as commercial flavors, fragnance compunds, colorants and terapeutical drugs. Their therapeutic potential is used for production of anticancer or antimalarial drugs, such as artemisnin. The most numerous and diverse class of natural products used in production for antimalarial drug are isoprenoids or more corectly their subclass, terpenoids. Malaria represent a major health problem in tropic and subtropic. Majority of death occur in children living in Africa. But the problem is that terpenoids are in plants produced in very small quantites and consequently not very economically feasible. For that reason the drugs for treatin malaria, artemisnin, is in short supply and is still unaffordable for most people living in malaria-endanger countries. Scientists are now focusing on alternative and cheaper way for terpenoid synthesis that can be enchanched and became economically acceptable. The alternative way is the expression of synthetic amorphadiene, precursor to artemisnin, and mevalonate isoprenoid way from yeas Saccharomyces cerevisiae in bacteria. In this paper I will summerize the project made in the production of amorphadiene by biotehnology and synthetic biology approach [1,2].

=== Artemisnin ===

Artemisnin is a substance that has antimalarial potential. Malaria is a infectious disease caused by parasitic protozan of the Plasmodium species. In the certain parts of the world malaria takes lives of more people worldwide than any other infecious disease, so it is a global problem. Death to malaria has been nowdays reduced thanks to the use of artemisnin-based combination therapy. Artemisnin is a natural compoud from plant ''Artemisia annua''. Chemically it is a sesquiterpene lactone, amorphadiene. The total sythesis of artemisnin is difficult and not very economical,so the new aproach is to put the hopes in sythetic biology and develop the new way, synthetic way, for its production [1,3].

=== Biology of terpenoids ===

For initiation of any synthetic biology process in metabolic engineering in that matter it is important to have some basic understanding of biosynthesis and regulation of terpenoids. Terpenoids, a class of isoprenoids, are natural products often sythesized by plants. They represent a large and diverse class and have many functions as primarly metabolites involved in growth and development of plants, or as secundary metabolites that optimize the interaction between the plant and the environment. Eventhought they are structural diverse, the share a common biosynthetic origin and follow similar synthesis tracks. All terpenoids are derived from repetitive fusion of isoprene units (C5 H8) [3]. The number of isoprene units determines their classification. For isoprenoid synthesis plants use mevalonate-dependent (MEV) pathway and mevalonte-independent pathway or alternative deoxyxylulose 5-phosphate pathway (DXP). In higher plants the biosynthesis begins with the generation of isopentenyl pyrophosphate (IPP). IPP is derived via the classic mevalonic acid pathway from acetly-coenzime A (acetyl-CoA). The IPP is isomerized to its allylic isomer dimethylallyl pyrophosphate (DMAPP) [3]. The continous condensation of IPP and DMAPP units leads to the formaton of prenylated pyrophosphates, the immediate precursors of the different terpenoid classes. So IPP and DMAPP are combined to form larger isoprenoids, such as farnesyl diphosphate (FPP). Condensation of IPP and DMAPP than units leads to the formation of precursors of the different terpenoid classes. Shematic production of terpenoids in ilustraded in this picture. Similary to plants, yeast Sacharomyces cerevisae uses the clasical mevalonate pathway for the synthesis of isoprenoids[3]. Prokariontes mostly use the DXP pathway for production IPP and DMAPP. This is an alternative pathway for IPP synthesis. This pathway uses Escherichia coli and some other bacteria for their terpenoid synthesis. IPP and DMAPP precurosr are essential to E. Coli for prenylation (addition of hydrophobic molecules to a protein or chemical compound) of tRNA and the synthesis of farnesyl pyrophosphate (FPP). FPP is then used for quinone (class of organic compounds) and cell wall synthesis. The DPX pathway begins with the glycolytic intermediates glyceraldehyde-3-phosphate (G3P) and pyruvate and continues through enzymatic steps for production of IPP and DMAPP. Therefore IPP and DMAPP are the end product in both way,MEV and DPX pathway [1,3].

== METABOLIC ENGINEERING FOR PRODUCTION OD TERPENOIDS ==

Metabolic engineering represents an improvement of production, construction, or cellular properties through the modification of specific biochemical reactions. It is described as the use of genetic engineering to reshape or modify the metabolism of organism. The aim is to optimize genetic and regulatory processes in cells to increase the production of a certain substance that we find in nature. It is based on optimisation of existing biochemical pathways or the introduction of natural pathway components in bacteria, yeast or plants. The main goal is the production of high-yield of specific metabolites that we can use in medicine or biotechnology. With progress in synthetic biology we can design a biologic funcion that do not exist in nature. Consequently, we can create and construct new biological components or redesign exisitng biological systems. In plants, most terpenoids are produced in a very small quantities in their natural sources. Extraction of this compouds from plants is expensive and therfore not economicaly accetable. So there is a big disagreement between the insistence for supply of terpenoids and therefore that leads to delay for their widespread application. With the understanding of terpenoid synthesis and progress in synthetic biology we are now able of redesign and modify the isoprenoid pathway to increase the terpenoid productivity [2,4]. ([http://www.disketa.si/media/uploads/2015-01/cd201ade6a167488767dfb1b118587bc.jpg See image here])

=== Microbal host ===

To elimante the need for plant extraction it is practical to produce terpenoid compouds at high yield in microbal host by introducing a heterlogous isoprenoid pathway into bacteria E.coli. Microorganisms, such as bacteria are attractive alternatives as hosts because they have rapid doubling time, they can achieve robussnes under process conditions and because of the simplicity of product putrification. And the costs are significantly lower than working with plants. With the engineering the DXP pathway we can increase the supply of isoprenoid precurosor needed for high level productio of isoprenoids in E.coli [1,3].

=== Operons ===

Operon is a a funcional unit of DNA that contains a cluster of genes under the control of a single promotor. The synthesis of natural or synthetic products involves the introduction of a large number of genes. In this case these genes were encoding the enzymes needed for metabolic pathway. So it is useful to put certain genes under the control of a single promotor. For that reason Plac promotor was used in this study [1,5].

== EXPERIMENT ==

=== Synthase gene asemby and amorphadiene production ===

For high level production and changing the difficulties in expressing teprene synthases they first synthesized and expressed a codon-optimized variants of ADS . ADS is a gene encoding amorphadiene synthase and is sequentially oxidised by citorchromeP450 CYP71AV1 and reduced by artemisinic aldehyde reductase (DBR2). Amorphadyene synthase is an enzyme and catalyse the first step in the anitmalarial drug artemisnin synthesis. It has an inportant role in catalysing the reaction of FPP to amorphadiene. Beacause of its importaint step towards artemisnin biosynthesis it was designed and improved for the purpuse to acheve high-level expression in E.coli. For ADS gene synthesis they used two step assemly and one step amplification PCR.(1) Assemby PCR is a useful technique for production novel gene sequences. With this method we can put together short fragments of DNA and it is also a good choice for gene construction. Reserches in this article analysed the sequence of three ADS genes from independet clones and identified mutations in each of the genes. Than, two site directed mutagenesis reaction was used for assembly and generation a functional ADS gene from two clones. Than they measure the amorphadiene production by the amorphadiene synthase in E.coli DH10B with natural (nonengineered DPX pathway) and in E.coli with engineered DPX pathway. E. coli with engineerd pathway was harboring the pSOE4 plasmid (DXP pathway operon with dxs,IppHp and ispA gene). The result sugested that FPP synthesis limited amorphadiene production in this engineered host [1,6]. ([http://www.disketa.si/media/uploads/2015-01/0bddf07f946e6f5be0638090dee56490.jpg See image here])

=== Egineering the mevalonate-dependet pathway in E.coli ===

The aim was to increase the intracellular concentration of FPP substrate. Genes encoding the MEV pathway from S.cerevisae was assembled into operons and expressed in E.coli. Because of the complexity of engineering numerous gene biosynthetic pathway, genes were divided into to operons, top and bottom.Top operon, MevT, consisted of three enzymes: acetoacetyl-CoA thiolase (''atoB''), HMG-CoA synthases (''HMGS'') and HMG-CoA reductase (''tHMGR''). This operon was responsible for transformation of acetyl-coA to mevalonate. Thre different bottom operons pMevB (''ERG12, ERG8,MVD1''), pMBI (''ERG12, ERG8, MVD1, idi'') and pMBIS (''ERG12, ERG8, MVD1, idi, ispA'') transformed mevalonate to IPP, DMAPP and consequently to FPP. The next step was to analyse and test the funcionality of this pathway. For this purpuse E.coli strand DYM1 with incoplemplete isoprenoid synthesis was transformed with plasmid expressing three bottom operon constuct- pMevB, pMBI and pMBI. E.coli strand DYM1 had a deletion in the ispC gene for encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase, and was not able to synthesise 2-C-methyl-D-erythritol 4-phosphate(MEP), which is important intermediate in isoprenoid biosynthesis[1]. Because of this deletion the synthesis of precursors IPP and DMAP is blocked. So then they tested what is going to happen if they grew the strain DYM1 in the presence of 2-C-methyl-D-ertihritol and in medium with mevalonate without methylerythritol since DYM1 strain was not capable to synthesisie MEP. As expected , all strains expressing operon from S.cerevisiae grew on the plates in the presence of 2-c-methyl-D-erythritol. But only strains with pMBI or pMBIS grew on plates with 1mM mevalonate in the absence of methylerythol. This was the conformation that synthetic MBI and MBIS operons were functional, because they were capable to support the growth of E.coli without ispC gene by supplying cells with IPP and DMAPP. Next they completed the mevalonate pathway by transforming the pMevT plasmid (expressing three genes mentioned before-atoB, HMGS and tHMGR) with pMBI or pMBIS. This coexpresion of two operons, pMevT and pMBI /pMBIS ,completed the mevlonate pathway and allowed the synthesis of sesquiterpene precursors from acetyl-CoA, IPP and DMAPP. Therefore, coexpression complemented the ispC deletion even in the absence of mevalonate, what was the proof, that MevT operon was functional [1,7].

=== Amorpadiene synthesis from mevalonate ===

The aim was to achieve high-level production of amorphadiene and to find out if the amount of FPP was limiting amoprhadiene output. As mentioned, E.coli was used as heterologous microorganism in which coupled mevalonate pathway with amorphadiene synthesis. Cells with ADS gene were coexpressed with MBIS operon and grown in medium with increasing amounts (0 to 40 mM) of exogenus mevalonate. The culture extract was analysed with gas chromatography-mass spectrometry, analytical method for identification different substances in a test sample. It combines the features of mass spectrometry and gas-liquid chromatography. Results showed that MBIS operon did not limit the amorphadiene production at the highest mevalonate concentration(40mM). ([http://www.disketa.si/media/uploads/2015-01/7711d592584a53282f2ecb0fd678ef40.jpg See image here]) Cultures with 40 mM mevalonate added produced much more amoprhadiene. With time, amorphadiene concentration was dropping beacause of the loss of the volatile terpene[1]. By adding more than 10 mM mevalonate in control cultures without ADS, the severe growth ihibition has been reported. ([http://www.disketa.si/media/uploads/2015-01/2a21f55f0fea31ffcbe89c866c9150f7.jpg See image here]) To discover the cause ofthis inhibition, the growth of E.coli DH10B from strains with empty, pMKPMK, pMevB, pMBI or pMBIS plasmid was measured in media with increasing concentrations of mevalonate. The addition of 5mM mevalonate to the medium inhibited the growth of cells with pMevB. But 5mM concentrations of mevalonate did not alter the growth of cells with pMKPMK, PMBI or pMBIS plasmid. This indicated that the accomulation of IPP is toxic and inhibits normal cell growth. The accmulation mostly occurs in cells with high flux through mevalonate pathway. The next step was comparison of intracellular prenyl pyrophosphate (FPP) in strains. This was done by feeding the cells harboring the different mevalonte operon construct with radiolabeled mevalonate. Than the labeled metabolites were tracked. For this experiment cell suspensions of E.coli harboring pMevB+pTrc99A, pMBI+ pTrc99A, pMBIS+ pTrc99A or pMBIS+pADS were used. pTRc99A is the bacterial expression vector with inducible to IPTG lacI promoter. The result showed that strains with MevB operon accumulated IPP but not FPP, when MBI and MBIS strains accumulated FPP. ([http://www.disketa.si/media/uploads/2015-01/31ad7a215f016dc26aceacf92eb46712.jpg See image here]) Because of the simultaneous expression of the amorphadiene synthase the waste of FPP that accumulated in the MBIS host was consumed. This was shown by a decrease in intracellular FPP. Cells expresing MBIS acumulated FPP and showed growth inhibition in the presence of 10 mM mevalonate. The prediction was that the coexpression of ADS would ease the growth inhibition by forwarding the prenyl pyrophosphate intermediates to volatile terpene olefin. For this test, the cell with pMBIS and empty expression vector pTrc99A (without ADS)and cells with pMBIS and pADS vectors were used. The medium was supplemented with mevalonate concentations ( 0mM, 5mM, 10mM, 20mM or 40mM). Two hours after addition of mevalonate and inducer IPTG, the growth inhibition was observed. ([http://www.disketa.si/media/uploads/2015-01/94b5f3d5636fe767316f770f3e2b0448.jpg See image here]) The result showed that the strains with pMBIS +ADS started to grow, but strain lacking the ADS still showed growth inhbition at higher concentrations of mevalonate. This supported the conclusion that converion of FPP to amophadiene has an important role in minimising growth inhibition. Briefly these result showed that engineered mevalonate patway produces high levels of prenyl pyrophosphate precursors. If the IPP isomerase, FPP synthase and terpen synthase is absent, IPP can acumulate and be toxic to the cells. The toxic levels of intracellular prenyl pyrphosphate can accumulate [1].

=== Amorphadiene synthesis from acetyl-Coa ===

The main goal was to achieve high amoprhaidene production from a simple and inexpensive carbon source. E.coli strains harboring pMevT,pMBIS and pADS plasmids were transformed with pMevT plasmid. Subsequently the strains were tested for ability to produce amoprhadiene in the absence of exogenous mevalonate. The comparison between E.coli expressing the native DPX pathway and engineered pathway as made. They tested native DXP pathway, engineered DPX pathway, mevalonate bottom pathway in the absence of mevalonate, mevlonate bottom pathway in medium suplemented with mevalonate, the complete mevalonate pathway and the complete mevalonate pathway supplemented with 0,8% glycerol. Glycerol was addded to investigate the effect of amorphadiene production of supplying as single carbon source and for prolongation of amorphadiene production. Breifly the resulut showed that expression of the mevalonate-dependent isoprenoid biosynthetic pathway delivers hight levels of isoprenoid precursor for the production of sesquiterpene [1]. ([http://www.disketa.si/media/uploads/2015-01/a31fe07a7c66854b1dc3ff36f8ed01ef.jpg See image here])

== DISSCUSION ==
The identification of genes encoding the enzymes involved in the artemisinin-biosynthesis pathway together with advances in metabolic engineering provides new options for engineering artemisinin biosynthesis. Heterologous production of artemisnin in microorganism such as E.coli. is an alternative way for production of artmeinsin in high yields. But eventhougt micoorganisms has shown great potential in that matter, optimisation of these heterlogous pathways still require maximal titer. Inequal gene expression can lead to accumulation of toxic metabolic intermediates and to over or under production of enzymes involved in pathway. (4). Conclusion based on these study shows that poor expression of the amoprhaidene synthase genes (ADS) limits high terpene olfein yields from the host. To achieve better results , in this study they chose to synthesiyse an amophaiene synthase gene (ADS). The comparison between E.coli expressing native sesquiterpene synthase genes and E.coli expressing the synthetic ADS gene showed big improvement in terpene synthesis due to synthetic ADS gene. The native DPX pathway showed poor supply of the prenyl pyrophosphate precuror. And that limited terpenoid -carotenoid yields in E.coli. With the metabolic engineering of the DPX pathway the flux of carotenoid accumulation showed signifcant increase. By using the mevalonate-dependent isoprenoid pathway from yeast S. cerevisae and engineering that pathway in bacteria E.coli, we avoid the native regulatory elements found in yeast while bypassing those of E.coli DXP pathway. In this study results showed growth inhibition in cells without ADS due to the vast excess of preny pyrophosphate. But the coexpression of a synthetic sesquiterpene synthase can contribute to reducing the excess pool of precursors and eliminate growth inhibition. In this study total biosynthesis of artemisnin was not completely achieved. But these results can help in further investigation for poduction of artemisnic acid. This acid can then be putrified and chemically converted into artemsinin. [1,7].

== CONCLUSIONS ==
To sum things up, artmeinsin, a sesquiterpene isolated from Artemisia annua,represent a novel drug for malaria treatment. The present artemisnin derivates are too expensive for general use and plants can not produce artmeinsin in big quantities. This fact inspired scientist to focus on developing the way for production of artemisnin in high quantities and to avoid big costs. The promising tool in alternative source of artmeinsin is the use of microoganisms. The aim is to transfer the genes encoding enzymes involved in biosynthetic pathway for artemisnin into a hight producing host organism. Several study have been reporting on engineering the metabolic pathway in S.cerevisae to produce the precursor for artmeisnic acid. With the effort in synthetic biology they used modified mevalonate pathway . With the help of syntehtic bilogy the yeast cells were engineerd to express ezymes needed for production of artmemisnic acid. But in this paper the main focus was on engineering a mevalonate pathway in bacteria E.coli. By engineering a new metabolic pathway in E.coli we can easily synthesise a precursor nedeed for artemisnin production. The goal is to produce drugs in better and cheape ways. But, why focus on artemisnin? Well, acording on recent studies artemisnin shows to be very sucesfull for the treatmen all strain of malaria. More than a milion people affected with malaria have already been cured by arteminsin. As already mentioned , because of very hight costs and limited production of arteminin in plants most populations in Africa can not afford it. Progress made in synthetic biology and ability to produce amorphadiene in microorganisms in big quantites opens a new possibility to produce low cost artemisnin and contributes to the brighter future od antimalaria drug deveopment, and therefore saving milions from suffering from malaria. [1,2,3].

[1]Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nature biotehnology; vol 21; pages 796-802, July 2003

[2] T. Mosses, J. Pollier, J. M. Thevelein, A. Goossens. Bioengineering of plant (tri)terpenoids: from metabolic engineering of plants to synthetic biology in vivo and in vitro. New Pathyologist. 200: 27-43, 2013

[3] M.Farhi, M. Kozin, S. Duchin, A. Vainstein. Metabolic engineering of plants for artemisinin synthesis. Biotehnology and genetic engineering rewiews.Vol.29,No.2: 135-148, 2013

[4] Synthetic biology ; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Synthetic_biology http://en.wikipedia.org/wiki/Synthetic_biology]

[5]Operon; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Operon http://en.wikipedia.org/wiki/Operon]

[6] M. K. Julsing, G. van Pouderoyen, J. van der Veen,H. J. Woerdenbag, O. Kayser, W. J. Quax. Amorphadiene synthase: probing the active site model with directed mutagenesis. Directed mutagenesis of AMDS.Chapter 4: 47-56

[7]J.R. Anthony, L. C. Anthony, F. Nowroozi, G. Kwon, J.D. Newman, J.D. Keasling. Optimization of themevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene. Metabolic Engineering. 11: 13-19, 2009

Engineering a mevalonate pathway in Escherichia coli for production of terpenoids

2015-01-19T23:51:47Z

Ana90: /* CONCLUSIONS */

'''Ana Kapraljević'''

[http://www2.lbl.gov/ttd/publications/2837pub1.pdf Engineering a mevalonate pathway in Escherichia coli for production of terpenoids]
Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling ;Nature biotehnology; July 2003; vol 21; pages 796-802

== INTRODUCTION ==

Plants can synthesise and accumulate a wide range of small molecules or natural products involved in psyhological an ecological process. With the knowledge we know about their biosyntesis we used them as commercial flavors, fragnance compunds, colorants and terapeutical drugs. Their therapeutic potential is used for production of anticancer or antimalarial drugs, such as artemisnin. The most numerous and diverse class of natural products used in production for antimalarial drug are isoprenoids or more corectly their subclass, terpenoids. Malaria represent a major health problem in tropic and subtropic. Majority of death occur in children living in Africa. But the problem is that terpenoids are in plants produced in very small quantites and consequently not very economically feasible. For that reason the drugs for treatin malaria, artemisnin, is in short supply and is still unaffordable for most people living in malaria-endanger countries. Scientists are now focusing on alternative and cheaper way for terpenoid synthesis that can be enchanched and became economically acceptable. The alternative way is the expression of synthetic amorphadiene, precursor to artemisnin, and mevalonate isoprenoid way from yeas Saccharomyces cerevisiae in bacteria. In this paper I will summerize the project made in the production of amorphadiene by biotehnology and synthetic biology approach [1,2].

=== Artemisnin ===

Artemisnin is a substance that has antimalarial potential. Malaria is a infectious disease caused by parasitic protozan of the Plasmodium species. In the certain parts of the world malaria takes lives of more people worldwide than any other infecious disease, so it is a global problem. Death to malaria has been nowdays reduced thanks to the use of artemisnin-based combination therapy. Artemisnin is a natural compoud from plant ''Artemisia annua''. Chemically it is a sesquiterpene lactone, amorphadiene. The total sythesis of artemisnin is difficult and not very economical,so the new aproach is to put the hopes in sythetic biology and develop the new way, synthetic way, for its production [1,3].

=== Biology of terpenoids ===

For initiation of any synthetic biology process in metabolic engineering in that matter it is important to have some basic understanding of biosynthesis and regulation of terpenoids. Terpenoids, a class of isoprenoids, are natural products often sythesized by plants. They represent a large and diverse class and have many functions as primarly metabolites involved in growth and development of plants, or as secundary metabolites that optimize the interaction between the plant and the environment. Eventhought they are structural diverse, the share a common biosynthetic origin and follow similar synthesis tracks. All terpenoids are derived from repetitive fusion of isoprene units (C5 H8) [3]. The number of isoprene units determines their classification. For isoprenoid synthesis plants use mevalonate-dependent (MEV) pathway and mevalonte-independent pathway or alternative deoxyxylulose 5-phosphate pathway (DXP). In higher plants the biosynthesis begins with the generation of isopentenyl pyrophosphate (IPP). IPP is derived via the classic mevalonic acid pathway from acetly-coenzime A (acetyl-CoA). The IPP is isomerized to its allylic isomer dimethylallyl pyrophosphate (DMAPP) [3]. The continous condensation of IPP and DMAPP units leads to the formaton of prenylated pyrophosphates, the immediate precursors of the different terpenoid classes. So IPP and DMAPP are combined to form larger isoprenoids, such as farnesyl diphosphate (FPP). Condensation of IPP and DMAPP than units leads to the formation of precursors of the different terpenoid classes. Shematic production of terpenoids in ilustraded in this picture. Similary to plants, yeast Sacharomyces cerevisae uses the clasical mevalonate pathway for the synthesis of isoprenoids[3]. Prokariontes mostly use the DXP pathway for production IPP and DMAPP. This is an alternative pathway for IPP synthesis. This pathway uses Escherichia coli and some other bacteria for their terpenoid synthesis. IPP and DMAPP precurosr are essential to E. Coli for prenylation (addition of hydrophobic molecules to a protein or chemical compound) of tRNA and the synthesis of farnesyl pyrophosphate (FPP). FPP is then used for quinone (class of organic compounds) and cell wall synthesis. The DPX pathway begins with the glycolytic intermediates glyceraldehyde-3-phosphate (G3P) and pyruvate and continues through enzymatic steps for production of IPP and DMAPP. Therefore IPP and DMAPP are the end product in both way,MEV and DPX pathway [1,3].

== METABOLIC ENGINEERING FOR PRODUCTION OD TERPENOIDS ==

Metabolic engineering represents an improvement of production, construction, or cellular properties through the modification of specific biochemical reactions. It is described as the use of genetic engineering to reshape or modify the metabolism of organism. The aim is to optimize genetic and regulatory processes in cells to increase the production of a certain substance that we find in nature. It is based on optimisation of existing biochemical pathways or the introduction of natural pathway components in bacteria, yeast or plants. The main goal is the production of high-yield of specific metabolites that we can use in medicine or biotechnology. With progress in synthetic biology we can design a biologic funcion that do not exist in nature. Consequently, we can create and construct new biological components or redesign exisitng biological systems. In plants, most terpenoids are produced in a very small quantities in their natural sources. Extraction of this compouds from plants is expensive and therfore not economicaly accetable. So there is a big disagreement between the insistence for supply of terpenoids and therefore that leads to delay for their widespread application. With the understanding of terpenoid synthesis and progress in synthetic biology we are now able of redesign and modify the isoprenoid pathway to increase the terpenoid productivity [2,4]. ([http://www.disketa.si/media/uploads/2015-01/cd201ade6a167488767dfb1b118587bc.jpg See image here])

=== Microbal host ===

To elimante the need for plant extraction it is practical to produce terpenoid compouds at high yield in microbal host by introducing a heterlogous isoprenoid pathway into bacteria E.coli. Microorganisms, such as bacteria are attractive alternatives as hosts because they have rapid doubling time, they can achieve robussnes under process conditions and because of the simplicity of product putrification. And the costs are significantly lower than working with plants. With the engineering the DXP pathway we can increase the supply of isoprenoid precurosor needed for high level productio of isoprenoids in E.coli [1,3].

=== Operons ===

Operon is a a funcional unit of DNA that contains a cluster of genes under the control of a single promotor. The synthesis of natural or synthetic products involves the introduction of a large number of genes. In this case these genes were encoding the enzymes needed for metabolic pathway. So it is useful to put certain genes under the control of a single promotor. For that reason Plac promotor was used in this study [1,5].

== EXPERIMENT ==

=== Synthase gene asemby and amorphadiene production ===

For high level production and changing the difficulties in expressing teprene synthases they first synthesized and expressed a codon-optimized variants of ADS . ADS is a gene encoding amorphadiene synthase and is sequentially oxidised by citorchromeP450 CYP71AV1 and reduced by artemisinic aldehyde reductase (DBR2). Amorphadyene synthase is an enzyme and catalyse the first step in the anitmalarial drug artemisnin synthesis. It has an inportant role in catalysing the reaction of FPP to amorphadiene. Beacause of its importaint step towards artemisnin biosynthesis it was designed and improved for the purpuse to acheve high-level expression in E.coli. For ADS gene synthesis they used two step assemly and one step amplification PCR.(1) Assemby PCR is a useful technique for production novel gene sequences. With this method we can put together short fragments of DNA and it is also a good choice for gene construction. Reserches in this article analysed the sequence of three ADS genes from independet clones and identified mutations in each of the genes. Than, two site directed mutagenesis reaction was used for assembly and generation a functional ADS gene from two clones. Than they measure the amorphadiene production by the amorphadiene synthase in E.coli DH10B with natural (nonengineered DPX pathway) and in E.coli with engineered DPX pathway. E. coli with engineerd pathway was harboring the pSOE4 plasmid (DXP pathway operon with dxs,IppHp and ispA gene). The result sugested that FPP synthesis limited amorphadiene production in this engineered host [1,6]. ([http://www.disketa.si/media/uploads/2015-01/0bddf07f946e6f5be0638090dee56490.jpg See image here])

=== Egineering the mevalonate-dependet pathway in E.coli ===

The aim was to increase the intracellular concentration of FPP substrate. Genes encoding the MEV pathway from S.cerevisae was assembled into operons and expressed in E.coli. Because of the complexity of engineering numerous gene biosynthetic pathway, genes were divided into to operons, top and bottom.Top operon, MevT, consisted of three enzymes: acetoacetyl-CoA thiolase (atoB), HMG-CoA synthases (HMGS) and HMG-CoA reductase (tHMGR). This operon was responsible for transformation of acetyl-coA to mevalonate. Thre different bottom operons pMevB (ERG12, ERG8,MVD1 ), pMBI (ERG12, ERG8, MVD1, idi) and pMBIS (ERG12, ERG8, MVD1, idi, ispA) transformed mevalonate to IPP, DMAPP and consequently to FPP. The next step was to analyse and test the funcionality of this pathway. For this purpuse E.coli strand DYM1 with incoplemplete isoprenoid synthesis was transformed with plasmid expressing three bottom operon constuct- pMevB, pMBI and pMBI. E.coli strand DYM1 had a deletion in the ispC gene for encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase, and was not able to synthesise 2-C-methyl-D-erythritol 4-phosphate(MEP), which is important intermediate in isoprenoid biosynthesis[1]. Because of this deletion the synthesis of precursors IPP and DMAP is blocked. So then they tested what is going to happen if they grew the strain DYM1 in the presence of 2-C-methyl-D-ertihritol and in medium with mevalonate without methylerythritol since DYM1 strain was not capable to synthesisie MEP. As expected , all strains expressing operon from S.cerevisiae grew on the plates in the presence of 2-c-methyl-D-erythritol. But only strains with pMBI or pMBIS grew on plates with 1mM mevalonate in the absence of methylerythol. This was the conformation that synthetic MBI and MBIS operons were functional, because they were capable to support the growth of E.coli without ispC gene by supplying cells with IPP and DMAPP. Next they completed the mevalonate pathway by transforming the pMevT plasmid (expressing three genes mentioned before-atoB, HMGS and tHMGR) with pMBI or pMBIS. This coexpresion of two operons, pMevT and pMBI /pMBIS ,completed the mevlonate pathway and allowed the synthesis of sesquiterpene precursors from acetyl-CoA, IPP and DMAPP. Therefore, coexpression complemented the ispC deletion even in the absence of mevalonate, what was the proof, that MevT operon was functional [1,7].

=== Amorpadiene synthesis from mevalonate ===

The aim was to achieve high-level production of amorphadiene and to find out if the amount of FPP was limiting amoprhadiene output. As mentioned, E.coli was used as heterologous microorganism in which coupled mevalonate pathway with amorphadiene synthesis. Cells with ADS gene were coexpressed with MBIS operon and grown in medium with increasing amounts (0 to 40 mM) of exogenus mevalonate. The culture extract was analysed with gas chromatography-mass spectrometry, analytical method for identification different substances in a test sample. It combines the features of mass spectrometry and gas-liquid chromatography. Results showed that MBIS operon did not limit the amorphadiene production at the highest mevalonate concentration(40mM). ([http://www.disketa.si/media/uploads/2015-01/7711d592584a53282f2ecb0fd678ef40.jpg See image here]) Cultures with 40 mM mevalonate added produced much more amoprhadiene. With time, amorphadiene concentration was dropping beacause of the loss of the volatile terpene[1]. By adding more than 10 mM mevalonate in control cultures without ADS, the severe growth ihibition has been reported. ([http://www.disketa.si/media/uploads/2015-01/2a21f55f0fea31ffcbe89c866c9150f7.jpg See image here]) To discover the cause ofthis inhibition, the growth of E.coli DH10B from strains with empty, pMKPMK, pMevB, pMBI or pMBIS plasmid was measured in media with increasing concentrations of mevalonate. The addition of 5mM mevalonate to the medium inhibited the growth of cells with pMevB. But 5mM concentrations of mevalonate did not alter the growth of cells with pMKPMK, PMBI or pMBIS plasmid. This indicated that the accomulation of IPP is toxic and inhibits normal cell growth. The accmulation mostly occurs in cells with high flux through mevalonate pathway. The next step was comparison of intracellular prenyl pyrophosphate (FPP) in strains. This was done by feeding the cells harboring the different mevalonte operon construct with radiolabeled mevalonate. Than the labeled metabolites were tracked. For this experiment cell suspensions of E.coli harboring pMevB+pTrc99A, pMBI+ pTrc99A, pMBIS+ pTrc99A or pMBIS+pADS were used. pTRc99A is the bacterial expression vector with inducible to IPTG lacI promoter. The result showed that strains with MevB operon accumulated IPP but not FPP, when MBI and MBIS strains accumulated FPP. ([http://www.disketa.si/media/uploads/2015-01/31ad7a215f016dc26aceacf92eb46712.jpg See image here]) Because of the simultaneous expression of the amorphadiene synthase the waste of FPP that accumulated in the MBIS host was consumed. This was shown by a decrease in intracellular FPP. Cells expresing MBIS acumulated FPP and showed growth inhibition in the presence of 10 mM mevalonate. The prediction was that the coexpression of ADS would ease the growth inhibition by forwarding the prenyl pyrophosphate intermediates to volatile terpene olefin. For this test, the cell with pMBIS and empty expression vector pTrc99A (without ADS)and cells with pMBIS and pADS vectors were used. The medium was supplemented with mevalonate concentations ( 0mM, 5mM, 10mM, 20mM or 40mM). Two hours after addition of mevalonate and inducer IPTG, the growth inhibition was observed. ([http://www.disketa.si/media/uploads/2015-01/94b5f3d5636fe767316f770f3e2b0448.jpg See image here]) The result showed that the strains with pMBIS +ADS started to grow, but strain lacking the ADS still showed growth inhbition at higher concentrations of mevalonate. This supported the conclusion that converion of FPP to amophadiene has an important role in minimising growth inhibition. Briefly these result showed that engineered mevalonate patway produces high levels of prenyl pyrophosphate precursors. If the IPP isomerase, FPP synthase and terpen synthase is absent, IPP can acumulate and be toxic to the cells. The toxic levels of intracellular prenyl pyrphosphate can accumulate [1].

=== Amorphadiene synthesis from acetyl-Coa ===

The main goal was to achieve high amoprhaidene production from a simple and inexpensive carbon source. E.coli strains harboring pMevT,pMBIS and pADS plasmids were transformed with pMevT plasmid. Subsequently the strains were tested for ability to produce amoprhadiene in the absence of exogenous mevalonate. The comparison between E.coli expressing the native DPX pathway and engineered pathway as made. They tested native DXP pathway, engineered DPX pathway, mevalonate bottom pathway in the absence of mevalonate, mevlonate bottom pathway in medium suplemented with mevalonate, the complete mevalonate pathway and the complete mevalonate pathway supplemented with 0,8% glycerol. Glycerol was addded to investigate the effect of amorphadiene production of supplying as single carbon source and for prolongation of amorphadiene production. Breifly the resulut showed that expression of the mevalonate-dependent isoprenoid biosynthetic pathway delivers hight levels of isoprenoid precursor for the production of sesquiterpene [1]. ([http://www.disketa.si/media/uploads/2015-01/a31fe07a7c66854b1dc3ff36f8ed01ef.jpg See image here])

== DISSCUSION ==
The identification of genes encoding the enzymes involved in the artemisinin-biosynthesis pathway together with advances in metabolic engineering provides new options for engineering artemisinin biosynthesis. Heterologous production of artemisnin in microorganism such as E.coli. is an alternative way for production of artmeinsin in high yields. But eventhougt micoorganisms has shown great potential in that matter, optimisation of these heterlogous pathways still require maximal titer. Inequal gene expression can lead to accumulation of toxic metabolic intermediates and to over or under production of enzymes involved in pathway. (4). Conclusion based on these study shows that poor expression of the amoprhaidene synthase genes (ADS) limits high terpene olfein yields from the host. To achieve better results , in this study they chose to synthesiyse an amophaiene synthase gene (ADS). The comparison between E.coli expressing native sesquiterpene synthase genes and E.coli expressing the synthetic ADS gene showed big improvement in terpene synthesis due to synthetic ADS gene. The native DPX pathway showed poor supply of the prenyl pyrophosphate precuror. And that limited terpenoid -carotenoid yields in E.coli. With the metabolic engineering of the DPX pathway the flux of carotenoid accumulation showed signifcant increase. By using the mevalonate-dependent isoprenoid pathway from yeast S. cerevisae and engineering that pathway in bacteria E.coli, we avoid the native regulatory elements found in yeast while bypassing those of E.coli DXP pathway. In this study results showed growth inhibition in cells without ADS due to the vast excess of preny pyrophosphate. But the coexpression of a synthetic sesquiterpene synthase can contribute to reducing the excess pool of precursors and eliminate growth inhibition. In this study total biosynthesis of artemisnin was not completely achieved. But these results can help in further investigation for poduction of artemisnic acid. This acid can then be putrified and chemically converted into artemsinin. [1,7].

== CONCLUSIONS ==
To sum things up, artmeinsin, a sesquiterpene isolated from Artemisia annua,represent a novel drug for malaria treatment. The present artemisnin derivates are too expensive for general use and plants can not produce artmeinsin in big quantities. This fact inspired scientist to focus on developing the way for production of artemisnin in high quantities and to avoid big costs. The promising tool in alternative source of artmeinsin is the use of microoganisms. The aim is to transfer the genes encoding enzymes involved in biosynthetic pathway for artemisnin into a hight producing host organism. Several study have been reporting on engineering the metabolic pathway in S.cerevisae to produce the precursor for artmeisnic acid. With the effort in synthetic biology they used modified mevalonate pathway . With the help of syntehtic bilogy the yeast cells were engineerd to express ezymes needed for production of artmemisnic acid. But in this paper the main focus was on engineering a mevalonate pathway in bacteria E.coli. By engineering a new metabolic pathway in E.coli we can easily synthesise a precursor nedeed for artemisnin production. The goal is to produce drugs in better and cheape ways. But, why focus on artemisnin? Well, acording on recent studies artemisnin shows to be very sucesfull for the treatmen all strain of malaria. More than a milion people affected with malaria have already been cured by arteminsin. As already mentioned , because of very hight costs and limited production of arteminin in plants most populations in Africa can not afford it. Progress made in synthetic biology and ability to produce amorphadiene in microorganisms in big quantites opens a new possibility to produce low cost artemisnin and contributes to the brighter future od antimalaria drug deveopment, and therefore saving milions from suffering from malaria. [1,2,3].

[1]Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nature biotehnology; vol 21; pages 796-802, July 2003

[2] T. Mosses, J. Pollier, J. M. Thevelein, A. Goossens. Bioengineering of plant (tri)terpenoids: from metabolic engineering of plants to synthetic biology in vivo and in vitro. New Pathyologist. 200: 27-43, 2013

[3] M.Farhi, M. Kozin, S. Duchin, A. Vainstein. Metabolic engineering of plants for artemisinin synthesis. Biotehnology and genetic engineering rewiews.Vol.29,No.2: 135-148, 2013

[4] Synthetic biology ; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Synthetic_biology http://en.wikipedia.org/wiki/Synthetic_biology]

[5]Operon; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Operon http://en.wikipedia.org/wiki/Operon]

[6] M. K. Julsing, G. van Pouderoyen, J. van der Veen,H. J. Woerdenbag, O. Kayser, W. J. Quax. Amorphadiene synthase: probing the active site model with directed mutagenesis. Directed mutagenesis of AMDS.Chapter 4: 47-56

[7]J.R. Anthony, L. C. Anthony, F. Nowroozi, G. Kwon, J.D. Newman, J.D. Keasling. Optimization of themevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene. Metabolic Engineering. 11: 13-19, 2009

Engineering a mevalonate pathway in Escherichia coli for production of terpenoids

2015-01-19T23:51:26Z

Ana90: /* CONCLUSIONS */

'''Ana Kapraljević'''

[http://www2.lbl.gov/ttd/publications/2837pub1.pdf Engineering a mevalonate pathway in Escherichia coli for production of terpenoids]
Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling ;Nature biotehnology; July 2003; vol 21; pages 796-802

== INTRODUCTION ==

Plants can synthesise and accumulate a wide range of small molecules or natural products involved in psyhological an ecological process. With the knowledge we know about their biosyntesis we used them as commercial flavors, fragnance compunds, colorants and terapeutical drugs. Their therapeutic potential is used for production of anticancer or antimalarial drugs, such as artemisnin. The most numerous and diverse class of natural products used in production for antimalarial drug are isoprenoids or more corectly their subclass, terpenoids. Malaria represent a major health problem in tropic and subtropic. Majority of death occur in children living in Africa. But the problem is that terpenoids are in plants produced in very small quantites and consequently not very economically feasible. For that reason the drugs for treatin malaria, artemisnin, is in short supply and is still unaffordable for most people living in malaria-endanger countries. Scientists are now focusing on alternative and cheaper way for terpenoid synthesis that can be enchanched and became economically acceptable. The alternative way is the expression of synthetic amorphadiene, precursor to artemisnin, and mevalonate isoprenoid way from yeas Saccharomyces cerevisiae in bacteria. In this paper I will summerize the project made in the production of amorphadiene by biotehnology and synthetic biology approach [1,2].

=== Artemisnin ===

Artemisnin is a substance that has antimalarial potential. Malaria is a infectious disease caused by parasitic protozan of the Plasmodium species. In the certain parts of the world malaria takes lives of more people worldwide than any other infecious disease, so it is a global problem. Death to malaria has been nowdays reduced thanks to the use of artemisnin-based combination therapy. Artemisnin is a natural compoud from plant ''Artemisia annua''. Chemically it is a sesquiterpene lactone, amorphadiene. The total sythesis of artemisnin is difficult and not very economical,so the new aproach is to put the hopes in sythetic biology and develop the new way, synthetic way, for its production [1,3].

=== Biology of terpenoids ===

For initiation of any synthetic biology process in metabolic engineering in that matter it is important to have some basic understanding of biosynthesis and regulation of terpenoids. Terpenoids, a class of isoprenoids, are natural products often sythesized by plants. They represent a large and diverse class and have many functions as primarly metabolites involved in growth and development of plants, or as secundary metabolites that optimize the interaction between the plant and the environment. Eventhought they are structural diverse, the share a common biosynthetic origin and follow similar synthesis tracks. All terpenoids are derived from repetitive fusion of isoprene units (C5 H8) [3]. The number of isoprene units determines their classification. For isoprenoid synthesis plants use mevalonate-dependent (MEV) pathway and mevalonte-independent pathway or alternative deoxyxylulose 5-phosphate pathway (DXP). In higher plants the biosynthesis begins with the generation of isopentenyl pyrophosphate (IPP). IPP is derived via the classic mevalonic acid pathway from acetly-coenzime A (acetyl-CoA). The IPP is isomerized to its allylic isomer dimethylallyl pyrophosphate (DMAPP) [3]. The continous condensation of IPP and DMAPP units leads to the formaton of prenylated pyrophosphates, the immediate precursors of the different terpenoid classes. So IPP and DMAPP are combined to form larger isoprenoids, such as farnesyl diphosphate (FPP). Condensation of IPP and DMAPP than units leads to the formation of precursors of the different terpenoid classes. Shematic production of terpenoids in ilustraded in this picture. Similary to plants, yeast Sacharomyces cerevisae uses the clasical mevalonate pathway for the synthesis of isoprenoids[3]. Prokariontes mostly use the DXP pathway for production IPP and DMAPP. This is an alternative pathway for IPP synthesis. This pathway uses Escherichia coli and some other bacteria for their terpenoid synthesis. IPP and DMAPP precurosr are essential to E. Coli for prenylation (addition of hydrophobic molecules to a protein or chemical compound) of tRNA and the synthesis of farnesyl pyrophosphate (FPP). FPP is then used for quinone (class of organic compounds) and cell wall synthesis. The DPX pathway begins with the glycolytic intermediates glyceraldehyde-3-phosphate (G3P) and pyruvate and continues through enzymatic steps for production of IPP and DMAPP. Therefore IPP and DMAPP are the end product in both way,MEV and DPX pathway [1,3].

== METABOLIC ENGINEERING FOR PRODUCTION OD TERPENOIDS ==

Metabolic engineering represents an improvement of production, construction, or cellular properties through the modification of specific biochemical reactions. It is described as the use of genetic engineering to reshape or modify the metabolism of organism. The aim is to optimize genetic and regulatory processes in cells to increase the production of a certain substance that we find in nature. It is based on optimisation of existing biochemical pathways or the introduction of natural pathway components in bacteria, yeast or plants. The main goal is the production of high-yield of specific metabolites that we can use in medicine or biotechnology. With progress in synthetic biology we can design a biologic funcion that do not exist in nature. Consequently, we can create and construct new biological components or redesign exisitng biological systems. In plants, most terpenoids are produced in a very small quantities in their natural sources. Extraction of this compouds from plants is expensive and therfore not economicaly accetable. So there is a big disagreement between the insistence for supply of terpenoids and therefore that leads to delay for their widespread application. With the understanding of terpenoid synthesis and progress in synthetic biology we are now able of redesign and modify the isoprenoid pathway to increase the terpenoid productivity [2,4]. ([http://www.disketa.si/media/uploads/2015-01/cd201ade6a167488767dfb1b118587bc.jpg See image here])

=== Microbal host ===

To elimante the need for plant extraction it is practical to produce terpenoid compouds at high yield in microbal host by introducing a heterlogous isoprenoid pathway into bacteria E.coli. Microorganisms, such as bacteria are attractive alternatives as hosts because they have rapid doubling time, they can achieve robussnes under process conditions and because of the simplicity of product putrification. And the costs are significantly lower than working with plants. With the engineering the DXP pathway we can increase the supply of isoprenoid precurosor needed for high level productio of isoprenoids in E.coli [1,3].

=== Operons ===

Operon is a a funcional unit of DNA that contains a cluster of genes under the control of a single promotor. The synthesis of natural or synthetic products involves the introduction of a large number of genes. In this case these genes were encoding the enzymes needed for metabolic pathway. So it is useful to put certain genes under the control of a single promotor. For that reason Plac promotor was used in this study [1,5].

== EXPERIMENT ==

=== Synthase gene asemby and amorphadiene production ===

For high level production and changing the difficulties in expressing teprene synthases they first synthesized and expressed a codon-optimized variants of ADS . ADS is a gene encoding amorphadiene synthase and is sequentially oxidised by citorchromeP450 CYP71AV1 and reduced by artemisinic aldehyde reductase (DBR2). Amorphadyene synthase is an enzyme and catalyse the first step in the anitmalarial drug artemisnin synthesis. It has an inportant role in catalysing the reaction of FPP to amorphadiene. Beacause of its importaint step towards artemisnin biosynthesis it was designed and improved for the purpuse to acheve high-level expression in E.coli. For ADS gene synthesis they used two step assemly and one step amplification PCR.(1) Assemby PCR is a useful technique for production novel gene sequences. With this method we can put together short fragments of DNA and it is also a good choice for gene construction. Reserches in this article analysed the sequence of three ADS genes from independet clones and identified mutations in each of the genes. Than, two site directed mutagenesis reaction was used for assembly and generation a functional ADS gene from two clones. Than they measure the amorphadiene production by the amorphadiene synthase in E.coli DH10B with natural (nonengineered DPX pathway) and in E.coli with engineered DPX pathway. E. coli with engineerd pathway was harboring the pSOE4 plasmid (DXP pathway operon with dxs,IppHp and ispA gene). The result sugested that FPP synthesis limited amorphadiene production in this engineered host [1,6]. ([http://www.disketa.si/media/uploads/2015-01/0bddf07f946e6f5be0638090dee56490.jpg See image here])

=== Egineering the mevalonate-dependet pathway in E.coli ===

The aim was to increase the intracellular concentration of FPP substrate. Genes encoding the MEV pathway from S.cerevisae was assembled into operons and expressed in E.coli. Because of the complexity of engineering numerous gene biosynthetic pathway, genes were divided into to operons, top and bottom.Top operon, MevT, consisted of three enzymes: acetoacetyl-CoA thiolase (atoB), HMG-CoA synthases (HMGS) and HMG-CoA reductase (tHMGR). This operon was responsible for transformation of acetyl-coA to mevalonate. Thre different bottom operons pMevB (ERG12, ERG8,MVD1 ), pMBI (ERG12, ERG8, MVD1, idi) and pMBIS (ERG12, ERG8, MVD1, idi, ispA) transformed mevalonate to IPP, DMAPP and consequently to FPP. The next step was to analyse and test the funcionality of this pathway. For this purpuse E.coli strand DYM1 with incoplemplete isoprenoid synthesis was transformed with plasmid expressing three bottom operon constuct- pMevB, pMBI and pMBI. E.coli strand DYM1 had a deletion in the ispC gene for encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase, and was not able to synthesise 2-C-methyl-D-erythritol 4-phosphate(MEP), which is important intermediate in isoprenoid biosynthesis[1]. Because of this deletion the synthesis of precursors IPP and DMAP is blocked. So then they tested what is going to happen if they grew the strain DYM1 in the presence of 2-C-methyl-D-ertihritol and in medium with mevalonate without methylerythritol since DYM1 strain was not capable to synthesisie MEP. As expected , all strains expressing operon from S.cerevisiae grew on the plates in the presence of 2-c-methyl-D-erythritol. But only strains with pMBI or pMBIS grew on plates with 1mM mevalonate in the absence of methylerythol. This was the conformation that synthetic MBI and MBIS operons were functional, because they were capable to support the growth of E.coli without ispC gene by supplying cells with IPP and DMAPP. Next they completed the mevalonate pathway by transforming the pMevT plasmid (expressing three genes mentioned before-atoB, HMGS and tHMGR) with pMBI or pMBIS. This coexpresion of two operons, pMevT and pMBI /pMBIS ,completed the mevlonate pathway and allowed the synthesis of sesquiterpene precursors from acetyl-CoA, IPP and DMAPP. Therefore, coexpression complemented the ispC deletion even in the absence of mevalonate, what was the proof, that MevT operon was functional [1,7].

=== Amorpadiene synthesis from mevalonate ===

The aim was to achieve high-level production of amorphadiene and to find out if the amount of FPP was limiting amoprhadiene output. As mentioned, E.coli was used as heterologous microorganism in which coupled mevalonate pathway with amorphadiene synthesis. Cells with ADS gene were coexpressed with MBIS operon and grown in medium with increasing amounts (0 to 40 mM) of exogenus mevalonate. The culture extract was analysed with gas chromatography-mass spectrometry, analytical method for identification different substances in a test sample. It combines the features of mass spectrometry and gas-liquid chromatography. Results showed that MBIS operon did not limit the amorphadiene production at the highest mevalonate concentration(40mM). ([http://www.disketa.si/media/uploads/2015-01/7711d592584a53282f2ecb0fd678ef40.jpg See image here]) Cultures with 40 mM mevalonate added produced much more amoprhadiene. With time, amorphadiene concentration was dropping beacause of the loss of the volatile terpene[1]. By adding more than 10 mM mevalonate in control cultures without ADS, the severe growth ihibition has been reported. ([http://www.disketa.si/media/uploads/2015-01/2a21f55f0fea31ffcbe89c866c9150f7.jpg See image here]) To discover the cause ofthis inhibition, the growth of E.coli DH10B from strains with empty, pMKPMK, pMevB, pMBI or pMBIS plasmid was measured in media with increasing concentrations of mevalonate. The addition of 5mM mevalonate to the medium inhibited the growth of cells with pMevB. But 5mM concentrations of mevalonate did not alter the growth of cells with pMKPMK, PMBI or pMBIS plasmid. This indicated that the accomulation of IPP is toxic and inhibits normal cell growth. The accmulation mostly occurs in cells with high flux through mevalonate pathway. The next step was comparison of intracellular prenyl pyrophosphate (FPP) in strains. This was done by feeding the cells harboring the different mevalonte operon construct with radiolabeled mevalonate. Than the labeled metabolites were tracked. For this experiment cell suspensions of E.coli harboring pMevB+pTrc99A, pMBI+ pTrc99A, pMBIS+ pTrc99A or pMBIS+pADS were used. pTRc99A is the bacterial expression vector with inducible to IPTG lacI promoter. The result showed that strains with MevB operon accumulated IPP but not FPP, when MBI and MBIS strains accumulated FPP. ([http://www.disketa.si/media/uploads/2015-01/31ad7a215f016dc26aceacf92eb46712.jpg See image here]) Because of the simultaneous expression of the amorphadiene synthase the waste of FPP that accumulated in the MBIS host was consumed. This was shown by a decrease in intracellular FPP. Cells expresing MBIS acumulated FPP and showed growth inhibition in the presence of 10 mM mevalonate. The prediction was that the coexpression of ADS would ease the growth inhibition by forwarding the prenyl pyrophosphate intermediates to volatile terpene olefin. For this test, the cell with pMBIS and empty expression vector pTrc99A (without ADS)and cells with pMBIS and pADS vectors were used. The medium was supplemented with mevalonate concentations ( 0mM, 5mM, 10mM, 20mM or 40mM). Two hours after addition of mevalonate and inducer IPTG, the growth inhibition was observed. ([http://www.disketa.si/media/uploads/2015-01/94b5f3d5636fe767316f770f3e2b0448.jpg See image here]) The result showed that the strains with pMBIS +ADS started to grow, but strain lacking the ADS still showed growth inhbition at higher concentrations of mevalonate. This supported the conclusion that converion of FPP to amophadiene has an important role in minimising growth inhibition. Briefly these result showed that engineered mevalonate patway produces high levels of prenyl pyrophosphate precursors. If the IPP isomerase, FPP synthase and terpen synthase is absent, IPP can acumulate and be toxic to the cells. The toxic levels of intracellular prenyl pyrphosphate can accumulate [1].

=== Amorphadiene synthesis from acetyl-Coa ===

The main goal was to achieve high amoprhaidene production from a simple and inexpensive carbon source. E.coli strains harboring pMevT,pMBIS and pADS plasmids were transformed with pMevT plasmid. Subsequently the strains were tested for ability to produce amoprhadiene in the absence of exogenous mevalonate. The comparison between E.coli expressing the native DPX pathway and engineered pathway as made. They tested native DXP pathway, engineered DPX pathway, mevalonate bottom pathway in the absence of mevalonate, mevlonate bottom pathway in medium suplemented with mevalonate, the complete mevalonate pathway and the complete mevalonate pathway supplemented with 0,8% glycerol. Glycerol was addded to investigate the effect of amorphadiene production of supplying as single carbon source and for prolongation of amorphadiene production. Breifly the resulut showed that expression of the mevalonate-dependent isoprenoid biosynthetic pathway delivers hight levels of isoprenoid precursor for the production of sesquiterpene [1]. ([http://www.disketa.si/media/uploads/2015-01/a31fe07a7c66854b1dc3ff36f8ed01ef.jpg See image here])

== DISSCUSION ==
The identification of genes encoding the enzymes involved in the artemisinin-biosynthesis pathway together with advances in metabolic engineering provides new options for engineering artemisinin biosynthesis. Heterologous production of artemisnin in microorganism such as E.coli. is an alternative way for production of artmeinsin in high yields. But eventhougt micoorganisms has shown great potential in that matter, optimisation of these heterlogous pathways still require maximal titer. Inequal gene expression can lead to accumulation of toxic metabolic intermediates and to over or under production of enzymes involved in pathway. (4). Conclusion based on these study shows that poor expression of the amoprhaidene synthase genes (ADS) limits high terpene olfein yields from the host. To achieve better results , in this study they chose to synthesiyse an amophaiene synthase gene (ADS). The comparison between E.coli expressing native sesquiterpene synthase genes and E.coli expressing the synthetic ADS gene showed big improvement in terpene synthesis due to synthetic ADS gene. The native DPX pathway showed poor supply of the prenyl pyrophosphate precuror. And that limited terpenoid -carotenoid yields in E.coli. With the metabolic engineering of the DPX pathway the flux of carotenoid accumulation showed signifcant increase. By using the mevalonate-dependent isoprenoid pathway from yeast S. cerevisae and engineering that pathway in bacteria E.coli, we avoid the native regulatory elements found in yeast while bypassing those of E.coli DXP pathway. In this study results showed growth inhibition in cells without ADS due to the vast excess of preny pyrophosphate. But the coexpression of a synthetic sesquiterpene synthase can contribute to reducing the excess pool of precursors and eliminate growth inhibition. In this study total biosynthesis of artemisnin was not completely achieved. But these results can help in further investigation for poduction of artemisnic acid. This acid can then be putrified and chemically converted into artemsinin. [1,7].

== CONCLUSIONS ==
To sum things up, artmeinsin, a sesquiterpene isolated from Artemisia annua,represent a novel drug for malaria treatment. The present artemisnin derivates are too expensive for general use and plants can not produce artmeinsin in big quantities. This fact inspired scientist to focus on developing the way for production of artemisnin in high quantities and to avoid big costs. The promising tool in alternative source of artmeinsin is the use of microoganisms. The aim is to transfer the genes encoding enzymes involved in biosynthetic pathway for artemisnin into a hight producing host organism. Several study have been reporting on engineering the metabolic pathway in S.cerevisae to produce the precursor for artmeisnic acid. With the effort in synthetic biology they used modified mevalonate pathway . With the help of syntehtic bilogy the yeast cells were engineerd to express ezymes needed for production of artmemisnic acid. But in this paper the main focus was on engineering a mevalonate pathway in bacteria E.coli. By engineering a new metabolic pathway in E.coli we can easily synthesise a precursor nedeed for artemisnin production. The goal is to produce drugs in better and cheape ways. But, why focus on artemisnin? Well, acording on recent studies artemisnin shows to be very sucesfull for the treatmen all strain of malaria. More than a milion people affected with malaria have already been cured by arteminsin. As already mentioned , because of very hight costs and limited production of arteminin in plants most populations in Africa can not afford it. Progress made in synthetic biology and ability to produce amorphadiene in microorganisms in big quantites opens a new possibility to produce low cost artemisnin and contributes to the brighter future od antimalaria drug deveopment, and therefore saving milions from suffering from malaria. [1, 2, 3].

[1]Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nature biotehnology; vol 21; pages 796-802, July 2003

[2] T. Mosses, J. Pollier, J. M. Thevelein, A. Goossens. Bioengineering of plant (tri)terpenoids: from metabolic engineering of plants to synthetic biology in vivo and in vitro. New Pathyologist. 200: 27-43, 2013

[3] M.Farhi, M. Kozin, S. Duchin, A. Vainstein. Metabolic engineering of plants for artemisinin synthesis. Biotehnology and genetic engineering rewiews.Vol.29,No.2: 135-148, 2013

[4] Synthetic biology ; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Synthetic_biology http://en.wikipedia.org/wiki/Synthetic_biology]

[5]Operon; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Operon http://en.wikipedia.org/wiki/Operon]

[6] M. K. Julsing, G. van Pouderoyen, J. van der Veen,H. J. Woerdenbag, O. Kayser, W. J. Quax. Amorphadiene synthase: probing the active site model with directed mutagenesis. Directed mutagenesis of AMDS.Chapter 4: 47-56

[7]J.R. Anthony, L. C. Anthony, F. Nowroozi, G. Kwon, J.D. Newman, J.D. Keasling. Optimization of themevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene. Metabolic Engineering. 11: 13-19, 2009

Engineering a mevalonate pathway in Escherichia coli for production of terpenoids

2015-01-19T23:51:04Z

Ana90: /* Microbal host */

'''Ana Kapraljević'''

[http://www2.lbl.gov/ttd/publications/2837pub1.pdf Engineering a mevalonate pathway in Escherichia coli for production of terpenoids]
Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling ;Nature biotehnology; July 2003; vol 21; pages 796-802

== INTRODUCTION ==

Plants can synthesise and accumulate a wide range of small molecules or natural products involved in psyhological an ecological process. With the knowledge we know about their biosyntesis we used them as commercial flavors, fragnance compunds, colorants and terapeutical drugs. Their therapeutic potential is used for production of anticancer or antimalarial drugs, such as artemisnin. The most numerous and diverse class of natural products used in production for antimalarial drug are isoprenoids or more corectly their subclass, terpenoids. Malaria represent a major health problem in tropic and subtropic. Majority of death occur in children living in Africa. But the problem is that terpenoids are in plants produced in very small quantites and consequently not very economically feasible. For that reason the drugs for treatin malaria, artemisnin, is in short supply and is still unaffordable for most people living in malaria-endanger countries. Scientists are now focusing on alternative and cheaper way for terpenoid synthesis that can be enchanched and became economically acceptable. The alternative way is the expression of synthetic amorphadiene, precursor to artemisnin, and mevalonate isoprenoid way from yeas Saccharomyces cerevisiae in bacteria. In this paper I will summerize the project made in the production of amorphadiene by biotehnology and synthetic biology approach [1,2].

=== Artemisnin ===

Artemisnin is a substance that has antimalarial potential. Malaria is a infectious disease caused by parasitic protozan of the Plasmodium species. In the certain parts of the world malaria takes lives of more people worldwide than any other infecious disease, so it is a global problem. Death to malaria has been nowdays reduced thanks to the use of artemisnin-based combination therapy. Artemisnin is a natural compoud from plant ''Artemisia annua''. Chemically it is a sesquiterpene lactone, amorphadiene. The total sythesis of artemisnin is difficult and not very economical,so the new aproach is to put the hopes in sythetic biology and develop the new way, synthetic way, for its production [1,3].

=== Biology of terpenoids ===

For initiation of any synthetic biology process in metabolic engineering in that matter it is important to have some basic understanding of biosynthesis and regulation of terpenoids. Terpenoids, a class of isoprenoids, are natural products often sythesized by plants. They represent a large and diverse class and have many functions as primarly metabolites involved in growth and development of plants, or as secundary metabolites that optimize the interaction between the plant and the environment. Eventhought they are structural diverse, the share a common biosynthetic origin and follow similar synthesis tracks. All terpenoids are derived from repetitive fusion of isoprene units (C5 H8) [3]. The number of isoprene units determines their classification. For isoprenoid synthesis plants use mevalonate-dependent (MEV) pathway and mevalonte-independent pathway or alternative deoxyxylulose 5-phosphate pathway (DXP). In higher plants the biosynthesis begins with the generation of isopentenyl pyrophosphate (IPP). IPP is derived via the classic mevalonic acid pathway from acetly-coenzime A (acetyl-CoA). The IPP is isomerized to its allylic isomer dimethylallyl pyrophosphate (DMAPP) [3]. The continous condensation of IPP and DMAPP units leads to the formaton of prenylated pyrophosphates, the immediate precursors of the different terpenoid classes. So IPP and DMAPP are combined to form larger isoprenoids, such as farnesyl diphosphate (FPP). Condensation of IPP and DMAPP than units leads to the formation of precursors of the different terpenoid classes. Shematic production of terpenoids in ilustraded in this picture. Similary to plants, yeast Sacharomyces cerevisae uses the clasical mevalonate pathway for the synthesis of isoprenoids[3]. Prokariontes mostly use the DXP pathway for production IPP and DMAPP. This is an alternative pathway for IPP synthesis. This pathway uses Escherichia coli and some other bacteria for their terpenoid synthesis. IPP and DMAPP precurosr are essential to E. Coli for prenylation (addition of hydrophobic molecules to a protein or chemical compound) of tRNA and the synthesis of farnesyl pyrophosphate (FPP). FPP is then used for quinone (class of organic compounds) and cell wall synthesis. The DPX pathway begins with the glycolytic intermediates glyceraldehyde-3-phosphate (G3P) and pyruvate and continues through enzymatic steps for production of IPP and DMAPP. Therefore IPP and DMAPP are the end product in both way,MEV and DPX pathway [1,3].

== METABOLIC ENGINEERING FOR PRODUCTION OD TERPENOIDS ==

Metabolic engineering represents an improvement of production, construction, or cellular properties through the modification of specific biochemical reactions. It is described as the use of genetic engineering to reshape or modify the metabolism of organism. The aim is to optimize genetic and regulatory processes in cells to increase the production of a certain substance that we find in nature. It is based on optimisation of existing biochemical pathways or the introduction of natural pathway components in bacteria, yeast or plants. The main goal is the production of high-yield of specific metabolites that we can use in medicine or biotechnology. With progress in synthetic biology we can design a biologic funcion that do not exist in nature. Consequently, we can create and construct new biological components or redesign exisitng biological systems. In plants, most terpenoids are produced in a very small quantities in their natural sources. Extraction of this compouds from plants is expensive and therfore not economicaly accetable. So there is a big disagreement between the insistence for supply of terpenoids and therefore that leads to delay for their widespread application. With the understanding of terpenoid synthesis and progress in synthetic biology we are now able of redesign and modify the isoprenoid pathway to increase the terpenoid productivity [2,4]. ([http://www.disketa.si/media/uploads/2015-01/cd201ade6a167488767dfb1b118587bc.jpg See image here])

=== Microbal host ===

To elimante the need for plant extraction it is practical to produce terpenoid compouds at high yield in microbal host by introducing a heterlogous isoprenoid pathway into bacteria E.coli. Microorganisms, such as bacteria are attractive alternatives as hosts because they have rapid doubling time, they can achieve robussnes under process conditions and because of the simplicity of product putrification. And the costs are significantly lower than working with plants. With the engineering the DXP pathway we can increase the supply of isoprenoid precurosor needed for high level productio of isoprenoids in E.coli [1,3].

=== Operons ===

Operon is a a funcional unit of DNA that contains a cluster of genes under the control of a single promotor. The synthesis of natural or synthetic products involves the introduction of a large number of genes. In this case these genes were encoding the enzymes needed for metabolic pathway. So it is useful to put certain genes under the control of a single promotor. For that reason Plac promotor was used in this study [1,5].

== EXPERIMENT ==

=== Synthase gene asemby and amorphadiene production ===

For high level production and changing the difficulties in expressing teprene synthases they first synthesized and expressed a codon-optimized variants of ADS . ADS is a gene encoding amorphadiene synthase and is sequentially oxidised by citorchromeP450 CYP71AV1 and reduced by artemisinic aldehyde reductase (DBR2). Amorphadyene synthase is an enzyme and catalyse the first step in the anitmalarial drug artemisnin synthesis. It has an inportant role in catalysing the reaction of FPP to amorphadiene. Beacause of its importaint step towards artemisnin biosynthesis it was designed and improved for the purpuse to acheve high-level expression in E.coli. For ADS gene synthesis they used two step assemly and one step amplification PCR.(1) Assemby PCR is a useful technique for production novel gene sequences. With this method we can put together short fragments of DNA and it is also a good choice for gene construction. Reserches in this article analysed the sequence of three ADS genes from independet clones and identified mutations in each of the genes. Than, two site directed mutagenesis reaction was used for assembly and generation a functional ADS gene from two clones. Than they measure the amorphadiene production by the amorphadiene synthase in E.coli DH10B with natural (nonengineered DPX pathway) and in E.coli with engineered DPX pathway. E. coli with engineerd pathway was harboring the pSOE4 plasmid (DXP pathway operon with dxs,IppHp and ispA gene). The result sugested that FPP synthesis limited amorphadiene production in this engineered host [1,6]. ([http://www.disketa.si/media/uploads/2015-01/0bddf07f946e6f5be0638090dee56490.jpg See image here])

=== Egineering the mevalonate-dependet pathway in E.coli ===

The aim was to increase the intracellular concentration of FPP substrate. Genes encoding the MEV pathway from S.cerevisae was assembled into operons and expressed in E.coli. Because of the complexity of engineering numerous gene biosynthetic pathway, genes were divided into to operons, top and bottom.Top operon, MevT, consisted of three enzymes: acetoacetyl-CoA thiolase (atoB), HMG-CoA synthases (HMGS) and HMG-CoA reductase (tHMGR). This operon was responsible for transformation of acetyl-coA to mevalonate. Thre different bottom operons pMevB (ERG12, ERG8,MVD1 ), pMBI (ERG12, ERG8, MVD1, idi) and pMBIS (ERG12, ERG8, MVD1, idi, ispA) transformed mevalonate to IPP, DMAPP and consequently to FPP. The next step was to analyse and test the funcionality of this pathway. For this purpuse E.coli strand DYM1 with incoplemplete isoprenoid synthesis was transformed with plasmid expressing three bottom operon constuct- pMevB, pMBI and pMBI. E.coli strand DYM1 had a deletion in the ispC gene for encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase, and was not able to synthesise 2-C-methyl-D-erythritol 4-phosphate(MEP), which is important intermediate in isoprenoid biosynthesis[1]. Because of this deletion the synthesis of precursors IPP and DMAP is blocked. So then they tested what is going to happen if they grew the strain DYM1 in the presence of 2-C-methyl-D-ertihritol and in medium with mevalonate without methylerythritol since DYM1 strain was not capable to synthesisie MEP. As expected , all strains expressing operon from S.cerevisiae grew on the plates in the presence of 2-c-methyl-D-erythritol. But only strains with pMBI or pMBIS grew on plates with 1mM mevalonate in the absence of methylerythol. This was the conformation that synthetic MBI and MBIS operons were functional, because they were capable to support the growth of E.coli without ispC gene by supplying cells with IPP and DMAPP. Next they completed the mevalonate pathway by transforming the pMevT plasmid (expressing three genes mentioned before-atoB, HMGS and tHMGR) with pMBI or pMBIS. This coexpresion of two operons, pMevT and pMBI /pMBIS ,completed the mevlonate pathway and allowed the synthesis of sesquiterpene precursors from acetyl-CoA, IPP and DMAPP. Therefore, coexpression complemented the ispC deletion even in the absence of mevalonate, what was the proof, that MevT operon was functional [1,7].

=== Amorpadiene synthesis from mevalonate ===

The aim was to achieve high-level production of amorphadiene and to find out if the amount of FPP was limiting amoprhadiene output. As mentioned, E.coli was used as heterologous microorganism in which coupled mevalonate pathway with amorphadiene synthesis. Cells with ADS gene were coexpressed with MBIS operon and grown in medium with increasing amounts (0 to 40 mM) of exogenus mevalonate. The culture extract was analysed with gas chromatography-mass spectrometry, analytical method for identification different substances in a test sample. It combines the features of mass spectrometry and gas-liquid chromatography. Results showed that MBIS operon did not limit the amorphadiene production at the highest mevalonate concentration(40mM). ([http://www.disketa.si/media/uploads/2015-01/7711d592584a53282f2ecb0fd678ef40.jpg See image here]) Cultures with 40 mM mevalonate added produced much more amoprhadiene. With time, amorphadiene concentration was dropping beacause of the loss of the volatile terpene[1]. By adding more than 10 mM mevalonate in control cultures without ADS, the severe growth ihibition has been reported. ([http://www.disketa.si/media/uploads/2015-01/2a21f55f0fea31ffcbe89c866c9150f7.jpg See image here]) To discover the cause ofthis inhibition, the growth of E.coli DH10B from strains with empty, pMKPMK, pMevB, pMBI or pMBIS plasmid was measured in media with increasing concentrations of mevalonate. The addition of 5mM mevalonate to the medium inhibited the growth of cells with pMevB. But 5mM concentrations of mevalonate did not alter the growth of cells with pMKPMK, PMBI or pMBIS plasmid. This indicated that the accomulation of IPP is toxic and inhibits normal cell growth. The accmulation mostly occurs in cells with high flux through mevalonate pathway. The next step was comparison of intracellular prenyl pyrophosphate (FPP) in strains. This was done by feeding the cells harboring the different mevalonte operon construct with radiolabeled mevalonate. Than the labeled metabolites were tracked. For this experiment cell suspensions of E.coli harboring pMevB+pTrc99A, pMBI+ pTrc99A, pMBIS+ pTrc99A or pMBIS+pADS were used. pTRc99A is the bacterial expression vector with inducible to IPTG lacI promoter. The result showed that strains with MevB operon accumulated IPP but not FPP, when MBI and MBIS strains accumulated FPP. ([http://www.disketa.si/media/uploads/2015-01/31ad7a215f016dc26aceacf92eb46712.jpg See image here]) Because of the simultaneous expression of the amorphadiene synthase the waste of FPP that accumulated in the MBIS host was consumed. This was shown by a decrease in intracellular FPP. Cells expresing MBIS acumulated FPP and showed growth inhibition in the presence of 10 mM mevalonate. The prediction was that the coexpression of ADS would ease the growth inhibition by forwarding the prenyl pyrophosphate intermediates to volatile terpene olefin. For this test, the cell with pMBIS and empty expression vector pTrc99A (without ADS)and cells with pMBIS and pADS vectors were used. The medium was supplemented with mevalonate concentations ( 0mM, 5mM, 10mM, 20mM or 40mM). Two hours after addition of mevalonate and inducer IPTG, the growth inhibition was observed. ([http://www.disketa.si/media/uploads/2015-01/94b5f3d5636fe767316f770f3e2b0448.jpg See image here]) The result showed that the strains with pMBIS +ADS started to grow, but strain lacking the ADS still showed growth inhbition at higher concentrations of mevalonate. This supported the conclusion that converion of FPP to amophadiene has an important role in minimising growth inhibition. Briefly these result showed that engineered mevalonate patway produces high levels of prenyl pyrophosphate precursors. If the IPP isomerase, FPP synthase and terpen synthase is absent, IPP can acumulate and be toxic to the cells. The toxic levels of intracellular prenyl pyrphosphate can accumulate [1].

=== Amorphadiene synthesis from acetyl-Coa ===

The main goal was to achieve high amoprhaidene production from a simple and inexpensive carbon source. E.coli strains harboring pMevT,pMBIS and pADS plasmids were transformed with pMevT plasmid. Subsequently the strains were tested for ability to produce amoprhadiene in the absence of exogenous mevalonate. The comparison between E.coli expressing the native DPX pathway and engineered pathway as made. They tested native DXP pathway, engineered DPX pathway, mevalonate bottom pathway in the absence of mevalonate, mevlonate bottom pathway in medium suplemented with mevalonate, the complete mevalonate pathway and the complete mevalonate pathway supplemented with 0,8% glycerol. Glycerol was addded to investigate the effect of amorphadiene production of supplying as single carbon source and for prolongation of amorphadiene production. Breifly the resulut showed that expression of the mevalonate-dependent isoprenoid biosynthetic pathway delivers hight levels of isoprenoid precursor for the production of sesquiterpene [1]. ([http://www.disketa.si/media/uploads/2015-01/a31fe07a7c66854b1dc3ff36f8ed01ef.jpg See image here])

== DISSCUSION ==
The identification of genes encoding the enzymes involved in the artemisinin-biosynthesis pathway together with advances in metabolic engineering provides new options for engineering artemisinin biosynthesis. Heterologous production of artemisnin in microorganism such as E.coli. is an alternative way for production of artmeinsin in high yields. But eventhougt micoorganisms has shown great potential in that matter, optimisation of these heterlogous pathways still require maximal titer. Inequal gene expression can lead to accumulation of toxic metabolic intermediates and to over or under production of enzymes involved in pathway. (4). Conclusion based on these study shows that poor expression of the amoprhaidene synthase genes (ADS) limits high terpene olfein yields from the host. To achieve better results , in this study they chose to synthesiyse an amophaiene synthase gene (ADS). The comparison between E.coli expressing native sesquiterpene synthase genes and E.coli expressing the synthetic ADS gene showed big improvement in terpene synthesis due to synthetic ADS gene. The native DPX pathway showed poor supply of the prenyl pyrophosphate precuror. And that limited terpenoid -carotenoid yields in E.coli. With the metabolic engineering of the DPX pathway the flux of carotenoid accumulation showed signifcant increase. By using the mevalonate-dependent isoprenoid pathway from yeast S. cerevisae and engineering that pathway in bacteria E.coli, we avoid the native regulatory elements found in yeast while bypassing those of E.coli DXP pathway. In this study results showed growth inhibition in cells without ADS due to the vast excess of preny pyrophosphate. But the coexpression of a synthetic sesquiterpene synthase can contribute to reducing the excess pool of precursors and eliminate growth inhibition. In this study total biosynthesis of artemisnin was not completely achieved. But these results can help in further investigation for poduction of artemisnic acid. This acid can then be putrified and chemically converted into artemsinin. [1,7].

== CONCLUSIONS ==
To sum things up, artmeinsin, a sesquiterpene isolated from Artemisia annua,represent a novel drug for malaria treatment. The present artemisnin derivates are too expensive for general use and plants can not produce artmeinsin in big quantities. This fact inspired scientist to focus on developing the way for production of artemisnin in high quantities and to avoid big costs. The promising tool in alternative source of artmeinsin is the use of microoganisms. The aim is to transfer the genes encoding enzymes involved in biosynthetic pathway for artemisnin into a hight producing host organism. Several study have been reporting on engineering the metabolic pathway in S.cerevisae to produce the precursor for artmeisnic acid. With the effort in synthetic biology they used modified mevalonate pathway . With the help of syntehtic bilogy the yeast cells were engineerd to express ezymes needed for production of artmemisnic acid. But in this paper the main focus was on engineering a mevalonate pathway in bacteria E.coli. By engineering a new metabolic pathway in E.coli we can easily synthesise a precursor nedeed for artemisnin production. The goal is to produce drugs in better and cheape ways. But, why focus on artemisnin? Well, acording on recent studies artemisnin shows to be very sucesfull for the treatmen all strain of malaria. More than a milion people affected with malaria have already been cured by arteminsin. As already mentioned , because of very hight costs and limited production of arteminin in plants most populations in Africa can not afford it. Progress made in synthetic biology and ability to produce amorphadiene in microorganisms in big quantites opens a new possibility to produce low cost artemisnin and contributes to the brighter future od antimalaria drug deveopment, and therefore saving milions from suffering from malaria. [1,2, 3].

[1]Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nature biotehnology; vol 21; pages 796-802, July 2003

[2] T. Mosses, J. Pollier, J. M. Thevelein, A. Goossens. Bioengineering of plant (tri)terpenoids: from metabolic engineering of plants to synthetic biology in vivo and in vitro. New Pathyologist. 200: 27-43, 2013

[3] M.Farhi, M. Kozin, S. Duchin, A. Vainstein. Metabolic engineering of plants for artemisinin synthesis. Biotehnology and genetic engineering rewiews.Vol.29,No.2: 135-148, 2013

[4] Synthetic biology ; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Synthetic_biology http://en.wikipedia.org/wiki/Synthetic_biology]

[5]Operon; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Operon http://en.wikipedia.org/wiki/Operon]

[6] M. K. Julsing, G. van Pouderoyen, J. van der Veen,H. J. Woerdenbag, O. Kayser, W. J. Quax. Amorphadiene synthase: probing the active site model with directed mutagenesis. Directed mutagenesis of AMDS.Chapter 4: 47-56

[7]J.R. Anthony, L. C. Anthony, F. Nowroozi, G. Kwon, J.D. Newman, J.D. Keasling. Optimization of themevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene. Metabolic Engineering. 11: 13-19, 2009

Engineering a mevalonate pathway in Escherichia coli for production of terpenoids

2015-01-19T23:50:29Z

Ana90:

'''Ana Kapraljević'''

[http://www2.lbl.gov/ttd/publications/2837pub1.pdf Engineering a mevalonate pathway in Escherichia coli for production of terpenoids]
Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling ;Nature biotehnology; July 2003; vol 21; pages 796-802

== INTRODUCTION ==

Plants can synthesise and accumulate a wide range of small molecules or natural products involved in psyhological an ecological process. With the knowledge we know about their biosyntesis we used them as commercial flavors, fragnance compunds, colorants and terapeutical drugs. Their therapeutic potential is used for production of anticancer or antimalarial drugs, such as artemisnin. The most numerous and diverse class of natural products used in production for antimalarial drug are isoprenoids or more corectly their subclass, terpenoids. Malaria represent a major health problem in tropic and subtropic. Majority of death occur in children living in Africa. But the problem is that terpenoids are in plants produced in very small quantites and consequently not very economically feasible. For that reason the drugs for treatin malaria, artemisnin, is in short supply and is still unaffordable for most people living in malaria-endanger countries. Scientists are now focusing on alternative and cheaper way for terpenoid synthesis that can be enchanched and became economically acceptable. The alternative way is the expression of synthetic amorphadiene, precursor to artemisnin, and mevalonate isoprenoid way from yeas Saccharomyces cerevisiae in bacteria. In this paper I will summerize the project made in the production of amorphadiene by biotehnology and synthetic biology approach [1,2].

=== Artemisnin ===

Artemisnin is a substance that has antimalarial potential. Malaria is a infectious disease caused by parasitic protozan of the Plasmodium species. In the certain parts of the world malaria takes lives of more people worldwide than any other infecious disease, so it is a global problem. Death to malaria has been nowdays reduced thanks to the use of artemisnin-based combination therapy. Artemisnin is a natural compoud from plant ''Artemisia annua''. Chemically it is a sesquiterpene lactone, amorphadiene. The total sythesis of artemisnin is difficult and not very economical,so the new aproach is to put the hopes in sythetic biology and develop the new way, synthetic way, for its production [1,3].

=== Biology of terpenoids ===

For initiation of any synthetic biology process in metabolic engineering in that matter it is important to have some basic understanding of biosynthesis and regulation of terpenoids. Terpenoids, a class of isoprenoids, are natural products often sythesized by plants. They represent a large and diverse class and have many functions as primarly metabolites involved in growth and development of plants, or as secundary metabolites that optimize the interaction between the plant and the environment. Eventhought they are structural diverse, the share a common biosynthetic origin and follow similar synthesis tracks. All terpenoids are derived from repetitive fusion of isoprene units (C5 H8) [3]. The number of isoprene units determines their classification. For isoprenoid synthesis plants use mevalonate-dependent (MEV) pathway and mevalonte-independent pathway or alternative deoxyxylulose 5-phosphate pathway (DXP). In higher plants the biosynthesis begins with the generation of isopentenyl pyrophosphate (IPP). IPP is derived via the classic mevalonic acid pathway from acetly-coenzime A (acetyl-CoA). The IPP is isomerized to its allylic isomer dimethylallyl pyrophosphate (DMAPP) [3]. The continous condensation of IPP and DMAPP units leads to the formaton of prenylated pyrophosphates, the immediate precursors of the different terpenoid classes. So IPP and DMAPP are combined to form larger isoprenoids, such as farnesyl diphosphate (FPP). Condensation of IPP and DMAPP than units leads to the formation of precursors of the different terpenoid classes. Shematic production of terpenoids in ilustraded in this picture. Similary to plants, yeast Sacharomyces cerevisae uses the clasical mevalonate pathway for the synthesis of isoprenoids[3]. Prokariontes mostly use the DXP pathway for production IPP and DMAPP. This is an alternative pathway for IPP synthesis. This pathway uses Escherichia coli and some other bacteria for their terpenoid synthesis. IPP and DMAPP precurosr are essential to E. Coli for prenylation (addition of hydrophobic molecules to a protein or chemical compound) of tRNA and the synthesis of farnesyl pyrophosphate (FPP). FPP is then used for quinone (class of organic compounds) and cell wall synthesis. The DPX pathway begins with the glycolytic intermediates glyceraldehyde-3-phosphate (G3P) and pyruvate and continues through enzymatic steps for production of IPP and DMAPP. Therefore IPP and DMAPP are the end product in both way,MEV and DPX pathway [1,3].

== METABOLIC ENGINEERING FOR PRODUCTION OD TERPENOIDS ==

Metabolic engineering represents an improvement of production, construction, or cellular properties through the modification of specific biochemical reactions. It is described as the use of genetic engineering to reshape or modify the metabolism of organism. The aim is to optimize genetic and regulatory processes in cells to increase the production of a certain substance that we find in nature. It is based on optimisation of existing biochemical pathways or the introduction of natural pathway components in bacteria, yeast or plants. The main goal is the production of high-yield of specific metabolites that we can use in medicine or biotechnology. With progress in synthetic biology we can design a biologic funcion that do not exist in nature. Consequently, we can create and construct new biological components or redesign exisitng biological systems. In plants, most terpenoids are produced in a very small quantities in their natural sources. Extraction of this compouds from plants is expensive and therfore not economicaly accetable. So there is a big disagreement between the insistence for supply of terpenoids and therefore that leads to delay for their widespread application. With the understanding of terpenoid synthesis and progress in synthetic biology we are now able of redesign and modify the isoprenoid pathway to increase the terpenoid productivity [2,4]. ([http://www.disketa.si/media/uploads/2015-01/cd201ade6a167488767dfb1b118587bc.jpg See image here])

=== Microbal host ===

To elimante the need for plant extraction it is practical to produce terpenoid compouds at high yield in microbal host by introducing a heterlogous isoprenoid pathway into bacteria E.coli. Microorganisms, such as bacteria are attractive alternatives as hosts because they have rapid doubling time, they can achieve robussnes under process conditions and because of the simplicity of product putrification. And the costs are significantly lower than working with plants. With the engineering the DXP pathway we can increase the supply of isoprenoid precurosor needed for high level productio of isoprenoids in E.coli. [1,3]

=== Operons ===

Operon is a a funcional unit of DNA that contains a cluster of genes under the control of a single promotor. The synthesis of natural or synthetic products involves the introduction of a large number of genes. In this case these genes were encoding the enzymes needed for metabolic pathway. So it is useful to put certain genes under the control of a single promotor. For that reason Plac promotor was used in this study [1,5].

== EXPERIMENT ==

=== Synthase gene asemby and amorphadiene production ===

For high level production and changing the difficulties in expressing teprene synthases they first synthesized and expressed a codon-optimized variants of ADS . ADS is a gene encoding amorphadiene synthase and is sequentially oxidised by citorchromeP450 CYP71AV1 and reduced by artemisinic aldehyde reductase (DBR2). Amorphadyene synthase is an enzyme and catalyse the first step in the anitmalarial drug artemisnin synthesis. It has an inportant role in catalysing the reaction of FPP to amorphadiene. Beacause of its importaint step towards artemisnin biosynthesis it was designed and improved for the purpuse to acheve high-level expression in E.coli. For ADS gene synthesis they used two step assemly and one step amplification PCR.(1) Assemby PCR is a useful technique for production novel gene sequences. With this method we can put together short fragments of DNA and it is also a good choice for gene construction. Reserches in this article analysed the sequence of three ADS genes from independet clones and identified mutations in each of the genes. Than, two site directed mutagenesis reaction was used for assembly and generation a functional ADS gene from two clones. Than they measure the amorphadiene production by the amorphadiene synthase in E.coli DH10B with natural (nonengineered DPX pathway) and in E.coli with engineered DPX pathway. E. coli with engineerd pathway was harboring the pSOE4 plasmid (DXP pathway operon with dxs,IppHp and ispA gene). The result sugested that FPP synthesis limited amorphadiene production in this engineered host [1,6]. ([http://www.disketa.si/media/uploads/2015-01/0bddf07f946e6f5be0638090dee56490.jpg See image here])

=== Egineering the mevalonate-dependet pathway in E.coli ===

The aim was to increase the intracellular concentration of FPP substrate. Genes encoding the MEV pathway from S.cerevisae was assembled into operons and expressed in E.coli. Because of the complexity of engineering numerous gene biosynthetic pathway, genes were divided into to operons, top and bottom.Top operon, MevT, consisted of three enzymes: acetoacetyl-CoA thiolase (atoB), HMG-CoA synthases (HMGS) and HMG-CoA reductase (tHMGR). This operon was responsible for transformation of acetyl-coA to mevalonate. Thre different bottom operons pMevB (ERG12, ERG8,MVD1 ), pMBI (ERG12, ERG8, MVD1, idi) and pMBIS (ERG12, ERG8, MVD1, idi, ispA) transformed mevalonate to IPP, DMAPP and consequently to FPP. The next step was to analyse and test the funcionality of this pathway. For this purpuse E.coli strand DYM1 with incoplemplete isoprenoid synthesis was transformed with plasmid expressing three bottom operon constuct- pMevB, pMBI and pMBI. E.coli strand DYM1 had a deletion in the ispC gene for encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase, and was not able to synthesise 2-C-methyl-D-erythritol 4-phosphate(MEP), which is important intermediate in isoprenoid biosynthesis[1]. Because of this deletion the synthesis of precursors IPP and DMAP is blocked. So then they tested what is going to happen if they grew the strain DYM1 in the presence of 2-C-methyl-D-ertihritol and in medium with mevalonate without methylerythritol since DYM1 strain was not capable to synthesisie MEP. As expected , all strains expressing operon from S.cerevisiae grew on the plates in the presence of 2-c-methyl-D-erythritol. But only strains with pMBI or pMBIS grew on plates with 1mM mevalonate in the absence of methylerythol. This was the conformation that synthetic MBI and MBIS operons were functional, because they were capable to support the growth of E.coli without ispC gene by supplying cells with IPP and DMAPP. Next they completed the mevalonate pathway by transforming the pMevT plasmid (expressing three genes mentioned before-atoB, HMGS and tHMGR) with pMBI or pMBIS. This coexpresion of two operons, pMevT and pMBI /pMBIS ,completed the mevlonate pathway and allowed the synthesis of sesquiterpene precursors from acetyl-CoA, IPP and DMAPP. Therefore, coexpression complemented the ispC deletion even in the absence of mevalonate, what was the proof, that MevT operon was functional [1,7].

=== Amorpadiene synthesis from mevalonate ===

The aim was to achieve high-level production of amorphadiene and to find out if the amount of FPP was limiting amoprhadiene output. As mentioned, E.coli was used as heterologous microorganism in which coupled mevalonate pathway with amorphadiene synthesis. Cells with ADS gene were coexpressed with MBIS operon and grown in medium with increasing amounts (0 to 40 mM) of exogenus mevalonate. The culture extract was analysed with gas chromatography-mass spectrometry, analytical method for identification different substances in a test sample. It combines the features of mass spectrometry and gas-liquid chromatography. Results showed that MBIS operon did not limit the amorphadiene production at the highest mevalonate concentration(40mM). ([http://www.disketa.si/media/uploads/2015-01/7711d592584a53282f2ecb0fd678ef40.jpg See image here]) Cultures with 40 mM mevalonate added produced much more amoprhadiene. With time, amorphadiene concentration was dropping beacause of the loss of the volatile terpene[1]. By adding more than 10 mM mevalonate in control cultures without ADS, the severe growth ihibition has been reported. ([http://www.disketa.si/media/uploads/2015-01/2a21f55f0fea31ffcbe89c866c9150f7.jpg See image here]) To discover the cause ofthis inhibition, the growth of E.coli DH10B from strains with empty, pMKPMK, pMevB, pMBI or pMBIS plasmid was measured in media with increasing concentrations of mevalonate. The addition of 5mM mevalonate to the medium inhibited the growth of cells with pMevB. But 5mM concentrations of mevalonate did not alter the growth of cells with pMKPMK, PMBI or pMBIS plasmid. This indicated that the accomulation of IPP is toxic and inhibits normal cell growth. The accmulation mostly occurs in cells with high flux through mevalonate pathway. The next step was comparison of intracellular prenyl pyrophosphate (FPP) in strains. This was done by feeding the cells harboring the different mevalonte operon construct with radiolabeled mevalonate. Than the labeled metabolites were tracked. For this experiment cell suspensions of E.coli harboring pMevB+pTrc99A, pMBI+ pTrc99A, pMBIS+ pTrc99A or pMBIS+pADS were used. pTRc99A is the bacterial expression vector with inducible to IPTG lacI promoter. The result showed that strains with MevB operon accumulated IPP but not FPP, when MBI and MBIS strains accumulated FPP. ([http://www.disketa.si/media/uploads/2015-01/31ad7a215f016dc26aceacf92eb46712.jpg See image here]) Because of the simultaneous expression of the amorphadiene synthase the waste of FPP that accumulated in the MBIS host was consumed. This was shown by a decrease in intracellular FPP. Cells expresing MBIS acumulated FPP and showed growth inhibition in the presence of 10 mM mevalonate. The prediction was that the coexpression of ADS would ease the growth inhibition by forwarding the prenyl pyrophosphate intermediates to volatile terpene olefin. For this test, the cell with pMBIS and empty expression vector pTrc99A (without ADS)and cells with pMBIS and pADS vectors were used. The medium was supplemented with mevalonate concentations ( 0mM, 5mM, 10mM, 20mM or 40mM). Two hours after addition of mevalonate and inducer IPTG, the growth inhibition was observed. ([http://www.disketa.si/media/uploads/2015-01/94b5f3d5636fe767316f770f3e2b0448.jpg See image here]) The result showed that the strains with pMBIS +ADS started to grow, but strain lacking the ADS still showed growth inhbition at higher concentrations of mevalonate. This supported the conclusion that converion of FPP to amophadiene has an important role in minimising growth inhibition. Briefly these result showed that engineered mevalonate patway produces high levels of prenyl pyrophosphate precursors. If the IPP isomerase, FPP synthase and terpen synthase is absent, IPP can acumulate and be toxic to the cells. The toxic levels of intracellular prenyl pyrphosphate can accumulate [1].

=== Amorphadiene synthesis from acetyl-Coa ===

The main goal was to achieve high amoprhaidene production from a simple and inexpensive carbon source. E.coli strains harboring pMevT,pMBIS and pADS plasmids were transformed with pMevT plasmid. Subsequently the strains were tested for ability to produce amoprhadiene in the absence of exogenous mevalonate. The comparison between E.coli expressing the native DPX pathway and engineered pathway as made. They tested native DXP pathway, engineered DPX pathway, mevalonate bottom pathway in the absence of mevalonate, mevlonate bottom pathway in medium suplemented with mevalonate, the complete mevalonate pathway and the complete mevalonate pathway supplemented with 0,8% glycerol. Glycerol was addded to investigate the effect of amorphadiene production of supplying as single carbon source and for prolongation of amorphadiene production. Breifly the resulut showed that expression of the mevalonate-dependent isoprenoid biosynthetic pathway delivers hight levels of isoprenoid precursor for the production of sesquiterpene [1]. ([http://www.disketa.si/media/uploads/2015-01/a31fe07a7c66854b1dc3ff36f8ed01ef.jpg See image here])

== DISSCUSION ==
The identification of genes encoding the enzymes involved in the artemisinin-biosynthesis pathway together with advances in metabolic engineering provides new options for engineering artemisinin biosynthesis. Heterologous production of artemisnin in microorganism such as E.coli. is an alternative way for production of artmeinsin in high yields. But eventhougt micoorganisms has shown great potential in that matter, optimisation of these heterlogous pathways still require maximal titer. Inequal gene expression can lead to accumulation of toxic metabolic intermediates and to over or under production of enzymes involved in pathway. (4). Conclusion based on these study shows that poor expression of the amoprhaidene synthase genes (ADS) limits high terpene olfein yields from the host. To achieve better results , in this study they chose to synthesiyse an amophaiene synthase gene (ADS). The comparison between E.coli expressing native sesquiterpene synthase genes and E.coli expressing the synthetic ADS gene showed big improvement in terpene synthesis due to synthetic ADS gene. The native DPX pathway showed poor supply of the prenyl pyrophosphate precuror. And that limited terpenoid -carotenoid yields in E.coli. With the metabolic engineering of the DPX pathway the flux of carotenoid accumulation showed signifcant increase. By using the mevalonate-dependent isoprenoid pathway from yeast S. cerevisae and engineering that pathway in bacteria E.coli, we avoid the native regulatory elements found in yeast while bypassing those of E.coli DXP pathway. In this study results showed growth inhibition in cells without ADS due to the vast excess of preny pyrophosphate. But the coexpression of a synthetic sesquiterpene synthase can contribute to reducing the excess pool of precursors and eliminate growth inhibition. In this study total biosynthesis of artemisnin was not completely achieved. But these results can help in further investigation for poduction of artemisnic acid. This acid can then be putrified and chemically converted into artemsinin. [1,7].

== CONCLUSIONS ==
To sum things up, artmeinsin, a sesquiterpene isolated from Artemisia annua,represent a novel drug for malaria treatment. The present artemisnin derivates are too expensive for general use and plants can not produce artmeinsin in big quantities. This fact inspired scientist to focus on developing the way for production of artemisnin in high quantities and to avoid big costs. The promising tool in alternative source of artmeinsin is the use of microoganisms. The aim is to transfer the genes encoding enzymes involved in biosynthetic pathway for artemisnin into a hight producing host organism. Several study have been reporting on engineering the metabolic pathway in S.cerevisae to produce the precursor for artmeisnic acid. With the effort in synthetic biology they used modified mevalonate pathway . With the help of syntehtic bilogy the yeast cells were engineerd to express ezymes needed for production of artmemisnic acid. But in this paper the main focus was on engineering a mevalonate pathway in bacteria E.coli. By engineering a new metabolic pathway in E.coli we can easily synthesise a precursor nedeed for artemisnin production. The goal is to produce drugs in better and cheape ways. But, why focus on artemisnin? Well, acording on recent studies artemisnin shows to be very sucesfull for the treatmen all strain of malaria. More than a milion people affected with malaria have already been cured by arteminsin. As already mentioned , because of very hight costs and limited production of arteminin in plants most populations in Africa can not afford it. Progress made in synthetic biology and ability to produce amorphadiene in microorganisms in big quantites opens a new possibility to produce low cost artemisnin and contributes to the brighter future od antimalaria drug deveopment, and therefore saving milions from suffering from malaria. [1,2, 3].

[1]Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nature biotehnology; vol 21; pages 796-802, July 2003

[2] T. Mosses, J. Pollier, J. M. Thevelein, A. Goossens. Bioengineering of plant (tri)terpenoids: from metabolic engineering of plants to synthetic biology in vivo and in vitro. New Pathyologist. 200: 27-43, 2013

[3] M.Farhi, M. Kozin, S. Duchin, A. Vainstein. Metabolic engineering of plants for artemisinin synthesis. Biotehnology and genetic engineering rewiews.Vol.29,No.2: 135-148, 2013

[4] Synthetic biology ; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Synthetic_biology http://en.wikipedia.org/wiki/Synthetic_biology]

[5]Operon; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Operon http://en.wikipedia.org/wiki/Operon]

[6] M. K. Julsing, G. van Pouderoyen, J. van der Veen,H. J. Woerdenbag, O. Kayser, W. J. Quax. Amorphadiene synthase: probing the active site model with directed mutagenesis. Directed mutagenesis of AMDS.Chapter 4: 47-56

[7]J.R. Anthony, L. C. Anthony, F. Nowroozi, G. Kwon, J.D. Newman, J.D. Keasling. Optimization of themevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene. Metabolic Engineering. 11: 13-19, 2009

Engineering a mevalonate pathway in Escherichia coli for production of terpenoids

2015-01-19T23:49:39Z

Ana90:

'''Ana Kapraljević'''

[http://www2.lbl.gov/ttd/publications/2837pub1.pdf Engineering a mevalonate pathway in Escherichia coli for production of terpenoids]
Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling ;Nature biotehnology; July 2003; vol 21; pages 796-802

== INTRODUCTION ==

Plants can synthesise and accumulate a wide range of small molecules or natural products involved in psyhological an ecological process. With the knowledge we know about their biosyntesis we used them as commercial flavors, fragnance compunds, colorants and terapeutical drugs. Their therapeutic potential is used for production of anticancer or antimalarial drugs, such as artemisnin. The most numerous and diverse class of natural products used in production for antimalarial drug are isoprenoids or more corectly their subclass, terpenoids. Malaria represent a major health problem in tropic and subtropic. Majority of death occur in children living in Africa. But the problem is that terpenoids are in plants produced in very small quantites and consequently not very economically feasible. For that reason the drugs for treatin malaria, artemisnin, is in short supply and is still unaffordable for most people living in malaria-endanger countries. Scientists are now focusing on alternative and cheaper way for terpenoid synthesis that can be enchanched and became economically acceptable. The alternative way is the expression of synthetic amorphadiene, precursor to artemisnin, and mevalonate isoprenoid way from yeas Saccharomyces cerevisiae in bacteria. In this paper I will summerize the project made in the production of amorphadiene by biotehnology and synthetic biology approach [1,2].

=== Artemisnin ===

Artemisnin is a substance that has antimalarial potential. Malaria is a infectious disease caused by parasitic protozan of the Plasmodium species. In the certain parts of the world malaria takes lives of more people worldwide than any other infecious disease, so it is a global problem. Death to malaria has been nowdays reduced thanks to the use of artemisnin-based combination therapy. Artemisnin is a natural compoud from plant ''Artemisia annua''. Chemically it is a sesquiterpene lactone, amorphadiene. The total sythesis of artemisnin is difficult and not very economical,so the new aproach is to put the hopes in sythetic biology and develop the new way, synthetic way, for its production [1,3].

=== Biology of terpenoids ===

For initiation of any synthetic biology process in metabolic engineering in that matter it is important to have some basic understanding of biosynthesis and regulation of terpenoids. Terpenoids, a class of isoprenoids, are natural products often sythesized by plants. They represent a large and diverse class and have many functions as primarly metabolites involved in growth and development of plants, or as secundary metabolites that optimize the interaction between the plant and the environment. Eventhought they are structural diverse, the share a common biosynthetic origin and follow similar synthesis tracks. All terpenoids are derived from repetitive fusion of isoprene units (C5 H8) [3]. The number of isoprene units determines their classification. For isoprenoid synthesis plants use mevalonate-dependent (MEV) pathway and mevalonte-independent pathway or alternative deoxyxylulose 5-phosphate pathway (DXP). In higher plants the biosynthesis begins with the generation of isopentenyl pyrophosphate (IPP). IPP is derived via the classic mevalonic acid pathway from acetly-coenzime A (acetyl-CoA). The IPP is isomerized to its allylic isomer dimethylallyl pyrophosphate (DMAPP) [3]. The continous condensation of IPP and DMAPP units leads to the formaton of prenylated pyrophosphates, the immediate precursors of the different terpenoid classes. So IPP and DMAPP are combined to form larger isoprenoids, such as farnesyl diphosphate (FPP). Condensation of IPP and DMAPP than units leads to the formation of precursors of the different terpenoid classes. Shematic production of terpenoids in ilustraded in this picture. Similary to plants, yeast Sacharomyces cerevisae uses the clasical mevalonate pathway for the synthesis of isoprenoids[3]. Prokariontes mostly use the DXP pathway for production IPP and DMAPP. This is an alternative pathway for IPP synthesis. This pathway uses Escherichia coli and some other bacteria for their terpenoid synthesis. IPP and DMAPP precurosr are essential to E. Coli for prenylation (addition of hydrophobic molecules to a protein or chemical compound) of tRNA and the synthesis of farnesyl pyrophosphate (FPP). FPP is then used for quinone (class of organic compounds) and cell wall synthesis. The DPX pathway begins with the glycolytic intermediates glyceraldehyde-3-phosphate (G3P) and pyruvate and continues through enzymatic steps for production of IPP and DMAPP. Therefore IPP and DMAPP are the end product in both way,MEV and DPX pathway [1,3].

== METABOLIC ENGINEERING FOR PRODUCTION OD TERPENOIDS ==

Metabolic engineering represents an improvement of production, construction, or cellular properties through the modification of specific biochemical reactions. It is described as the use of genetic engineering to reshape or modify the metabolism of organism. The aim is to optimize genetic and regulatory processes in cells to increase the production of a certain substance that we find in nature. It is based on optimisation of existing biochemical pathways or the introduction of natural pathway components in bacteria, yeast or plants. The main goal is the production of high-yield of specific metabolites that we can use in medicine or biotechnology. With progress in synthetic biology we can design a biologic funcion that do not exist in nature. Consequently, we can create and construct new biological components or redesign exisitng biological systems. In plants, most terpenoids are produced in a very small quantities in their natural sources. Extraction of this compouds from plants is expensive and therfore not economicaly accetable. So there is a big disagreement between the insistence for supply of terpenoids and therefore that leads to delay for their widespread application. With the understanding of terpenoid synthesis and progress in synthetic biology we are now able of redesign and modify the isoprenoid pathway to increase the terpenoid productivity [2,4]. ([http://www.disketa.si/media/uploads/2015-01/cd201ade6a167488767dfb1b118587bc.jpg See image here])

=== Microbal host ===

To elimante the need for plant extraction it is practical to produce terpenoid compouds at high yield in microbal host by introducing a heterlogous isoprenoid pathway into bacteria E.coli. Microorganisms, such as bacteria are attractive alternatives as hosts because they have rapid doubling time, they can achieve robussnes under process conditions and because of the simplicity of product putrification. And the costs are significantly lower than working with plants. With the engineering the DXP pathway we can increase the supply of isoprenoid precurosor needed for high level productio of isoprenoids in E.coli. [1,3]

=== Operons ===

Operon is a a funcional unit of DNA that contains a cluster of genes under the control of a single promotor. The synthesis of natural or synthetic products involves the introduction of a large number of genes. In this case these genes were encoding the enzymes needed for metabolic pathway. So it is useful to put certain genes under the control of a single promotor. For that reason Plac promotor was used in this study [1,5].

== EXPERIMENT ==

=== Synthase gene asemby and amorphadiene production ===

For high level production and changing the difficulties in expressing teprene synthases they first synthesized and expressed a codon-optimized variants of ADS . ADS is a gene encoding amorphadiene synthase and is sequentially oxidised by citorchromeP450 CYP71AV1 and reduced by artemisinic aldehyde reductase (DBR2). Amorphadyene synthase is an enzyme and catalyse the first step in the anitmalarial drug artemisnin synthesis. It has an inportant role in catalysing the reaction of FPP to amorphadiene. Beacause of its importaint step towards artemisnin biosynthesis it was designed and improved for the purpuse to acheve high-level expression in E.coli. For ADS gene synthesis they used two step assemly and one step amplification PCR.(1) Assemby PCR is a useful technique for production novel gene sequences. With this method we can put together short fragments of DNA and it is also a good choice for gene construction. Reserches in this article analysed the sequence of three ADS genes from independet clones and identified mutations in each of the genes. Than, two site directed mutagenesis reaction was used for assembly and generation a functional ADS gene from two clones. Than they measure the amorphadiene production by the amorphadiene synthase in E.coli DH10B with natural (nonengineered DPX pathway) and in E.coli with engineered DPX pathway. E. coli with engineerd pathway was harboring the pSOE4 plasmid (DXP pathway operon with dxs,IppHp and ispA gene). The result sugested that FPP synthesis limited amorphadiene production in this engineered host [1,6]. ([http://www.disketa.si/media/uploads/2015-01/0bddf07f946e6f5be0638090dee56490.jpg See image here])

=== Egineering the mevalonate-dependet pathway in E.coli ===

The aim was to increase the intracellular concentration of FPP substrate. Genes encoding the MEV pathway from S.cerevisae was assembled into operons and expressed in E.coli. Because of the complexity of engineering numerous gene biosynthetic pathway, genes were divided into to operons, top and bottom.Top operon, MevT, consisted of three enzymes: acetoacetyl-CoA thiolase (atoB), HMG-CoA synthases (HMGS) and HMG-CoA reductase (tHMGR). This operon was responsible for transformation of acetyl-coA to mevalonate. Thre different bottom operons pMevB (ERG12, ERG8,MVD1 ), pMBI (ERG12, ERG8, MVD1, idi) and pMBIS (ERG12, ERG8, MVD1, idi, ispA) transformed mevalonate to IPP, DMAPP and consequently to FPP. The next step was to analyse and test the funcionality of this pathway. For this purpuse E.coli strand DYM1 with incoplemplete isoprenoid synthesis was transformed with plasmid expressing three bottom operon constuct- pMevB, pMBI and pMBI. E.coli strand DYM1 had a deletion in the ispC gene for encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase, and was not able to synthesise 2-C-methyl-D-erythritol 4-phosphate(MEP), which is important intermediate in isoprenoid biosynthesis[1]. Because of this deletion the synthesis of precursors IPP and DMAP is blocked. So then they tested what is going to happen if they grew the strain DYM1 in the presence of 2-C-methyl-D-ertihritol and in medium with mevalonate without methylerythritol since DYM1 strain was not capable to synthesisie MEP. As expected , all strains expressing operon from S.cerevisiae grew on the plates in the presence of 2-c-methyl-D-erythritol. But only strains with pMBI or pMBIS grew on plates with 1mM mevalonate in the absence of methylerythol. This was the conformation that synthetic MBI and MBIS operons were functional, because they were capable to support the growth of E.coli without ispC gene by supplying cells with IPP and DMAPP. Next they completed the mevalonate pathway by transforming the pMevT plasmid (expressing three genes mentioned before-atoB, HMGS and tHMGR) with pMBI or pMBIS. This coexpresion of two operons, pMevT and pMBI /pMBIS ,completed the mevlonate pathway and allowed the synthesis of sesquiterpene precursors from acetyl-CoA, IPP and DMAPP. Therefore, coexpression complemented the ispC deletion even in the absence of mevalonate, what was the proof, that MevT operon was functional [1,7].

=== Amorpadiene synthesis from mevalonate ===

The aim was to achieve high-level production of amorphadiene and to find out if the amount of FPP was limiting amoprhadiene output. As mentioned, E.coli was used as heterologous microorganism in which coupled mevalonate pathway with amorphadiene synthesis. Cells with ADS gene were coexpressed with MBIS operon and grown in medium with increasing amounts (0 to 40 mM) of exogenus mevalonate. The culture extract was analysed with gas chromatography-mass spectrometry, analytical method for identification different substances in a test sample. It combines the features of mass spectrometry and gas-liquid chromatography. Results showed that MBIS operon did not limit the amorphadiene production at the highest mevalonate concentration(40mM). ([http://www.disketa.si/media/uploads/2015-01/7711d592584a53282f2ecb0fd678ef40.jpg See image 1 here]) Cultures with 40 mM mevalonate added produced much more amoprhadiene. With time, amorphadiene concentration was dropping beacause of the loss of the volatile terpene[1]. By adding more than 10 mM mevalonate in control cultures without ADS, the severe growth ihibition has been reported. ([http://www.disketa.si/media/uploads/2015-01/2a21f55f0fea31ffcbe89c866c9150f7.jpg See image here]) To discover the cause ofthis inhibition, the growth of E.coli DH10B from strains with empty, pMKPMK, pMevB, pMBI or pMBIS plasmid was measured in media with increasing concentrations of mevalonate. The addition of 5mM mevalonate to the medium inhibited the growth of cells with pMevB. But 5mM concentrations of mevalonate did not alter the growth of cells with pMKPMK, PMBI or pMBIS plasmid. This indicated that the accomulation of IPP is toxic and inhibits normal cell growth. The accmulation mostly occurs in cells with high flux through mevalonate pathway. The next step was comparison of intracellular prenyl pyrophosphate (FPP) in strains. This was done by feeding the cells harboring the different mevalonte operon construct with radiolabeled mevalonate. Than the labeled metabolites were tracked. For this experiment cell suspensions of E.coli harboring pMevB+pTrc99A, pMBI+ pTrc99A, pMBIS+ pTrc99A or pMBIS+pADS were used. pTRc99A is the bacterial expression vector with inducible to IPTG lacI promoter. The result showed that strains with MevB operon accumulated IPP but not FPP, when MBI and MBIS strains accumulated FPP. ([http://www.disketa.si/media/uploads/2015-01/31ad7a215f016dc26aceacf92eb46712.jpg See image here]) Because of the simultaneous expression of the amorphadiene synthase the waste of FPP that accumulated in the MBIS host was consumed. This was shown by a decrease in intracellular FPP. Cells expresing MBIS acumulated FPP and showed growth inhibition in the presence of 10 mM mevalonate. The prediction was that the coexpression of ADS would ease the growth inhibition by forwarding the prenyl pyrophosphate intermediates to volatile terpene olefin. For this test, the cell with pMBIS and empty expression vector pTrc99A (without ADS)and cells with pMBIS and pADS vectors were used. The medium was supplemented with mevalonate concentations ( 0mM, 5mM, 10mM, 20mM or 40mM). Two hours after addition of mevalonate and inducer IPTG, the growth inhibition was observed. ([http://www.disketa.si/media/uploads/2015-01/94b5f3d5636fe767316f770f3e2b0448.jpg See image here]) The result showed that the strains with pMBIS +ADS started to grow, but strain lacking the ADS still showed growth inhbition at higher concentrations of mevalonate. This supported the conclusion that converion of FPP to amophadiene has an important role in minimising growth inhibition. Briefly these result showed that engineered mevalonate patway produces high levels of prenyl pyrophosphate precursors. If the IPP isomerase, FPP synthase and terpen synthase is absent, IPP can acumulate and be toxic to the cells. The toxic levels of intracellular prenyl pyrphosphate can accumulate [1].

=== Amorphadiene synthesis from acetyl-Coa ===

The main goal was to achieve high amoprhaidene production from a simple and inexpensive carbon source. E.coli strains harboring pMevT,pMBIS and pADS plasmids were transformed with pMevT plasmid. Subsequently the strains were tested for ability to produce amoprhadiene in the absence of exogenous mevalonate. The comparison between E.coli expressing the native DPX pathway and engineered pathway as made. They tested native DXP pathway, engineered DPX pathway, mevalonate bottom pathway in the absence of mevalonate, mevlonate bottom pathway in medium suplemented with mevalonate, the complete mevalonate pathway and the complete mevalonate pathway supplemented with 0,8% glycerol. Glycerol was addded to investigate the effect of amorphadiene production of supplying as single carbon source and for prolongation of amorphadiene production. Breifly the resulut showed that expression of the mevalonate-dependent isoprenoid biosynthetic pathway delivers hight levels of isoprenoid precursor for the production of sesquiterpene [1]. ([http://www.disketa.si/media/uploads/2015-01/a31fe07a7c66854b1dc3ff36f8ed01ef.jpg See image here])

== DISSCUSION ==
The identification of genes encoding the enzymes involved in the artemisinin-biosynthesis pathway together with advances in metabolic engineering provides new options for engineering artemisinin biosynthesis. Heterologous production of artemisnin in microorganism such as E.coli. is an alternative way for production of artmeinsin in high yields. But eventhougt micoorganisms has shown great potential in that matter, optimisation of these heterlogous pathways still require maximal titer. Inequal gene expression can lead to accumulation of toxic metabolic intermediates and to over or under production of enzymes involved in pathway. (4). Conclusion based on these study shows that poor expression of the amoprhaidene synthase genes (ADS) limits high terpene olfein yields from the host. To achieve better results , in this study they chose to synthesiyse an amophaiene synthase gene (ADS). The comparison between E.coli expressing native sesquiterpene synthase genes and E.coli expressing the synthetic ADS gene showed big improvement in terpene synthesis due to synthetic ADS gene. The native DPX pathway showed poor supply of the prenyl pyrophosphate precuror. And that limited terpenoid -carotenoid yields in E.coli. With the metabolic engineering of the DPX pathway the flux of carotenoid accumulation showed signifcant increase. By using the mevalonate-dependent isoprenoid pathway from yeast S. cerevisae and engineering that pathway in bacteria E.coli, we avoid the native regulatory elements found in yeast while bypassing those of E.coli DXP pathway. In this study results showed growth inhibition in cells without ADS due to the vast excess of preny pyrophosphate. But the coexpression of a synthetic sesquiterpene synthase can contribute to reducing the excess pool of precursors and eliminate growth inhibition. In this study total biosynthesis of artemisnin was not completely achieved. But these results can help in further investigation for poduction of artemisnic acid. This acid can then be putrified and chemically converted into artemsinin. [1,7].

== CONCLUSIONS ==
To sum things up, artmeinsin, a sesquiterpene isolated from Artemisia annua,represent a novel drug for malaria treatment. The present artemisnin derivates are too expensive for general use and plants can not produce artmeinsin in big quantities. This fact inspired scientist to focus on developing the way for production of artemisnin in high quantities and to avoid big costs. The promising tool in alternative source of artmeinsin is the use of microoganisms. The aim is to transfer the genes encoding enzymes involved in biosynthetic pathway for artemisnin into a hight producing host organism. Several study have been reporting on engineering the metabolic pathway in S.cerevisae to produce the precursor for artmeisnic acid. With the effort in synthetic biology they used modified mevalonate pathway . With the help of syntehtic bilogy the yeast cells were engineerd to express ezymes needed for production of artmemisnic acid. But in this paper the main focus was on engineering a mevalonate pathway in bacteria E.coli. By engineering a new metabolic pathway in E.coli we can easily synthesise a precursor nedeed for artemisnin production. The goal is to produce drugs in better and cheape ways. But, why focus on artemisnin? Well, acording on recent studies artemisnin shows to be very sucesfull for the treatmen all strain of malaria. More than a milion people affected with malaria have already been cured by arteminsin. As already mentioned , because of very hight costs and limited production of arteminin in plants most populations in Africa can not afford it. Progress made in synthetic biology and ability to produce amorphadiene in microorganisms in big quantites opens a new possibility to produce low cost artemisnin and contributes to the brighter future od antimalaria drug deveopment, and therefore saving milions from suffering from malaria. [1,2, 3].

[1]Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nature biotehnology; vol 21; pages 796-802, July 2003

[2] T. Mosses, J. Pollier, J. M. Thevelein, A. Goossens. Bioengineering of plant (tri)terpenoids: from metabolic engineering of plants to synthetic biology in vivo and in vitro. New Pathyologist. 200: 27-43, 2013

[3] M.Farhi, M. Kozin, S. Duchin, A. Vainstein. Metabolic engineering of plants for artemisinin synthesis. Biotehnology and genetic engineering rewiews.Vol.29,No.2: 135-148, 2013

[4] Synthetic biology ; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Synthetic_biology http://en.wikipedia.org/wiki/Synthetic_biology]

[5]Operon; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Operon http://en.wikipedia.org/wiki/Operon]

[6] M. K. Julsing, G. van Pouderoyen, J. van der Veen,H. J. Woerdenbag, O. Kayser, W. J. Quax. Amorphadiene synthase: probing the active site model with directed mutagenesis. Directed mutagenesis of AMDS.Chapter 4: 47-56

[7]J.R. Anthony, L. C. Anthony, F. Nowroozi, G. Kwon, J.D. Newman, J.D. Keasling. Optimization of themevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene. Metabolic Engineering. 11: 13-19, 2009

Engineering a mevalonate pathway in Escherichia coli for production of terpenoids

2015-01-19T23:49:04Z

Ana90:

'''Ana Kapraljević'''

[http://www2.lbl.gov/ttd/publications/2837pub1.pdf Engineering a mevalonate pathway in Escherichia coli for production of terpenoids]
Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling ;Nature biotehnology; July 2003; vol 21; pages 796-802

== INTRODUCTION ==

Plants can synthesise and accumulate a wide range of small molecules or natural products involved in psyhological an ecological process. With the knowledge we know about their biosyntesis we used them as commercial flavors, fragnance compunds, colorants and terapeutical drugs. Their therapeutic potential is used for production of anticancer or antimalarial drugs, such as artemisnin. The most numerous and diverse class of natural products used in production for antimalarial drug are isoprenoids or more corectly their subclass, terpenoids. Malaria represent a major health problem in tropic and subtropic. Majority of death occur in children living in Africa. But the problem is that terpenoids are in plants produced in very small quantites and consequently not very economically feasible. For that reason the drugs for treatin malaria, artemisnin, is in short supply and is still unaffordable for most people living in malaria-endanger countries. Scientists are now focusing on alternative and cheaper way for terpenoid synthesis that can be enchanched and became economically acceptable. The alternative way is the expression of synthetic amorphadiene, precursor to artemisnin, and mevalonate isoprenoid way from yeas Saccharomyces cerevisiae in bacteria. In this paper I will summerize the project made in the production of amorphadiene by biotehnology and synthetic biology approach [1,2].

=== Artemisnin ===

Artemisnin is a substance that has antimalarial potential. Malaria is a infectious disease caused by parasitic protozan of the Plasmodium species. In the certain parts of the world malaria takes lives of more people worldwide than any other infecious disease, so it is a global problem. Death to malaria has been nowdays reduced thanks to the use of artemisnin-based combination therapy. Artemisnin is a natural compoud from plant ''Artemisia annua''. Chemically it is a sesquiterpene lactone, amorphadiene. The total sythesis of artemisnin is difficult and not very economical,so the new aproach is to put the hopes in sythetic biology and develop the new way, synthetic way, for its production [1,3].

=== Biology of terpenoids ===

For initiation of any synthetic biology process in metabolic engineering in that matter it is important to have some basic understanding of biosynthesis and regulation of terpenoids. Terpenoids, a class of isoprenoids, are natural products often sythesized by plants. They represent a large and diverse class and have many functions as primarly metabolites involved in growth and development of plants, or as secundary metabolites that optimize the interaction between the plant and the environment. Eventhought they are structural diverse, the share a common biosynthetic origin and follow similar synthesis tracks. All terpenoids are derived from repetitive fusion of isoprene units (C5 H8) [3]. The number of isoprene units determines their classification. For isoprenoid synthesis plants use mevalonate-dependent (MEV) pathway and mevalonte-independent pathway or alternative deoxyxylulose 5-phosphate pathway (DXP). In higher plants the biosynthesis begins with the generation of isopentenyl pyrophosphate (IPP). IPP is derived via the classic mevalonic acid pathway from acetly-coenzime A (acetyl-CoA). The IPP is isomerized to its allylic isomer dimethylallyl pyrophosphate (DMAPP) [3]. The continous condensation of IPP and DMAPP units leads to the formaton of prenylated pyrophosphates, the immediate precursors of the different terpenoid classes. So IPP and DMAPP are combined to form larger isoprenoids, such as farnesyl diphosphate (FPP). Condensation of IPP and DMAPP than units leads to the formation of precursors of the different terpenoid classes. Shematic production of terpenoids in ilustraded in this picture. Similary to plants, yeast Sacharomyces cerevisae uses the clasical mevalonate pathway for the synthesis of isoprenoids[3]. Prokariontes mostly use the DXP pathway for production IPP and DMAPP. This is an alternative pathway for IPP synthesis. This pathway uses Escherichia coli and some other bacteria for their terpenoid synthesis. IPP and DMAPP precurosr are essential to E. Coli for prenylation (addition of hydrophobic molecules to a protein or chemical compound) of tRNA and the synthesis of farnesyl pyrophosphate (FPP). FPP is then used for quinone (class of organic compounds) and cell wall synthesis. The DPX pathway begins with the glycolytic intermediates glyceraldehyde-3-phosphate (G3P) and pyruvate and continues through enzymatic steps for production of IPP and DMAPP. Therefore IPP and DMAPP are the end product in both way,MEV and DPX pathway [1,3].

== METABOLIC ENGINEERING FOR PRODUCTION OD TERPENOIDS ==

Metabolic engineering represents an improvement of production, construction, or cellular properties through the modification of specific biochemical reactions. It is described as the use of genetic engineering to reshape or modify the metabolism of organism. The aim is to optimize genetic and regulatory processes in cells to increase the production of a certain substance that we find in nature. It is based on optimisation of existing biochemical pathways or the introduction of natural pathway components in bacteria, yeast or plants. The main goal is the production of high-yield of specific metabolites that we can use in medicine or biotechnology. With progress in synthetic biology we can design a biologic funcion that do not exist in nature. Consequently, we can create and construct new biological components or redesign exisitng biological systems. In plants, most terpenoids are produced in a very small quantities in their natural sources. Extraction of this compouds from plants is expensive and therfore not economicaly accetable. So there is a big disagreement between the insistence for supply of terpenoids and therefore that leads to delay for their widespread application. With the understanding of terpenoid synthesis and progress in synthetic biology we are now able of redesign and modify the isoprenoid pathway to increase the terpenoid productivity [2,4]. ([http://www.disketa.si/media/uploads/2015-01/cd201ade6a167488767dfb1b118587bc.jpg See image here])

=== Microbal host ===

To elimante the need for plant extraction it is practical to produce terpenoid compouds at high yield in microbal host by introducing a heterlogous isoprenoid pathway into bacteria E.coli. Microorganisms, such as bacteria are attractive alternatives as hosts because they have rapid doubling time, they can achieve robussnes under process conditions and because of the simplicity of product putrification. And the costs are significantly lower than working with plants. With the engineering the DXP pathway we can increase the supply of isoprenoid precurosor needed for high level productio of isoprenoids in E.coli. [1,3]

=== Operons ===

Operon is a a funcional unit of DNA that contains a cluster of genes under the control of a single promotor. The synthesis of natural or synthetic products involves the introduction of a large number of genes. In this case these genes were encoding the enzymes needed for metabolic pathway. So it is useful to put certain genes under the control of a single promotor. For that reason Plac promotor was used in this study [1,5].

== EXPERIMENT ==

=== Synthase gene asemby and amorphadiene production ===

For high level production and changing the difficulties in expressing teprene synthases they first synthesized and expressed a codon-optimized variants of ADS . ADS is a gene encoding amorphadiene synthase and is sequentially oxidised by citorchromeP450 CYP71AV1 and reduced by artemisinic aldehyde reductase (DBR2). Amorphadyene synthase is an enzyme and catalyse the first step in the anitmalarial drug artemisnin synthesis. It has an inportant role in catalysing the reaction of FPP to amorphadiene. Beacause of its importaint step towards artemisnin biosynthesis it was designed and improved for the purpuse to acheve high-level expression in E.coli. For ADS gene synthesis they used two step assemly and one step amplification PCR.(1) Assemby PCR is a useful technique for production novel gene sequences. With this method we can put together short fragments of DNA and it is also a good choice for gene construction. Reserches in this article analysed the sequence of three ADS genes from independet clones and identified mutations in each of the genes. Than, two site directed mutagenesis reaction was used for assembly and generation a functional ADS gene from two clones. Than they measure the amorphadiene production by the amorphadiene synthase in E.coli DH10B with natural (nonengineered DPX pathway) and in E.coli with engineered DPX pathway. E. coli with engineerd pathway was harboring the pSOE4 plasmid (DXP pathway operon with dxs,IppHp and ispA gene). The result sugested that FPP synthesis limited amorphadiene production in this engineered host [1,6]. ([http://www.disketa.si/media/uploads/2015-01/0bddf07f946e6f5be0638090dee56490.jpg See image here])

=== Egineering the mevalonate-dependet pathway in E.coli ===

The aim was to increase the intracellular concentration of FPP substrate. Genes encoding the MEV pathway from S.cerevisae was assembled into operons and expressed in E.coli. Because of the complexity of engineering numerous gene biosynthetic pathway, genes were divided into to operons, top and bottom.Top operon, MevT, consisted of three enzymes: acetoacetyl-CoA thiolase (atoB), HMG-CoA synthases (HMGS) and HMG-CoA reductase (tHMGR). This operon was responsible for transformation of acetyl-coA to mevalonate. Thre different bottom operons pMevB (ERG12, ERG8,MVD1 ), pMBI (ERG12, ERG8, MVD1, idi) and pMBIS (ERG12, ERG8, MVD1, idi, ispA) transformed mevalonate to IPP, DMAPP and consequently to FPP. The next step was to analyse and test the funcionality of this pathway. For this purpuse E.coli strand DYM1 with incoplemplete isoprenoid synthesis was transformed with plasmid expressing three bottom operon constuct- pMevB, pMBI and pMBI. E.coli strand DYM1 had a deletion in the ispC gene for encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase, and was not able to synthesise 2-C-methyl-D-erythritol 4-phosphate(MEP), which is important intermediate in isoprenoid biosynthesis[1]. Because of this deletion the synthesis of precursors IPP and DMAP is blocked. So then they tested what is going to happen if they grew the strain DYM1 in the presence of 2-C-methyl-D-ertihritol and in medium with mevalonate without methylerythritol since DYM1 strain was not capable to synthesisie MEP. As expected , all strains expressing operon from S.cerevisiae grew on the plates in the presence of 2-c-methyl-D-erythritol. But only strains with pMBI or pMBIS grew on plates with 1mM mevalonate in the absence of methylerythol. This was the conformation that synthetic MBI and MBIS operons were functional, because they were capable to support the growth of E.coli without ispC gene by supplying cells with IPP and DMAPP. Next they completed the mevalonate pathway by transforming the pMevT plasmid (expressing three genes mentioned before-atoB, HMGS and tHMGR) with pMBI or pMBIS. This coexpresion of two operons, pMevT and pMBI /pMBIS ,completed the mevlonate pathway and allowed the synthesis of sesquiterpene precursors from acetyl-CoA, IPP and DMAPP. Therefore, coexpression complemented the ispC deletion even in the absence of mevalonate, what was the proof, that MevT operon was functional [1,7].

=== Amorpadiene synthesis from mevalonate ===

The aim was to achieve high-level production of amorphadiene and to find out if the amount of FPP was limiting amoprhadiene output. As mentioned, E.coli was used as heterologous microorganism in which coupled mevalonate pathway with amorphadiene synthesis. Cells with ADS gene were coexpressed with MBIS operon and grown in medium with increasing amounts (0 to 40 mM) of exogenus mevalonate. The culture extract was analysed with gas chromatography-mass spectrometry, analytical method for identification different substances in a test sample. It combines the features of mass spectrometry and gas-liquid chromatography. Results showed that MBIS operon did not limit the amorphadiene production at the highest mevalonate concentration(40mM). ([http://www.disketa.si/media/uploads/2015-01/7711d592584a53282f2ecb0fd678ef40.jpg See image 1 here]) Cultures with 40 mM mevalonate added produced much more amoprhadiene. With time, amorphadiene concentration was dropping beacause of the loss of the volatile terpene[1]. By adding more than 10 mM mevalonate in control cultures without ADS, the severe growth ihibition has been reported. ([http://www.disketa.si/media/uploads/2015-01/2a21f55f0fea31ffcbe89c866c9150f7.jpg See image here]) To discover the cause ofthis inhibition, the growth of E.coli DH10B from strains with empty, pMKPMK, pMevB, pMBI or pMBIS plasmid was measured in media with increasing concentrations of mevalonate. The addition of 5mM mevalonate to the medium inhibited the growth of cells with pMevB. But 5mM concentrations of mevalonate did not alter the growth of cells with pMKPMK, PMBI or pMBIS plasmid. This indicated that the accomulation of IPP is toxic and inhibits normal cell growth. The accmulation mostly occurs in cells with high flux through mevalonate pathway. The next step was comparison of intracellular prenyl pyrophosphate (FPP) in strains. This was done by feeding the cells harboring the different mevalonte operon construct with radiolabeled mevalonate. Than the labeled metabolites were tracked. For this experiment cell suspensions of E.coli harboring pMevB+pTrc99A, pMBI+ pTrc99A, pMBIS+ pTrc99A or pMBIS+pADS were used. pTRc99A is the bacterial expression vector with inducible to IPTG lacI promoter. The result showed that strains with MevB operon accumulated IPP but not FPP, when MBI and MBIS strains accumulated FPP. ([http://www.disketa.si/media/uploads/2015-01/31ad7a215f016dc26aceacf92eb46712.jpg See image here]) Because of the simultaneous expression of the amorphadiene synthase the waste of FPP that accumulated in the MBIS host was consumed. This was shown by a decrease in intracellular FPP. Cells expresing MBIS acumulated FPP and showed growth inhibition in the presence of 10 mM mevalonate. The prediction was that the coexpression of ADS would ease the growth inhibition by forwarding the prenyl pyrophosphate intermediates to volatile terpene olefin. For this test, the cell with pMBIS and empty expression vector pTrc99A (without ADS)and cells with pMBIS and pADS vectors were used. The medium was supplemented with mevalonate concentations ( 0mM, 5mM, 10mM, 20mM or 40mM). Two hours after addition of mevalonate and inducer IPTG, the growth inhibition was observed. ([http://www.disketa.si/media/uploads/2015-01/94b5f3d5636fe767316f770f3e2b0448.jpg See image here]) The result showed that the strains with pMBIS +ADS started to grow, but strain lacking the ADS still showed growth inhbition at higher concentrations of mevalonate. This supported the conclusion that converion of FPP to amophadiene has an important role in minimising growth inhibition. Briefly these result showed that engineered mevalonate patway produces high levels of prenyl pyrophosphate precursors. If the IPP isomerase, FPP synthase and terpen synthase is absent, IPP can acumulate and be toxic to the cells. The toxic levels of intracellular prenyl pyrphosphate can accumulate [1].

=== Amorphadiene synthesis from acetyl-Coa ===

The main goal was to achieve high amoprhaidene production from a simple and inexpensive carbon source. E.coli strains harboring pMevT,pMBIS and pADS plasmids were transformed with pMevT plasmid. Subsequently the strains were tested for ability to produce amoprhadiene in the absence of exogenous mevalonate. The comparison between E.coli expressing the native DPX pathway and engineered pathway as made. They tested native DXP pathway, engineered DPX pathway, mevalonate bottom pathway in the absence of mevalonate, mevlonate bottom pathway in medium suplemented with mevalonate, the complete mevalonate pathway and the complete mevalonate pathway supplemented with 0,8% glycerol. Glycerol was addded to investigate the effect of amorphadiene production of supplying as single carbon source and for prolongation of amorphadiene production. Breifly the resulut showed that expression of the mevalonate-dependent isoprenoid biosynthetic pathway delivers hight levels of isoprenoid precursor for the production of sesquiterpene [1]. ([http://www.disketa.si/media/uploads/2015-01/a31fe07a7c66854b1dc3ff36f8ed01ef.jpg See image here])

== DISSCUSION ==
The identification of genes encoding the enzymes involved in the artemisinin-biosynthesis pathway together with advances in metabolic engineering provides new options for engineering artemisinin biosynthesis. Heterologous production of artemisnin in microorganism such as E.coli. is an alternative way for production of artmeinsin in high yields. But eventhougt micoorganisms has shown great potential in that matter, optimisation of these heterlogous pathways still require maximal titer. Inequal gene expression can lead to accumulation of toxic metabolic intermediates and to over or under production of enzymes involved in pathway. (4). Conclusion based on these study shows that poor expression of the amoprhaidene synthase genes (ADS) limits high terpene olfein yields from the host. To achieve better results , in this study they chose to synthesiyse an amophaiene synthase gene (ADS). The comparison between E.coli expressing native sesquiterpene synthase genes and E.coli expressing the synthetic ADS gene showed big improvement in terpene synthesis due to synthetic ADS gene. The native DPX pathway showed poor supply of the prenyl pyrophosphate precuror. And that limited terpenoid -carotenoid yields in E.coli. With the metabolic engineering of the DPX pathway the flux of carotenoid accumulation showed signifcant increase. By using the mevalonate-dependent isoprenoid pathway from yeast S. cerevisae and engineering that pathway in bacteria E.coli, we avoid the native regulatory elements found in yeast while bypassing those of E.coli DXP pathway. In this study results showed growth inhibition in cells without ADS due to the vast excess of preny pyrophosphate. But the coexpression of a synthetic sesquiterpene synthase can contribute to reducing the excess pool of precursors and eliminate growth inhibition. In this study total biosynthesis of artemisnin was not completely achieved. But these results can help in further investigation for poduction of artemisnic acid. This acid can then be putrified and chemically converted into artemsinin. [1,7].

== CONCLUSIONS ==
To sum things up, artmeinsin, a sesquiterpene isolated from Artemisia annua,represent a novel drug for malaria treatment. The present artemisnin derivates are too expensive for general use and plants can not produce artmeinsin in big quantities. This fact inspired scientist to focus on developing the way for production of artemisnin in high quantities and to avoid big costs. The promising tool in alternative source of artmeinsin is the use of microoganisms. The aim is to transfer the genes encoding enzymes involved in biosynthetic pathway for artemisnin into a hight producing host organism. Several study have been reporting on engineering the metabolic pathway in S.cerevisae to produce the precursor for artmeisnic acid. With the effort in synthetic biology they used modified mevalonate pathway . With the help of syntehtic bilogy the yeast cells were engineerd to express ezymes needed for production of artmemisnic acid. But in this paper the main focus was on engineering a mevalonate pathway in bacteria E.coli. By engineering a new metabolic pathway in E.coli we can easily synthesise a precursor nedeed for artemisnin production. The goal is to produce drugs in better and cheape ways. But, why focus on artemisnin? Well, acording on recent studies artemisnin shows to be very sucesfull for the treatmen all strain of malaria. More than a milion people affected with malaria have already been cured by arteminsin. As already mentioned , because of very hight costs and limited production of arteminin in plants most populations in Africa can not afford it. Progress made in synthetic biology and ability to produce amorphadiene in microorganisms in big quantites opens a new possibility to produce low cost artemisnin and contributes to the brighter future od antimalaria drug deveopment, and therefore saving milions from suffering from malaria. [1,2, 3].

[1]Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nature biotehnology; vol 21; pages 796-802, July 2003

[2] T. Mosses, J. Pollier, J. M. Thevelein, A. Goossens. Bioengineering of plant (tri)terpenoids: from metabolic engineering of plants to synthetic biology in vivo and in vitro. New Pathyologist. 200: 27-43, 2013

[3] M.Farhi, M. Kozin, S. Duchin, A. Vainstein. Metabolic engineering of plants for artemisinin synthesis. Biotehnology and genetic engineering rewiews.Vol.29,No.2: 135-148, 2013

[4] Synthetic biology ; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Synthetic_biology http://en.wikipedia.org/wiki/Synthetic_biology]

[5]Operon; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Operon http://en.wikipedia.org/wiki/Operon]

[6] M. K. Julsing, G. van Pouderoyen, J. van der Veen,H. J. Woerdenbag, O. Kayser, W. J. Quax. Amorphadiene synthase: probing the active site model with directed mutagenesis. Directed mutagenesis of AMDS.Chapter 4: 47-56

[7]J.R. Anthony, L. C. Anthony, F. Nowroozi, G. Kwon, J.D. Newman, J.D. Keasling. Optimization of themevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene. Metabolic Engineering. 11: 13-19, 2009

Engineering a mevalonate pathway in Escherichia coli for production of terpenoids

2015-01-19T23:48:34Z

Ana90:

'''Ana Kapraljević'''

[http://www2.lbl.gov/ttd/publications/2837pub1.pdf Engineering a mevalonate pathway in Escherichia coli for production of terpenoids]
Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling ;Nature biotehnology; July 2003; vol 21; pages 796-802

== INTRODUCTION ==

Plants can synthesise and accumulate a wide range of small molecules or natural products involved in psyhological an ecological process. With the knowledge we know about their biosyntesis we used them as commercial flavors, fragnance compunds, colorants and terapeutical drugs. Their therapeutic potential is used for production of anticancer or antimalarial drugs, such as artemisnin. The most numerous and diverse class of natural products used in production for antimalarial drug are isoprenoids or more corectly their subclass, terpenoids. Malaria represent a major health problem in tropic and subtropic. Majority of death occur in children living in Africa. But the problem is that terpenoids are in plants produced in very small quantites and consequently not very economically feasible. For that reason the drugs for treatin malaria, artemisnin, is in short supply and is still unaffordable for most people living in malaria-endanger countries. Scientists are now focusing on alternative and cheaper way for terpenoid synthesis that can be enchanched and became economically acceptable. The alternative way is the expression of synthetic amorphadiene, precursor to artemisnin, and mevalonate isoprenoid way from yeas Saccharomyces cerevisiae in bacteria. In this paper I will summerize the project made in the production of amorphadiene by biotehnology and synthetic biology approach [1,2].

=== Artemisnin ===

Artemisnin is a substance that has antimalarial potential. Malaria is a infectious disease caused by parasitic protozan of the Plasmodium species. In the certain parts of the world malaria takes lives of more people worldwide than any other infecious disease, so it is a global problem. Death to malaria has been nowdays reduced thanks to the use of artemisnin-based combination therapy. Artemisnin is a natural compoud from plant ''Artemisia annua''. Chemically it is a sesquiterpene lactone, amorphadiene. The total sythesis of artemisnin is difficult and not very economical,so the new aproach is to put the hopes in sythetic biology and develop the new way, synthetic way, for its production [1,3].

=== Biology of terpenoids ===

For initiation of any synthetic biology process in metabolic engineering in that matter it is important to have some basic understanding of biosynthesis and regulation of terpenoids. Terpenoids, a class of isoprenoids, are natural products often sythesized by plants. They represent a large and diverse class and have many functions as primarly metabolites involved in growth and development of plants, or as secundary metabolites that optimize the interaction between the plant and the environment. Eventhought they are structural diverse, the share a common biosynthetic origin and follow similar synthesis tracks. All terpenoids are derived from repetitive fusion of isoprene units (C5 H8) [3]. The number of isoprene units determines their classification. For isoprenoid synthesis plants use mevalonate-dependent (MEV) pathway and mevalonte-independent pathway or alternative deoxyxylulose 5-phosphate pathway (DXP). In higher plants the biosynthesis begins with the generation of isopentenyl pyrophosphate (IPP). IPP is derived via the classic mevalonic acid pathway from acetly-coenzime A (acetyl-CoA). The IPP is isomerized to its allylic isomer dimethylallyl pyrophosphate (DMAPP) [3]. The continous condensation of IPP and DMAPP units leads to the formaton of prenylated pyrophosphates, the immediate precursors of the different terpenoid classes. So IPP and DMAPP are combined to form larger isoprenoids, such as farnesyl diphosphate (FPP). Condensation of IPP and DMAPP than units leads to the formation of precursors of the different terpenoid classes. Shematic production of terpenoids in ilustraded in this picture. Similary to plants, yeast Sacharomyces cerevisae uses the clasical mevalonate pathway for the synthesis of isoprenoids[3]. Prokariontes mostly use the DXP pathway for production IPP and DMAPP. This is an alternative pathway for IPP synthesis. This pathway uses Escherichia coli and some other bacteria for their terpenoid synthesis. IPP and DMAPP precurosr are essential to E. Coli for prenylation (addition of hydrophobic molecules to a protein or chemical compound) of tRNA and the synthesis of farnesyl pyrophosphate (FPP). FPP is then used for quinone (class of organic compounds) and cell wall synthesis. The DPX pathway begins with the glycolytic intermediates glyceraldehyde-3-phosphate (G3P) and pyruvate and continues through enzymatic steps for production of IPP and DMAPP. Therefore IPP and DMAPP are the end product in both way,MEV and DPX pathway [1,3].

== METABOLIC ENGINEERING FOR PRODUCTION OD TERPENOIDS ==

Metabolic engineering represents an improvement of production, construction, or cellular properties through the modification of specific biochemical reactions. It is described as the use of genetic engineering to reshape or modify the metabolism of organism. The aim is to optimize genetic and regulatory processes in cells to increase the production of a certain substance that we find in nature. It is based on optimisation of existing biochemical pathways or the introduction of natural pathway components in bacteria, yeast or plants. The main goal is the production of high-yield of specific metabolites that we can use in medicine or biotechnology. With progress in synthetic biology we can design a biologic funcion that do not exist in nature. Consequently, we can create and construct new biological components or redesign exisitng biological systems. In plants, most terpenoids are produced in a very small quantities in their natural sources. Extraction of this compouds from plants is expensive and therfore not economicaly accetable. So there is a big disagreement between the insistence for supply of terpenoids and therefore that leads to delay for their widespread application. With the understanding of terpenoid synthesis and progress in synthetic biology we are now able of redesign and modify the isoprenoid pathway to increase the terpenoid productivity [2,4]. ([http://www.disketa.si/media/uploads/2015-01/cd201ade6a167488767dfb1b118587bc.jpg See image here])

=== Microbal host ===

To elimante the need for plant extraction it is practical to produce terpenoid compouds at high yield in microbal host by introducing a heterlogous isoprenoid pathway into bacteria E.coli. Microorganisms, such as bacteria are attractive alternatives as hosts because they have rapid doubling time, they can achieve robussnes under process conditions and because of the simplicity of product putrification. And the costs are significantly lower than working with plants. With the engineering the DXP pathway we can increase the supply of isoprenoid precurosor needed for high level productio of isoprenoids in E.coli. [1,3]

=== Operons ===

Operon is a a funcional unit of DNA that contains a cluster of genes under the control of a single promotor. The synthesis of natural or synthetic products involves the introduction of a large number of genes. In this case these genes were encoding the enzymes needed for metabolic pathway. So it is useful to put certain genes under the control of a single promotor. For that reason Plac promotor was used in this study [1,5].

== EXPERIMENT ==

=== Synthase gene asemby and amorphadiene production ===

For high level production and changing the difficulties in expressing teprene synthases they first synthesized and expressed a codon-optimized variants of ADS . ADS is a gene encoding amorphadiene synthase and is sequentially oxidised by citorchromeP450 CYP71AV1 and reduced by artemisinic aldehyde reductase (DBR2). Amorphadyene synthase is an enzyme and catalyse the first step in the anitmalarial drug artemisnin synthesis. It has an inportant role in catalysing the reaction of FPP to amorphadiene. Beacause of its importaint step towards artemisnin biosynthesis it was designed and improved for the purpuse to acheve high-level expression in E.coli. For ADS gene synthesis they used two step assemly and one step amplification PCR.(1) Assemby PCR is a useful technique for production novel gene sequences. With this method we can put together short fragments of DNA and it is also a good choice for gene construction. Reserches in this article analysed the sequence of three ADS genes from independet clones and identified mutations in each of the genes. Than, two site directed mutagenesis reaction was used for assembly and generation a functional ADS gene from two clones. Than they measure the amorphadiene production by the amorphadiene synthase in E.coli DH10B with natural (nonengineered DPX pathway) and in E.coli with engineered DPX pathway. E. coli with engineerd pathway was harboring the pSOE4 plasmid (DXP pathway operon with dxs,IppHp and ispA gene). The result sugested that FPP synthesis limited amorphadiene production in this engineered host [1,6]. ([http://www.disketa.si/media/uploads/2015-01/0bddf07f946e6f5be0638090dee56490.jpg See image here])

=== Egineering the mevalonate-dependet pathway in E.coli ===

The aim was to increase the intracellular concentration of FPP substrate. Genes encoding the MEV pathway from S.cerevisae was assembled into operons and expressed in E.coli. Because of the complexity of engineering numerous gene biosynthetic pathway, genes were divided into to operons, top and bottom.Top operon, MevT, consisted of three enzymes: acetoacetyl-CoA thiolase (atoB), HMG-CoA synthases (HMGS) and HMG-CoA reductase (tHMGR). This operon was responsible for transformation of acetyl-coA to mevalonate. Thre different bottom operons pMevB (ERG12, ERG8,MVD1 ), pMBI (ERG12, ERG8, MVD1, idi) and pMBIS (ERG12, ERG8, MVD1, idi, ispA) transformed mevalonate to IPP, DMAPP and consequently to FPP. The next step was to analyse and test the funcionality of this pathway. For this purpuse E.coli strand DYM1 with incoplemplete isoprenoid synthesis was transformed with plasmid expressing three bottom operon constuct- pMevB, pMBI and pMBI. E.coli strand DYM1 had a deletion in the ispC gene for encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase, and was not able to synthesise 2-C-methyl-D-erythritol 4-phosphate(MEP), which is important intermediate in isoprenoid biosynthesis[1]. Because of this deletion the synthesis of precursors IPP and DMAP is blocked. So then they tested what is going to happen if they grew the strain DYM1 in the presence of 2-C-methyl-D-ertihritol and in medium with mevalonate without methylerythritol since DYM1 strain was not capable to synthesisie MEP. As expected , all strains expressing operon from S.cerevisiae grew on the plates in the presence of 2-c-methyl-D-erythritol. But only strains with pMBI or pMBIS grew on plates with 1mM mevalonate in the absence of methylerythol. This was the conformation that synthetic MBI and MBIS operons were functional, because they were capable to support the growth of E.coli without ispC gene by supplying cells with IPP and DMAPP. Next they completed the mevalonate pathway by transforming the pMevT plasmid (expressing three genes mentioned before-atoB, HMGS and tHMGR) with pMBI or pMBIS. This coexpresion of two operons, pMevT and pMBI /pMBIS ,completed the mevlonate pathway and allowed the synthesis of sesquiterpene precursors from acetyl-CoA, IPP and DMAPP. Therefore, coexpression complemented the ispC deletion even in the absence of mevalonate, what was the proof, that MevT operon was functional [1,7].

=== Amorpadiene synthesis from mevalonate ===

The aim was to achieve high-level production of amorphadiene and to find out if the amount of FPP was limiting amoprhadiene output. As mentioned, E.coli was used as heterologous microorganism in which coupled mevalonate pathway with amorphadiene synthesis. Cells with ADS gene were coexpressed with MBIS operon and grown in medium with increasing amounts (0 to 40 mM) of exogenus mevalonate. The culture extract was analysed with gas chromatography-mass spectrometry, analytical method for identification different substances in a test sample. It combines the features of mass spectrometry and gas-liquid chromatography. Results showed that MBIS operon did not limit the amorphadiene production at the highest mevalonate concentration(40mM). ([http://www.disketa.si/media/uploads/2015-01/7711d592584a53282f2ecb0fd678ef40.jpg See image 1 here]) Cultures with 40 mM mevalonate added produced much more amoprhadiene. With time, amorphadiene concentration was dropping beacause of the loss of the volatile terpene[1]. By adding more than 10 mM mevalonate in control cultures without ADS, the severe growth ihibition has been reported. ([http://www.disketa.si/media/uploads/2015-01/2a21f55f0fea31ffcbe89c866c9150f7.jpg See image here]) To discover the cause ofthis inhibition, the growth of E.coli DH10B from strains with empty, pMKPMK, pMevB, pMBI or pMBIS plasmid was measured in media with increasing concentrations of mevalonate. The addition of 5mM mevalonate to the medium inhibited the growth of cells with pMevB. But 5mM concentrations of mevalonate did not alter the growth of cells with pMKPMK, PMBI or pMBIS plasmid. This indicated that the accomulation of IPP is toxic and inhibits normal cell growth. The accmulation mostly occurs in cells with high flux through mevalonate pathway. The next step was comparison of intracellular prenyl pyrophosphate (FPP) in strains. This was done by feeding the cells harboring the different mevalonte operon construct with radiolabeled mevalonate. Than the labeled metabolites were tracked. For this experiment cell suspensions of E.coli harboring pMevB+pTrc99A, pMBI+ pTrc99A, pMBIS+ pTrc99A or pMBIS+pADS were used. pTRc99A is the bacterial expression vector with inducible to IPTG lacI promoter. The result showed that strains with MevB operon accumulated IPP but not FPP, when MBI and MBIS strains accumulated FPP. ([http://www.disketa.si/media/uploads/2015-01/31ad7a215f016dc26aceacf92eb46712.jpg See image here]) Because of the simultaneous expression of the amorphadiene synthase the waste of FPP that accumulated in the MBIS host was consumed. This was shown by a decrease in intracellular FPP. Cells expresing MBIS acumulated FPP and showed growth inhibition in the presence of 10 mM mevalonate. The prediction was that the coexpression of ADS would ease the growth inhibition by forwarding the prenyl pyrophosphate intermediates to volatile terpene olefin. For this test, the cell with pMBIS and empty expression vector pTrc99A (without ADS)and cells with pMBIS and pADS vectors were used. The medium was supplemented with mevalonate concentations ( 0mM, 5mM, 10mM, 20mM or 40mM). Two hours after addition of mevalonate and inducer IPTG, the growth inhibition was observed. ([http://www.disketa.si/media/uploads/2015-01/94b5f3d5636fe767316f770f3e2b0448.jpg See image here]) The result showed that the strains with pMBIS +ADS started to grow, but strain lacking the ADS still showed growth inhbition at higher concentrations of mevalonate. This supported the conclusion that converion of FPP to amophadiene has an important role in minimising growth inhibition. Briefly these result showed that engineered mevalonate patway produces high levels of prenyl pyrophosphate precursors. If the IPP isomerase, FPP synthase and terpen synthase is absent, IPP can acumulate and be toxic to the cells. The toxic levels of intracellular prenyl pyrphosphate can accumulate [1].

=== Amorphadiene synthesis from acetyl-Coa ===

The main goal was to achieve high amoprhaidene production from a simple and inexpensive carbon source. E.coli strains harboring pMevT,pMBIS and pADS plasmids were transformed with pMevT plasmid. Subsequently the strains were tested for ability to produce amoprhadiene in the absence of exogenous mevalonate. The comparison between E.coli expressing the native DPX pathway and engineered pathway as made. They tested native DXP pathway, engineered DPX pathway, mevalonate bottom pathway in the absence of mevalonate, mevlonate bottom pathway in medium suplemented with mevalonate, the complete mevalonate pathway and the complete mevalonate pathway supplemented with 0,8% glycerol. Glycerol was addded to investigate the effect of amorphadiene production of supplying as single carbon source and for prolongation of amorphadiene production. Breifly the resulut showed that expression of the mevalonate-dependent isoprenoid biosynthetic pathway delivers hight levels of isoprenoid precursor for the production of sesquiterpene [1]. ([http://www.disketa.si/media/uploads/2015-01/a31fe07a7c66854b1dc3ff36f8ed01ef.jpg See image here])

== DISSCUSION ==
The identification of genes encoding the enzymes involved in the artemisinin-biosynthesis pathway together with advances in metabolic engineering provides new options for engineering artemisinin biosynthesis. Heterologous production of artemisnin in microorganism such as E.coli. is an alternative way for production of artmeinsin in high yields. But eventhougt micoorganisms has shown great potential in that matter, optimisation of these heterlogous pathways still require maximal titer. Inequal gene expression can lead to accumulation of toxic metabolic intermediates and to over or under production of enzymes involved in pathway. (4). Conclusion based on these study shows that poor expression of the amoprhaidene synthase genes (ADS) limits high terpene olfein yields from the host. To achieve better results , in this study they chose to synthesiyse an amophaiene synthase gene (ADS). The comparison between E.coli expressing native sesquiterpene synthase genes and E.coli expressing the synthetic ADS gene showed big improvement in terpene synthesis due to synthetic ADS gene. The native DPX pathway showed poor supply of the prenyl pyrophosphate precuror. And that limited terpenoid -carotenoid yields in E.coli. With the metabolic engineering of the DPX pathway the flux of carotenoid accumulation showed signifcant increase. By using the mevalonate-dependent isoprenoid pathway from yeast S. cerevisae and engineering that pathway in bacteria E.coli, we avoid the native regulatory elements found in yeast while bypassing those of E.coli DXP pathway. In this study results showed growth inhibition in cells without ADS due to the vast excess of preny pyrophosphate. But the coexpression of a synthetic sesquiterpene synthase can contribute to reducing the excess pool of precursors and eliminate growth inhibition. In this study total biosynthesis of artemisnin was not completely achieved. But these results can help in further investigation for poduction of artemisnic acid. This acid can then be putrified and chemically converted into artemsinin. [1,7].

== CONCLUSIONS ==
To sum things up, artmeinsin, a sesquiterpene isolated from Artemisia annua,represent a novel drug for malaria treatment. The present artemisnin derivates are too expensive for general use and plants can not produce artmeinsin in big quantities. This fact inspired scientist to focus on developing the way for production of artemisnin in high quantities and to avoid big costs. The promising tool in alternative source of artmeinsin is the use of microoganisms. The aim is to transfer the genes encoding enzymes involved in biosynthetic pathway for artemisnin into a hight producing host organism. Several study have been reporting on engineering the metabolic pathway in S.cerevisae to produce the precursor for artmeisnic acid. With the effort in synthetic biology they used modified mevalonate pathway . With the help of syntehtic bilogy the yeast cells were engineerd to express ezymes needed for production of artmemisnic acid. But in this paper the main focus was on engineering a mevalonate pathway in bacteria E.coli. By engineering a new metabolic pathway in E.coli we can easily synthesise a precursor nedeed for artemisnin production. The goal is to produce drugs in better and cheape ways. But, why focus on artemisnin? Well, acording on recent studies artemisnin shows to be very sucesfull for the treatmen all strain of malaria. More than a milion people affected with malaria have already been cured by arteminsin. As already mentioned , because of very hight costs and limited production of arteminin in plants most populations in Africa can not afford it. Progress made in synthetic biology and ability to produce amorphadiene in microorganisms in big quantites opens a new possibility to produce low cost artemisnin and contributes to the brighter future od antimalaria drug deveopment, and therefore saving milions from suffering from malaria. [1,2, 3].

[1]Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nature biotehnology; vol 21; pages 796-802, July 2003

[2] T. Mosses, J. Pollier, J. M. Thevelein, A. Goossens. Bioengineering of plant (tri)terpenoids: from metabolic engineering of plants to synthetic biology in vivo and in vitro. New Pathyologist. 200: 27-43, 2013

[3] M.Farhi, M. Kozin, S. Duchin, A. Vainstein. Metabolic engineering of plants for artemisinin synthesis. Biotehnology and genetic engineering rewiews.Vol.29,No.2: 135-148, 2013

[4] Synthetic biology ; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Synthetic_biology http://en.wikipedia.org/wiki/Synthetic_biology]

[5]Operon; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Operon http://en.wikipedia.org/wiki/Operon]

[6] M. K. Julsing, G. van Pouderoyen, J. van der Veen,H. J. Woerdenbag, O. Kayser, W. J. Quax. Amorphadiene synthase: probing the active site model with directed mutagenesis. Directed mutagenesis of AMDS.Chapter 4: 47-56

[7]J.R. Anthony, L. C. Anthony, F. Nowroozi, G. Kwon, J.D. Newman, J.D. Keasling. Optimization of themevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene. Metabolic Engineering. 11: 13-19, 2009

Engineering a mevalonate pathway in Escherichia coli for production of terpenoids

2015-01-19T23:44:09Z

Ana90:

'''Ana Kapraljević'''

[http://www2.lbl.gov/ttd/publications/2837pub1.pdf Engineering a mevalonate pathway in Escherichia coli for production of terpenoids]
Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling ;Nature biotehnology; July 2003; vol 21; pages 796-802

=== INTRODUCTION ===

Plants can synthesise and accumulate a wide range of small molecules or natural products involved in psyhological an ecological process. With the knowledge we know about their biosyntesis we used them as commercial flavors, fragnance compunds, colorants and terapeutical drugs. Their therapeutic potential is used for production of anticancer or antimalarial drugs, such as artemisnin. The most numerous and diverse class of natural products used in production for antimalarial drug are isoprenoids or more corectly their subclass, terpenoids. Malaria represent a major health problem in tropic and subtropic. Majority of death occur in children living in Africa. But the problem is that terpenoids are in plants produced in very small quantites and consequently not very economically feasible. For that reason the drugs for treatin malaria, artemisnin, is in short supply and is still unaffordable for most people living in malaria-endanger countries. Scientists are now focusing on alternative and cheaper way for terpenoid synthesis that can be enchanched and became economically acceptable. The alternative way is the expression of synthetic amorphadiene, precursor to artemisnin, and mevalonate isoprenoid way from yeas Saccharomyces cerevisiae in bacteria. In this paper I will summerize the project made in the production of amorphadiene by biotehnology and synthetic biology approach [1,2].

'''Artemisnin'''

Artemisnin is a substance that has antimalarial potential. Malaria is a infectious disease caused by parasitic protozan of the Plasmodium species. In the certain parts of the world malaria takes lives of more people worldwide than any other infecious disease, so it is a global problem. Death to malaria has been nowdays reduced thanks to the use of artemisnin-based combination therapy. Artemisnin is a natural compoud from plant ''Artemisia annua''. Chemically it is a sesquiterpene lactone, amorphadiene. The total sythesis of artemisnin is difficult and not very economical,so the new aproach is to put the hopes in sythetic biology and develop the new way, synthetic way, for its production [1,3].

'''Biology of terpenoids'''

For initiation of any synthetic biology process in metabolic engineering in that matter it is important to have some basic understanding of biosynthesis and regulation of terpenoids. Terpenoids, a class of isoprenoids, are natural products often sythesized by plants. They represent a large and diverse class and have many functions as primarly metabolites involved in growth and development of plants, or as secundary metabolites that optimize the interaction between the plant and the environment. Eventhought they are structural diverse, the share a common biosynthetic origin and follow similar synthesis tracks. All terpenoids are derived from repetitive fusion of isoprene units (C5 H8) [3]. The number of isoprene units determines their classification. For isoprenoid synthesis plants use mevalonate-dependent (MEV) pathway and mevalonte-independent pathway or alternative deoxyxylulose 5-phosphate pathway (DXP). In higher plants the biosynthesis begins with the generation of isopentenyl pyrophosphate (IPP). IPP is derived via the classic mevalonic acid pathway from acetly-coenzime A (acetyl-CoA). The IPP is isomerized to its allylic isomer dimethylallyl pyrophosphate (DMAPP) [3]. The continous condensation of IPP and DMAPP units leads to the formaton of prenylated pyrophosphates, the immediate precursors of the different terpenoid classes. So IPP and DMAPP are combined to form larger isoprenoids, such as farnesyl diphosphate (FPP). Condensation of IPP and DMAPP than units leads to the formation of precursors of the different terpenoid classes. Shematic production of terpenoids in ilustraded in this picture. Similary to plants, yeast Sacharomyces cerevisae uses the clasical mevalonate pathway for the synthesis of isoprenoids[3]. Prokariontes mostly use the DXP pathway for production IPP and DMAPP. This is an alternative pathway for IPP synthesis. This pathway uses Escherichia coli and some other bacteria for their terpenoid synthesis. IPP and DMAPP precurosr are essential to E. Coli for prenylation (addition of hydrophobic molecules to a protein or chemical compound) of tRNA and the synthesis of farnesyl pyrophosphate (FPP). FPP is then used for quinone (class of organic compounds) and cell wall synthesis. The DPX pathway begins with the glycolytic intermediates glyceraldehyde-3-phosphate (G3P) and pyruvate and continues through enzymatic steps for production of IPP and DMAPP. Therefore IPP and DMAPP are the end product in both way,MEV and DPX pathway [1,3].

=== METABOLIC ENGINEERING FOR PRODUCTION OD TERPENOIDS ===

Metabolic engineering represents an improvement of production, construction, or cellular properties through the modification of specific biochemical reactions. It is described as the use of genetic engineering to reshape or modify the metabolism of organism. The aim is to optimize genetic and regulatory processes in cells to increase the production of a certain substance that we find in nature. It is based on optimisation of existing biochemical pathways or the introduction of natural pathway components in bacteria, yeast or plants. The main goal is the production of high-yield of specific metabolites that we can use in medicine or biotechnology. With progress in synthetic biology we can design a biologic funcion that do not exist in nature. Consequently, we can create and construct new biological components or redesign exisitng biological systems. In plants, most terpenoids are produced in a very small quantities in their natural sources. Extraction of this compouds from plants is expensive and therfore not economicaly accetable. So there is a big disagreement between the insistence for supply of terpenoids and therefore that leads to delay for their widespread application. With the understanding of terpenoid synthesis and progress in synthetic biology we are now able of redesign and modify the isoprenoid pathway to increase the terpenoid productivity [2,4]. ([http://www.disketa.si/media/uploads/2015-01/cd201ade6a167488767dfb1b118587bc.jpg See image here])

'''Microbal host'''

To elimante the need for plant extraction it is practical to produce terpenoid compouds at high yield in microbal host by introducing a heterlogous isoprenoid pathway into bacteria E.coli. Microorganisms, such as bacteria are attractive alternatives as hosts because they have rapid doubling time, they can achieve robussnes under process conditions and because of the simplicity of product putrification. And the costs are significantly lower than working with plants. With the engineering the DXP pathway we can increase the supply of isoprenoid precurosor needed for high level productio of isoprenoids in E.coli. [1,3]

'''Operons'''

Operon is a a funcional unit of DNA that contains a cluster of genes under the control of a single promotor. The synthesis of natural or synthetic products involves the introduction of a large number of genes. In this case these genes were encoding the enzymes needed for metabolic pathway. So it is useful to put certain genes under the control of a single promotor. For that reason Plac promotor was used in this study [1,5].

=== EXPERIMENT ===

'''Synthase gene asemby and amorphadiene production'''

For high level production and changing the difficulties in expressing teprene synthases they first synthesized and expressed a codon-optimized variants of ADS . ADS is a gene encoding amorphadiene synthase and is sequentially oxidised by citorchromeP450 CYP71AV1 and reduced by artemisinic aldehyde reductase (DBR2). Amorphadyene synthase is an enzyme and catalyse the first step in the anitmalarial drug artemisnin synthesis. It has an inportant role in catalysing the reaction of FPP to amorphadiene. Beacause of its importaint step towards artemisnin biosynthesis it was designed and improved for the purpuse to acheve high-level expression in E.coli. For ADS gene synthesis they used two step assemly and one step amplification PCR.(1) Assemby PCR is a useful technique for production novel gene sequences. With this method we can put together short fragments of DNA and it is also a good choice for gene construction. Reserches in this article analysed the sequence of three ADS genes from independet clones and identified mutations in each of the genes. Than, two site directed mutagenesis reaction was used for assembly and generation a functional ADS gene from two clones. Than they measure the amorphadiene production by the amorphadiene synthase in E.coli DH10B with natural (nonengineered DPX pathway) and in E.coli with engineered DPX pathway. E. coli with engineerd pathway was harboring the pSOE4 plasmid (DXP pathway operon with dxs,IppHp and ispA gene). The result sugested that FPP synthesis limited amorphadiene production in this engineered host [1,6]. ([http://www.disketa.si/media/uploads/2015-01/0bddf07f946e6f5be0638090dee56490.jpg See image here])

'''Egineering the mevalonate-dependet pathway in E.coli'''

The aim was to increase the intracellular concentration of FPP substrate. Genes encoding the MEV pathway from S.cerevisae was assembled into operons and expressed in E.coli. Because of the complexity of engineering numerous gene biosynthetic pathway, genes were divided into to operons, top and bottom.Top operon, MevT, consisted of three enzymes: acetoacetyl-CoA thiolase (atoB), HMG-CoA synthases (HMGS) and HMG-CoA reductase (tHMGR). This operon was responsible for transformation of acetyl-coA to mevalonate. Thre different bottom operons pMevB (ERG12, ERG8,MVD1 ), pMBI (ERG12, ERG8, MVD1, idi) and pMBIS (ERG12, ERG8, MVD1, idi, ispA) transformed mevalonate to IPP, DMAPP and consequently to FPP. The next step was to analyse and test the funcionality of this pathway. For this purpuse E.coli strand DYM1 with incoplemplete isoprenoid synthesis was transformed with plasmid expressing three bottom operon constuct- pMevB, pMBI and pMBI. E.coli strand DYM1 had a deletion in the ispC gene for encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase, and was not able to synthesise 2-C-methyl-D-erythritol 4-phosphate(MEP), which is important intermediate in isoprenoid biosynthesis[1]. Because of this deletion the synthesis of precursors IPP and DMAP is blocked. So then they tested what is going to happen if they grew the strain DYM1 in the presence of 2-C-methyl-D-ertihritol and in medium with mevalonate without methylerythritol since DYM1 strain was not capable to synthesisie MEP. As expected , all strains expressing operon from S.cerevisiae grew on the plates in the presence of 2-c-methyl-D-erythritol. But only strains with pMBI or pMBIS grew on plates with 1mM mevalonate in the absence of methylerythol. This was the conformation that synthetic MBI and MBIS operons were functional, because they were capable to support the growth of E.coli without ispC gene by supplying cells with IPP and DMAPP. Next they completed the mevalonate pathway by transforming the pMevT plasmid (expressing three genes mentioned before-atoB, HMGS and tHMGR) with pMBI or pMBIS. This coexpresion of two operons, pMevT and pMBI /pMBIS ,completed the mevlonate pathway and allowed the synthesis of sesquiterpene precursors from acetyl-CoA, IPP and DMAPP. Therefore, coexpression complemented the ispC deletion even in the absence of mevalonate, what was the proof, that MevT operon was functional [1,7].

'''Amorpadiene synthesis from mevalonate'''

The aim was to achieve high-level production of amorphadiene and to find out if the amount of FPP was limiting amoprhadiene output. As mentioned, E.coli was used as heterologous microorganism in which coupled mevalonate pathway with amorphadiene synthesis. Cells with ADS gene were coexpressed with MBIS operon and grown in medium with increasing amounts (0 to 40 mM) of exogenus mevalonate. The culture extract was analysed with gas chromatography-mass spectrometry, analytical method for identification different substances in a test sample. It combines the features of mass spectrometry and gas-liquid chromatography. Results showed that MBIS operon did not limit the amorphadiene production at the highest mevalonate concentration(40mM). ([http://www.disketa.si/media/uploads/2015-01/7711d592584a53282f2ecb0fd678ef40.jpg See image 1 here]) Cultures with 40 mM mevalonate added produced much more amoprhadiene. With time, amorphadiene concentration was dropping beacause of the loss of the volatile terpene[1]. By adding more than 10 mM mevalonate in control cultures without ADS, the severe growth ihibition has been reported. ([http://www.disketa.si/media/uploads/2015-01/2a21f55f0fea31ffcbe89c866c9150f7.jpg See image here]) To discover the cause ofthis inhibition, the growth of E.coli DH10B from strains with empty, pMKPMK, pMevB, pMBI or pMBIS plasmid was measured in media with increasing concentrations of mevalonate. The addition of 5mM mevalonate to the medium inhibited the growth of cells with pMevB. But 5mM concentrations of mevalonate did not alter the growth of cells with pMKPMK, PMBI or pMBIS plasmid. This indicated that the accomulation of IPP is toxic and inhibits normal cell growth. The accmulation mostly occurs in cells with high flux through mevalonate pathway. The next step was comparison of intracellular prenyl pyrophosphate (FPP) in strains. This was done by feeding the cells harboring the different mevalonte operon construct with radiolabeled mevalonate. Than the labeled metabolites were tracked. For this experiment cell suspensions of E.coli harboring pMevB+pTrc99A, pMBI+ pTrc99A, pMBIS+ pTrc99A or pMBIS+pADS were used. pTRc99A is the bacterial expression vector with inducible to IPTG lacI promoter. The result showed that strains with MevB operon accumulated IPP but not FPP, when MBI and MBIS strains accumulated FPP. ([http://www.disketa.si/media/uploads/2015-01/31ad7a215f016dc26aceacf92eb46712.jpg See image here]) Because of the simultaneous expression of the amorphadiene synthase the waste of FPP that accumulated in the MBIS host was consumed. This was shown by a decrease in intracellular FPP. Cells expresing MBIS acumulated FPP and showed growth inhibition in the presence of 10 mM mevalonate. The prediction was that the coexpression of ADS would ease the growth inhibition by forwarding the prenyl pyrophosphate intermediates to volatile terpene olefin. For this test, the cell with pMBIS and empty expression vector pTrc99A (without ADS)and cells with pMBIS and pADS vectors were used. The medium was supplemented with mevalonate concentations ( 0mM, 5mM, 10mM, 20mM or 40mM). Two hours after addition of mevalonate and inducer IPTG, the growth inhibition was observed. ([http://www.disketa.si/media/uploads/2015-01/94b5f3d5636fe767316f770f3e2b0448.jpg See image here]) The result showed that the strains with pMBIS +ADS started to grow, but strain lacking the ADS still showed growth inhbition at higher concentrations of mevalonate. This supported the conclusion that converion of FPP to amophadiene has an important role in minimising growth inhibition. Briefly these result showed that engineered mevalonate patway produces high levels of prenyl pyrophosphate precursors. If the IPP isomerase, FPP synthase and terpen synthase is absent, IPP can acumulate and be toxic to the cells. The toxic levels of intracellular prenyl pyrphosphate can accumulate [1].

'''Amorphadiene synthesis from acetyl-Coa'''

The main goal was to achieve high amoprhaidene production from a simple and inexpensive carbon source. E.coli strains harboring pMevT,pMBIS and pADS plasmids were transformed with pMevT plasmid. Subsequently the strains were tested for ability to produce amoprhadiene in the absence of exogenous mevalonate. The comparison between E.coli expressing the native DPX pathway and engineered pathway as made. They tested native DXP pathway, engineered DPX pathway, mevalonate bottom pathway in the absence of mevalonate, mevlonate bottom pathway in medium suplemented with mevalonate, the complete mevalonate pathway and the complete mevalonate pathway supplemented with 0,8% glycerol. Glycerol was addded to investigate the effect of amorphadiene production of supplying as single carbon source and for prolongation of amorphadiene production. Breifly the resulut showed that expression of the mevalonate-dependent isoprenoid biosynthetic pathway delivers hight levels of isoprenoid precursor for the production of sesquiterpene [1]. ([http://www.disketa.si/media/uploads/2015-01/a31fe07a7c66854b1dc3ff36f8ed01ef.jpg See image here])

=== DISSCUSION ===
The identification of genes encoding the enzymes involved in the artemisinin-biosynthesis pathway together with advances in metabolic engineering provides new options for engineering artemisinin biosynthesis. Heterologous production of artemisnin in microorganism such as E.coli. is an alternative way for production of artmeinsin in high yields. But eventhougt micoorganisms has shown great potential in that matter, optimisation of these heterlogous pathways still require maximal titer. Inequal gene expression can lead to accumulation of toxic metabolic intermediates and to over or under production of enzymes involved in pathway. (4). Conclusion based on these study shows that poor expression of the amoprhaidene synthase genes (ADS) limits high terpene olfein yields from the host. To achieve better results , in this study they chose to synthesiyse an amophaiene synthase gene (ADS). The comparison between E.coli expressing native sesquiterpene synthase genes and E.coli expressing the synthetic ADS gene showed big improvement in terpene synthesis due to synthetic ADS gene. The native DPX pathway showed poor supply of the prenyl pyrophosphate precuror. And that limited terpenoid -carotenoid yields in E.coli. With the metabolic engineering of the DPX pathway the flux of carotenoid accumulation showed signifcant increase. By using the mevalonate-dependent isoprenoid pathway from yeast S. cerevisae and engineering that pathway in bacteria E.coli, we avoid the native regulatory elements found in yeast while bypassing those of E.coli DXP pathway. In this study results showed growth inhibition in cells without ADS due to the vast excess of preny pyrophosphate. But the coexpression of a synthetic sesquiterpene synthase can contribute to reducing the excess pool of precursors and eliminate growth inhibition. In this study total biosynthesis of artemisnin was not completely achieved. But these results can help in further investigation for poduction of artemisnic acid. This acid can then be putrified and chemically converted into artemsinin. [1,7].

=== CONCLUSIONS ===
To sum things up, artmeinsin, a sesquiterpene isolated from Artemisia annua,represent a novel drug for malaria treatment. The present artemisnin derivates are too expensive for general use and plants can not produce artmeinsin in big quantities. This fact inspired scientist to focus on developing the way for production of artemisnin in high quantities and to avoid big costs. The promising tool in alternative source of artmeinsin is the use of microoganisms. The aim is to transfer the genes encoding enzymes involved in biosynthetic pathway for artemisnin into a hight producing host organism. Several study have been reporting on engineering the metabolic pathway in S.cerevisae to produce the precursor for artmeisnic acid. With the effort in synthetic biology they used modified mevalonate pathway . With the help of syntehtic bilogy the yeast cells were engineerd to express ezymes needed for production of artmemisnic acid. But in this paper the main focus was on engineering a mevalonate pathway in bacteria E.coli. By engineering a new metabolic pathway in E.coli we can easily synthesise a precursor nedeed for artemisnin production. The goal is to produce drugs in better and cheape ways. But, why focus on artemisnin? Well, acording on recent studies artemisnin shows to be very sucesfull for the treatmen all strain of malaria. More than a milion people affected with malaria have already been cured by arteminsin. As already mentioned , because of very hight costs and limited production of arteminin in plants most populations in Africa can not afford it. Progress made in synthetic biology and ability to produce amorphadiene in microorganisms in big quantites opens a new possibility to produce low cost artemisnin and contributes to the brighter future od antimalaria drug deveopment, and therefore saving milions from suffering from malaria. [1,2, 3].

[1]Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nature biotehnology; vol 21; pages 796-802, July 2003

[2] T. Mosses, J. Pollier, J. M. Thevelein, A. Goossens. Bioengineering of plant (tri)terpenoids: from metabolic engineering of plants to synthetic biology in vivo and in vitro. New Pathyologist. 200: 27-43, 2013

[3] M.Farhi, M. Kozin, S. Duchin, A. Vainstein. Metabolic engineering of plants for artemisinin synthesis. Biotehnology and genetic engineering rewiews.Vol.29,No.2: 135-148, 2013

[4] Synthetic biology ; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Synthetic_biology http://en.wikipedia.org/wiki/Synthetic_biology]

[5]Operon; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Operon http://en.wikipedia.org/wiki/Operon]

[6] M. K. Julsing, G. van Pouderoyen, J. van der Veen,H. J. Woerdenbag, O. Kayser, W. J. Quax. Amorphadiene synthase: probing the active site model with directed mutagenesis. Directed mutagenesis of AMDS.Chapter 4: 47-56

[7]J.R. Anthony, L. C. Anthony, F. Nowroozi, G. Kwon, J.D. Newman, J.D. Keasling. Optimization of themevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene. Metabolic Engineering. 11: 13-19, 2009

Engineering a mevalonate pathway in Escherichia coli for production of terpenoids

2015-01-19T23:43:43Z

Ana90:

'''Ana Kapraljević'''

[http://www2.lbl.gov/ttd/publications/2837pub1.pdf Engineering a mevalonate pathway in Escherichia coli for production of terpenoids]
Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling ;Nature biotehnology; July 2003; vol 21; pages 796-802

=== INTRODUCTION ===

Plants can synthesise and accumulate a wide range of small molecules or natural products involved in psyhological an ecological process. With the knowledge we know about their biosyntesis we used them as commercial flavors, fragnance compunds, colorants and terapeutical drugs. Their therapeutic potential is used for production of anticancer or antimalarial drugs, such as artemisnin. The most numerous and diverse class of natural products used in production for antimalarial drug are isoprenoids or more corectly their subclass, terpenoids. Malaria represent a major health problem in tropic and subtropic. Majority of death occur in children living in Africa. But the problem is that terpenoids are in plants produced in very small quantites and consequently not very economically feasible. For that reason the drugs for treatin malaria, artemisnin, is in short supply and is still unaffordable for most people living in malaria-endanger countries. Scientists are now focusing on alternative and cheaper way for terpenoid synthesis that can be enchanched and became economically acceptable. The alternative way is the expression of synthetic amorphadiene, precursor to artemisnin, and mevalonate isoprenoid way from yeas Saccharomyces cerevisiae in bacteria. In this paper I will summerize the project made in the production of amorphadiene by biotehnology and synthetic biology approach [1,2].

'''Artemisnin'''

Artemisnin is a substance that has antimalarial potential. Malaria is a infectious disease caused by parasitic protozan of the Plasmodium species. In the certain parts of the world malaria takes lives of more people worldwide than any other infecious disease, so it is a global problem. Death to malaria has been nowdays reduced thanks to the use of artemisnin-based combination therapy. Artemisnin is a natural compoud from plant ''Artemisia annua''. Chemically it is a sesquiterpene lactone, amorphadiene. The total sythesis of artemisnin is difficult and not very economical,so the new aproach is to put the hopes in sythetic biology and develop the new way, synthetic way, for its production [1,3].

'''Biology of terpenoids'''

For initiation of any synthetic biology process in metabolic engineering in that matter it is important to have some basic understanding of biosynthesis and regulation of terpenoids. Terpenoids, a class of isoprenoids, are natural products often sythesized by plants. They represent a large and diverse class and have many functions as primarly metabolites involved in growth and development of plants, or as secundary metabolites that optimize the interaction between the plant and the environment. Eventhought they are structural diverse, the share a common biosynthetic origin and follow similar synthesis tracks. All terpenoids are derived from repetitive fusion of isoprene units (C5 H8) [3]. The number of isoprene units determines their classification. For isoprenoid synthesis plants use mevalonate-dependent (MEV) pathway and mevalonte-independent pathway or alternative deoxyxylulose 5-phosphate pathway (DXP). In higher plants the biosynthesis begins with the generation of isopentenyl pyrophosphate (IPP). IPP is derived via the classic mevalonic acid pathway from acetly-coenzime A (acetyl-CoA). The IPP is isomerized to its allylic isomer dimethylallyl pyrophosphate (DMAPP) [3]. The continous condensation of IPP and DMAPP units leads to the formaton of prenylated pyrophosphates, the immediate precursors of the different terpenoid classes. So IPP and DMAPP are combined to form larger isoprenoids, such as farnesyl diphosphate (FPP). Condensation of IPP and DMAPP than units leads to the formation of precursors of the different terpenoid classes. Shematic production of terpenoids in ilustraded in this picture. Similary to plants, yeast Sacharomyces cerevisae uses the clasical mevalonate pathway for the synthesis of isoprenoids[3]. Prokariontes mostly use the DXP pathway for production IPP and DMAPP. This is an alternative pathway for IPP synthesis. This pathway uses Escherichia coli and some other bacteria for their terpenoid synthesis. IPP and DMAPP precurosr are essential to E. Coli for prenylation (addition of hydrophobic molecules to a protein or chemical compound) of tRNA and the synthesis of farnesyl pyrophosphate (FPP). FPP is then used for quinone (class of organic compounds) and cell wall synthesis. The DPX pathway begins with the glycolytic intermediates glyceraldehyde-3-phosphate (G3P) and pyruvate and continues through enzymatic steps for production of IPP and DMAPP. Therefore IPP and DMAPP are the end product in both way,MEV and DPX pathway [1,3].

=== METABOLIC ENGINEERING FOR PRODUCTION OD TERPENOIDS ===

Metabolic engineering represents an improvement of production, construction, or cellular properties through the modification of specific biochemical reactions. It is described as the use of genetic engineering to reshape or modify the metabolism of organism. The aim is to optimize genetic and regulatory processes in cells to increase the production of a certain substance that we find in nature. It is based on optimisation of existing biochemical pathways or the introduction of natural pathway components in bacteria, yeast or plants. The main goal is the production of high-yield of specific metabolites that we can use in medicine or biotechnology. With progress in synthetic biology we can design a biologic funcion that do not exist in nature. Consequently, we can create and construct new biological components or redesign exisitng biological systems. In plants, most terpenoids are produced in a very small quantities in their natural sources. Extraction of this compouds from plants is expensive and therfore not economicaly accetable. So there is a big disagreement between the insistence for supply of terpenoids and therefore that leads to delay for their widespread application. With the understanding of terpenoid synthesis and progress in synthetic biology we are now able of redesign and modify the isoprenoid pathway to increase the terpenoid productivity [2,4]. ([http://www.disketa.si/media/uploads/2015-01/cd201ade6a167488767dfb1b118587bc.jpg See image here])

'''Microbal host'''

To elimante the need for plant extraction it is practical to produce terpenoid compouds at high yield in microbal host by introducing a heterlogous isoprenoid pathway into bacteria E.coli. Microorganisms, such as bacteria are attractive alternatives as hosts because they have rapid doubling time, they can achieve robussnes under process conditions and because of the simplicity of product putrification. And the costs are significantly lower than working with plants. With the engineering the DXP pathway we can increase the supply of isoprenoid precurosor needed for high level productio of isoprenoids in E.coli. [1,3]

'''Operons'''

Operon is a a funcional unit of DNA that contains a cluster of genes under the control of a single promotor. The synthesis of natural or synthetic products involves the introduction of a large number of genes. In this case these genes were encoding the enzymes needed for metabolic pathway. So it is useful to put certain genes under the control of a single promotor. For that reason Plac promotor was used in this study [1,5].

=== EXPERIMENT ===

'''Synthase gene asemby and amorphadiene production'''

For high level production and changing the difficulties in expressing teprene synthases they first synthesized and expressed a codon-optimized variants of ADS . ADS is a gene encoding amorphadiene synthase and is sequentially oxidised by citorchromeP450 CYP71AV1 and reduced by artemisinic aldehyde reductase (DBR2). Amorphadyene synthase is an enzyme and catalyse the first step in the anitmalarial drug artemisnin synthesis. It has an inportant role in catalysing the reaction of FPP to amorphadiene. Beacause of its importaint step towards artemisnin biosynthesis it was designed and improved for the purpuse to acheve high-level expression in E.coli. For ADS gene synthesis they used two step assemly and one step amplification PCR.(1) Assemby PCR is a useful technique for production novel gene sequences. With this method we can put together short fragments of DNA and it is also a good choice for gene construction. Reserches in this article analysed the sequence of three ADS genes from independet clones and identified mutations in each of the genes. Than, two site directed mutagenesis reaction was used for assembly and generation a functional ADS gene from two clones. Than they measure the amorphadiene production by the amorphadiene synthase in E.coli DH10B with natural (nonengineered DPX pathway) and in E.coli with engineered DPX pathway. E. coli with engineerd pathway was harboring the pSOE4 plasmid (DXP pathway operon with dxs,IppHp and ispA gene). The result sugested that FPP synthesis limited amorphadiene production in this engineered host [1,6]. ([http://www.disketa.si/media/uploads/2015-01/0bddf07f946e6f5be0638090dee56490.jpg See image here])

'''Egineering the mevalonate-dependet pathway in E.coli'''

The aim was to increase the intracellular concentration of FPP substrate. Genes encoding the MEV pathway from S.cerevisae was assembled into operons and expressed in E.coli. Because of the complexity of engineering numerous gene biosynthetic pathway, genes were divided into to operons, top and bottom.Top operon, MevT, consisted of three enzymes: acetoacetyl-CoA thiolase (atoB), HMG-CoA synthases (HMGS) and HMG-CoA reductase (tHMGR). This operon was responsible for transformation of acetyl-coA to mevalonate. Thre different bottom operons pMevB (ERG12, ERG8,MVD1 ), pMBI (ERG12, ERG8, MVD1, idi) and pMBIS (ERG12, ERG8, MVD1, idi, ispA) transformed mevalonate to IPP, DMAPP and consequently to FPP. The next step was to analyse and test the funcionality of this pathway. For this purpuse E.coli strand DYM1 with incoplemplete isoprenoid synthesis was transformed with plasmid expressing three bottom operon constuct- pMevB, pMBI and pMBI. E.coli strand DYM1 had a deletion in the ispC gene for encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase, and was not able to synthesise 2-C-methyl-D-erythritol 4-phosphate(MEP), which is important intermediate in isoprenoid biosynthesis[1]. Because of this deletion the synthesis of precursors IPP and DMAP is blocked. So then they tested what is going to happen if they grew the strain DYM1 in the presence of 2-C-methyl-D-ertihritol and in medium with mevalonate without methylerythritol since DYM1 strain was not capable to synthesisie MEP. As expected , all strains expressing operon from S.cerevisiae grew on the plates in the presence of 2-c-methyl-D-erythritol. But only strains with pMBI or pMBIS grew on plates with 1mM mevalonate in the absence of methylerythol. This was the conformation that synthetic MBI and MBIS operons were functional, because they were capable to support the growth of E.coli without ispC gene by supplying cells with IPP and DMAPP. Next they completed the mevalonate pathway by transforming the pMevT plasmid (expressing three genes mentioned before-atoB, HMGS and tHMGR) with pMBI or pMBIS. This coexpresion of two operons, pMevT and pMBI /pMBIS ,completed the mevlonate pathway and allowed the synthesis of sesquiterpene precursors from acetyl-CoA, IPP and DMAPP. Therefore, coexpression complemented the ispC deletion even in the absence of mevalonate, what was the proof, that MevT operon was functional [1,7].

'''Amorpadiene synthesis from mevalonate'''

The aim was to achieve high-level production of amorphadiene and to find out if the amount of FPP was limiting amoprhadiene output. As mentioned, E.coli was used as heterologous microorganism in which coupled mevalonate pathway with amorphadiene synthesis. Cells with ADS gene were coexpressed with MBIS operon and grown in medium with increasing amounts (0 to 40 mM) of exogenus mevalonate. The culture extract was analysed with gas chromatography-mass spectrometry, analytical method for identification different substances in a test sample. It combines the features of mass spectrometry and gas-liquid chromatography. Results showed that MBIS operon did not limit the amorphadiene production at the highest mevalonate concentration(40mM). ([http://www.disketa.si/media/uploads/2015-01/7711d592584a53282f2ecb0fd678ef40.jpg See image 1 here]) Cultures with 40 mM mevalonate added produced much more amoprhadiene. With time, amorphadiene concentration was dropping beacause of the loss of the volatile terpene[1]. By adding more than 10 mM mevalonate in control cultures without ADS, the severe growth ihibition has been reported. ([http://www.disketa.si/media/uploads/2015-01/2a21f55f0fea31ffcbe89c866c9150f7.jpg See image here]) To discover the cause ofthis inhibition, the growth of E.coli DH10B from strains with empty, pMKPMK, pMevB, pMBI or pMBIS plasmid was measured in media with increasing concentrations of mevalonate. The addition of 5mM mevalonate to the medium inhibited the growth of cells with pMevB. But 5mM concentrations of mevalonate did not alter the growth of cells with pMKPMK, PMBI or pMBIS plasmid. This indicated that the accomulation of IPP is toxic and inhibits normal cell growth. The accmulation mostly occurs in cells with high flux through mevalonate pathway. The next step was comparison of intracellular prenyl pyrophosphate (FPP) in strains. This was done by feeding the cells harboring the different mevalonte operon construct with radiolabeled mevalonate. Than the labeled metabolites were tracked. For this experiment cell suspensions of E.coli harboring pMevB+pTrc99A, pMBI+ pTrc99A, pMBIS+ pTrc99A or pMBIS+pADS were used. pTRc99A is the bacterial expression vector with inducible to IPTG lacI promoter. The result showed that strains with MevB operon accumulated IPP but not FPP, when MBI and MBIS strains accumulated FPP. ([http://www.disketa.si/media/uploads/2015-01/31ad7a215f016dc26aceacf92eb46712.jpg See image here]) Because of the simultaneous expression of the amorphadiene synthase the waste of FPP that accumulated in the MBIS host was consumed. This was shown by a decrease in intracellular FPP. Cells expresing MBIS acumulated FPP and showed growth inhibition in the presence of 10 mM mevalonate. The prediction was that the coexpression of ADS would ease the growth inhibition by forwarding the prenyl pyrophosphate intermediates to volatile terpene olefin. For this test, the cell with pMBIS and empty expression vector pTrc99A (without ADS)and cells with pMBIS and pADS vectors were used. The medium was supplemented with mevalonate concentations ( 0mM, 5mM, 10mM, 20mM or 40mM). Two hours after addition of mevalonate and inducer IPTG, the growth inhibition was observed. ([http://www.disketa.si/media/uploads/2015-01/94b5f3d5636fe767316f770f3e2b0448.jpg See image here]) The result showed that the strains with pMBIS +ADS started to grow, but strain lacking the ADS still showed growth inhbition at higher concentrations of mevalonate. This supported the conclusion that converion of FPP to amophadiene has an important role in minimising growth inhibition. Briefly these result showed that engineered mevalonate patway produces high levels of prenyl pyrophosphate precursors. If the IPP isomerase, FPP synthase and terpen synthase is absent, IPP can acumulate and be toxic to the cells. The toxic levels of intracellular prenyl pyrphosphate can accumulate [1].

'''Amorphadiene synthesis from acetyl-Coa'''

The main goal was to achieve high amoprhaidene production from a simple and inexpensive carbon source. E.coli strains harboring pMevT,pMBIS and pADS plasmids were transformed with pMevT plasmid. Subsequently the strains were tested for ability to produce amoprhadiene in the absence of exogenous mevalonate. The comparison between E.coli expressing the native DPX pathway and engineered pathway as made. They tested native DXP pathway, engineered DPX pathway, mevalonate bottom pathway in the absence of mevalonate, mevlonate bottom pathway in medium suplemented with mevalonate, the complete mevalonate pathway and the complete mevalonate pathway supplemented with 0,8% glycerol. Glycerol was addded to investigate the effect of amorphadiene production of supplying as single carbon source and for prolongation of amorphadiene production. Breifly the resulut showed that expression of the mevalonate-dependent isoprenoid biosynthetic pathway delivers hight levels of isoprenoid precursor for the production of sesquiterpene [1]. ([http://www.disketa.si/media/uploads/2015-01/a31fe07a7c66854b1dc3ff36f8ed01ef.jpg See image here])

=== DISSCUSION ===
The identification of genes encoding the enzymes involved in the artemisinin-biosynthesis pathway together with advances in metabolic engineering provides new options for engineering artemisinin biosynthesis. Heterologous production of artemisnin in microorganism such as E.coli. is an alternative way for production of artmeinsin in high yields. But eventhougt micoorganisms has shown great potential in that matter, optimisation of these heterlogous pathways still require maximal titer. Inequal gene expression can lead to accumulation of toxic metabolic intermediates and to over or under production of enzymes involved in pathway. (4). Conclusion based on these study shows that poor expression of the amoprhaidene synthase genes (ADS) limits high terpene olfein yields from the host. To achieve better results , in this study they chose to synthesiyse an amophaiene synthase gene (ADS). The comparison between E.coli expressing native sesquiterpene synthase genes and E.coli expressing the synthetic ADS gene showed big improvement in terpene synthesis due to synthetic ADS gene. The native DPX pathway showed poor supply of the prenyl pyrophosphate precuror. And that limited terpenoid -carotenoid yields in E.coli. With the metabolic engineering of the DPX pathway the flux of carotenoid accumulation showed signifcant increase. By using the mevalonate-dependent isoprenoid pathway from yeast S. cerevisae and engineering that pathway in bacteria E.coli, we avoid the native regulatory elements found in yeast while bypassing those of E.coli DXP pathway. In this study results showed growth inhibition in cells without ADS due to the vast excess of preny pyrophosphate. But the coexpression of a synthetic sesquiterpene synthase can contribute to reducing the excess pool of precursors and eliminate growth inhibition. In this study total biosynthesis of artemisnin was not completely achieved. But these results can help in further investigation for poduction of artemisnic acid. This acid can then be putrified and chemically converted into artemsinin. [1,7].

=== CONCLUSIONS ===
To sum things up, artmeinsin, a sesquiterpene isolated from Artemisia annua,represent a novel drug for malaria treatment. The present artemisnin derivates are too expensive for general use and plants can not produce artmeinsin in big quantities. This fact inspired scientist to focus on developing the way for production of artemisnin in high quantities and to avoid big costs. The promising tool in alternative source of artmeinsin is the use of microoganisms. The aim is to transfer the genes encoding enzymes involved in biosynthetic pathway for artemisnin into a hight producing host organism. Several study have been reporting on engineering the metabolic pathway in S.cerevisae to produce the precursor for artmeisnic acid. With the effort in synthetic biology they used modified mevalonate pathway . With the help of syntehtic bilogy the yeast cells were engineerd to express ezymes needed for production of artmemisnic acid. But in this paper the main focus was on engineering a mevalonate pathway in bacteria E.coli. By engineering a new metabolic pathway in E.coli we can easily synthesise a precursor nedeed for artemisnin production. The goal is to produce drugs in better and cheape ways. But, why focus on artemisnin? Well, acording on recent studies artemisnin shows to be very sucesfull for the treatmen all strain of malaria. More than a milion people affected with malaria have already been cured by arteminsin. As already mentioned , because of very hight costs and limited production of arteminin in plants most populations in Africa can not afford it. Progress made in synthetic biology and ability to produce amorphadiene in microorganisms in big quantites opens a new possibility to produce low cost artemisnin and contributes to the brighter future od antimalaria drug deveopment, and therefore saving milions from suffering from malaria. [1,2, 3].

[1]Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nature biotehnology; vol 21; pages 796-802, July 2003

[2] T. Mosses, J. Pollier, J. M. Thevelein, A. Goossens. Bioengineering of plant (tri)terpenoids: from metabolic engineering of plants to synthetic biology in vivo and in vitro. New Pathyologist. 200: 27-43, 2013

[3] M.Farhi, M. Kozin, S. Duchin, A. Vainstein. Metabolic engineering of plants for artemisinin synthesis. Biotehnology and genetic engineering rewiews.Vol.29,No.2: 135-148, 2013

[4] Synthetic biology ; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Synthetic_biology http://en.wikipedia.org/wiki/Synthetic_biology]

[5]Operon; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Operon http://en.wikipedia.org/wiki/Operon]

[6] M. K. Julsing, G. van Pouderoyen, J. van der Veen,H. J. Woerdenbag, O. Kayser, W. J. Quax. Amorphadiene synthase: probing the active site model with directed mutagenesis. Directed mutagenesis of AMDS.Chapter 4: 47-56

[7]J.R. Anthony, L. C. Anthony, F. Nowroozi, G. Kwon, J.D. Newman, J.D. Keasling. Optimization of themevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene. Metabolic Engineering. 11: 13-19, 2009

Engineering a mevalonate pathway in Escherichia coli for production of terpenoids

2015-01-19T23:43:19Z

Ana90:

'''Ana Kapraljević'''

[http://www2.lbl.gov/ttd/publications/2837pub1.pdf Engineering a mevalonate pathway in Escherichia coli for production of terpenoids]
Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling ;Nature biotehnology; July 2003; vol 21; pages 796-802

=== INTRODUCTION ===

Plants can synthesise and accumulate a wide range of small molecules or natural products involved in psyhological an ecological process. With the knowledge we know about their biosyntesis we used them as commercial flavors, fragnance compunds, colorants and terapeutical drugs. Their therapeutic potential is used for production of anticancer or antimalarial drugs, such as artemisnin. The most numerous and diverse class of natural products used in production for antimalarial drug are isoprenoids or more corectly their subclass, terpenoids. Malaria represent a major health problem in tropic and subtropic. Majority of death occur in children living in Africa. But the problem is that terpenoids are in plants produced in very small quantites and consequently not very economically feasible. For that reason the drugs for treatin malaria, artemisnin, is in short supply and is still unaffordable for most people living in malaria-endanger countries. Scientists are now focusing on alternative and cheaper way for terpenoid synthesis that can be enchanched and became economically acceptable. The alternative way is the expression of synthetic amorphadiene, precursor to artemisnin, and mevalonate isoprenoid way from yeas Saccharomyces cerevisiae in bacteria. In this paper I will summerize the project made in the production of amorphadiene by biotehnology and synthetic biology approach [1,2].

'''Artemisnin'''

Artemisnin is a substance that has antimalarial potential. Malaria is a infectious disease caused by parasitic protozan of the Plasmodium species. In the certain parts of the world malaria takes lives of more people worldwide than any other infecious disease, so it is a global problem. Death to malaria has been nowdays reduced thanks to the use of artemisnin-based combination therapy. Artemisnin is a natural compoud from plant ''Artemisia annua''. Chemically it is a sesquiterpene lactone, amorphadiene. The total sythesis of artemisnin is difficult and not very economical,so the new aproach is to put the hopes in sythetic biology and develop the new way, synthetic way, for its production [1,3].

'''Biology of terpenoids'''

For initiation of any synthetic biology process in metabolic engineering in that matter it is important to have some basic understanding of biosynthesis and regulation of terpenoids. Terpenoids, a class of isoprenoids, are natural products often sythesized by plants. They represent a large and diverse class and have many functions as primarly metabolites involved in growth and development of plants, or as secundary metabolites that optimize the interaction between the plant and the environment. Eventhought they are structural diverse, the share a common biosynthetic origin and follow similar synthesis tracks. All terpenoids are derived from repetitive fusion of isoprene units (C5 H8) [3]. The number of isoprene units determines their classification. For isoprenoid synthesis plants use mevalonate-dependent (MEV) pathway and mevalonte-independent pathway or alternative deoxyxylulose 5-phosphate pathway (DXP). In higher plants the biosynthesis begins with the generation of isopentenyl pyrophosphate (IPP). IPP is derived via the classic mevalonic acid pathway from acetly-coenzime A (acetyl-CoA). The IPP is isomerized to its allylic isomer dimethylallyl pyrophosphate (DMAPP) [3]. The continous condensation of IPP and DMAPP units leads to the formaton of prenylated pyrophosphates, the immediate precursors of the different terpenoid classes. So IPP and DMAPP are combined to form larger isoprenoids, such as farnesyl diphosphate (FPP). Condensation of IPP and DMAPP than units leads to the formation of precursors of the different terpenoid classes. Shematic production of terpenoids in ilustraded in this picture. Similary to plants, yeast Sacharomyces cerevisae uses the clasical mevalonate pathway for the synthesis of isoprenoids[3]. Prokariontes mostly use the DXP pathway for production IPP and DMAPP. This is an alternative pathway for IPP synthesis. This pathway uses Escherichia coli and some other bacteria for their terpenoid synthesis. IPP and DMAPP precurosr are essential to E. Coli for prenylation (addition of hydrophobic molecules to a protein or chemical compound) of tRNA and the synthesis of farnesyl pyrophosphate (FPP). FPP is then used for quinone (class of organic compounds) and cell wall synthesis. The DPX pathway begins with the glycolytic intermediates glyceraldehyde-3-phosphate (G3P) and pyruvate and continues through enzymatic steps for production of IPP and DMAPP. Therefore IPP and DMAPP are the end product in both way,MEV and DPX pathway [1,3].

=== METABOLIC ENGINEERING FOR PRODUCTION OD TERPENOIDS ===

Metabolic engineering represents an improvement of production, construction, or cellular properties through the modification of specific biochemical reactions. It is described as the use of genetic engineering to reshape or modify the metabolism of organism. The aim is to optimize genetic and regulatory processes in cells to increase the production of a certain substance that we find in nature. It is based on optimisation of existing biochemical pathways or the introduction of natural pathway components in bacteria, yeast or plants. The main goal is the production of high-yield of specific metabolites that we can use in medicine or biotechnology. With progress in synthetic biology we can design a biologic funcion that do not exist in nature. Consequently, we can create and construct new biological components or redesign exisitng biological systems. In plants, most terpenoids are produced in a very small quantities in their natural sources. Extraction of this compouds from plants is expensive and therfore not economicaly accetable. So there is a big disagreement between the insistence for supply of terpenoids and therefore that leads to delay for their widespread application. With the understanding of terpenoid synthesis and progress in synthetic biology we are now able of redesign and modify the isoprenoid pathway to increase the terpenoid productivity [2,4]. ([http://www.disketa.si/media/uploads/2015-01/cd201ade6a167488767dfb1b118587bc.jpg See image here])

'''Microbal host'''

To elimante the need for plant extraction it is practical to produce terpenoid compouds at high yield in microbal host by introducing a heterlogous isoprenoid pathway into bacteria E.coli. Microorganisms, such as bacteria are attractive alternatives as hosts because they have rapid doubling time, they can achieve robussnes under process conditions and because of the simplicity of product putrification. And the costs are significantly lower than working with plants. With the engineering the DXP pathway we can increase the supply of isoprenoid precurosor needed for high level productio of isoprenoids in E.coli. [1,3]

'''Operons'''

Operon is a a funcional unit of DNA that contains a cluster of genes under the control of a single promotor. The synthesis of natural or synthetic products involves the introduction of a large number of genes. In this case these genes were encoding the enzymes needed for metabolic pathway. So it is useful to put certain genes under the control of a single promotor. For that reason Plac promotor was used in this study [1,5].

=== EXPERIMENT ===

'''Synthase gene asemby and amorphadiene production'''

For high level production and changing the difficulties in expressing teprene synthases they first synthesized and expressed a codon-optimized variants of ADS . ADS is a gene encoding amorphadiene synthase and is sequentially oxidised by citorchromeP450 CYP71AV1 and reduced by artemisinic aldehyde reductase (DBR2). Amorphadyene synthase is an enzyme and catalyse the first step in the anitmalarial drug artemisnin synthesis. It has an inportant role in catalysing the reaction of FPP to amorphadiene. Beacause of its importaint step towards artemisnin biosynthesis it was designed and improved for the purpuse to acheve high-level expression in E.coli. For ADS gene synthesis they used two step assemly and one step amplification PCR.(1) Assemby PCR is a useful technique for production novel gene sequences. With this method we can put together short fragments of DNA and it is also a good choice for gene construction. Reserches in this article analysed the sequence of three ADS genes from independet clones and identified mutations in each of the genes. Than, two site directed mutagenesis reaction was used for assembly and generation a functional ADS gene from two clones. Than they measure the amorphadiene production by the amorphadiene synthase in E.coli DH10B with natural (nonengineered DPX pathway) and in E.coli with engineered DPX pathway. E. coli with engineerd pathway was harboring the pSOE4 plasmid (DXP pathway operon with dxs,IppHp and ispA gene). The result sugested that FPP synthesis limited amorphadiene production in this engineered host [1,6]. ([http://www.disketa.si/media/uploads/2015-01/0bddf07f946e6f5be0638090dee56490.jpg See image here])

'''Egineering the mevalonate-dependet pathway in E.coli'''

The aim was to increase the intracellular concentration of FPP substrate. Genes encoding the MEV pathway from S.cerevisae was assembled into operons and expressed in E.coli. Because of the complexity of engineering numerous gene biosynthetic pathway, genes were divided into to operons, top and bottom.Top operon, MevT, consisted of three enzymes: acetoacetyl-CoA thiolase (atoB), HMG-CoA synthases (HMGS) and HMG-CoA reductase (tHMGR). This operon was responsible for transformation of acetyl-coA to mevalonate. Thre different bottom operons pMevB (ERG12, ERG8,MVD1 ), pMBI (ERG12, ERG8, MVD1, idi) and pMBIS (ERG12, ERG8, MVD1, idi, ispA) transformed mevalonate to IPP, DMAPP and consequently to FPP. The next step was to analyse and test the funcionality of this pathway. For this purpuse E.coli strand DYM1 with incoplemplete isoprenoid synthesis was transformed with plasmid expressing three bottom operon constuct- pMevB, pMBI and pMBI. E.coli strand DYM1 had a deletion in the ispC gene for encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase, and was not able to synthesise 2-C-methyl-D-erythritol 4-phosphate(MEP), which is important intermediate in isoprenoid biosynthesis[1]. Because of this deletion the synthesis of precursors IPP and DMAP is blocked. So then they tested what is going to happen if they grew the strain DYM1 in the presence of 2-C-methyl-D-ertihritol and in medium with mevalonate without methylerythritol since DYM1 strain was not capable to synthesisie MEP. As expected , all strains expressing operon from S.cerevisiae grew on the plates in the presence of 2-c-methyl-D-erythritol. But only strains with pMBI or pMBIS grew on plates with 1mM mevalonate in the absence of methylerythol. This was the conformation that synthetic MBI and MBIS operons were functional, because they were capable to support the growth of E.coli without ispC gene by supplying cells with IPP and DMAPP. Next they completed the mevalonate pathway by transforming the pMevT plasmid (expressing three genes mentioned before-atoB, HMGS and tHMGR) with pMBI or pMBIS. This coexpresion of two operons, pMevT and pMBI /pMBIS ,completed the mevlonate pathway and allowed the synthesis of sesquiterpene precursors from acetyl-CoA, IPP and DMAPP. Therefore, coexpression complemented the ispC deletion even in the absence of mevalonate, what was the proof, that MevT operon was functional [1,7].

'''Amorpadiene synthesis from mevalonate'''

The aim was to achieve high-level production of amorphadiene and to find out if the amount of FPP was limiting amoprhadiene output. As mentioned, E.coli was used as heterologous microorganism in which coupled mevalonate pathway with amorphadiene synthesis. Cells with ADS gene were coexpressed with MBIS operon and grown in medium with increasing amounts (0 to 40 mM) of exogenus mevalonate. The culture extract was analysed with gas chromatography-mass spectrometry, analytical method for identification different substances in a test sample. It combines the features of mass spectrometry and gas-liquid chromatography. Results showed that MBIS operon did not limit the amorphadiene production at the highest mevalonate concentration(40mM). ([http://www.disketa.si/media/uploads/2015-01/7711d592584a53282f2ecb0fd678ef40.jpg See image 1 here]) Cultures with 40 mM mevalonate added produced much more amoprhadiene. With time, amorphadiene concentration was dropping beacause of the loss of the volatile terpene[1]. By adding more than 10 mM mevalonate in control cultures without ADS, the severe growth ihibition has been reported. ([http://www.disketa.si/media/uploads/2015-01/2a21f55f0fea31ffcbe89c866c9150f7.jpg See image here]) To discover the cause ofthis inhibition, the growth of E.coli DH10B from strains with empty, pMKPMK, pMevB, pMBI or pMBIS plasmid was measured in media with increasing concentrations of mevalonate. The addition of 5mM mevalonate to the medium inhibited the growth of cells with pMevB. But 5mM concentrations of mevalonate did not alter the growth of cells with pMKPMK, PMBI or pMBIS plasmid. This indicated that the accomulation of IPP is toxic and inhibits normal cell growth. The accmulation mostly occurs in cells with high flux through mevalonate pathway. The next step was comparison of intracellular prenyl pyrophosphate (FPP) in strains. This was done by feeding the cells harboring the different mevalonte operon construct with radiolabeled mevalonate. Than the labeled metabolites were tracked. For this experiment cell suspensions of E.coli harboring pMevB+pTrc99A, pMBI+ pTrc99A, pMBIS+ pTrc99A or pMBIS+pADS were used. pTRc99A is the bacterial expression vector with inducible to IPTG lacI promoter. The result showed that strains with MevB operon accumulated IPP but not FPP, when MBI and MBIS strains accumulated FPP. ([http://www.disketa.si/media/uploads/2015-01/31ad7a215f016dc26aceacf92eb46712.jpg See image here]) Because of the simultaneous expression of the amorphadiene synthase the waste of FPP that accumulated in the MBIS host was consumed. This was shown by a decrease in intracellular FPP. Cells expresing MBIS acumulated FPP and showed growth inhibition in the presence of 10 mM mevalonate. The prediction was that the coexpression of ADS would ease the growth inhibition by forwarding the prenyl pyrophosphate intermediates to volatile terpene olefin. For this test, the cell with pMBIS and empty expression vector pTrc99A (without ADS)and cells with pMBIS and pADS vectors were used. The medium was supplemented with mevalonate concentations ( 0mM, 5mM, 10mM, 20mM or 40mM). Two hours after addition of mevalonate and inducer IPTG, the growth inhibition was observed. ([http://www.disketa.si/media/uploads/2015-01/94b5f3d5636fe767316f770f3e2b0448.jpg See image here]) The result showed that the strains with pMBIS +ADS started to grow, but strain lacking the ADS still showed growth inhbition at higher concentrations of mevalonate. This supported the conclusion that converion of FPP to amophadiene has an important role in minimising growth inhibition. Briefly these result showed that engineered mevalonate patway produces high levels of prenyl pyrophosphate precursors. If the IPP isomerase, FPP synthase and terpen synthase is absent, IPP can acumulate and be toxic to the cells. The toxic levels of intracellular prenyl pyrphosphate can accumulate [1].

'''Amorphadiene synthesis from acetyl-Coa'''

The main goal was to achieve high amoprhaidene production from a simple and inexpensive carbon source. E.coli strains harboring pMevT,pMBIS and pADS plasmids were transformed with pMevT plasmid. Subsequently the strains were tested for ability to produce amoprhadiene in the absence of exogenous mevalonate. The comparison between E.coli expressing the native DPX pathway and engineered pathway as made. They tested native DXP pathway, engineered DPX pathway, mevalonate bottom pathway in the absence of mevalonate, mevlonate bottom pathway in medium suplemented with mevalonate, the complete mevalonate pathway and the complete mevalonate pathway supplemented with 0,8% glycerol. Glycerol was addded to investigate the effect of amorphadiene production of supplying as single carbon source and for prolongation of amorphadiene production. Breifly the resulut showed that expression of the mevalonate-dependent isoprenoid biosynthetic pathway delivers hight levels of isoprenoid precursor for the production of sesquiterpene [1]. ([http://www.disketa.si/media/uploads/2015-01/a31fe07a7c66854b1dc3ff36f8ed01ef.jpg See image here])

=== DISSCUSION ===
The identification of genes encoding the enzymes involved in the artemisinin-biosynthesis pathway together with advances in metabolic engineering provides new options for engineering artemisinin biosynthesis. Heterologous production of artemisnin in microorganism such as E.coli. is an alternative way for production of artmeinsin in high yields. But eventhougt micoorganisms has shown great potential in that matter, optimisation of these heterlogous pathways still require maximal titer. Inequal gene expression can lead to accumulation of toxic metabolic intermediates and to over or under production of enzymes involved in pathway. (4). Conclusion based on these study shows that poor expression of the amoprhaidene synthase genes (ADS) limits high terpene olfein yields from the host. To achieve better results , in this study they chose to synthesiyse an amophaiene synthase gene (ADS). The comparison between E.coli expressing native sesquiterpene synthase genes and E.coli expressing the synthetic ADS gene showed big improvement in terpene synthesis due to synthetic ADS gene. The native DPX pathway showed poor supply of the prenyl pyrophosphate precuror. And that limited terpenoid -carotenoid yields in E.coli. With the metabolic engineering of the DPX pathway the flux of carotenoid accumulation showed signifcant increase. By using the mevalonate-dependent isoprenoid pathway from yeast S. cerevisae and engineering that pathway in bacteria E.coli, we avoid the native regulatory elements found in yeast while bypassing those of E.coli DXP pathway. In this study results showed growth inhibition in cells without ADS due to the vast excess of preny pyrophosphate. But the coexpression of a synthetic sesquiterpene synthase can contribute to reducing the excess pool of precursors and eliminate growth inhibition. In this study total biosynthesis of artemisnin was not completely achieved. But these results can help in further investigation for poduction of artemisnic acid. This acid can then be putrified and chemically converted into artemsinin. [1,7].

=== CONCLUSIONS ===
To sum things up, artmeinsin, a sesquiterpene isolated from Artemisia annua,represent a novel drug for malaria treatment. The present artemisnin derivates are too expensive for general use and plants can not produce artmeinsin in big quantities. This fact inspired scientist to focus on developing the way for production of artemisnin in high quantities and to avoid big costs. The promising tool in alternative source of artmeinsin is the use of microoganisms. The aim is to transfer the genes encoding enzymes involved in biosynthetic pathway for artemisnin into a hight producing host organism. Several study have been reporting on engineering the metabolic pathway in S.cerevisae to produce the precursor for artmeisnic acid. With the effort in synthetic biology they used modified mevalonate pathway . With the help of syntehtic bilogy the yeast cells were engineerd to express ezymes needed for production of artmemisnic acid. But in this paper the main focus was on engineering a mevalonate pathway in bacteria E.coli. By engineering a new metabolic pathway in E.coli we can easily synthesise a precursor nedeed for artemisnin production. The goal is to produce drugs in better and cheape ways. But, why focus on artemisnin? Well, acording on recent studies artemisnin shows to be very sucesfull for the treatmen all strain of malaria. More than a milion people affected with malaria have already been cured by arteminsin. As already mentioned , because of very hight costs and limited production of arteminin in plants most populations in Africa can not afford it. Progress made in synthetic biology and ability to produce amorphadiene in microorganisms in big quantites opens a new possibility to produce low cost artemisnin and contributes to the brighter future od antimalaria drug deveopment, and therefore saving milions from suffering from malaria. [1,2, 3].

[1]Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nature biotehnology; vol 21; pages 796-802, July 2003

[2] T. Mosses, J. Pollier, J. M. Thevelein, A. Goossens. Bioengineering of plant (tri)terpenoids: from metabolic engineering of plants to synthetic biology in vivo and in vitro. New Pathyologist. 200: 27-43, 2013

[3] M.Farhi, M. Kozin, S. Duchin, A. Vainstein. Metabolic engineering of plants for artemisinin synthesis. Biotehnology and genetic engineering rewiews.Vol.29,No.2: 135-148, 2013

[4] Synthetic biology ; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Synthetic_biology http://en.wikipedia.org/wiki/Synthetic_biology]

[5]Operon; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Operon http://en.wikipedia.org/wiki/Operon]

[6] M. K. Julsing, G. van Pouderoyen, J. van der Veen,H. J. Woerdenbag, O. Kayser, W. J. Quax. Amorphadiene synthase: probing the active site model with directed mutagenesis. Directed mutagenesis of AMDS.Chapter 4: 47-56

[7]J.R. Anthony, L. C. Anthony, F. Nowroozi, G. Kwon, J.D. Newman, J.D. Keasling. Optimization of themevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene. Metabolic Engineering. 11: 13-19, 2009

Engineering a mevalonate pathway in Escherichia coli for production of terpenoids

2015-01-19T23:41:05Z

Ana90: New page: '''Ana Kapraljević''' [http://www2.lbl.gov/ttd/publications/2837pub1.pdf Engineering a mevalonate pathway in Escherichia coli for production of terpenoids] Vincent JJ Martin, Douglas J P...

'''Ana Kapraljević'''

[http://www2.lbl.gov/ttd/publications/2837pub1.pdf Engineering a mevalonate pathway in Escherichia coli for production of terpenoids]
Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling ;Nature biotehnology; July 2003; vol 21; pages 796-802

=== INTRODUCTION ===
Plants can synthesise and accumulate a wide range of small molecules or natural products involved in psyhological an ecological process. With the knowledge we know about their biosyntesis we used them as commercial flavors, fragnance compunds, colorants and terapeutical drugs. Their therapeutic potential is used for production of anticancer or antimalarial drugs, such as artemisnin. The most numerous and diverse class of natural products used in production for antimalarial drug are isoprenoids or more corectly their subclass, terpenoids. Malaria represent a major health problem in tropic and subtropic. Majority of death occur in children living in Africa. But the problem is that terpenoids are in plants produced in very small quantites and consequently not very economically feasible. For that reason the drugs for treatin malaria, artemisnin, is in short supply and is still unaffordable for most people living in malaria-endanger countries. Scientists are now focusing on alternative and cheaper way for terpenoid synthesis that can be enchanched and became economically acceptable. The alternative way is the expression of synthetic amorphadiene, precursor to artemisnin, and mevalonate isoprenoid way from yeas Saccharomyces cerevisiae in bacteria. In this paper I will summerize the project made in the production of amorphadiene by biotehnology and synthetic biology approach [1,2].

'''Artemisnin'''
Artemisnin is a substance that has antimalarial potential. Malaria is a infectious disease caused by parasitic protozan of the Plasmodium species. In the certain parts of the world malaria takes lives of more people worldwide than any other infecious disease, so it is a global problem. Death to malaria has been nowdays reduced thanks to the use of artemisnin-based combination therapy. Artemisnin is a natural compoud from plant ''Artemisia annua''. Chemically it is a sesquiterpene lactone, amorphadiene. The total sythesis of artemisnin is difficult and not very economical,so the new aproach is to put the hopes in sythetic biology and develop the new way, synthetic way, for its production [1,3].

'''Biology of terpenoids'''
For initiation of any synthetic biology process in metabolic engineering in that matter it is important to have some basic understanding of biosynthesis and regulation of terpenoids. Terpenoids, a class of isoprenoids, are natural products often sythesized by plants. They represent a large and diverse class and have many functions as primarly metabolites involved in growth and development of plants, or as secundary metabolites that optimize the interaction between the plant and the environment. Eventhought they are structural diverse, the share a common biosynthetic origin and follow similar synthesis tracks. All terpenoids are derived from repetitive fusion of isoprene units (C5 H8) [3]. The number of isoprene units determines their classification. For isoprenoid synthesis plants use mevalonate-dependent (MEV) pathway and mevalonte-independent pathway or alternative deoxyxylulose 5-phosphate pathway (DXP). In higher plants the biosynthesis begins with the generation of isopentenyl pyrophosphate (IPP). IPP is derived via the classic mevalonic acid pathway from acetly-coenzime A (acetyl-CoA). The IPP is isomerized to its allylic isomer dimethylallyl pyrophosphate (DMAPP) [3]. The continous condensation of IPP and DMAPP units leads to the formaton of prenylated pyrophosphates, the immediate precursors of the different terpenoid classes. So IPP and DMAPP are combined to form larger isoprenoids, such as farnesyl diphosphate (FPP). Condensation of IPP and DMAPP than units leads to the formation of precursors of the different terpenoid classes. Shematic production of terpenoids in ilustraded in this picture. Similary to plants, yeast Sacharomyces cerevisae uses the clasical mevalonate pathway for the synthesis of isoprenoids[3]. Prokariontes mostly use the DXP pathway for production IPP and DMAPP. This is an alternative pathway for IPP synthesis. This pathway uses Escherichia coli and some other bacteria for their terpenoid synthesis. IPP and DMAPP precurosr are essential to E. Coli for prenylation (addition of hydrophobic molecules to a protein or chemical compound) of tRNA and the synthesis of farnesyl pyrophosphate (FPP). FPP is then used for quinone (class of organic compounds) and cell wall synthesis. The DPX pathway begins with the glycolytic intermediates glyceraldehyde-3-phosphate (G3P) and pyruvate and continues through enzymatic steps for production of IPP and DMAPP. Therefore IPP and DMAPP are the end product in both way,MEV and DPX pathway [1,3].

=== METABOLIC ENGINEERING FOR PRODUCTION OD TERPENOIDS ===
Metabolic engineering represents an improvement of production, construction, or cellular properties through the modification of specific biochemical reactions. It is described as the use of genetic engineering to reshape or modify the metabolism of organism. The aim is to optimize genetic and regulatory processes in cells to increase the production of a certain substance that we find in nature. It is based on optimisation of existing biochemical pathways or the introduction of natural pathway components in bacteria, yeast or plants. The main goal is the production of high-yield of specific metabolites that we can use in medicine or biotechnology. With progress in synthetic biology we can design a biologic funcion that do not exist in nature. Consequently, we can create and construct new biological components or redesign exisitng biological systems. In plants, most terpenoids are produced in a very small quantities in their natural sources. Extraction of this compouds from plants is expensive and therfore not economicaly accetable. So there is a big disagreement between the insistence for supply of terpenoids and therefore that leads to delay for their widespread application. With the understanding of terpenoid synthesis and progress in synthetic biology we are now able of redesign and modify the isoprenoid pathway to increase the terpenoid productivity [2,4]. ([http://www.disketa.si/media/uploads/2015-01/cd201ade6a167488767dfb1b118587bc.jpg See image here])

'''Microbal host'''
To elimante the need for plant extraction it is practical to produce terpenoid compouds at high yield in microbal host by introducing a heterlogous isoprenoid pathway into bacteria E.coli. Microorganisms, such as bacteria are attractive alternatives as hosts because they have rapid doubling time, they can achieve robussnes under process conditions and because of the simplicity of product putrification. And the costs are significantly lower than working with plants. With the engineering the DXP pathway we can increase the supply of isoprenoid precurosor needed for high level productio of isoprenoids in E.coli. [1,3]

'''Operons'''
Operon is a a funcional unit of DNA that contains a cluster of genes under the control of a single promotor. The synthesis of natural or synthetic products involves the introduction of a large number of genes. In this case these genes were encoding the enzymes needed for metabolic pathway. So it is useful to put certain genes under the control of a single promotor. For that reason Plac promotor was used in this study [1,5].

=== EXPERIMENT ===

'''Synthase gene asemby and amorphadiene production'''
For high level production and changing the difficulties in expressing teprene synthases they first synthesized and expressed a codon-optimized variants of ADS . ADS is a gene encoding amorphadiene synthase and is sequentially oxidised by citorchromeP450 CYP71AV1 and reduced by artemisinic aldehyde reductase (DBR2). Amorphadyene synthase is an enzyme and catalyse the first step in the anitmalarial drug artemisnin synthesis. It has an inportant role in catalysing the reaction of FPP to amorphadiene. Beacause of its importaint step towards artemisnin biosynthesis it was designed and improved for the purpuse to acheve high-level expression in E.coli. For ADS gene synthesis they used two step assemly and one step amplification PCR.(1) Assemby PCR is a useful technique for production novel gene sequences. With this method we can put together short fragments of DNA and it is also a good choice for gene construction. Reserches in this article analysed the sequence of three ADS genes from independet clones and identified mutations in each of the genes. Than, two site directed mutagenesis reaction was used for assembly and generation a functional ADS gene from two clones. Than they measure the amorphadiene production by the amorphadiene synthase in E.coli DH10B with natural (nonengineered DPX pathway) and in E.coli with engineered DPX pathway. E. coli with engineerd pathway was harboring the pSOE4 plasmid (DXP pathway operon with dxs,IppHp and ispA gene). The result sugested that FPP synthesis limited amorphadiene production in this engineered host [1,6]. ([http://www.disketa.si/media/uploads/2015-01/0bddf07f946e6f5be0638090dee56490.jpg See image here])

'''Egineering the mevalonate-dependet pathway in E.coli'''
The aim was to increase the intracellular concentration of FPP substrate. Genes encoding the MEV pathway from S.cerevisae was assembled into operons and expressed in E.coli. Because of the complexity of engineering numerous gene biosynthetic pathway, genes were divided into to operons, top and bottom.Top operon, MevT, consisted of three enzymes: acetoacetyl-CoA thiolase (atoB), HMG-CoA synthases (HMGS) and HMG-CoA reductase (tHMGR). This operon was responsible for transformation of acetyl-coA to mevalonate. Thre different bottom operons pMevB (ERG12, ERG8,MVD1 ), pMBI (ERG12, ERG8, MVD1, idi) and pMBIS (ERG12, ERG8, MVD1, idi, ispA) transformed mevalonate to IPP, DMAPP and consequently to FPP. The next step was to analyse and test the funcionality of this pathway. For this purpuse E.coli strand DYM1 with incoplemplete isoprenoid synthesis was transformed with plasmid expressing three bottom operon constuct- pMevB, pMBI and pMBI. E.coli strand DYM1 had a deletion in the ispC gene for encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase, and was not able to synthesise 2-C-methyl-D-erythritol 4-phosphate(MEP), which is important intermediate in isoprenoid biosynthesis[1]. Because of this deletion the synthesis of precursors IPP and DMAP is blocked. So then they tested what is going to happen if they grew the strain DYM1 in the presence of 2-C-methyl-D-ertihritol and in medium with mevalonate without methylerythritol since DYM1 strain was not capable to synthesisie MEP. As expected , all strains expressing operon from S.cerevisiae grew on the plates in the presence of 2-c-methyl-D-erythritol. But only strains with pMBI or pMBIS grew on plates with 1mM mevalonate in the absence of methylerythol. This was the conformation that synthetic MBI and MBIS operons were functional, because they were capable to support the growth of E.coli without ispC gene by supplying cells with IPP and DMAPP. Next they completed the mevalonate pathway by transforming the pMevT plasmid (expressing three genes mentioned before-atoB, HMGS and tHMGR) with pMBI or pMBIS. This coexpresion of two operons, pMevT and pMBI /pMBIS ,completed the mevlonate pathway and allowed the synthesis of sesquiterpene precursors from acetyl-CoA, IPP and DMAPP. Therefore, coexpression complemented the ispC deletion even in the absence of mevalonate, what was the proof, that MevT operon was functional [1,7].

'''Amorpadiene synthesis from mevalonate'''
The aim was to achieve high-level production of amorphadiene and to find out if the amount of FPP was limiting amoprhadiene output. As mentioned, E.coli was used as heterologous microorganism in which coupled mevalonate pathway with amorphadiene synthesis. Cells with ADS gene were coexpressed with MBIS operon and grown in medium with increasing amounts (0 to 40 mM) of exogenus mevalonate. The culture extract was analysed with gas chromatography-mass spectrometry, analytical method for identification different substances in a test sample. It combines the features of mass spectrometry and gas-liquid chromatography. Results showed that MBIS operon did not limit the amorphadiene production at the highest mevalonate concentration(40mM). ([http://www.disketa.si/media/uploads/2015-01/7711d592584a53282f2ecb0fd678ef40.jpg See image 1 here]) Cultures with 40 mM mevalonate added produced much more amoprhadiene. With time, amorphadiene concentration was dropping beacause of the loss of the volatile terpene[1]. By adding more than 10 mM mevalonate in control cultures without ADS, the severe growth ihibition has been reported. ([http://www.disketa.si/media/uploads/2015-01/2a21f55f0fea31ffcbe89c866c9150f7.jpg See image here]) To discover the cause ofthis inhibition, the growth of E.coli DH10B from strains with empty, pMKPMK, pMevB, pMBI or pMBIS plasmid was measured in media with increasing concentrations of mevalonate. The addition of 5mM mevalonate to the medium inhibited the growth of cells with pMevB. But 5mM concentrations of mevalonate did not alter the growth of cells with pMKPMK, PMBI or pMBIS plasmid. This indicated that the accomulation of IPP is toxic and inhibits normal cell growth. The accmulation mostly occurs in cells with high flux through mevalonate pathway. The next step was comparison of intracellular prenyl pyrophosphate (FPP) in strains. This was done by feeding the cells harboring the different mevalonte operon construct with radiolabeled mevalonate. Than the labeled metabolites were tracked. For this experiment cell suspensions of E.coli harboring pMevB+pTrc99A, pMBI+ pTrc99A, pMBIS+ pTrc99A or pMBIS+pADS were used. pTRc99A is the bacterial expression vector with inducible to IPTG lacI promoter. The result showed that strains with MevB operon accumulated IPP but not FPP, when MBI and MBIS strains accumulated FPP. ([http://www.disketa.si/media/uploads/2015-01/31ad7a215f016dc26aceacf92eb46712.jpg See image here]) Because of the simultaneous expression of the amorphadiene synthase the waste of FPP that accumulated in the MBIS host was consumed. This was shown by a decrease in intracellular FPP. Cells expresing MBIS acumulated FPP and showed growth inhibition in the presence of 10 mM mevalonate. The prediction was that the coexpression of ADS would ease the growth inhibition by forwarding the prenyl pyrophosphate intermediates to volatile terpene olefin. For this test, the cell with pMBIS and empty expression vector pTrc99A (without ADS)and cells with pMBIS and pADS vectors were used. The medium was supplemented with mevalonate concentations ( 0mM, 5mM, 10mM, 20mM or 40mM). Two hours after addition of mevalonate and inducer IPTG, the growth inhibition was observed. ([http://www.disketa.si/media/uploads/2015-01/94b5f3d5636fe767316f770f3e2b0448.jpg See image here]) The result showed that the strains with pMBIS +ADS started to grow, but strain lacking the ADS still showed growth inhbition at higher concentrations of mevalonate. This supported the conclusion that converion of FPP to amophadiene has an important role in minimising growth inhibition. Briefly these result showed that engineered mevalonate patway produces high levels of prenyl pyrophosphate precursors. If the IPP isomerase, FPP synthase and terpen synthase is absent, IPP can acumulate and be toxic to the cells. The toxic levels of intracellular prenyl pyrphosphate can accumulate [1].

'''Amorphadiene synthesis from acetyl-Coa'''
The main goal was to achieve high amoprhaidene production from a simple and inexpensive carbon source. E.coli strains harboring pMevT,pMBIS and pADS plasmids were transformed with pMevT plasmid. Subsequently the strains were tested for ability to produce amoprhadiene in the absence of exogenous mevalonate. The comparison between E.coli expressing the native DPX pathway and engineered pathway as made. They tested native DXP pathway, engineered DPX pathway, mevalonate bottom pathway in the absence of mevalonate, mevlonate bottom pathway in medium suplemented with mevalonate, the complete mevalonate pathway and the complete mevalonate pathway supplemented with 0,8% glycerol. Glycerol was addded to investigate the effect of amorphadiene production of supplying as single carbon source and for prolongation of amorphadiene production. Breifly the resulut showed that expression of the mevalonate-dependent isoprenoid biosynthetic pathway delivers hight levels of isoprenoid precursor for the production of sesquiterpene [1]. ([http://www.disketa.si/media/uploads/2015-01/a31fe07a7c66854b1dc3ff36f8ed01ef.jpg See image here])

=== DISSCUSION ===
The identification of genes encoding the enzymes involved in the artemisinin-biosynthesis pathway together with advances in metabolic engineering provides new options for engineering artemisinin biosynthesis. Heterologous production of artemisnin in microorganism such as E.coli. is an alternative way for production of artmeinsin in high yields. But eventhougt micoorganisms has shown great potential in that matter, optimisation of these heterlogous pathways still require maximal titer. Inequal gene expression can lead to accumulation of toxic metabolic intermediates and to over or under production of enzymes involved in pathway. (4). Conclusion based on these study shows that poor expression of the amoprhaidene synthase genes (ADS) limits high terpene olfein yields from the host. To achieve better results , in this study they chose to synthesiyse an amophaiene synthase gene (ADS). The comparison between E.coli expressing native sesquiterpene synthase genes and E.coli expressing the synthetic ADS gene showed big improvement in terpene synthesis due to synthetic ADS gene. The native DPX pathway showed poor supply of the prenyl pyrophosphate precuror. And that limited terpenoid -carotenoid yields in E.coli. With the metabolic engineering of the DPX pathway the flux of carotenoid accumulation showed signifcant increase. By using the mevalonate-dependent isoprenoid pathway from yeast S. cerevisae and engineering that pathway in bacteria E.coli, we avoid the native regulatory elements found in yeast while bypassing those of E.coli DXP pathway. In this study results showed growth inhibition in cells without ADS due to the vast excess of preny pyrophosphate. But the coexpression of a synthetic sesquiterpene synthase can contribute to reducing the excess pool of precursors and eliminate growth inhibition. In this study total biosynthesis of artemisnin was not completely achieved. But these results can help in further investigation for poduction of artemisnic acid. This acid can then be putrified and chemically converted into artemsinin. [1,7].

=== CONCLUSIONS ===
To sum things up, artmeinsin, a sesquiterpene isolated from Artemisia annua,represent a novel drug for malaria treatment. The present artemisnin derivates are too expensive for general use and plants can not produce artmeinsin in big quantities. This fact inspired scientist to focus on developing the way for production of artemisnin in high quantities and to avoid big costs. The promising tool in alternative source of artmeinsin is the use of microoganisms. The aim is to transfer the genes encoding enzymes involved in biosynthetic pathway for artemisnin into a hight producing host organism. Several study have been reporting on engineering the metabolic pathway in S.cerevisae to produce the precursor for artmeisnic acid. With the effort in synthetic biology they used modified mevalonate pathway . With the help of syntehtic bilogy the yeast cells were engineerd to express ezymes needed for production of artmemisnic acid. But in this paper the main focus was on engineering a mevalonate pathway in bacteria E.coli. By engineering a new metabolic pathway in E.coli we can easily synthesise a precursor nedeed for artemisnin production. The goal is to produce drugs in better and cheape ways. But, why focus on artemisnin? Well, acording on recent studies artemisnin shows to be very sucesfull for the treatmen all strain of malaria. More than a milion people affected with malaria have already been cured by arteminsin. As already mentioned , because of very hight costs and limited production of arteminin in plants most populations in Africa can not afford it. Progress made in synthetic biology and ability to produce amorphadiene in microorganisms in big quantites opens a new possibility to produce low cost artemisnin and contributes to the brighter future od antimalaria drug deveopment, and therefore saving milions from suffering from malaria. [1,2, 3].

[1]Vincent JJ Martin, Douglas J Pitera, Sydnor T Whithers, Jack D Newman & Jay D Keasling. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nature biotehnology; vol 21; pages 796-802, July 2003
[2] T. Mosses, J. Pollier, J. M. Thevelein, A. Goossens. Bioengineering of plant (tri)terpenoids: from metabolic engineering of plants to synthetic biology in vivo and in vitro. New Pathyologist. 200: 27-43, 2013
[3] M.Farhi, M. Kozin, S. Duchin, A. Vainstein. Metabolic engineering of plants for artemisinin synthesis. Biotehnology and genetic engineering rewiews.Vol.29,No.2: 135-148, 2013
[4] Synthetic biology ; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Synthetic_biology http://en.wikipedia.org/wiki/Synthetic_biology]
[5]Operon; Wikipedia the free encyclopedia [citied 17.1.2015]. [http://en.wikipedia.org/wiki/Operon http://en.wikipedia.org/wiki/Operon]
[6] M. K. Julsing, G. van Pouderoyen, J. van der Veen,H. J. Woerdenbag, O. Kayser, W. J. Quax. Amorphadiene synthase: probing the active site model with directed mutagenesis. Directed mutagenesis of AMDS.Chapter 4: 47-56
[7]J.R. Anthony, L. C. Anthony, F. Nowroozi, G. Kwon, J.D. Newman, J.D. Keasling. Optimization of themevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene. Metabolic Engineering. 11: 13-19, 2009

SB students resources

2015-01-19T22:33:21Z

Ana90: /* List of articles for presentation */

===Introduction to our students resources in Synthetic Biology===
(Marko Dolinar)

Synthetic biology made a vast progress in good 10 years since it established itself as an interdisciplinary field of research on the interface of molecular biology and engineering. University of Ljubljana Faculty of Chemistry and Chemical Technology has introduced a Synthetic Biology course as a part od Biochemistry MSc programme only in 2013/14. This is relatively late, considering a great success of Slovenian students at iGEM competitions since their first attendance in 2006. On the other hand, the field is still in its first stages if development and a complete textbook for a MSc level course is still missing. This is the reason why our students collaborated on the preparation of a Synthetic Biology textbook with the working title Synthetic Biology - A Students Textbook. It exists as a draft that is not publicly available and is actually part 1 of a (to be) 2-volumes title. Part I is subtitled Engineering Biology, while Part II (that currently doesn't exisist yet) will be subtitled Synthetic Biology Applications.

As in all highly competitive fields of science and technology, students should be following recent progress by reading articles in high quality journals. However, this is often a very difficult task, especially at the BSc level. Specificities of the scientific and technical language, push of publishers towards very short methodological chapters and limited knowledge studens might have about advanced techniques make understanding papers a very challenging task. Therefore, I decided to face MSc students with the challenge to explain selected SB articles in a manner that would make the content of these articles understandable to BSc level students and non-experts.

In 2014/15, seminars in Synthetic Biology include explanations and presentations of some of the top-cited articles from the field of Synthetic Biology. I compiled a list of 95 articles published between 2000 and 2014 having the highest number of citations according to the Web of Science database. The list ended with the paper just exceeding the 100 citations limit. Not included in the list were reviews. With 20 students enrolled in the course, the list has been further reduced to top 40 papers in the field. Students have been asked to check for content (they further eliminated 3 papers which proved to be reviews) and availabitly (they all seemed to be available as full texts with our university subscriptions). My suggestion was to avoid selecting for presentation papers with very similar content. Especially in the field of genome editing there has been a very rapid progress in the past few years resulting in a number of highly-cited articles which could appear very similar in content for a non-specialist. From the shortlist of 37 articles, students selected a topic they believed would be most interesting or easiest to explain. Presentations will be both written (in English, which is not the mother tongue of my students) and oral (in Slovenian, to establish and maintain Slovenian terminology in the field).

===List of articles for presentation===

This is the list of top-cited papers from the broader field of Synthetic Biology that students chose for explanation in 2014/15 (sorted by year of publication):

#[[A synthetic oscillatory network of transcriptional regulators]], Michael B. Elowitz & Stanislas Leibler, Letters to Nature, 2000 - Valter Bergant
#[[Construction of a genetic toggle switch in Escherichia coli]]. Gardner ''et al''., Nature, 2000 - Urban Bezeljak
#[[Positive feedback in eukaryotic gene networks: cell differentiation by graded to binary response conversion]]. Becskei ''et al''., EMBO J, 2001 - Andreja Bratovš
#[[Chemical synthesis of poliovirus cDNA: Generation of infectious virus in the absence of natural template]]. Jeronimo Cello ''et al''., Science,2002 - Veronika Jarc
#[[Combinatorial synthesis of genetic networks]]. Guet C.C. ''et al'', Science, 2002 - Maja Remškar
#[[Engineering a mevalonate pathway in Escherichia coli for production of terpenoids]] (2003) - Ana Kapraljević
#Programmed population control by cell-cell communication and regulated killing. You et al, Nature (2004)[http://wiki.fkkt.uni-lj.si/index.php/7.Programmed_population_control_by_cell-cell_communication_and_regulated_killing] - Alja Zottel
#Gene regulation at the single-cell level (2005) - Katarina Uršič
#[[A synthetic multicellular system for programmed pattern formation]]. (2005) - Mitja Crček
#[[Long-term monitoring of bacteria undergoing programmed population control in a microchemostat]]. Balagadde ''et al.'', ''Science'', 2005 - Jana Verbančič
#[[Tuning genetic control through promoter engineering]], Hal Alper ''et al''., PNAS, 2005 - Špela Pohleven
#[[Production of the antimalarial drug precursor artemisinic acid in engineered yeast ]]. Ro ''et al''., ''Nature''., 2006- Živa Marsetič
#[[An improved zinc-finger nuclease architecture for highly specific genome editing]], Miller ''et al''., ''Nature Biotechnol''., 2007 - Eva Knapič
#[[Establishment of HIV-1 resistance in CD4(+) T cells by genome editing using zinc-finger nucleases]] (2008) - Tamara Marić
#[[Synthetic protein scaffolds provide modular control over metabolic flux]]. Dueber ''et al''., Nature Biotechnology, 2009. - Ana Dolinar
#[[Creation of a bacterial cell controlled by a chemically synthesized genome]]. Gibson, D. G. ''et al.'', Science, 2010 - Eva Lucija Kozak
#[[A TALE nuclease architecture for efficient genome editing]], Miller ''et al'', ''Nature Biotechnol''., 2011 - Jernej Mustar
#Multiplex genome engineering using CRISPR/Cas systems (2013) - Uroš Stupar
#[[RNA-guided human genome engineering via Cas9]]. Mali ''et al''., Science, 2013 - Luka Smole
#[[One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering (2013)]] - Andrej Vrankar

''Please link the title of each paper with your written seminar wiki page. Expand the citation according to the following example:
''
#Emergent bistability by a growth-modulating positive feedback circuit. Tan et al., Nature Chem. Biol., 2009

ZVIŠEVANJE OCENE

2013-12-19T09:25:22Z

Ana90:

30017949

30017952

30017941

Gene silencing shRNA

2013-11-01T21:16:57Z

Ana90:

'''OD RNA POSREDOVANO UTIŠANJE GENOV N-NIKOTINAMID METILTRANSFERAZE, POVEZANO Z ZMANJŠANO TUMORGENOSTJO V CELICAH HUMANEGA ORALNEGA KARCINOMA'''

Oralni skvamozni celični karcino(OSCC) je pogosta in dokaj smrtna oblika tumorja. Smrtnost je visoka zaradi njegove zapoznele diagnoze. Encim N-nikotinamid metiltransferaza katalizira N-metilacijo nikotinamida, pirimidinov in drugih strukturnih analogov,ki imajo pomembno vlogo pri biotransformaciji in detoksikaciji ksenobiotikov. N-metilacija je tudi metabolna pot izločanja nikotinamida. Nikotinamid je normalno prisoten v celicah in je močan inhibitor encimov, kot so histonske deacetilaze, povezane z odgovorom na stres, regulacijo apoptoze in poli (-adenozin difosfat riboze) polimeraze (PARP), ki je pomembna pri DNA odzivu na poškodbe. NNMT pa zniža nikotinamidni intracelularni nivo in tako vpliva na celično rast in kemorezistenco. Njegovo povečano ekspresijo so ugotovili pri široki različici tumorjev, ter so tako hoteli raziskovati njegov prispevek h karcinogenosti OSCC. Ekspresijo NNMT so v študiji preučevali na sedmih humanih rakavih celičnih linijah(PE/CA-PJ15, PE/CA-PJ34, PE/CA-PJ41, PE/CA-PJ46, PE/CA-PJ49, HSC-2, HSC-3) z RealTime PCR, Western Blot analizo in testom katalitične aktivnosti. Posledično so ocenili tudi efekt z shRNA povezanega utišanja gena NNMT na celično proliferacijo. NNMT so detektirali v vseh testiranih celičnih linijah, največji ekpresijski level pa so zaznali v PE/CA-PJ15 celicah.

'''shRNA'''

Shor hairpin(sh) RNA je dvoverižna RNA, ki tvori zanko in se lahko uporabi za utišanje genske ekpresije preko RNA interference. Ekspresijo v celicah dosežemo z dostavo plazmidov, lahko pa tudi preko bakterijskih celic. Ko se vektor integrira v gostiteljski genom poteče shRNA transkripcija v jedru. Pre-shRNA se eksportira iz jedra v citoplazmo. Protein Dicer procesira shRNA, odstrani se zanka in nastane siRNA.Nato se poveže v RNA induciran utiševalni kompleks (RICS). Sense veriga se razgradi, antisense veriga pa usmeri RICS do mRNA, ki ima komlementarno sekvenco. RICS represira translacijo mRNA. ShRNA pripelje do utišanja genov.

'''Metode'''

Količino kvantitativne NNMT mRNA so določili z RealTime PCR. Naredili so tudi Western Blot analizo pri kateri so hoteli oceniti NNMT proteinski ekspresijski nivo in tudi NNMT encimski test, ki temelji na metodi HPLC s katerim so analizirali NNMT aktivnost (test temelji na merjenju količine nastalega N-metilnikotinamida). S testi so zaznali NNMT v vseh testiranih celičnih linijah, največjo ekspresijo pa so zaznali pri PE/CA-PJ15 celicah.

'''ShRNA plazmidna transfekcija'''

Uporabili so set vektorjev pLKO.1, ki so vsebovali kasete z lasnično zanko(hairpin loop), ki kodirajo shRNA s tarčo na humani NNMT. Celice PE/CA-PJ15 so transfecirali s štirimi shRNA plazmidi(pLKO.1-330, 1-448, 1-164, 1-711) proti NNMT. Kontrolne celice so tretirali le s transfekcijskim agentom (mock ). 48 ur po transfekciji so celice nacepili na medij s puromicinom, saj je plazmidu vseboval rezistenco proti puromicinu. Učinkovitost genskega utišanja so zaznali z Real Time PCR in Western Blot analizo. Ugotovili so, da je bila ekspresija NNMT zmanjšana v transfeciranih celicah. V primerjavi s kontrolnimi celicami(mock) je bila ekspresija 6,25 krat nižja.

Izvedli so še kolorimetrični MMT test, s katerim so ocenili celično proliferacijo. Test s 3-(4,5-dimethylthiazol-2-yl)-2,5-difenil tetrazolin bromidom (MTT), meri spremembo MMT v netopni formazan. Število nastalih kristalov korelira z številom vaiabilnih celic. Reakcijski produkt so pomerili še z absorbanco pri 595. Inhibicijo celične proliferacije so potrdili s testom na mehkem agarju rastočih kolonij, kjer je bilo opazno, da so bile kolonije z NNMT, pri katerih so plazmidi utišali gen za proliferacijo manj številne v primerjavi s kontrolnim celicami.
S testom tumorgenosti so ocenli efekt razkapanja (knockdown)NNMT. Subkutano so vnesli transfekcijske in kontrolne PE/CA-PJ15 celice v miške na levo in desno stran hrbta.Uporabili so miške brez timusa. Velikost tumorjev so tedensko merili in računali volumen tumorja po formuli :dolžina x širina x višina. Ugotovili so da je bila rast tumorjev zmanjšana v miškah s transfekcijskimi celicami, v primerjavi s tistimi,v katere s vnesli kontrolne celice.

'''Zaključek'''

Ugotovili so, da zmanjšano izražanje NNMT vodi do inhibicije proliferacije in tudi do zmanjšane rakaste aktivnosti pri OSCC. Pri miškah so opazili, da ko je prišlo do utišanja genov, je tudi prišlo do redukcije tumorskega volumna.
Z RNA interference knockdow DNA predstavlja novo orodje za študij biološke vloge NNMT in njegove uporabe pri terapevtskem zdravljenju ter zgodnjemu prepoznavanju OSCC. Slabost metode shRNA plazmidne transfekcije je v tem, da se ne more uporabiti ''in vivo'' in ima dokaj omejeno uporabo

Gene silencing shRNA

2013-11-01T16:14:49Z

Ana90:

'''OD RNA POSREDOVANO UTIŠANJE GENOV N-NIKOTINAMID METILTRANSFERAZE, POVEZANO Z ZMANJŠANO TUMORGENOSTJO V CELICAH HUMANEGA ORALNEGA KARCINOMA'''
Oralni skvamozni celični karcino(OSCC) je pogosta in dokaj smrtna oblika tumorja. Smrtnost je visoka zaradi njegove zapoznele diagnoze. Encim N-nikotinamid metiltransferaza katalizira N-metilacijo nikotinamida, pirimidinov in drugih strukturnih analogov,ki imajo pomembno vlogo pri biotransformaciji in detoksikaciji ksenobiotikov. N-metilacija je tudi metabolna pot izločanja nikotinamida. Nikotinamid je normalno prisoten v celicah in je močan inhibitor encimov, kot so histonske deacetilaze, povezane z odgovorom na stres, regulacijo apoptoze in poli (-adenozin difosfat riboze) polimeraze (PARP), ki je pomembna pri DNA odzivu na poškodbe. NNMT pa zniža nikotinamidni intracelularni nivo in tako vpliva na celično rast in kemorezistenco. Njegovo povečano ekspresijo so ugotovili pri široki različici tumorjev, ter so tako hoteli raziskovati njegov prispevek h karcinogenosti OSCC. Ekspresijo NNMT so v študiji preučevali na sedmih humanih rakavih celičnih linijah(PE/CA-PJ15, PE/CA-PJ34, PE/CA-PJ41, PE/CA-PJ46, PE/CA-PJ49, HSC-2, HSC-3) z RealTime PCR, Western Blot analizo in testom katalitične aktivnosti. Posledično so ocenili tudi efekt z shRNA povezanega utišanja gena NNMT na celično proliferacijo. NNMT so detektirali v vseh testiranih celičnih linijah, največji ekpresijski level pa so zaznali v PE/CA-PJ15 celicah.

'''shRNA'''
Shor hairpin(sh) RNA je dvoverižna RNA, ki tvori zanko in se lahko uporabi za utišanje genske ekpresije preko RNA interference. Ekspresijo v celicah dosežemo z dostavo plazmidov, lahko pa tudi preko bakterijskih celic. Ko se vektor integrira v gostiteljski genom poteče shRNA transkripcija v jedru. Pre-shRNA se eksportira iz jedra v citoplazmo. Protein Dicer procesira shRNA, odstrani se zanka in nastane siRNA.Nato se poveže v RNA induciran utiševalni kompleks (RICS). Sense veriga se razgradi, antisense veriga pa usmeri RICS do mRNA, ki ima komlementarno sekvenco. RICS represira translacijo mRNA. ShRNA pripelje do utišanja genov.

'''Metode'''
Količino kvantitativne NNMT mRNA so določili z RealTime PCR. Naredili so tudi Western Blot analizo pri kateri so hoteli oceniti NNMT proteinski ekspresijski nivo in tudi NNMT encimski test, ki temelji na metodi HPLC s katerim so analizirali NNMT aktivnost (test temelji na merjenju količine nastalega N-metilnikotinamida). S testi so zaznali NNMT v vseh testiranih celičnih linijah, največjo ekspresijo pa so zaznali pri PE/CA-PJ15 celicah.

'''ShRNA plazmidna transfekcija'''
Uporabili so set vektorjev pLKO.1, ki so vsebovali kasete z lasnično zanko(hairpin loop), ki kodirajo shRNA s tarčo na humani NNMT. Celice PE/CA-PJ15 so transfecirali s štirimi shRNA plazmidi(pLKO.1-330, 1-448, 1-164, 1-711) proti NNMT. Kontrolne celice so tretirali le s transfekcijskim agentom (mock ). 48 ur po transfekciji so celice nacepili na medij s puromicinom, saj je plazmidu vseboval rezistenco proti puromicinu. Učinkovitost genskega utišanja so zaznali z Real Time PCR in Western Blot analizo. Ugotovili so, da je bila ekspresija NNMT zmanjšana v transfeciranih celicah. V primerjavi s kontrolnimi celicami(mock) je bila ekspresija 6,25 krat nižja.

Izvedli so še kolorimetrični MMT test, s katerim so ocenili celično proliferacijo. Test s 3-(4,5-dimethylthiazol-2-yl)-2,5-difenil tetrazolin bromidom (MTT), meri spremembo MMT v netopni formazan. Število nastalih kristalov korelira z številom vaiabilnih celic. Reakcijski produkt so pomerili še z absorbanco pri 595. Inhibicijo celične proliferacije so potrdili s testom na mehkem agarju rastočih kolonij, kjer je bilo opazno, da so bile kolonije z NNMT, pri katerih so plazmidi utišali gen za proliferacijo manj številne v primerjavi s kontrolnim celicami.
S testom tumorgenosti so ocenli efekt razkapanja (knockdown)NNMT. Subkutano so vnesli transfekcijske in kontrolne PE/CA-PJ15 celice v miške na levo in desno stran hrbta.Uporabili so miške brez timusa. Velikost tumorjev so tedensko merili in računali volumen tumorja po formuli :dolžina x širina x višina. Ugotovili so da je bila rast tumorjev zmanjšana v miškah s transfekcijskimi celicami, v primerjavi s tistimi,v katere s vnesli kontrolne celice.

'''Zaključek'''
Ugotovili so, da zmanjšano izražanje NNMT vodi do inhibicije proliferacije in tudi do zmanjšane rakaste aktivnosti pri OSCC. Pri miškah so opazili, da ko je prišlo do utišanja genov, je tudi prišlo do redukcije tumorskega volumna.
Z RNA interference knockdow DNA predstavlja novo orodje za študij biološke vloge NNMT in njegove uporabe pri terapevtskem zdravljenju ter zgodnjemu prepoznavanju OSCC. Slabost metode shRNA plazmidne transfekcije je v tem, da se ne more uporabiti ''in vivo'' in ima dokaj omejeno uporabo

Gene silencing shRNA

2013-11-01T16:13:25Z

Ana90:

'''OD RNA POSREDOVANO UTIŠANJE GENOV N-NIKOTINAMID METILTRANSFERAZE, POVEZANO Z ZMANJŠANO TUMORGENOSTJO V CELICAH HUMANEGA ORALNEGA KARCINOMA'''
Oralni skvamozni celični karcino(OSCC) je pogosta in dokaj smrtna oblika tumorja. Smrtnost je visoka zaradi njegove zapoznele diagnoze. Encim N-nikotinamid metiltransferaza katalizira N-metilacijo nikotinamida, pirimidinov in drugih strukturnih analogov,ki imajo pomembno vlogo pri biotransformaciji in detoksikaciji ksenobiotikov. N-metilacija je tudi metabolna pot izločanja nikotinamida. Nikotinamid je normalno prisoten v celicah in je močan inhibitor encimov, kot so histonske deacetilaze, povezane z odgovorom na stres, regulacijo apoptoze in poli (-adenozin difosfat riboze) polimeraze (PARP) , ki je pomembna pri DNA odzivu na poškodbe. NNMT pa zniža nikotinamidni intracelularni nivo in tako vpliva na celično rast in kemorezistenco. Njegovo povečano ekspresijo so ugotovili pri široki različici tumorjev, ter so tako hoteli raziskovati njegov prispevek h karcinogenosti OSCC. Ekspresijo NNMT so v študiji preučevali na sedmih humanih rakavih celičnih linijah(PE/CA-PJ15, PE/CA-PJ34, PE/CA-PJ41, PE/CA-PJ46, PE/CA-PJ49, HSC-2, HSC-3) z RealTime PCR, Western Blot analizo in testom katalitične aktivnosti. Posledično so ocenili tudi efekt z rhRNA povezanega utišanja gena NNMT na celično proliferacijo. NNMT so detektirali v vseh testiranih celičnih linijah, največji ekpresijski level pa so zaznali v PE/CA-PJ15 celicah.

'''shRNA'''
Shor hairpin(sh) RNA je dvoverižna RNA, ki tvori zanko in se lahko uporabi za utišanje genske ekpresije preko RNA interference. Ekspresijo v celicah dosežemo z dostavo plazmidov, lahko pa tudi preko bakterijskih celic. Ko se vektor integrira v gostiteljski genom poteče shRNA transkripcija v jedru. Pre-shRNA se eksportira iz jedra v citoplazmo. Protein Dicer procesira shRNA, odstrani se zanka in nastane siRNA.Nato se poveže v RNA induciran utiševalni kompleks (RICS). Sense veriga se razgradi, antisense veriga pa usmeri RICS do mRNA, ki ima komlementarno sekvenco. RICS represira translacijo mRNA. ShRNA pripelje do utišanja genov.

'''Metode'''
Količino kvantitativne NNMT mRNA so določili z RealTime PCR. Naredili so tudi Western Blot analizo pri kateri so hoteli oceniti NNMT proteinski ekspresijski nivo in tudi NNMT encimski test, ki temelji na metodi HPLC s katerim so analizirali NNMT aktivnost (test temelji na merjenju količine nastalega N-metilnikotinamida). S testi so zaznali NNMT v vseh testiranih celičnih linijah, največjo ekspresijo pa so zaznali pri PE/CA-PJ15 celicah.

'''ShRNA plazmidna transfekcija'''
Uporabili so set vektorjev pLKO.1, ki so vsebovali kasete z lasnično zanko(hairpin loop), ki kodirajo shRNA s tarčo na humani NNMT. Celice PE/CA-PJ15 so transfecirali s štirimi shRNA plazmidi(pLKO.1-330, 1-448, 1-164, 1-711) proti NNMT. Kontrolne celice so tretirali le s transfekcijskim agentom (mock ). 48 ur po transfekciji so celice nacepili na medij s puromicinom, saj je plazmidu vseboval rezistenco proti puromicinu. Učinkovitost genskega utišanja so zaznali z Real Time PCR in Western Blot analizo. Ugotovili so, da je bila ekspresija NNMT zmanjšana v transfeciranih celicah. V primerjavi s kontrolnimi celicami(mock) je bila ekspresija 6,25 krat nižja.

Izvedli so še kolorimetrični MMT test, s katerim so ocenili celično proliferacijo. Test s 3-(4,5-dimethylthiazol-2-yl)-2,5-difenil tetrazolin bromidom (MTT), meri spremembo MMT v netopni formazan. Število nastalih kristalov korelira z številom vaiabilnih celic. Reakcijski produkt so pomerili še z absorbanco pri 595. Inhibicijo celične proliferacije so potrdili s testom na mehkem agarju rastočih kolonij, kjer je bilo opazno, da so bile kolonije z NNMT, pri katerih so plazmidi utišali gen za proliferacijo manj številne v primerjavi s kontrolnim celicami.
S testom tumorgenosti so ocenli efekt razkapanja (knockdown)NNMT. Subkutano so vnesli transfekcijske in kontrolne PE/CA-PJ15 celice v miške na levo in desno stran hrbta.Uporabili so miške brez timusa. Velikost tumorjev so tedensko merili in računali volumen tumorja po formuli :dolžina x širina x višina. Ugotovili so da je bila rast tumorjev zmanjšana v miškah s transfekcijskimi celicami, v primerjavi s tistimi,v katere s vnesli kontrolne celice.

'''Zaključek'''
Ugotovili so, da zmanjšano izražanje NNMT vodi do inhibicije proliferacije in tudi do zmanjšane rakaste aktivnosti pri OSCC. Pri miškah so opazili, da ko je prišlo do utišanja genov, je tudi prišlo do redukcije tumorskega volumna.
Z RNA interference knockdow DNA predstavlja novo orodje za študij biološke vloge NNMT in njegove uporabe pri terapevtskem zdravljenju ter zgodnjemu prepoznavanju OSCC. Slabost metode shRNA plazmidne transfekcije je v tem, da se ne more uporabiti ''in vivo'' in ima dokaj omejeno uporabo

Gene silencing shRNA

2013-11-01T16:07:52Z

Ana90: New page: '''OD RNA POSREDOVANO UTIŠANJE GENOV N-NIKOTINAMID METILTRANSFERAZE, POVEZANO Z ZMANJŠANO TUMORGENOSTJO V CELICAH HUMANEGA ORALNEGA KARCINOMA''' Oralni skvamozni celični karcino(OSCC) j...

'''OD RNA POSREDOVANO UTIŠANJE GENOV N-NIKOTINAMID METILTRANSFERAZE, POVEZANO Z ZMANJŠANO TUMORGENOSTJO V CELICAH HUMANEGA ORALNEGA KARCINOMA'''
Oralni skvamozni celični karcino(OSCC) je pogosta in dokaj smrtna oblika tumorja. Smrtnost je visoka zaradi njegove zapoznele diagnoze. Encim N-nikotinamid metiltransferaza katalizira N-metilacijo nikotinamida, pirimidinov in drugih strukturnih analogov,ki imajo pomembno vlogo pri biotransformaciji in detoksikaciji ksenobiotikov. N-metilacija je tudi metabolna pot izločanja nikotinamida. Nikotinamid je normalno prisoten v celicah in je močan inhibitor encimov, kot so histonske deacetilaze, povezane z odgovorom na stres, regulacijo apoptoze in poli (-adenozin difosfat riboze) polimeraze (PARP) , ki je pomembna pri DNA odzivu na poškodbe. NNMT pa zniža nikotinamidni intracelularni nivo in tako vpliva na celično rast in kemorezistenco. Njegovo povečano ekspresijo so ugotovili pri široki različici tumorjev, ter so tako hoteli raziskovati njegov prispevek h karcinogenosti OSCC. Ekspresijo NNMT so v študiji preučevali na sedmih humanih rakavih celičnih linijah(PE/CA-PJ15, PE/CA-PJ34, PE/CA-PJ41, PE/CA-PJ46, PE/CA-PJ49, HSC-2, HSC-3) z RealTime PCR, Western Blot analizo in testom katalitične aktivnosti. Posledično so ocenili tudi efekt z rhRNA povezanega utišanja gena NNMT na celično proliferacijo. NNMT so detektirali v vseh testiranih celičnih linijah, največji ekpresijski level pa so zaznali v PE/CA-PJ15 celicah.

shRNA
Shor hairpin(sh) RNA je dvoverižna RNA, ki tvori zanko in se lahko uporabi za utišanje genske ekpresije preko RNA interference. Ekspresijo v celicah dosežemo z dostavo plazmidov, lahko pa tudi preko bakterijskih celic. Ko se vektor integrira v gostiteljski genom poteče shRNA trasnkripcija v jedru. Pre-shRNA se eksportira iz jedra v citoplazmo. Protein Dicer procesira shRNA, odstrani se zanka in nastane siRNA.Nato se poveže v RNA induciran utiševalni kompleks (RICS). Sense veriga se razgradi, antisense veriga pa usmeri RICS do mRNA, ki ima komlementarno sekvenco. RICS represira translacijo mRNA. ShRNA pripelje do utišanja genov.

'''Metode'''
Količino kvantitativne NNMT mRNA so določili z RealTime PCR. Naredili so tudi Western Blot analizo pri kateri so hoteli oceniti NNMT proteinski ekspresijski nivo in tudi NNMT encimski test, ki temelji na metodi HPLC s katerim so analizirali NNMT aktivnost (test temelji na merjenju količine nastalega N-metilnikotinamida). Z testi so zaznali NNMT v vseh testiranih celičnih linijah, naječjo ekspresijo pa so zaznali pri PE/CA-PJ15 celicah.
ShRNA plazmidna transfekcija
Uporabili so set vektorjev pLKO.1, ki so vsebovali kasete z lasnično zanko(hairpin loop), ki kodirajo shRNA s tarčo na humani NNMT. Celice PE/CA-PJ15 so transfekcirali s štirimi shRNA plazmidi(pLKO.1-330, 1-448, 1-164, 1-711) proti NNMT. Kontrolne celice so tretirali le s transfekcijskim agentom (mock ). 48 ur po transfekciji so celice nacepili na medij s puromicinom, saj je plazmidu vseboval rezistenco proti puromicinu. Učinkovitost genskega utišanja so zaznali z Real Time PCR in Western Blot analizo. Ugotovili so, da je bila ekspresija NNMT zmanjšana v transfekciranih celicah. V primerjavi s kontrolnimi celicami(mock) je bila ekspresija 6,25 krat nižja.

Izvedli so še kolorimetrični MMT test, s katerim so ocenili celično proliferacijo. Test s 3-(4,5-dimethylthiazol-2-yl)-2,5-difenil tetrazolin bromidom (MTT), meri spremembo MMT v netopni formazan. Število nastalih kristalov korelira z številom vaiabilnih celic. Reakcijski produkt so pomerili še z absorbanco pri 595. Inhibicijo celične proliferacije so potrdili s testom na mehkem agarju rastočih kolonij, kjer je bilo opazno, da so bile kolonije z NNMT, pri katerih so plazmidi utišali gen za priliferacijo manj številne v primerjavi s kontrolnim celicami.
S testom tumorgenosti so ocenli efekt razkapanja (knockdown)NNMT. Subkutano so injecirali transfekcijske in kontrolne PE/CA-PJ15 celice v miške na levo in desno stran hrbta.Uporabili so miške brez timusa. Velikost tumorjev so tedensko merili in računali volumen tumorja po formuli :dolžina x širina x višina. Ugotovili so da je bila rast tumorjev zmanjšana v miškah s transfekcijskimi celicami, v primerjavi s tistimi,v katere s vnesli kontrolne celice.

'''Zaključek'''
Ugotovili so, da zmanjšano izražanje NNMT vodi do inhibicije priliferacije in tudi do zmanjšane rakaste aktivnosti pri OSCC. Pri miškah so opazili, da ko je prišlo do utišanja genov, je tudi prišlo do redukcije tumorskega volumna. Z RNA interference razklapanje(knockdown) DNA predstavlja novo orodje za študij biološke vloge NNMT in njegove uporabe pri terapevtskem zdravljenju ter zgodnjemu prepoznavanju OSCC. Slabost metode plazmidne shRNA transfekcije je v tem, da se ne more uporabiti in vivo in ima dokaj omejeno uporabo