Wiki FKKT - User contributions [en]

Production of the antimalarial drug precursor artemisinic acid in engineered yeast

2015-01-20T04:45:39Z

Živa Mersetič: /* References */

This coursework is my attempt to paraphrase and explain in simple words the article of Ro DK, Paradise EM, Ouellet M, Fisher KJ, Newman KL, Ndungu JM, Ho KA, Eachus RA, Ham TS, Kirby J, Chang MC, Withers ST, Shiba Y, Sarpong R, Keasling JD with the title Production of the antimalarial drug precursor artemisinic acid in engineered yeast. The article was published in 2006 in Nature and it is one of the most cited articles in history os synthetic biology.
The article is one of many articles contributing to the realization of one of the biggest practical application of synthetic biology. The authors describe how they managed to prepare a yeast strain ‘’Saccharomyces cerevisiae’’, also known as Baker’s yeast, which was able to produce artemisinic acid. The production of anti-malarial drugs Artemisinic acid can be afterwards chemically transformed into artemisinin, which is today the first line treatment for malaria worldwide.
I divided this coursework in three parts. In the first part some aspects of malaria as disease and history of treatment are described. Second part is trying to explain the work behind the article. The third part is highlighting what happened after happy ending of the article in 2006, in other words, how things went on until today.
==Malaria and treatment==
=== Malaria===
Malaria is an emerging epidemic disease, occurring in warmer parts of the world. Because of its occurrence in tropical and subtropical areas where a lot of moisture is present its name is derived from Italian words for “bad air” [2]. It is endemic in a broad band around the equator in America, Asia and Africa. Most of deaths (85–90%) are misled in Sub-Saharan Africa (Layne SP. 2006).
It is caused by four species of sporozoa, but mostly by ‘’Plasmodium vivax ‘’and ‘’Plasmidium falciparum’’. This parasite performs part if its life cycle in human and part of it in the mosquito called ‘’Anopheles’’. Female mosquitoes of genus ‘’Anopheles’’ transmit protozoan ‘’Plasmodium falciparum’’ from person to person. The life cycle of ‘’Plasmidium falciparum’’ is complex. When mosquito injects saliva together with ‘’Plasmidium falciparum’’ sporozoites into a human host, those small, elongated cells travel through the bloodstream to the liver, where they convert into larger cells called schizont. Those cells divide into many small cells called merozoites, which enter bloodstream and infects erythrocytes. In red blood cells merozites grow, divide and exit cell by cell lysis. This asexual reproduction cycle takes approximately 48 hours. During this 48-hour period, malaria specific syndromes occur, such as chills, followed by fever up to 40◦C when ‘’Plasmidium falciparum’’ cells are released from cells. Because of the loss of red blood cells, malaria generally causes anemia.
Not all protozoal cells liberated from red blood cells are able to infect other erythrocytes. The protozoan cells, that cannot infect red blood cells are named gametocytes and are infective only for mosquito. When another mosquito feeds with infected blood gametocytes enter its digestive tract. In mosquito sexual production occurs and zygote is formed, which forms number of sporozoites. Some of these reach the salivary gland of the mosquito, and are injected into next human reservoir by the bite of mosquito (Madigan et al. 2006).

Picture of Plasmodium life cycle: http://www.uni-tuebingen.de/modeling/Mod_Malaria_Cycle_en.html

===Treatment===
Until now no antimalarial vaccine had been developed. Consequently all the treatment of malaria relies upon antilamarial drugs. First drug against malaria was quinine. It was extracted from the bark of the Cinchona tree in 1820 and was the only drug used for malaria treatment until 1930s (Foley et al. 1998) . Due to the occurrence of resistance of plasmodium species against quinine, search of new active pharmaceutical substances against malaria started. In the context of those researches chloroquinine and other synthetic guinoline antimalarials such as mefloquine were developed (Liu et al. 1979) . Unfortunately, plasmodium species developed resistance against those drugs, too (Warhurst 2001) .

In China another antimalarial drug was discovered in glandular trichomes on leaves and floral buds of plant called sweet wormwood or ‘’Artemisia annua’’. It was called artemisinin. Molecules of artemisinin contain a peroxide (O-O) group. In the presence of iron from damaged blood cells, the peroxide group is assumed to generate reactive free radicals which could destroy the DNA of the plasmodium (Liu et al. 1979) .
Today World Health Organisation recommends artemisinin-based combination therapies.
== Production of the antimalarial drug precursor artemisinic acid in engineered yeast==

===Mevalonate pathway and artemisinin synthesis pathway===

Even though artemisinin can be extracted from ‘’A. annua’’ plants, this way of obtaining the drug faces multiple obstacles. One of the major obstacle in direct extraction from plants lies in dependence of artemisinin assay in plant tissue upon natural environmental variability. Contamination by other plant trepenes, can cause problems in steps of purification (Zeng et al. 2008) .
In order to avoid those problems, which in effect cause major fluctuations in artemisinin price and thereby make it unaffordable for third world patients, Ro and coworkers published an article describing production of artemisinic acid in yeast ‘’Saccharomyces cerevisiae’’. Their idea was to rewrite the genome of ordinary brewer’s yeast to encourage it to make artemisinic acid. ‘’Saccharomyces cerevisiae’’ in an eukaryotic microorganism and can be easily grown in large bioreactors in industrial environment, thus making artemisinin production and especially purification cheaper.

Artemisinin has been categorized as a terpenoid or isoprenoid (Connolly et al. 1991). Ro and coworkers knew, that artemisinin biosynthesis pathway consists of two stages.

In the first stage linear isoprene precursors, such as GPP ( geranyl pyrophosphate ) later converted into FPP (farnesyl pyrophosphate) are synthesized from Acetyl-CoA via mevalonate pathway. This mevalonate pathway is present in all organisms including ‘’Saccharomices cerevisiae’’. Here should be mentioned that in ‘’Saccharomices cerevisiae’’ mevalonate pathway which starts from Acetyl-CoA and goes through intermediates such as HMG-CoA, mevalonate, IPP, GPP and FPP continues into synthesis of Squalene, which is then used for the synthesis of ergosterol (Covello et al. 2008) . Ergosterol is found in cell membranes of fungi and protozoa where stabilizes the membrane an makes it less flexible (Madigan et al. 2006).

In the second stage cyclic trepenes are synthesized from linear isoprene precursors ( GPP, FPP ). In case of artemisinic acid synthesis cyclic trepene is amorpha – 4, 11 diene from ‘’A. annua’’. This intermediate is in next steps transformed into artemisinic acid.

Schematic representation of the mevalonate pathway in ‘’S. cerevisiae’’ and its crossroad with atremisinic acid synthesis pathway can be seen following this link: http://www.nature.com/nature/journal/v440/n7086/fig_tab/nature04640_F1.html#figure-title

The involvement of mevalonate pathway in artemisinin biosynthesis has been already proven (Akhila et al. 1987). It was also known, that amorpha – 4, 11 diene, the first intermediate in the second stage of artemisinin is cyclized FPP by enzyme amorpha – 4, 11 diene synthase (ADS) (Bouwmeester et al. 1999). Gene encoding this enzyme was already known. However, the gene and amino acid sequence of the next enzyme in pathway was still unknown. This at the time unknown enzyme should be able to catalyze reaction of oxidation of amorpha – 4, 11 diene into artemisinic acid via artemisinic aldehyde and artemisinic alcohol intermediates. Ro and coworkers made great breakthrough and identified a cytochrome P450 monooxygenase (CYP71AV1) / amorpha-4,11-diene oxidase (AMO) for the oxidation of amorpha - 4,11-diene.

=== Isolating genes encoding enzymes responsible for oxidizing amorphadiene to artemisinic acid in ‘’A. annua ‘’===

Previous research demonstrated, that enzyme catalyzing the first regiospecific hydroxylation of amorphadiene in A. annua belongs to group on enzymes called cytochrome P450 monooxygenase (P450) (Bertea et al. 2005). Cytochrome P450 enzymes (CYPs) are, in general, oxidase enzymes. The most common reaction catalyzed by those enzymes is an insertion of one atom of oxygen into the aliphatic position of an organic substrate while the other oxygen atom is reduced to water. Most CYPs require a protein partner to deliver one or more electrons (Siegel et al. 2007).

In order to find this specific enzyme they collected P450-expressed-sequence tags (ESTs) belonging to sunflower and lettuce from the Asteraceae EST-database (http://www.cgpdb.ucdavis.edu).
EST is abbreviation for an expressed sequence tag. They are short sub-sequences of a cDNA sequence. ESTs have a relatively low quality of sequence. Their length is limited by current technology to approximately 500 to 800 nucleotides. They may be used to identify gene transcripts, to unravel new genes or to determine gene sequences. They also represent portions of expressed genes (Adams et al. 1991).

With known amino acid sequences of cytochrome P450 monooxygenases from sunflowers and lettuce (CYP71 subfamily), they designed degenerated primers, which would in PCR reaction amplify even cytochrome P450 monooxygenases from different species, in our case from’’ A. annua’’.

At the same time total RNA from trichome enriched cells of ‘’A.anua’’ was extracted. Using total RNA, cDNA pool was prepared.

Researchers were hoping to find enzymes from P450 family that existed and were transcribed and translated in ‘’A. annua’’, because there was a strong possibility , that one of those enzymes was the enzyme they were looking for.

Retrieved cDNA pool and synthesized degenerative primers were used in PCR reaction. Result of this pcr reaction was the isolation of several unique P450 fragments from an ‘’A. annua’’ trichome-enriched complementary DNA pool. Comparing DNA sequences of those fragments single ‘’A. annua’’ P450 gene fragment was spotted. It had 85–88% identity at the amino-acid level to ESTs of unknown function from both sunflower and lettuce. Sequence identity of this ‘’A. annua’’ P450 fragment to other P450 fragments outside the Asteraceae family was much lower. This P450 gene was therefore a suitable candidate for a cytochrome P450 monooxygenase (CYP71AV1)/amorpha-4,11-diene oxidase (AMO) enzyme.

With DNA sequence, isolation of open reading frame of 495 amino acids of CYP71AV1 enzyme from ‘’A. annua’’ was possible.

As previously stated CYP71AV1 as all cytochrome P450 enzyme needs its native redoxs partner. They used ‘’A. annua’’ cytochrome P450 oxidoreductase (CPR) as a redox partner.

===Rewriting the genome of ordinary ‘’Saccharomyces cerevisiae’’ to encourage it to make artemisinic acid===

With missing genes discovered Ro and coworkers could start developing synthetic yeast strain which could produce atremisinic acid. Since yeast is eukaryotic microbe problems such as lack of heterologous gene expression and nonfunctional membrane – bound proteins were excluded.

What they needed to do was:

1. To engineer the farnesyl pyrophosphate (FPP) biosynthetic pathway to increase FPP production and decrease its use for sterols

2. To introduce the amorphadiene synthase gene (ADS) from ‘’A. annua’’ into the high FPP producer to convert FPP to amorphadiene, and

3. To clone a novel cytochrome P450 that performs a three-step oxidation of amorphadiene to artemisinic acid from’’ A. annua’’ and expressing it in the amorphadiene producer

First step was to upregulate the expression of some genes encoding for enzymes present in mevalonate pathway and to downregulate enzyme ERG9, which catalyzes conversion of FPP into squalene (encodes squalene synthase,which catalyses the competing reaction of joining two farnesyl diphosphate moieties to form squalene (Paddon et al. 2013)). Thereby total concentration of FPP in the cell would increase providing more substrate (FPP) for amorphadiene synthase to convert FPP to amorphadiene.

Using Yeast Integrating plasmids - These plasmids lack an ORI and must be integrated directly into the host chromosome via homologous recombination – Ro and coworkers inserted following genes under strong GAL1 promoters into yeast genome one by one. They used standard lithium acetate method. After every successful transformation three to ten colonies from each transformation were tested for the highest amorphadiene producing transformation.

ADS:

The first of many steps was transfection with plasmid harboring amorphadiene synthase gene (ADS) from ‘’A. annua’’. Subsequently changes in mevalonate pathway were needed.

tHMGR:

Firstly they added gene coding tHMGR enzyme. Overexpression of a truncated, soluble form of 3 - hydroy – 3 - methylglutaryl - coenzyme A reductase (tHMGR) – enzyme catalyzing conversion of HMG-CoA into mevalonate, improved amorphadiene production approximately fivefold.

upc2-1:

After that upc2-1, asemi-dominant mutant allele that enhances the activity of UPC2 was added. UPC2 is a global transcription factor regulating the biosynthesis of sterols in ‘’S. cerevisiae’’ (Davies et al. 2005).

ERG9:

To downregulate enzyme ERG9, which catalyzes conversion of FPP into squalene (the first step after FPP in the sterol biosynthetic pathway), its promoter was changed from its original promotor into Pmet3, a methionine-represible promoter. This increased amorphadiene production an additional twofold.

tHMGR:

Additionally they inserted another copy of tHMGR gene into the chromosome. This insertion increased amorphadiene
production by 50% .

ERG20:

Finally they put ERG20, gene encoding FPP synthase, under strong GAL1 promotor.

Result of all these insertions and modifications was strain called EPY224 which was able to produce 153 mg per litre of amorphadiene.

Of course, both previously mentioned; CYP71AV1 and its cytochrome P450 oxidoreductase (CPR) as a redox partner, were still needed to be placed between yeast genes. Vector with CPR gene and CYP71AV1 gene under the control of galactose-inducible promoters was inserted via transformation into transgenic yeast strain EPY224.

To detect whether desired substance (artemisinic acid) was produced with synthetic yeast strain, yeast coulture medium and cell pellet were investigated via gas chromatography followed by mass spectrophotometer to detect newly synthesized substances. Artemisinic acid from ‘’A. annua’’ was used as reference for retention time. Results showed, that more than 95% of this novel compound was associated with the cell pellet. Authors claim, that easy removal of artemisinic acid by alkaline buffer confirms the idea that artemisinic acid is efficiently transported out of yeast cells but remains bound to the cell surface when it is protonated under acidic culture conditions.

Using single silica gel column chromatographic separation, artemisinic acid was purified. NMR spectra also confirmed that artemisinic acid produced by yeast strain matches artemisinic acid extracted from ‘’A. annua’’.

== Artemisinic acid production in yeast today==
Even though results in 2006 were very promising some problems still remained unsolved. Besides, in attempts to scale up the process new issues emerged. One of the biggest was low viability of synthetic yeast strains due to rapid accumulation of artemisinic acid and poor pairing of cytochrome P450 and their reductases, which resulted in formation of reactive oxygen species.

In 2013 Paddon and coworkers publish an article where a significantly improved, re-engineered yeast strain that is able to produce much more artemisinic acid is described. There are three enzymes added: CYB5, ADH1 and ALDH1, all of them are involved in the oxidation of amorphadiene to artemisinic acid in ‘’A. annua’’ plants. Also MET3 promoter is replaced with the copper-regulated CTR3 promoter, enabling restriction of ERG9 expression by addition of the inexpensive repressor CuSO4 to the medium rather than the more expensive methionine (Paddon et al. 2013) .

Commercial production of semisynthetic artemisinin is now underway at Sanofi's site in Garessio, Italy. The innovative industrial process to produce semisynthetic artemisinin consists in the production of artemisinic acid through fermentation—which is performed by Huvepharma, in Bulgaria—followed by a synthetic transformation of the artemisinic acid into artemisinin via photochemistry, which will be performed at Sanofi’s Garessio site. The production goal is set at 35 tons for 2013. It is expected to increase to 50-60 tons per year in 2014, supplying approximately 1/3 of the global annual need for artemisinin (Pantjushenko. 2013) .

==References==
Adams MD, Kelley JM, Gocayne JD, et al. (1991). "Complementary DNA sequencing: expressed sequence tags and human genome project". Science 252 (5013): 1651–6. doi:10.1126/science.2047873.

Akhila A, Thakur RS, Popli SP (1987) Biosynthesis of artemisininin Artemisia annua. Phytochemistry 26:1927–1930

Bertea, C. M. et al. (2005) Identification of intermediates and enzymes involved in the early steps of artemisinin biosynthesis in Artemisia annua. Planta Med. 71, 40–-47.

Boumeester HJ, Wallaart TE, Janssen MH, van Loo B,Jansen BJ, Posthumus MA, Schmidt CO, de Kraker JW, Knig WA, Franssen MC (1999) Amorpha-4,11-diene synthase catalyze the first probable step in artemisinin biosynthesis. Phytochemistry 52:843–854

Brock Biology of Microorganisms (11th Edition) (2006) by Michael T. Madigan, John M. Martinko, David Stahl, David P. Clark
Connolly JD, Hill RA (1991) Dictionary of terpenoids. Vol 1, Mono- and Sesquiterpenoids. Chapman and Hall, London

Covello PS, Teoh KH, Polichuk DR, Reed DW, Nowak G (2007) Functional genomics and the biosynthesis of artemisinin. Phytochemistry 68:1864–1871

Davies B S J, Wang H S, Rine J (2005) Dual activators of the sterol biosynthetic pathway of Saccharomyces cerevisiae: Similar activation/regulatory domains but different response mechanisms. Mol. Cell. Biol. 25, 7375–-7385

Foley M, Tilley L (1998) Quinoline antimalarials:Mechanisms of action and resistance and prospects for new agents. Pharmacol Ther 79:55–87

Layne SP (2006) "Principles of Infectious Disease Epidemiology" (PDF). EPI 220. UCLA Department of Epidemiology. Archived from the original on 2006-02-20. Retrieved 2007-06-15
Liu JM, Ni MY, Fan JF, Tu YY, Wu ZH, Wu YL, Chou WS (1979) Structure and reaction of arteannuin. Acta Chim Sin 37:129–143

Pantjushenko E (2013) "Sanofi and PATH announce the launch of large-scale production of semisynthetic artemisinin against malaria". PATH.
Sigel R, Sigel A, Sigel H(2007). The Ubiquitous Roles of Cytochrome P450 Proteins: Metal Ions in Life Sciences. New York: Wiley.

Warhurst D. (2001) New developments: Chloroquine-resistance in Plasmodium falciparum. Drug Resistance Updates 4:141–144

Zeng Q, Qiu F, Yuan L. (2008) Production of artemisinin by genetically-modified microbes. Biotechnol Lett. 30:581–592.

Paddon CJ, Westfall PJ, Pitera DJ, Benjamin K, Fisher K, McPhee D, Leavell MD, Tai A, Main A, Eng D, Polichuk DR, Teoh KH, Reed DW, Treynor T, Lenihan J, Fleck M, Bajad S, Dang G, Dengrove D, Diola D, Dorin G, Ellens KW, Fickes S, Galazzo J, Gaucher SP, Geistlinger T, Henry R, Hepp M, Horning T, Iqbal T, Jiang H, Kizer L, Lieu B, Melis D, Moss N, Regentin R, Secrest S, Tsuruta H, Vazquez R, Westblade LF, Xu L, Yu M, Zhang Y, Zhao L, Lievense J, Covello PS, Keasling JD, Reiling KK, Renninger NS, Newman JD (2013) High-level semi-synthetic production of the potent antimalarial artemisinin. Nature; 496(7446):528-32

Production of the antimalarial drug precursor artemisinic acid in engineered yeast

2015-01-20T04:44:24Z

Živa Mersetič: /* Mevalonate pathway and artemisinin synthesis pathway */

This coursework is my attempt to paraphrase and explain in simple words the article of Ro DK, Paradise EM, Ouellet M, Fisher KJ, Newman KL, Ndungu JM, Ho KA, Eachus RA, Ham TS, Kirby J, Chang MC, Withers ST, Shiba Y, Sarpong R, Keasling JD with the title Production of the antimalarial drug precursor artemisinic acid in engineered yeast. The article was published in 2006 in Nature and it is one of the most cited articles in history os synthetic biology.
The article is one of many articles contributing to the realization of one of the biggest practical application of synthetic biology. The authors describe how they managed to prepare a yeast strain ‘’Saccharomyces cerevisiae’’, also known as Baker’s yeast, which was able to produce artemisinic acid. The production of anti-malarial drugs Artemisinic acid can be afterwards chemically transformed into artemisinin, which is today the first line treatment for malaria worldwide.
I divided this coursework in three parts. In the first part some aspects of malaria as disease and history of treatment are described. Second part is trying to explain the work behind the article. The third part is highlighting what happened after happy ending of the article in 2006, in other words, how things went on until today.
==Malaria and treatment==
=== Malaria===
Malaria is an emerging epidemic disease, occurring in warmer parts of the world. Because of its occurrence in tropical and subtropical areas where a lot of moisture is present its name is derived from Italian words for “bad air” [2]. It is endemic in a broad band around the equator in America, Asia and Africa. Most of deaths (85–90%) are misled in Sub-Saharan Africa (Layne SP. 2006).
It is caused by four species of sporozoa, but mostly by ‘’Plasmodium vivax ‘’and ‘’Plasmidium falciparum’’. This parasite performs part if its life cycle in human and part of it in the mosquito called ‘’Anopheles’’. Female mosquitoes of genus ‘’Anopheles’’ transmit protozoan ‘’Plasmodium falciparum’’ from person to person. The life cycle of ‘’Plasmidium falciparum’’ is complex. When mosquito injects saliva together with ‘’Plasmidium falciparum’’ sporozoites into a human host, those small, elongated cells travel through the bloodstream to the liver, where they convert into larger cells called schizont. Those cells divide into many small cells called merozoites, which enter bloodstream and infects erythrocytes. In red blood cells merozites grow, divide and exit cell by cell lysis. This asexual reproduction cycle takes approximately 48 hours. During this 48-hour period, malaria specific syndromes occur, such as chills, followed by fever up to 40◦C when ‘’Plasmidium falciparum’’ cells are released from cells. Because of the loss of red blood cells, malaria generally causes anemia.
Not all protozoal cells liberated from red blood cells are able to infect other erythrocytes. The protozoan cells, that cannot infect red blood cells are named gametocytes and are infective only for mosquito. When another mosquito feeds with infected blood gametocytes enter its digestive tract. In mosquito sexual production occurs and zygote is formed, which forms number of sporozoites. Some of these reach the salivary gland of the mosquito, and are injected into next human reservoir by the bite of mosquito (Madigan et al. 2006).

Picture of Plasmodium life cycle: http://www.uni-tuebingen.de/modeling/Mod_Malaria_Cycle_en.html

===Treatment===
Until now no antimalarial vaccine had been developed. Consequently all the treatment of malaria relies upon antilamarial drugs. First drug against malaria was quinine. It was extracted from the bark of the Cinchona tree in 1820 and was the only drug used for malaria treatment until 1930s (Foley et al. 1998) . Due to the occurrence of resistance of plasmodium species against quinine, search of new active pharmaceutical substances against malaria started. In the context of those researches chloroquinine and other synthetic guinoline antimalarials such as mefloquine were developed (Liu et al. 1979) . Unfortunately, plasmodium species developed resistance against those drugs, too (Warhurst 2001) .

In China another antimalarial drug was discovered in glandular trichomes on leaves and floral buds of plant called sweet wormwood or ‘’Artemisia annua’’. It was called artemisinin. Molecules of artemisinin contain a peroxide (O-O) group. In the presence of iron from damaged blood cells, the peroxide group is assumed to generate reactive free radicals which could destroy the DNA of the plasmodium (Liu et al. 1979) .
Today World Health Organisation recommends artemisinin-based combination therapies.
== Production of the antimalarial drug precursor artemisinic acid in engineered yeast==

===Mevalonate pathway and artemisinin synthesis pathway===

Even though artemisinin can be extracted from ‘’A. annua’’ plants, this way of obtaining the drug faces multiple obstacles. One of the major obstacle in direct extraction from plants lies in dependence of artemisinin assay in plant tissue upon natural environmental variability. Contamination by other plant trepenes, can cause problems in steps of purification (Zeng et al. 2008) .
In order to avoid those problems, which in effect cause major fluctuations in artemisinin price and thereby make it unaffordable for third world patients, Ro and coworkers published an article describing production of artemisinic acid in yeast ‘’Saccharomyces cerevisiae’’. Their idea was to rewrite the genome of ordinary brewer’s yeast to encourage it to make artemisinic acid. ‘’Saccharomyces cerevisiae’’ in an eukaryotic microorganism and can be easily grown in large bioreactors in industrial environment, thus making artemisinin production and especially purification cheaper.

Artemisinin has been categorized as a terpenoid or isoprenoid (Connolly et al. 1991). Ro and coworkers knew, that artemisinin biosynthesis pathway consists of two stages.

In the first stage linear isoprene precursors, such as GPP ( geranyl pyrophosphate ) later converted into FPP (farnesyl pyrophosphate) are synthesized from Acetyl-CoA via mevalonate pathway. This mevalonate pathway is present in all organisms including ‘’Saccharomices cerevisiae’’. Here should be mentioned that in ‘’Saccharomices cerevisiae’’ mevalonate pathway which starts from Acetyl-CoA and goes through intermediates such as HMG-CoA, mevalonate, IPP, GPP and FPP continues into synthesis of Squalene, which is then used for the synthesis of ergosterol (Covello et al. 2008) . Ergosterol is found in cell membranes of fungi and protozoa where stabilizes the membrane an makes it less flexible (Madigan et al. 2006).

In the second stage cyclic trepenes are synthesized from linear isoprene precursors ( GPP, FPP ). In case of artemisinic acid synthesis cyclic trepene is amorpha – 4, 11 diene from ‘’A. annua’’. This intermediate is in next steps transformed into artemisinic acid.

Schematic representation of the mevalonate pathway in ‘’S. cerevisiae’’ and its crossroad with atremisinic acid synthesis pathway can be seen following this link: http://www.nature.com/nature/journal/v440/n7086/fig_tab/nature04640_F1.html#figure-title

The involvement of mevalonate pathway in artemisinin biosynthesis has been already proven (Akhila et al. 1987). It was also known, that amorpha – 4, 11 diene, the first intermediate in the second stage of artemisinin is cyclized FPP by enzyme amorpha – 4, 11 diene synthase (ADS) (Bouwmeester et al. 1999). Gene encoding this enzyme was already known. However, the gene and amino acid sequence of the next enzyme in pathway was still unknown. This at the time unknown enzyme should be able to catalyze reaction of oxidation of amorpha – 4, 11 diene into artemisinic acid via artemisinic aldehyde and artemisinic alcohol intermediates. Ro and coworkers made great breakthrough and identified a cytochrome P450 monooxygenase (CYP71AV1) / amorpha-4,11-diene oxidase (AMO) for the oxidation of amorpha - 4,11-diene.

=== Isolating genes encoding enzymes responsible for oxidizing amorphadiene to artemisinic acid in ‘’A. annua ‘’===

Previous research demonstrated, that enzyme catalyzing the first regiospecific hydroxylation of amorphadiene in A. annua belongs to group on enzymes called cytochrome P450 monooxygenase (P450) (Bertea et al. 2005). Cytochrome P450 enzymes (CYPs) are, in general, oxidase enzymes. The most common reaction catalyzed by those enzymes is an insertion of one atom of oxygen into the aliphatic position of an organic substrate while the other oxygen atom is reduced to water. Most CYPs require a protein partner to deliver one or more electrons (Siegel et al. 2007).

In order to find this specific enzyme they collected P450-expressed-sequence tags (ESTs) belonging to sunflower and lettuce from the Asteraceae EST-database (http://www.cgpdb.ucdavis.edu).
EST is abbreviation for an expressed sequence tag. They are short sub-sequences of a cDNA sequence. ESTs have a relatively low quality of sequence. Their length is limited by current technology to approximately 500 to 800 nucleotides. They may be used to identify gene transcripts, to unravel new genes or to determine gene sequences. They also represent portions of expressed genes (Adams et al. 1991).

With known amino acid sequences of cytochrome P450 monooxygenases from sunflowers and lettuce (CYP71 subfamily), they designed degenerated primers, which would in PCR reaction amplify even cytochrome P450 monooxygenases from different species, in our case from’’ A. annua’’.

At the same time total RNA from trichome enriched cells of ‘’A.anua’’ was extracted. Using total RNA, cDNA pool was prepared.

Researchers were hoping to find enzymes from P450 family that existed and were transcribed and translated in ‘’A. annua’’, because there was a strong possibility , that one of those enzymes was the enzyme they were looking for.

Retrieved cDNA pool and synthesized degenerative primers were used in PCR reaction. Result of this pcr reaction was the isolation of several unique P450 fragments from an ‘’A. annua’’ trichome-enriched complementary DNA pool. Comparing DNA sequences of those fragments single ‘’A. annua’’ P450 gene fragment was spotted. It had 85–88% identity at the amino-acid level to ESTs of unknown function from both sunflower and lettuce. Sequence identity of this ‘’A. annua’’ P450 fragment to other P450 fragments outside the Asteraceae family was much lower. This P450 gene was therefore a suitable candidate for a cytochrome P450 monooxygenase (CYP71AV1)/amorpha-4,11-diene oxidase (AMO) enzyme.

With DNA sequence, isolation of open reading frame of 495 amino acids of CYP71AV1 enzyme from ‘’A. annua’’ was possible.

As previously stated CYP71AV1 as all cytochrome P450 enzyme needs its native redoxs partner. They used ‘’A. annua’’ cytochrome P450 oxidoreductase (CPR) as a redox partner.

===Rewriting the genome of ordinary ‘’Saccharomyces cerevisiae’’ to encourage it to make artemisinic acid===

With missing genes discovered Ro and coworkers could start developing synthetic yeast strain which could produce atremisinic acid. Since yeast is eukaryotic microbe problems such as lack of heterologous gene expression and nonfunctional membrane – bound proteins were excluded.

What they needed to do was:

1. To engineer the farnesyl pyrophosphate (FPP) biosynthetic pathway to increase FPP production and decrease its use for sterols

2. To introduce the amorphadiene synthase gene (ADS) from ‘’A. annua’’ into the high FPP producer to convert FPP to amorphadiene, and

3. To clone a novel cytochrome P450 that performs a three-step oxidation of amorphadiene to artemisinic acid from’’ A. annua’’ and expressing it in the amorphadiene producer

First step was to upregulate the expression of some genes encoding for enzymes present in mevalonate pathway and to downregulate enzyme ERG9, which catalyzes conversion of FPP into squalene (encodes squalene synthase,which catalyses the competing reaction of joining two farnesyl diphosphate moieties to form squalene (Paddon et al. 2013)). Thereby total concentration of FPP in the cell would increase providing more substrate (FPP) for amorphadiene synthase to convert FPP to amorphadiene.

Using Yeast Integrating plasmids - These plasmids lack an ORI and must be integrated directly into the host chromosome via homologous recombination – Ro and coworkers inserted following genes under strong GAL1 promoters into yeast genome one by one. They used standard lithium acetate method. After every successful transformation three to ten colonies from each transformation were tested for the highest amorphadiene producing transformation.

ADS:

The first of many steps was transfection with plasmid harboring amorphadiene synthase gene (ADS) from ‘’A. annua’’. Subsequently changes in mevalonate pathway were needed.

tHMGR:

Firstly they added gene coding tHMGR enzyme. Overexpression of a truncated, soluble form of 3 - hydroy – 3 - methylglutaryl - coenzyme A reductase (tHMGR) – enzyme catalyzing conversion of HMG-CoA into mevalonate, improved amorphadiene production approximately fivefold.

upc2-1:

After that upc2-1, asemi-dominant mutant allele that enhances the activity of UPC2 was added. UPC2 is a global transcription factor regulating the biosynthesis of sterols in ‘’S. cerevisiae’’ (Davies et al. 2005).

ERG9:

To downregulate enzyme ERG9, which catalyzes conversion of FPP into squalene (the first step after FPP in the sterol biosynthetic pathway), its promoter was changed from its original promotor into Pmet3, a methionine-represible promoter. This increased amorphadiene production an additional twofold.

tHMGR:

Additionally they inserted another copy of tHMGR gene into the chromosome. This insertion increased amorphadiene
production by 50% .

ERG20:

Finally they put ERG20, gene encoding FPP synthase, under strong GAL1 promotor.

Result of all these insertions and modifications was strain called EPY224 which was able to produce 153 mg per litre of amorphadiene.

Of course, both previously mentioned; CYP71AV1 and its cytochrome P450 oxidoreductase (CPR) as a redox partner, were still needed to be placed between yeast genes. Vector with CPR gene and CYP71AV1 gene under the control of galactose-inducible promoters was inserted via transformation into transgenic yeast strain EPY224.

To detect whether desired substance (artemisinic acid) was produced with synthetic yeast strain, yeast coulture medium and cell pellet were investigated via gas chromatography followed by mass spectrophotometer to detect newly synthesized substances. Artemisinic acid from ‘’A. annua’’ was used as reference for retention time. Results showed, that more than 95% of this novel compound was associated with the cell pellet. Authors claim, that easy removal of artemisinic acid by alkaline buffer confirms the idea that artemisinic acid is efficiently transported out of yeast cells but remains bound to the cell surface when it is protonated under acidic culture conditions.

Using single silica gel column chromatographic separation, artemisinic acid was purified. NMR spectra also confirmed that artemisinic acid produced by yeast strain matches artemisinic acid extracted from ‘’A. annua’’.

== Artemisinic acid production in yeast today==
Even though results in 2006 were very promising some problems still remained unsolved. Besides, in attempts to scale up the process new issues emerged. One of the biggest was low viability of synthetic yeast strains due to rapid accumulation of artemisinic acid and poor pairing of cytochrome P450 and their reductases, which resulted in formation of reactive oxygen species.

In 2013 Paddon and coworkers publish an article where a significantly improved, re-engineered yeast strain that is able to produce much more artemisinic acid is described. There are three enzymes added: CYB5, ADH1 and ALDH1, all of them are involved in the oxidation of amorphadiene to artemisinic acid in ‘’A. annua’’ plants. Also MET3 promoter is replaced with the copper-regulated CTR3 promoter, enabling restriction of ERG9 expression by addition of the inexpensive repressor CuSO4 to the medium rather than the more expensive methionine (Paddon et al. 2013) .

Commercial production of semisynthetic artemisinin is now underway at Sanofi's site in Garessio, Italy. The innovative industrial process to produce semisynthetic artemisinin consists in the production of artemisinic acid through fermentation—which is performed by Huvepharma, in Bulgaria—followed by a synthetic transformation of the artemisinic acid into artemisinin via photochemistry, which will be performed at Sanofi’s Garessio site. The production goal is set at 35 tons for 2013. It is expected to increase to 50-60 tons per year in 2014, supplying approximately 1/3 of the global annual need for artemisinin (Pantjushenko. 2013) .

==References==
Adams MD, Kelley JM, Gocayne JD, et al. (1991). "Complementary DNA sequencing: expressed sequence tags and human genome project". Science 252 (5013): 1651–6. doi:10.1126/science.2047873.

Akhila A, Thakur RS, Popli SP (1987) Biosynthesis of artemisininin Artemisia annua. Phytochemistry 26:1927–1930

Bertea, C. M. et al. (2005) Identification of intermediates and enzymes involved in the early steps of artemisinin biosynthesis in Artemisia annua. Planta Med. 71, 40–-47.

Boumeester HJ, Wallaart TE, Janssen MH, van Loo B,Jansen BJ, Posthumus MA, Schmidt CO, de Kraker JW, Knig WA, Franssen MC (1999) Amorpha-4,11-diene synthase catalyze the first probable step in artemisinin biosynthesis. Phytochemistry 52:843–854

Brock Biology of Microorganisms (11th Edition) (2006) by Michael T. Madigan, John M. Martinko, David Stahl, David P. Clark
Connolly JD, Hill RA (1991) Dictionary of terpenoids. Vol 1, Mono- and Sesquiterpenoids. Chapman and Hall, London

Covello PS, Teoh KH, Polichuk DR, Reed DW, Nowak G (2007) Functional genomics and the biosynthesis of artemisinin. Phytochemistry 68:1864–1871

Davies B S J, Wang H S, Rine J (2005) Dual activators of the sterol biosynthetic pathway of Saccharomyces cerevisiae: Similar activation/regulatory domains but different response mechanisms. Mol. Cell. Biol. 25, 7375–-7385

Foley M, Tilley L (1998) Quinoline antimalarials:Mechanisms of action and resistance and prospects for new agents. Pharmacol Ther 79:55–87

Layne SP (2006) "Principles of Infectious Disease Epidemiology" (PDF). EPI 220. UCLA Department of Epidemiology. Archived from the original on 2006-02-20. Retrieved 2007-06-15
Liu JM, Ni MY, Fan JF, Tu YY, Wu ZH, Wu YL, Chou WS (1979) Structure and reaction of arteannuin. Acta Chim Sin 37:129–143
Pantjushenko E (2013) "Sanofi and PATH announce the launch of large-scale production of semisynthetic artemisinin against malaria". PATH.
Sigel R, Sigel A, Sigel H(2007). The Ubiquitous Roles of Cytochrome P450 Proteins: Metal Ions in Life Sciences. New York: Wiley.

Warhurst D. (2001) New developments: Chloroquine-resistance in Plasmodium falciparum. Drug Resistance Updates 4:141–144

Zeng Q, Qiu F, Yuan L. (2008) Production of artemisinin by genetically-modified microbes. Biotechnol Lett. 30:581–592.
Paddon CJ, Westfall PJ, Pitera DJ, Benjamin K, Fisher K, McPhee D, Leavell MD, Tai A, Main A, Eng D, Polichuk DR, Teoh KH, Reed DW, Treynor T, Lenihan J, Fleck M, Bajad S, Dang G, Dengrove D, Diola D, Dorin G, Ellens KW, Fickes S, Galazzo J, Gaucher SP, Geistlinger T, Henry R, Hepp M, Horning T, Iqbal T, Jiang H, Kizer L, Lieu B, Melis D, Moss N, Regentin R, Secrest S, Tsuruta H, Vazquez R, Westblade LF, Xu L, Yu M, Zhang Y, Zhao L, Lievense J, Covello PS, Keasling JD, Reiling KK, Renninger NS, Newman JD (2013) High-level semi-synthetic production of the potent antimalarial artemisinin. Nature; 496(7446):528-32

Production of the antimalarial drug precursor artemisinic acid in engineered yeast

2015-01-20T04:41:37Z

Živa Mersetič: /* Isolating genes encoding enzymes responsible for oxidizing amorphadiene to artemisinic acid in ‘’A. annua ‘’ */

This coursework is my attempt to paraphrase and explain in simple words the article of Ro DK, Paradise EM, Ouellet M, Fisher KJ, Newman KL, Ndungu JM, Ho KA, Eachus RA, Ham TS, Kirby J, Chang MC, Withers ST, Shiba Y, Sarpong R, Keasling JD with the title Production of the antimalarial drug precursor artemisinic acid in engineered yeast. The article was published in 2006 in Nature and it is one of the most cited articles in history os synthetic biology.
The article is one of many articles contributing to the realization of one of the biggest practical application of synthetic biology. The authors describe how they managed to prepare a yeast strain ‘’Saccharomyces cerevisiae’’, also known as Baker’s yeast, which was able to produce artemisinic acid. The production of anti-malarial drugs Artemisinic acid can be afterwards chemically transformed into artemisinin, which is today the first line treatment for malaria worldwide.
I divided this coursework in three parts. In the first part some aspects of malaria as disease and history of treatment are described. Second part is trying to explain the work behind the article. The third part is highlighting what happened after happy ending of the article in 2006, in other words, how things went on until today.
==Malaria and treatment==
=== Malaria===
Malaria is an emerging epidemic disease, occurring in warmer parts of the world. Because of its occurrence in tropical and subtropical areas where a lot of moisture is present its name is derived from Italian words for “bad air” [2]. It is endemic in a broad band around the equator in America, Asia and Africa. Most of deaths (85–90%) are misled in Sub-Saharan Africa (Layne SP. 2006).
It is caused by four species of sporozoa, but mostly by ‘’Plasmodium vivax ‘’and ‘’Plasmidium falciparum’’. This parasite performs part if its life cycle in human and part of it in the mosquito called ‘’Anopheles’’. Female mosquitoes of genus ‘’Anopheles’’ transmit protozoan ‘’Plasmodium falciparum’’ from person to person. The life cycle of ‘’Plasmidium falciparum’’ is complex. When mosquito injects saliva together with ‘’Plasmidium falciparum’’ sporozoites into a human host, those small, elongated cells travel through the bloodstream to the liver, where they convert into larger cells called schizont. Those cells divide into many small cells called merozoites, which enter bloodstream and infects erythrocytes. In red blood cells merozites grow, divide and exit cell by cell lysis. This asexual reproduction cycle takes approximately 48 hours. During this 48-hour period, malaria specific syndromes occur, such as chills, followed by fever up to 40◦C when ‘’Plasmidium falciparum’’ cells are released from cells. Because of the loss of red blood cells, malaria generally causes anemia.
Not all protozoal cells liberated from red blood cells are able to infect other erythrocytes. The protozoan cells, that cannot infect red blood cells are named gametocytes and are infective only for mosquito. When another mosquito feeds with infected blood gametocytes enter its digestive tract. In mosquito sexual production occurs and zygote is formed, which forms number of sporozoites. Some of these reach the salivary gland of the mosquito, and are injected into next human reservoir by the bite of mosquito (Madigan et al. 2006).

Picture of Plasmodium life cycle: http://www.uni-tuebingen.de/modeling/Mod_Malaria_Cycle_en.html

===Treatment===
Until now no antimalarial vaccine had been developed. Consequently all the treatment of malaria relies upon antilamarial drugs. First drug against malaria was quinine. It was extracted from the bark of the Cinchona tree in 1820 and was the only drug used for malaria treatment until 1930s (Foley et al. 1998) . Due to the occurrence of resistance of plasmodium species against quinine, search of new active pharmaceutical substances against malaria started. In the context of those researches chloroquinine and other synthetic guinoline antimalarials such as mefloquine were developed (Liu et al. 1979) . Unfortunately, plasmodium species developed resistance against those drugs, too (Warhurst 2001) .

In China another antimalarial drug was discovered in glandular trichomes on leaves and floral buds of plant called sweet wormwood or ‘’Artemisia annua’’. It was called artemisinin. Molecules of artemisinin contain a peroxide (O-O) group. In the presence of iron from damaged blood cells, the peroxide group is assumed to generate reactive free radicals which could destroy the DNA of the plasmodium (Liu et al. 1979) .
Today World Health Organisation recommends artemisinin-based combination therapies.
== Production of the antimalarial drug precursor artemisinic acid in engineered yeast==

===Mevalonate pathway and artemisinin synthesis pathway===

Even though artemisinin can be extracted from ‘’A. annua’’ plants, this way of obtaining the drug faces multiple obstacles. One of the major obstacle in direct extraction from plants lies in dependence of artemisinin assay in plant tissue upon natural environmental variability. Contamination by other plant trepenes, can cause problems in steps of purification (Zeng et al. 2008) .
In order to avoid those problems, which in effect cause major fluctuations in artemisinin price and thereby make it unaffordable for third world patients, Ro and coworkers published an article describing production of artemisinic acid in yeast ‘’Saccharomyces cerevisiae’’. Their idea was to rewrite the genome of ordinary brewer’s yeast to encourage it to make artemisinic acid. ‘’Saccharomyces cerevisiae’’ in an eukaryotic microorganism and can be easily grown in large bioreactors in industrial environment, thus making artemisinin production and especially purification cheaper.
Artemisinin has been categorized as a terpenoid or isoprenoid (Connolly et al. 1991) .

Ro and coworkers knew, that artemisinin biosynthesis pathway consists of two stages.

In the first stage linear isoprene precursors, such as GPP ( geranyl pyrophosphate ) later converted into FPP (farnesyl pyrophosphate) are synthesized from Acetyl-CoA via mevalonate pathway.

This mevalonate pathway is present in all organisms including ‘’Saccharomices cerevisiae’’. Here should be mentioned that in ‘’Saccharomices cerevisiae’’ mevalonate pathway which starts from Acetyl-CoA and goes through intermediates such as HMG-CoA, mevalonate, IPP, GPP and FPP continues into synthesis of Squalene, which is then used for the synthesis of ergosterol (Covello et al. 2008) . Ergosterol is found in cell membranes of fungi and protozoa where stabilizes the membrane an makes it less flexible (Madigan et al. 2006).

In the second stage cyclic trepenes are synthesized from linear isoprene precursors ( GPP, FPP ). In case of artemisinic acid synthesis cyclic trepene is amorpha – 4, 11 diene from ‘’A. annua’’. This intermediate is in next steps transformed into artemisinic acid.
Schematic representation of the mevalonate pathway in ‘’S. cerevisiae’’ and its crossroad with atremisinic acid synthesis pathway can be seen following this link: http://www.nature.com/nature/journal/v440/n7086/fig_tab/nature04640_F1.html#figure-title

The involvement of mevalonate pathway in artemisinin biosynthesis has been already proven (Akhila et al. 1987). It was also known, that amorpha – 4, 11 diene, the first intermediate in the second stage of artemisinin is cyclized FPP by enzyme amorpha – 4, 11 diene synthase (ADS) (Bouwmeester et al. 1999). Gene encoding this enzyme was already known.
However, the gene and amino acid sequence of the next enzyme in pathway was still unknown. This at the time unknown enzyme should be able to catalyze reaction of oxidation of amorpha – 4, 11 diene into artemisinic acid via artemisinic aldehyde and artemisinic alcohol intermediates.
Ro and coworkers made great breakthrough and identified a cytochrome P450 monooxygenase (CYP71AV1)/
amorpha-4,11-diene oxidase (AMO) for the oxidation of amorpha - 4,11-diene.

=== Isolating genes encoding enzymes responsible for oxidizing amorphadiene to artemisinic acid in ‘’A. annua ‘’===

Previous research demonstrated, that enzyme catalyzing the first regiospecific hydroxylation of amorphadiene in A. annua belongs to group on enzymes called cytochrome P450 monooxygenase (P450) (Bertea et al. 2005). Cytochrome P450 enzymes (CYPs) are, in general, oxidase enzymes. The most common reaction catalyzed by those enzymes is an insertion of one atom of oxygen into the aliphatic position of an organic substrate while the other oxygen atom is reduced to water. Most CYPs require a protein partner to deliver one or more electrons (Siegel et al. 2007).

In order to find this specific enzyme they collected P450-expressed-sequence tags (ESTs) belonging to sunflower and lettuce from the Asteraceae EST-database (http://www.cgpdb.ucdavis.edu).
EST is abbreviation for an expressed sequence tag. They are short sub-sequences of a cDNA sequence. ESTs have a relatively low quality of sequence. Their length is limited by current technology to approximately 500 to 800 nucleotides. They may be used to identify gene transcripts, to unravel new genes or to determine gene sequences. They also represent portions of expressed genes (Adams et al. 1991).

With known amino acid sequences of cytochrome P450 monooxygenases from sunflowers and lettuce (CYP71 subfamily), they designed degenerated primers, which would in PCR reaction amplify even cytochrome P450 monooxygenases from different species, in our case from’’ A. annua’’.

At the same time total RNA from trichome enriched cells of ‘’A.anua’’ was extracted. Using total RNA, cDNA pool was prepared.

Researchers were hoping to find enzymes from P450 family that existed and were transcribed and translated in ‘’A. annua’’, because there was a strong possibility , that one of those enzymes was the enzyme they were looking for.

Retrieved cDNA pool and synthesized degenerative primers were used in PCR reaction. Result of this pcr reaction was the isolation of several unique P450 fragments from an ‘’A. annua’’ trichome-enriched complementary DNA pool. Comparing DNA sequences of those fragments single ‘’A. annua’’ P450 gene fragment was spotted. It had 85–88% identity at the amino-acid level to ESTs of unknown function from both sunflower and lettuce. Sequence identity of this ‘’A. annua’’ P450 fragment to other P450 fragments outside the Asteraceae family was much lower. This P450 gene was therefore a suitable candidate for a cytochrome P450 monooxygenase (CYP71AV1)/amorpha-4,11-diene oxidase (AMO) enzyme.

With DNA sequence, isolation of open reading frame of 495 amino acids of CYP71AV1 enzyme from ‘’A. annua’’ was possible.

As previously stated CYP71AV1 as all cytochrome P450 enzyme needs its native redoxs partner. They used ‘’A. annua’’ cytochrome P450 oxidoreductase (CPR) as a redox partner.

===Rewriting the genome of ordinary ‘’Saccharomyces cerevisiae’’ to encourage it to make artemisinic acid===

With missing genes discovered Ro and coworkers could start developing synthetic yeast strain which could produce atremisinic acid. Since yeast is eukaryotic microbe problems such as lack of heterologous gene expression and nonfunctional membrane – bound proteins were excluded.

What they needed to do was:

1. To engineer the farnesyl pyrophosphate (FPP) biosynthetic pathway to increase FPP production and decrease its use for sterols

2. To introduce the amorphadiene synthase gene (ADS) from ‘’A. annua’’ into the high FPP producer to convert FPP to amorphadiene, and

3. To clone a novel cytochrome P450 that performs a three-step oxidation of amorphadiene to artemisinic acid from’’ A. annua’’ and expressing it in the amorphadiene producer

First step was to upregulate the expression of some genes encoding for enzymes present in mevalonate pathway and to downregulate enzyme ERG9, which catalyzes conversion of FPP into squalene (encodes squalene synthase,which catalyses the competing reaction of joining two farnesyl diphosphate moieties to form squalene (Paddon et al. 2013)). Thereby total concentration of FPP in the cell would increase providing more substrate (FPP) for amorphadiene synthase to convert FPP to amorphadiene.

Using Yeast Integrating plasmids - These plasmids lack an ORI and must be integrated directly into the host chromosome via homologous recombination – Ro and coworkers inserted following genes under strong GAL1 promoters into yeast genome one by one. They used standard lithium acetate method. After every successful transformation three to ten colonies from each transformation were tested for the highest amorphadiene producing transformation.

ADS:

The first of many steps was transfection with plasmid harboring amorphadiene synthase gene (ADS) from ‘’A. annua’’. Subsequently changes in mevalonate pathway were needed.

tHMGR:

Firstly they added gene coding tHMGR enzyme. Overexpression of a truncated, soluble form of 3 - hydroy – 3 - methylglutaryl - coenzyme A reductase (tHMGR) – enzyme catalyzing conversion of HMG-CoA into mevalonate, improved amorphadiene production approximately fivefold.

upc2-1:

After that upc2-1, asemi-dominant mutant allele that enhances the activity of UPC2 was added. UPC2 is a global transcription factor regulating the biosynthesis of sterols in ‘’S. cerevisiae’’ (Davies et al. 2005).

ERG9:

To downregulate enzyme ERG9, which catalyzes conversion of FPP into squalene (the first step after FPP in the sterol biosynthetic pathway), its promoter was changed from its original promotor into Pmet3, a methionine-represible promoter. This increased amorphadiene production an additional twofold.

tHMGR:

Additionally they inserted another copy of tHMGR gene into the chromosome. This insertion increased amorphadiene
production by 50% .

ERG20:

Finally they put ERG20, gene encoding FPP synthase, under strong GAL1 promotor.

Result of all these insertions and modifications was strain called EPY224 which was able to produce 153 mg per litre of amorphadiene.

Of course, both previously mentioned; CYP71AV1 and its cytochrome P450 oxidoreductase (CPR) as a redox partner, were still needed to be placed between yeast genes. Vector with CPR gene and CYP71AV1 gene under the control of galactose-inducible promoters was inserted via transformation into transgenic yeast strain EPY224.

To detect whether desired substance (artemisinic acid) was produced with synthetic yeast strain, yeast coulture medium and cell pellet were investigated via gas chromatography followed by mass spectrophotometer to detect newly synthesized substances. Artemisinic acid from ‘’A. annua’’ was used as reference for retention time. Results showed, that more than 95% of this novel compound was associated with the cell pellet. Authors claim, that easy removal of artemisinic acid by alkaline buffer confirms the idea that artemisinic acid is efficiently transported out of yeast cells but remains bound to the cell surface when it is protonated under acidic culture conditions.

Using single silica gel column chromatographic separation, artemisinic acid was purified. NMR spectra also confirmed that artemisinic acid produced by yeast strain matches artemisinic acid extracted from ‘’A. annua’’.

== Artemisinic acid production in yeast today==
Even though results in 2006 were very promising some problems still remained unsolved. Besides, in attempts to scale up the process new issues emerged. One of the biggest was low viability of synthetic yeast strains due to rapid accumulation of artemisinic acid and poor pairing of cytochrome P450 and their reductases, which resulted in formation of reactive oxygen species.

In 2013 Paddon and coworkers publish an article where a significantly improved, re-engineered yeast strain that is able to produce much more artemisinic acid is described. There are three enzymes added: CYB5, ADH1 and ALDH1, all of them are involved in the oxidation of amorphadiene to artemisinic acid in ‘’A. annua’’ plants. Also MET3 promoter is replaced with the copper-regulated CTR3 promoter, enabling restriction of ERG9 expression by addition of the inexpensive repressor CuSO4 to the medium rather than the more expensive methionine (Paddon et al. 2013) .

Commercial production of semisynthetic artemisinin is now underway at Sanofi's site in Garessio, Italy. The innovative industrial process to produce semisynthetic artemisinin consists in the production of artemisinic acid through fermentation—which is performed by Huvepharma, in Bulgaria—followed by a synthetic transformation of the artemisinic acid into artemisinin via photochemistry, which will be performed at Sanofi’s Garessio site. The production goal is set at 35 tons for 2013. It is expected to increase to 50-60 tons per year in 2014, supplying approximately 1/3 of the global annual need for artemisinin (Pantjushenko. 2013) .

==References==
Adams MD, Kelley JM, Gocayne JD, et al. (1991). "Complementary DNA sequencing: expressed sequence tags and human genome project". Science 252 (5013): 1651–6. doi:10.1126/science.2047873.

Akhila A, Thakur RS, Popli SP (1987) Biosynthesis of artemisininin Artemisia annua. Phytochemistry 26:1927–1930

Bertea, C. M. et al. (2005) Identification of intermediates and enzymes involved in the early steps of artemisinin biosynthesis in Artemisia annua. Planta Med. 71, 40–-47.

Boumeester HJ, Wallaart TE, Janssen MH, van Loo B,Jansen BJ, Posthumus MA, Schmidt CO, de Kraker JW, Knig WA, Franssen MC (1999) Amorpha-4,11-diene synthase catalyze the first probable step in artemisinin biosynthesis. Phytochemistry 52:843–854

Brock Biology of Microorganisms (11th Edition) (2006) by Michael T. Madigan, John M. Martinko, David Stahl, David P. Clark
Connolly JD, Hill RA (1991) Dictionary of terpenoids. Vol 1, Mono- and Sesquiterpenoids. Chapman and Hall, London

Covello PS, Teoh KH, Polichuk DR, Reed DW, Nowak G (2007) Functional genomics and the biosynthesis of artemisinin. Phytochemistry 68:1864–1871

Davies B S J, Wang H S, Rine J (2005) Dual activators of the sterol biosynthetic pathway of Saccharomyces cerevisiae: Similar activation/regulatory domains but different response mechanisms. Mol. Cell. Biol. 25, 7375–-7385

Foley M, Tilley L (1998) Quinoline antimalarials:Mechanisms of action and resistance and prospects for new agents. Pharmacol Ther 79:55–87

Layne SP (2006) "Principles of Infectious Disease Epidemiology" (PDF). EPI 220. UCLA Department of Epidemiology. Archived from the original on 2006-02-20. Retrieved 2007-06-15
Liu JM, Ni MY, Fan JF, Tu YY, Wu ZH, Wu YL, Chou WS (1979) Structure and reaction of arteannuin. Acta Chim Sin 37:129–143
Pantjushenko E (2013) "Sanofi and PATH announce the launch of large-scale production of semisynthetic artemisinin against malaria". PATH.
Sigel R, Sigel A, Sigel H(2007). The Ubiquitous Roles of Cytochrome P450 Proteins: Metal Ions in Life Sciences. New York: Wiley.

Warhurst D. (2001) New developments: Chloroquine-resistance in Plasmodium falciparum. Drug Resistance Updates 4:141–144

Zeng Q, Qiu F, Yuan L. (2008) Production of artemisinin by genetically-modified microbes. Biotechnol Lett. 30:581–592.
Paddon CJ, Westfall PJ, Pitera DJ, Benjamin K, Fisher K, McPhee D, Leavell MD, Tai A, Main A, Eng D, Polichuk DR, Teoh KH, Reed DW, Treynor T, Lenihan J, Fleck M, Bajad S, Dang G, Dengrove D, Diola D, Dorin G, Ellens KW, Fickes S, Galazzo J, Gaucher SP, Geistlinger T, Henry R, Hepp M, Horning T, Iqbal T, Jiang H, Kizer L, Lieu B, Melis D, Moss N, Regentin R, Secrest S, Tsuruta H, Vazquez R, Westblade LF, Xu L, Yu M, Zhang Y, Zhao L, Lievense J, Covello PS, Keasling JD, Reiling KK, Renninger NS, Newman JD (2013) High-level semi-synthetic production of the potent antimalarial artemisinin. Nature; 496(7446):528-32

Production of the antimalarial drug precursor artemisinic acid in engineered yeast

2015-01-20T04:38:35Z

Živa Mersetič: /* Rewriting the genome of ordinary ‘’Saccharomyces cerevisiae’’ to encourage it to make artemisinic acid */

This coursework is my attempt to paraphrase and explain in simple words the article of Ro DK, Paradise EM, Ouellet M, Fisher KJ, Newman KL, Ndungu JM, Ho KA, Eachus RA, Ham TS, Kirby J, Chang MC, Withers ST, Shiba Y, Sarpong R, Keasling JD with the title Production of the antimalarial drug precursor artemisinic acid in engineered yeast. The article was published in 2006 in Nature and it is one of the most cited articles in history os synthetic biology.
The article is one of many articles contributing to the realization of one of the biggest practical application of synthetic biology. The authors describe how they managed to prepare a yeast strain ‘’Saccharomyces cerevisiae’’, also known as Baker’s yeast, which was able to produce artemisinic acid. The production of anti-malarial drugs Artemisinic acid can be afterwards chemically transformed into artemisinin, which is today the first line treatment for malaria worldwide.
I divided this coursework in three parts. In the first part some aspects of malaria as disease and history of treatment are described. Second part is trying to explain the work behind the article. The third part is highlighting what happened after happy ending of the article in 2006, in other words, how things went on until today.
==Malaria and treatment==
=== Malaria===
Malaria is an emerging epidemic disease, occurring in warmer parts of the world. Because of its occurrence in tropical and subtropical areas where a lot of moisture is present its name is derived from Italian words for “bad air” [2]. It is endemic in a broad band around the equator in America, Asia and Africa. Most of deaths (85–90%) are misled in Sub-Saharan Africa (Layne SP. 2006).
It is caused by four species of sporozoa, but mostly by ‘’Plasmodium vivax ‘’and ‘’Plasmidium falciparum’’. This parasite performs part if its life cycle in human and part of it in the mosquito called ‘’Anopheles’’. Female mosquitoes of genus ‘’Anopheles’’ transmit protozoan ‘’Plasmodium falciparum’’ from person to person. The life cycle of ‘’Plasmidium falciparum’’ is complex. When mosquito injects saliva together with ‘’Plasmidium falciparum’’ sporozoites into a human host, those small, elongated cells travel through the bloodstream to the liver, where they convert into larger cells called schizont. Those cells divide into many small cells called merozoites, which enter bloodstream and infects erythrocytes. In red blood cells merozites grow, divide and exit cell by cell lysis. This asexual reproduction cycle takes approximately 48 hours. During this 48-hour period, malaria specific syndromes occur, such as chills, followed by fever up to 40◦C when ‘’Plasmidium falciparum’’ cells are released from cells. Because of the loss of red blood cells, malaria generally causes anemia.
Not all protozoal cells liberated from red blood cells are able to infect other erythrocytes. The protozoan cells, that cannot infect red blood cells are named gametocytes and are infective only for mosquito. When another mosquito feeds with infected blood gametocytes enter its digestive tract. In mosquito sexual production occurs and zygote is formed, which forms number of sporozoites. Some of these reach the salivary gland of the mosquito, and are injected into next human reservoir by the bite of mosquito (Madigan et al. 2006).

Picture of Plasmodium life cycle: http://www.uni-tuebingen.de/modeling/Mod_Malaria_Cycle_en.html

===Treatment===
Until now no antimalarial vaccine had been developed. Consequently all the treatment of malaria relies upon antilamarial drugs. First drug against malaria was quinine. It was extracted from the bark of the Cinchona tree in 1820 and was the only drug used for malaria treatment until 1930s (Foley et al. 1998) . Due to the occurrence of resistance of plasmodium species against quinine, search of new active pharmaceutical substances against malaria started. In the context of those researches chloroquinine and other synthetic guinoline antimalarials such as mefloquine were developed (Liu et al. 1979) . Unfortunately, plasmodium species developed resistance against those drugs, too (Warhurst 2001) .

In China another antimalarial drug was discovered in glandular trichomes on leaves and floral buds of plant called sweet wormwood or ‘’Artemisia annua’’. It was called artemisinin. Molecules of artemisinin contain a peroxide (O-O) group. In the presence of iron from damaged blood cells, the peroxide group is assumed to generate reactive free radicals which could destroy the DNA of the plasmodium (Liu et al. 1979) .
Today World Health Organisation recommends artemisinin-based combination therapies.
== Production of the antimalarial drug precursor artemisinic acid in engineered yeast==

===Mevalonate pathway and artemisinin synthesis pathway===

Even though artemisinin can be extracted from ‘’A. annua’’ plants, this way of obtaining the drug faces multiple obstacles. One of the major obstacle in direct extraction from plants lies in dependence of artemisinin assay in plant tissue upon natural environmental variability. Contamination by other plant trepenes, can cause problems in steps of purification (Zeng et al. 2008) .
In order to avoid those problems, which in effect cause major fluctuations in artemisinin price and thereby make it unaffordable for third world patients, Ro and coworkers published an article describing production of artemisinic acid in yeast ‘’Saccharomyces cerevisiae’’. Their idea was to rewrite the genome of ordinary brewer’s yeast to encourage it to make artemisinic acid. ‘’Saccharomyces cerevisiae’’ in an eukaryotic microorganism and can be easily grown in large bioreactors in industrial environment, thus making artemisinin production and especially purification cheaper.
Artemisinin has been categorized as a terpenoid or isoprenoid (Connolly et al. 1991) .

Ro and coworkers knew, that artemisinin biosynthesis pathway consists of two stages.

In the first stage linear isoprene precursors, such as GPP ( geranyl pyrophosphate ) later converted into FPP (farnesyl pyrophosphate) are synthesized from Acetyl-CoA via mevalonate pathway.

This mevalonate pathway is present in all organisms including ‘’Saccharomices cerevisiae’’. Here should be mentioned that in ‘’Saccharomices cerevisiae’’ mevalonate pathway which starts from Acetyl-CoA and goes through intermediates such as HMG-CoA, mevalonate, IPP, GPP and FPP continues into synthesis of Squalene, which is then used for the synthesis of ergosterol (Covello et al. 2008) . Ergosterol is found in cell membranes of fungi and protozoa where stabilizes the membrane an makes it less flexible (Madigan et al. 2006).

In the second stage cyclic trepenes are synthesized from linear isoprene precursors ( GPP, FPP ). In case of artemisinic acid synthesis cyclic trepene is amorpha – 4, 11 diene from ‘’A. annua’’. This intermediate is in next steps transformed into artemisinic acid.
Schematic representation of the mevalonate pathway in ‘’S. cerevisiae’’ and its crossroad with atremisinic acid synthesis pathway can be seen following this link: http://www.nature.com/nature/journal/v440/n7086/fig_tab/nature04640_F1.html#figure-title

The involvement of mevalonate pathway in artemisinin biosynthesis has been already proven (Akhila et al. 1987). It was also known, that amorpha – 4, 11 diene, the first intermediate in the second stage of artemisinin is cyclized FPP by enzyme amorpha – 4, 11 diene synthase (ADS) (Bouwmeester et al. 1999). Gene encoding this enzyme was already known.
However, the gene and amino acid sequence of the next enzyme in pathway was still unknown. This at the time unknown enzyme should be able to catalyze reaction of oxidation of amorpha – 4, 11 diene into artemisinic acid via artemisinic aldehyde and artemisinic alcohol intermediates.
Ro and coworkers made great breakthrough and identified a cytochrome P450 monooxygenase (CYP71AV1)/
amorpha-4,11-diene oxidase (AMO) for the oxidation of amorpha - 4,11-diene.

=== Isolating genes encoding enzymes responsible for oxidizing amorphadiene to artemisinic acid in ‘’A. annua ‘’===

Previous research demonstrated, that enzyme catalyzing the first regiospecific hydroxylation of amorphadiene in A. annua belongs to group on enzymes called cytochrome P450 monooxygenase (P450) (Bertea et al. 2005).

Cytochrome P450 enzymes (CYPs) are, in general, oxidase enzymes. The most common reaction catalyzed by those enzymes is an insertion of one atom of oxygen into the aliphatic position of an organic substrate while the other oxygen atom is reduced to water. Most CYPs require a protein partner to deliver one or more electrons (Siegel et al. 2007).

In order to find this specific enzyme they collected P450-expressed-sequence tags (ESTs) belonging to sunflower and lettuce from the Asteraceae EST-database (http://www.cgpdb.ucdavis.edu).
EST is abbreviation for an expressed sequence tag. They are short sub-sequences of a cDNA sequence. ESTs have a relatively low quality of sequence. Their length is limited by current technology to approximately 500 to 800 nucleotides. They may be used to identify gene transcripts, to unravel new genes or to determine gene sequences. They also represent portions of expressed genes (Adams et al. 1991).

With known amino acid sequences of cytochrome P450 monooxygenases from sunflowers and lettuce (CYP71 subfamily), they designed degenerated primers, which would in PCR reaction amplify even cytochrome P450 monooxygenases from different species, in our case from’’ A. annua’’.

At the same time total RNA from trichome enriched cells of ‘’A.anua’’ was extracted. Using total RNA, cDNA pool was prepared.

Researchers were hoping to find enzymes from P450 family that existed and were transcribed and translated in ‘’A. annua’’, because there was a strong possibility , that one of those enzymes was the enzyme they were looking for.

Retrieved cDNA pool and synthesized degenerative primers were used in PCR reaction. Result of this pcr reaction was the isolation of several unique P450 fragments from an ‘’A. annua’’ trichome-enriched complementary DNA pool.

Comparing DNA sequences of those fragments single ‘’A. annua’’ P450 gene fragment was spotted. It had 85–88% identity at the amino-acid level to ESTs of unknown function from both sunflower and lettuce. Sequence identity of this ‘’A. annua’’ P450 fragment to other P450 fragments outside the Asteraceae family was much lower. This P450 gene was therefore a suitable candidate for a cytochrome P450 monooxygenase (CYP71AV1)/amorpha-4,11-diene oxidase (AMO) enzyme.

With DNA sequence, isolation of open reading frame of 495 amino acids of CYP71AV1 enzyme from ‘’A. annua’’ was possible.

As previously stated CYP71AV1 as all cytochrome P450 enzyme needs its native redoxs partner. They used ‘’A. annua’’ cytochrome P450 oxidoreductase (CPR) as a redox partner.

===Rewriting the genome of ordinary ‘’Saccharomyces cerevisiae’’ to encourage it to make artemisinic acid===

With missing genes discovered Ro and coworkers could start developing synthetic yeast strain which could produce atremisinic acid. Since yeast is eukaryotic microbe problems such as lack of heterologous gene expression and nonfunctional membrane – bound proteins were excluded.

What they needed to do was:

1. To engineer the farnesyl pyrophosphate (FPP) biosynthetic pathway to increase FPP production and decrease its use for sterols

2. To introduce the amorphadiene synthase gene (ADS) from ‘’A. annua’’ into the high FPP producer to convert FPP to amorphadiene, and

3. To clone a novel cytochrome P450 that performs a three-step oxidation of amorphadiene to artemisinic acid from’’ A. annua’’ and expressing it in the amorphadiene producer

First step was to upregulate the expression of some genes encoding for enzymes present in mevalonate pathway and to downregulate enzyme ERG9, which catalyzes conversion of FPP into squalene (encodes squalene synthase,which catalyses the competing reaction of joining two farnesyl diphosphate moieties to form squalene (Paddon et al. 2013)). Thereby total concentration of FPP in the cell would increase providing more substrate (FPP) for amorphadiene synthase to convert FPP to amorphadiene.

Using Yeast Integrating plasmids - These plasmids lack an ORI and must be integrated directly into the host chromosome via homologous recombination – Ro and coworkers inserted following genes under strong GAL1 promoters into yeast genome one by one. They used standard lithium acetate method. After every successful transformation three to ten colonies from each transformation were tested for the highest amorphadiene producing transformation.

ADS:

The first of many steps was transfection with plasmid harboring amorphadiene synthase gene (ADS) from ‘’A. annua’’. Subsequently changes in mevalonate pathway were needed.

tHMGR:

Firstly they added gene coding tHMGR enzyme. Overexpression of a truncated, soluble form of 3 - hydroy – 3 - methylglutaryl - coenzyme A reductase (tHMGR) – enzyme catalyzing conversion of HMG-CoA into mevalonate, improved amorphadiene production approximately fivefold.

upc2-1:

After that upc2-1, asemi-dominant mutant allele that enhances the activity of UPC2 was added. UPC2 is a global transcription factor regulating the biosynthesis of sterols in ‘’S. cerevisiae’’ (Davies et al. 2005).

ERG9:

To downregulate enzyme ERG9, which catalyzes conversion of FPP into squalene (the first step after FPP in the sterol biosynthetic pathway), its promoter was changed from its original promotor into Pmet3, a methionine-represible promoter. This increased amorphadiene production an additional twofold.

tHMGR:

Additionally they inserted another copy of tHMGR gene into the chromosome. This insertion increased amorphadiene
production by 50% .

ERG20:

Finally they put ERG20, gene encoding FPP synthase, under strong GAL1 promotor.

Result of all these insertions and modifications was strain called EPY224 which was able to produce 153 mg per litre of amorphadiene.

Of course, both previously mentioned; CYP71AV1 and its cytochrome P450 oxidoreductase (CPR) as a redox partner, were still needed to be placed between yeast genes. Vector with CPR gene and CYP71AV1 gene under the control of galactose-inducible promoters was inserted via transformation into transgenic yeast strain EPY224.

To detect whether desired substance (artemisinic acid) was produced with synthetic yeast strain, yeast coulture medium and cell pellet were investigated via gas chromatography followed by mass spectrophotometer to detect newly synthesized substances. Artemisinic acid from ‘’A. annua’’ was used as reference for retention time. Results showed, that more than 95% of this novel compound was associated with the cell pellet. Authors claim, that easy removal of artemisinic acid by alkaline buffer confirms the idea that artemisinic acid is efficiently transported out of yeast cells but remains bound to the cell surface when it is protonated under acidic culture conditions.

Using single silica gel column chromatographic separation, artemisinic acid was purified. NMR spectra also confirmed that artemisinic acid produced by yeast strain matches artemisinic acid extracted from ‘’A. annua’’.

== Artemisinic acid production in yeast today==
Even though results in 2006 were very promising some problems still remained unsolved. Besides, in attempts to scale up the process new issues emerged. One of the biggest was low viability of synthetic yeast strains due to rapid accumulation of artemisinic acid and poor pairing of cytochrome P450 and their reductases, which resulted in formation of reactive oxygen species.

In 2013 Paddon and coworkers publish an article where a significantly improved, re-engineered yeast strain that is able to produce much more artemisinic acid is described. There are three enzymes added: CYB5, ADH1 and ALDH1, all of them are involved in the oxidation of amorphadiene to artemisinic acid in ‘’A. annua’’ plants. Also MET3 promoter is replaced with the copper-regulated CTR3 promoter, enabling restriction of ERG9 expression by addition of the inexpensive repressor CuSO4 to the medium rather than the more expensive methionine (Paddon et al. 2013) .

Commercial production of semisynthetic artemisinin is now underway at Sanofi's site in Garessio, Italy. The innovative industrial process to produce semisynthetic artemisinin consists in the production of artemisinic acid through fermentation—which is performed by Huvepharma, in Bulgaria—followed by a synthetic transformation of the artemisinic acid into artemisinin via photochemistry, which will be performed at Sanofi’s Garessio site. The production goal is set at 35 tons for 2013. It is expected to increase to 50-60 tons per year in 2014, supplying approximately 1/3 of the global annual need for artemisinin (Pantjushenko. 2013) .

==References==
Adams MD, Kelley JM, Gocayne JD, et al. (1991). "Complementary DNA sequencing: expressed sequence tags and human genome project". Science 252 (5013): 1651–6. doi:10.1126/science.2047873.

Akhila A, Thakur RS, Popli SP (1987) Biosynthesis of artemisininin Artemisia annua. Phytochemistry 26:1927–1930

Bertea, C. M. et al. (2005) Identification of intermediates and enzymes involved in the early steps of artemisinin biosynthesis in Artemisia annua. Planta Med. 71, 40–-47.

Boumeester HJ, Wallaart TE, Janssen MH, van Loo B,Jansen BJ, Posthumus MA, Schmidt CO, de Kraker JW, Knig WA, Franssen MC (1999) Amorpha-4,11-diene synthase catalyze the first probable step in artemisinin biosynthesis. Phytochemistry 52:843–854

Brock Biology of Microorganisms (11th Edition) (2006) by Michael T. Madigan, John M. Martinko, David Stahl, David P. Clark
Connolly JD, Hill RA (1991) Dictionary of terpenoids. Vol 1, Mono- and Sesquiterpenoids. Chapman and Hall, London

Covello PS, Teoh KH, Polichuk DR, Reed DW, Nowak G (2007) Functional genomics and the biosynthesis of artemisinin. Phytochemistry 68:1864–1871

Davies B S J, Wang H S, Rine J (2005) Dual activators of the sterol biosynthetic pathway of Saccharomyces cerevisiae: Similar activation/regulatory domains but different response mechanisms. Mol. Cell. Biol. 25, 7375–-7385

Foley M, Tilley L (1998) Quinoline antimalarials:Mechanisms of action and resistance and prospects for new agents. Pharmacol Ther 79:55–87

Layne SP (2006) "Principles of Infectious Disease Epidemiology" (PDF). EPI 220. UCLA Department of Epidemiology. Archived from the original on 2006-02-20. Retrieved 2007-06-15
Liu JM, Ni MY, Fan JF, Tu YY, Wu ZH, Wu YL, Chou WS (1979) Structure and reaction of arteannuin. Acta Chim Sin 37:129–143
Pantjushenko E (2013) "Sanofi and PATH announce the launch of large-scale production of semisynthetic artemisinin against malaria". PATH.
Sigel R, Sigel A, Sigel H(2007). The Ubiquitous Roles of Cytochrome P450 Proteins: Metal Ions in Life Sciences. New York: Wiley.

Warhurst D. (2001) New developments: Chloroquine-resistance in Plasmodium falciparum. Drug Resistance Updates 4:141–144

Zeng Q, Qiu F, Yuan L. (2008) Production of artemisinin by genetically-modified microbes. Biotechnol Lett. 30:581–592.
Paddon CJ, Westfall PJ, Pitera DJ, Benjamin K, Fisher K, McPhee D, Leavell MD, Tai A, Main A, Eng D, Polichuk DR, Teoh KH, Reed DW, Treynor T, Lenihan J, Fleck M, Bajad S, Dang G, Dengrove D, Diola D, Dorin G, Ellens KW, Fickes S, Galazzo J, Gaucher SP, Geistlinger T, Henry R, Hepp M, Horning T, Iqbal T, Jiang H, Kizer L, Lieu B, Melis D, Moss N, Regentin R, Secrest S, Tsuruta H, Vazquez R, Westblade LF, Xu L, Yu M, Zhang Y, Zhao L, Lievense J, Covello PS, Keasling JD, Reiling KK, Renninger NS, Newman JD (2013) High-level semi-synthetic production of the potent antimalarial artemisinin. Nature; 496(7446):528-32

Production of the antimalarial drug precursor artemisinic acid in engineered yeast

2015-01-20T04:37:55Z

Živa Mersetič: /* Rewriting the genome of ordinary ‘’Saccharomyces cerevisiae’’ to encourage it to make artemisinic acid */

This coursework is my attempt to paraphrase and explain in simple words the article of Ro DK, Paradise EM, Ouellet M, Fisher KJ, Newman KL, Ndungu JM, Ho KA, Eachus RA, Ham TS, Kirby J, Chang MC, Withers ST, Shiba Y, Sarpong R, Keasling JD with the title Production of the antimalarial drug precursor artemisinic acid in engineered yeast. The article was published in 2006 in Nature and it is one of the most cited articles in history os synthetic biology.
The article is one of many articles contributing to the realization of one of the biggest practical application of synthetic biology. The authors describe how they managed to prepare a yeast strain ‘’Saccharomyces cerevisiae’’, also known as Baker’s yeast, which was able to produce artemisinic acid. The production of anti-malarial drugs Artemisinic acid can be afterwards chemically transformed into artemisinin, which is today the first line treatment for malaria worldwide.
I divided this coursework in three parts. In the first part some aspects of malaria as disease and history of treatment are described. Second part is trying to explain the work behind the article. The third part is highlighting what happened after happy ending of the article in 2006, in other words, how things went on until today.
==Malaria and treatment==
=== Malaria===
Malaria is an emerging epidemic disease, occurring in warmer parts of the world. Because of its occurrence in tropical and subtropical areas where a lot of moisture is present its name is derived from Italian words for “bad air” [2]. It is endemic in a broad band around the equator in America, Asia and Africa. Most of deaths (85–90%) are misled in Sub-Saharan Africa (Layne SP. 2006).
It is caused by four species of sporozoa, but mostly by ‘’Plasmodium vivax ‘’and ‘’Plasmidium falciparum’’. This parasite performs part if its life cycle in human and part of it in the mosquito called ‘’Anopheles’’. Female mosquitoes of genus ‘’Anopheles’’ transmit protozoan ‘’Plasmodium falciparum’’ from person to person. The life cycle of ‘’Plasmidium falciparum’’ is complex. When mosquito injects saliva together with ‘’Plasmidium falciparum’’ sporozoites into a human host, those small, elongated cells travel through the bloodstream to the liver, where they convert into larger cells called schizont. Those cells divide into many small cells called merozoites, which enter bloodstream and infects erythrocytes. In red blood cells merozites grow, divide and exit cell by cell lysis. This asexual reproduction cycle takes approximately 48 hours. During this 48-hour period, malaria specific syndromes occur, such as chills, followed by fever up to 40◦C when ‘’Plasmidium falciparum’’ cells are released from cells. Because of the loss of red blood cells, malaria generally causes anemia.
Not all protozoal cells liberated from red blood cells are able to infect other erythrocytes. The protozoan cells, that cannot infect red blood cells are named gametocytes and are infective only for mosquito. When another mosquito feeds with infected blood gametocytes enter its digestive tract. In mosquito sexual production occurs and zygote is formed, which forms number of sporozoites. Some of these reach the salivary gland of the mosquito, and are injected into next human reservoir by the bite of mosquito (Madigan et al. 2006).

Picture of Plasmodium life cycle: http://www.uni-tuebingen.de/modeling/Mod_Malaria_Cycle_en.html

===Treatment===
Until now no antimalarial vaccine had been developed. Consequently all the treatment of malaria relies upon antilamarial drugs. First drug against malaria was quinine. It was extracted from the bark of the Cinchona tree in 1820 and was the only drug used for malaria treatment until 1930s (Foley et al. 1998) . Due to the occurrence of resistance of plasmodium species against quinine, search of new active pharmaceutical substances against malaria started. In the context of those researches chloroquinine and other synthetic guinoline antimalarials such as mefloquine were developed (Liu et al. 1979) . Unfortunately, plasmodium species developed resistance against those drugs, too (Warhurst 2001) .

In China another antimalarial drug was discovered in glandular trichomes on leaves and floral buds of plant called sweet wormwood or ‘’Artemisia annua’’. It was called artemisinin. Molecules of artemisinin contain a peroxide (O-O) group. In the presence of iron from damaged blood cells, the peroxide group is assumed to generate reactive free radicals which could destroy the DNA of the plasmodium (Liu et al. 1979) .
Today World Health Organisation recommends artemisinin-based combination therapies.
== Production of the antimalarial drug precursor artemisinic acid in engineered yeast==

===Mevalonate pathway and artemisinin synthesis pathway===

Even though artemisinin can be extracted from ‘’A. annua’’ plants, this way of obtaining the drug faces multiple obstacles. One of the major obstacle in direct extraction from plants lies in dependence of artemisinin assay in plant tissue upon natural environmental variability. Contamination by other plant trepenes, can cause problems in steps of purification (Zeng et al. 2008) .
In order to avoid those problems, which in effect cause major fluctuations in artemisinin price and thereby make it unaffordable for third world patients, Ro and coworkers published an article describing production of artemisinic acid in yeast ‘’Saccharomyces cerevisiae’’. Their idea was to rewrite the genome of ordinary brewer’s yeast to encourage it to make artemisinic acid. ‘’Saccharomyces cerevisiae’’ in an eukaryotic microorganism and can be easily grown in large bioreactors in industrial environment, thus making artemisinin production and especially purification cheaper.
Artemisinin has been categorized as a terpenoid or isoprenoid (Connolly et al. 1991) .

Ro and coworkers knew, that artemisinin biosynthesis pathway consists of two stages.

In the first stage linear isoprene precursors, such as GPP ( geranyl pyrophosphate ) later converted into FPP (farnesyl pyrophosphate) are synthesized from Acetyl-CoA via mevalonate pathway.

This mevalonate pathway is present in all organisms including ‘’Saccharomices cerevisiae’’. Here should be mentioned that in ‘’Saccharomices cerevisiae’’ mevalonate pathway which starts from Acetyl-CoA and goes through intermediates such as HMG-CoA, mevalonate, IPP, GPP and FPP continues into synthesis of Squalene, which is then used for the synthesis of ergosterol (Covello et al. 2008) . Ergosterol is found in cell membranes of fungi and protozoa where stabilizes the membrane an makes it less flexible (Madigan et al. 2006).

In the second stage cyclic trepenes are synthesized from linear isoprene precursors ( GPP, FPP ). In case of artemisinic acid synthesis cyclic trepene is amorpha – 4, 11 diene from ‘’A. annua’’. This intermediate is in next steps transformed into artemisinic acid.
Schematic representation of the mevalonate pathway in ‘’S. cerevisiae’’ and its crossroad with atremisinic acid synthesis pathway can be seen following this link: http://www.nature.com/nature/journal/v440/n7086/fig_tab/nature04640_F1.html#figure-title

The involvement of mevalonate pathway in artemisinin biosynthesis has been already proven (Akhila et al. 1987). It was also known, that amorpha – 4, 11 diene, the first intermediate in the second stage of artemisinin is cyclized FPP by enzyme amorpha – 4, 11 diene synthase (ADS) (Bouwmeester et al. 1999). Gene encoding this enzyme was already known.
However, the gene and amino acid sequence of the next enzyme in pathway was still unknown. This at the time unknown enzyme should be able to catalyze reaction of oxidation of amorpha – 4, 11 diene into artemisinic acid via artemisinic aldehyde and artemisinic alcohol intermediates.
Ro and coworkers made great breakthrough and identified a cytochrome P450 monooxygenase (CYP71AV1)/
amorpha-4,11-diene oxidase (AMO) for the oxidation of amorpha - 4,11-diene.

=== Isolating genes encoding enzymes responsible for oxidizing amorphadiene to artemisinic acid in ‘’A. annua ‘’===

Previous research demonstrated, that enzyme catalyzing the first regiospecific hydroxylation of amorphadiene in A. annua belongs to group on enzymes called cytochrome P450 monooxygenase (P450) (Bertea et al. 2005).

Cytochrome P450 enzymes (CYPs) are, in general, oxidase enzymes. The most common reaction catalyzed by those enzymes is an insertion of one atom of oxygen into the aliphatic position of an organic substrate while the other oxygen atom is reduced to water. Most CYPs require a protein partner to deliver one or more electrons (Siegel et al. 2007).

In order to find this specific enzyme they collected P450-expressed-sequence tags (ESTs) belonging to sunflower and lettuce from the Asteraceae EST-database (http://www.cgpdb.ucdavis.edu).
EST is abbreviation for an expressed sequence tag. They are short sub-sequences of a cDNA sequence. ESTs have a relatively low quality of sequence. Their length is limited by current technology to approximately 500 to 800 nucleotides. They may be used to identify gene transcripts, to unravel new genes or to determine gene sequences. They also represent portions of expressed genes (Adams et al. 1991).

With known amino acid sequences of cytochrome P450 monooxygenases from sunflowers and lettuce (CYP71 subfamily), they designed degenerated primers, which would in PCR reaction amplify even cytochrome P450 monooxygenases from different species, in our case from’’ A. annua’’.

At the same time total RNA from trichome enriched cells of ‘’A.anua’’ was extracted. Using total RNA, cDNA pool was prepared.

Researchers were hoping to find enzymes from P450 family that existed and were transcribed and translated in ‘’A. annua’’, because there was a strong possibility , that one of those enzymes was the enzyme they were looking for.

Retrieved cDNA pool and synthesized degenerative primers were used in PCR reaction. Result of this pcr reaction was the isolation of several unique P450 fragments from an ‘’A. annua’’ trichome-enriched complementary DNA pool.

Comparing DNA sequences of those fragments single ‘’A. annua’’ P450 gene fragment was spotted. It had 85–88% identity at the amino-acid level to ESTs of unknown function from both sunflower and lettuce. Sequence identity of this ‘’A. annua’’ P450 fragment to other P450 fragments outside the Asteraceae family was much lower. This P450 gene was therefore a suitable candidate for a cytochrome P450 monooxygenase (CYP71AV1)/amorpha-4,11-diene oxidase (AMO) enzyme.

With DNA sequence, isolation of open reading frame of 495 amino acids of CYP71AV1 enzyme from ‘’A. annua’’ was possible.

As previously stated CYP71AV1 as all cytochrome P450 enzyme needs its native redoxs partner. They used ‘’A. annua’’ cytochrome P450 oxidoreductase (CPR) as a redox partner.

===Rewriting the genome of ordinary ‘’Saccharomyces cerevisiae’’ to encourage it to make artemisinic acid===

With missing genes discovered Ro and coworkers could start developing synthetic yeast strain which could produce atremisinic acid. Since yeast is eukaryotic microbe problems such as lack of heterologous gene expression and nonfunctional membrane – bound proteins were excluded.

What they needed to do was:

1. To engineer the farnesyl pyrophosphate (FPP) biosynthetic pathway to increase FPP production and decrease its use for sterols

2. To introduce the amorphadiene synthase gene (ADS) from ‘’A. annua’’ into the high FPP producer to convert FPP to amorphadiene, and

3. To clone a novel cytochrome P450 that performs a three-step oxidation of amorphadiene to artemisinic acid from’’ A. annua’’ and expressing it in the amorphadiene producer

First step was to upregulate the expression of some genes encoding for enzymes present in mevalonate pathway and to downregulate enzyme ERG9, which catalyzes conversion of FPP into squalene (encodes squalene synthase,which catalyses the competing reaction of joining two farnesyl diphosphate moieties to form squalene (Paddon et al. 2013)). Thereby total concentration of FPP in the cell would increase providing more substrate (FPP) for amorphadiene synthase to convert FPP to amorphadiene.

Using Yeast Integrating plasmids - These plasmids lack an ORI and must be integrated directly into the host chromosome via homologous recombination – Ro and coworkers inserted following genes under strong GAL1 promoters into yeast genome one by one. They used standard lithium acetate method. After every successful transformation three to ten colonies from each transformation were tested for the highest amorphadiene producing transformation.

ADS:

The first of many steps was transfection with plasmid harboring amorphadiene synthase gene (ADS) from ‘’A. annua’’. Subsequently changes in mevalonate pathway were needed.

tHMGR:

Firstly they added gene coding tHMGR enzyme. Overexpression of a truncated, soluble form of 3 - hydroy – 3 - methylglutaryl - coenzyme A reductase (tHMGR) – enzyme catalyzing conversion of HMG-CoA into mevalonate, improved amorphadiene production approximately fivefold.

upc2-1:

After that upc2-1, asemi-dominant mutant allele that enhances the activity of UPC2 was added. UPC2 is a global transcription factor regulating the biosynthesis of sterols in ‘’S. cerevisiae’’ (Davies et al. 2005).

ERG9:

To downregulate enzyme ERG9, which catalyzes conversion of FPP into squalene (the first step after FPP in the sterol biosynthetic pathway), its promoter was changed from its original promotor into Pmet3, a methionine-represible promoter. This increased amorphadiene production an additional twofold.

tHMGR:

Additionally they inserted another copy of tHMGR gene into the chromosome. This insertion increased amorphadiene
production by 50% .

ERG20:

Finally they put ERG20, gene encoding FPP synthase, under strong GAL1 promotor.

Result of all these insertions and modifications was strain called EPY224 which was able to produce 153 mg per litre of amorphadiene.

Of course, both previously mentioned; CYP71AV1 and its cytochrome P450 oxidoreductase (CPR) as a redox partner, were still needed to be placed between yeast genes. Vector with CPR gene and CYP71AV1 gene under the control of galactose-inducible promoters was inserted via transformation into transgenic yeast strain EPY224.

To detect whether desired substance (artemisinic acid) was produced with synthetic yeast strain, yeast coulture medium and cell pellet were investigated via gas chromatography followed by mass spectrophotometer to detect newly synthesized substances. Artemisinic acid from ‘’A. annua’’ was used as reference for retention time. Results showed, that more than 95% of this novel compound was associated with the cell pellet. Authors claim, that easy removal of artemisinic acid by alkaline buffer confirms the idea that artemisinic acid is efficiently transported out of yeast cells but remains bound to the cell surface when it is protonated under acidic culture conditions.

Using single silica gel column chromatographic separation, artemisinic acid was purified. NMR spectra also confirmed that artemisinic acid produced by yeast strain matches artemisinic acid extracted from ‘’A. annua’’.

== Artemisinic acid production in yeast today==
Even though results in 2006 were very promising some problems still remained unsolved. Besides, in attempts to scale up the process new issues emerged. One of the biggest was low viability of synthetic yeast strains due to rapid accumulation of artemisinic acid and poor pairing of cytochrome P450 and their reductases, which resulted in formation of reactive oxygen species.

In 2013 Paddon and coworkers publish an article where a significantly improved, re-engineered yeast strain that is able to produce much more artemisinic acid is described. There are three enzymes added: CYB5, ADH1 and ALDH1, all of them are involved in the oxidation of amorphadiene to artemisinic acid in ‘’A. annua’’ plants. Also MET3 promoter is replaced with the copper-regulated CTR3 promoter, enabling restriction of ERG9 expression by addition of the inexpensive repressor CuSO4 to the medium rather than the more expensive methionine (Paddon et al. 2013) .

Commercial production of semisynthetic artemisinin is now underway at Sanofi's site in Garessio, Italy. The innovative industrial process to produce semisynthetic artemisinin consists in the production of artemisinic acid through fermentation—which is performed by Huvepharma, in Bulgaria—followed by a synthetic transformation of the artemisinic acid into artemisinin via photochemistry, which will be performed at Sanofi’s Garessio site. The production goal is set at 35 tons for 2013. It is expected to increase to 50-60 tons per year in 2014, supplying approximately 1/3 of the global annual need for artemisinin (Pantjushenko. 2013) .

==References==
Adams MD, Kelley JM, Gocayne JD, et al. (1991). "Complementary DNA sequencing: expressed sequence tags and human genome project". Science 252 (5013): 1651–6. doi:10.1126/science.2047873.

Akhila A, Thakur RS, Popli SP (1987) Biosynthesis of artemisininin Artemisia annua. Phytochemistry 26:1927–1930

Bertea, C. M. et al. (2005) Identification of intermediates and enzymes involved in the early steps of artemisinin biosynthesis in Artemisia annua. Planta Med. 71, 40–-47.

Boumeester HJ, Wallaart TE, Janssen MH, van Loo B,Jansen BJ, Posthumus MA, Schmidt CO, de Kraker JW, Knig WA, Franssen MC (1999) Amorpha-4,11-diene synthase catalyze the first probable step in artemisinin biosynthesis. Phytochemistry 52:843–854

Brock Biology of Microorganisms (11th Edition) (2006) by Michael T. Madigan, John M. Martinko, David Stahl, David P. Clark
Connolly JD, Hill RA (1991) Dictionary of terpenoids. Vol 1, Mono- and Sesquiterpenoids. Chapman and Hall, London

Covello PS, Teoh KH, Polichuk DR, Reed DW, Nowak G (2007) Functional genomics and the biosynthesis of artemisinin. Phytochemistry 68:1864–1871

Davies B S J, Wang H S, Rine J (2005) Dual activators of the sterol biosynthetic pathway of Saccharomyces cerevisiae: Similar activation/regulatory domains but different response mechanisms. Mol. Cell. Biol. 25, 7375–-7385

Foley M, Tilley L (1998) Quinoline antimalarials:Mechanisms of action and resistance and prospects for new agents. Pharmacol Ther 79:55–87

Layne SP (2006) "Principles of Infectious Disease Epidemiology" (PDF). EPI 220. UCLA Department of Epidemiology. Archived from the original on 2006-02-20. Retrieved 2007-06-15
Liu JM, Ni MY, Fan JF, Tu YY, Wu ZH, Wu YL, Chou WS (1979) Structure and reaction of arteannuin. Acta Chim Sin 37:129–143
Pantjushenko E (2013) "Sanofi and PATH announce the launch of large-scale production of semisynthetic artemisinin against malaria". PATH.
Sigel R, Sigel A, Sigel H(2007). The Ubiquitous Roles of Cytochrome P450 Proteins: Metal Ions in Life Sciences. New York: Wiley.

Warhurst D. (2001) New developments: Chloroquine-resistance in Plasmodium falciparum. Drug Resistance Updates 4:141–144

Zeng Q, Qiu F, Yuan L. (2008) Production of artemisinin by genetically-modified microbes. Biotechnol Lett. 30:581–592.
Paddon CJ, Westfall PJ, Pitera DJ, Benjamin K, Fisher K, McPhee D, Leavell MD, Tai A, Main A, Eng D, Polichuk DR, Teoh KH, Reed DW, Treynor T, Lenihan J, Fleck M, Bajad S, Dang G, Dengrove D, Diola D, Dorin G, Ellens KW, Fickes S, Galazzo J, Gaucher SP, Geistlinger T, Henry R, Hepp M, Horning T, Iqbal T, Jiang H, Kizer L, Lieu B, Melis D, Moss N, Regentin R, Secrest S, Tsuruta H, Vazquez R, Westblade LF, Xu L, Yu M, Zhang Y, Zhao L, Lievense J, Covello PS, Keasling JD, Reiling KK, Renninger NS, Newman JD (2013) High-level semi-synthetic production of the potent antimalarial artemisinin. Nature; 496(7446):528-32

Production of the antimalarial drug precursor artemisinic acid in engineered yeast

2015-01-20T04:34:56Z

Živa Mersetič: /* Rewriting the genome of ordinary ‘’Saccharomyces cerevisiae’’ to encourage it to make artemisinic acid */

This coursework is my attempt to paraphrase and explain in simple words the article of Ro DK, Paradise EM, Ouellet M, Fisher KJ, Newman KL, Ndungu JM, Ho KA, Eachus RA, Ham TS, Kirby J, Chang MC, Withers ST, Shiba Y, Sarpong R, Keasling JD with the title Production of the antimalarial drug precursor artemisinic acid in engineered yeast. The article was published in 2006 in Nature and it is one of the most cited articles in history os synthetic biology.
The article is one of many articles contributing to the realization of one of the biggest practical application of synthetic biology. The authors describe how they managed to prepare a yeast strain ‘’Saccharomyces cerevisiae’’, also known as Baker’s yeast, which was able to produce artemisinic acid. The production of anti-malarial drugs Artemisinic acid can be afterwards chemically transformed into artemisinin, which is today the first line treatment for malaria worldwide.
I divided this coursework in three parts. In the first part some aspects of malaria as disease and history of treatment are described. Second part is trying to explain the work behind the article. The third part is highlighting what happened after happy ending of the article in 2006, in other words, how things went on until today.
==Malaria and treatment==
=== Malaria===
Malaria is an emerging epidemic disease, occurring in warmer parts of the world. Because of its occurrence in tropical and subtropical areas where a lot of moisture is present its name is derived from Italian words for “bad air” [2]. It is endemic in a broad band around the equator in America, Asia and Africa. Most of deaths (85–90%) are misled in Sub-Saharan Africa (Layne SP. 2006).
It is caused by four species of sporozoa, but mostly by ‘’Plasmodium vivax ‘’and ‘’Plasmidium falciparum’’. This parasite performs part if its life cycle in human and part of it in the mosquito called ‘’Anopheles’’. Female mosquitoes of genus ‘’Anopheles’’ transmit protozoan ‘’Plasmodium falciparum’’ from person to person. The life cycle of ‘’Plasmidium falciparum’’ is complex. When mosquito injects saliva together with ‘’Plasmidium falciparum’’ sporozoites into a human host, those small, elongated cells travel through the bloodstream to the liver, where they convert into larger cells called schizont. Those cells divide into many small cells called merozoites, which enter bloodstream and infects erythrocytes. In red blood cells merozites grow, divide and exit cell by cell lysis. This asexual reproduction cycle takes approximately 48 hours. During this 48-hour period, malaria specific syndromes occur, such as chills, followed by fever up to 40◦C when ‘’Plasmidium falciparum’’ cells are released from cells. Because of the loss of red blood cells, malaria generally causes anemia.
Not all protozoal cells liberated from red blood cells are able to infect other erythrocytes. The protozoan cells, that cannot infect red blood cells are named gametocytes and are infective only for mosquito. When another mosquito feeds with infected blood gametocytes enter its digestive tract. In mosquito sexual production occurs and zygote is formed, which forms number of sporozoites. Some of these reach the salivary gland of the mosquito, and are injected into next human reservoir by the bite of mosquito (Madigan et al. 2006).

Picture of Plasmodium life cycle: http://www.uni-tuebingen.de/modeling/Mod_Malaria_Cycle_en.html

===Treatment===
Until now no antimalarial vaccine had been developed. Consequently all the treatment of malaria relies upon antilamarial drugs. First drug against malaria was quinine. It was extracted from the bark of the Cinchona tree in 1820 and was the only drug used for malaria treatment until 1930s (Foley et al. 1998) . Due to the occurrence of resistance of plasmodium species against quinine, search of new active pharmaceutical substances against malaria started. In the context of those researches chloroquinine and other synthetic guinoline antimalarials such as mefloquine were developed (Liu et al. 1979) . Unfortunately, plasmodium species developed resistance against those drugs, too (Warhurst 2001) .

In China another antimalarial drug was discovered in glandular trichomes on leaves and floral buds of plant called sweet wormwood or ‘’Artemisia annua’’. It was called artemisinin. Molecules of artemisinin contain a peroxide (O-O) group. In the presence of iron from damaged blood cells, the peroxide group is assumed to generate reactive free radicals which could destroy the DNA of the plasmodium (Liu et al. 1979) .
Today World Health Organisation recommends artemisinin-based combination therapies.
== Production of the antimalarial drug precursor artemisinic acid in engineered yeast==

===Mevalonate pathway and artemisinin synthesis pathway===

Even though artemisinin can be extracted from ‘’A. annua’’ plants, this way of obtaining the drug faces multiple obstacles. One of the major obstacle in direct extraction from plants lies in dependence of artemisinin assay in plant tissue upon natural environmental variability. Contamination by other plant trepenes, can cause problems in steps of purification (Zeng et al. 2008) .
In order to avoid those problems, which in effect cause major fluctuations in artemisinin price and thereby make it unaffordable for third world patients, Ro and coworkers published an article describing production of artemisinic acid in yeast ‘’Saccharomyces cerevisiae’’. Their idea was to rewrite the genome of ordinary brewer’s yeast to encourage it to make artemisinic acid. ‘’Saccharomyces cerevisiae’’ in an eukaryotic microorganism and can be easily grown in large bioreactors in industrial environment, thus making artemisinin production and especially purification cheaper.
Artemisinin has been categorized as a terpenoid or isoprenoid (Connolly et al. 1991) .

Ro and coworkers knew, that artemisinin biosynthesis pathway consists of two stages.

In the first stage linear isoprene precursors, such as GPP ( geranyl pyrophosphate ) later converted into FPP (farnesyl pyrophosphate) are synthesized from Acetyl-CoA via mevalonate pathway.

This mevalonate pathway is present in all organisms including ‘’Saccharomices cerevisiae’’. Here should be mentioned that in ‘’Saccharomices cerevisiae’’ mevalonate pathway which starts from Acetyl-CoA and goes through intermediates such as HMG-CoA, mevalonate, IPP, GPP and FPP continues into synthesis of Squalene, which is then used for the synthesis of ergosterol (Covello et al. 2008) . Ergosterol is found in cell membranes of fungi and protozoa where stabilizes the membrane an makes it less flexible (Madigan et al. 2006).

In the second stage cyclic trepenes are synthesized from linear isoprene precursors ( GPP, FPP ). In case of artemisinic acid synthesis cyclic trepene is amorpha – 4, 11 diene from ‘’A. annua’’. This intermediate is in next steps transformed into artemisinic acid.
Schematic representation of the mevalonate pathway in ‘’S. cerevisiae’’ and its crossroad with atremisinic acid synthesis pathway can be seen following this link: http://www.nature.com/nature/journal/v440/n7086/fig_tab/nature04640_F1.html#figure-title

The involvement of mevalonate pathway in artemisinin biosynthesis has been already proven (Akhila et al. 1987). It was also known, that amorpha – 4, 11 diene, the first intermediate in the second stage of artemisinin is cyclized FPP by enzyme amorpha – 4, 11 diene synthase (ADS) (Bouwmeester et al. 1999). Gene encoding this enzyme was already known.
However, the gene and amino acid sequence of the next enzyme in pathway was still unknown. This at the time unknown enzyme should be able to catalyze reaction of oxidation of amorpha – 4, 11 diene into artemisinic acid via artemisinic aldehyde and artemisinic alcohol intermediates.
Ro and coworkers made great breakthrough and identified a cytochrome P450 monooxygenase (CYP71AV1)/
amorpha-4,11-diene oxidase (AMO) for the oxidation of amorpha - 4,11-diene.

=== Isolating genes encoding enzymes responsible for oxidizing amorphadiene to artemisinic acid in ‘’A. annua ‘’===

Previous research demonstrated, that enzyme catalyzing the first regiospecific hydroxylation of amorphadiene in A. annua belongs to group on enzymes called cytochrome P450 monooxygenase (P450) (Bertea et al. 2005).

Cytochrome P450 enzymes (CYPs) are, in general, oxidase enzymes. The most common reaction catalyzed by those enzymes is an insertion of one atom of oxygen into the aliphatic position of an organic substrate while the other oxygen atom is reduced to water. Most CYPs require a protein partner to deliver one or more electrons (Siegel et al. 2007).

In order to find this specific enzyme they collected P450-expressed-sequence tags (ESTs) belonging to sunflower and lettuce from the Asteraceae EST-database (http://www.cgpdb.ucdavis.edu).
EST is abbreviation for an expressed sequence tag. They are short sub-sequences of a cDNA sequence. ESTs have a relatively low quality of sequence. Their length is limited by current technology to approximately 500 to 800 nucleotides. They may be used to identify gene transcripts, to unravel new genes or to determine gene sequences. They also represent portions of expressed genes (Adams et al. 1991).

With known amino acid sequences of cytochrome P450 monooxygenases from sunflowers and lettuce (CYP71 subfamily), they designed degenerated primers, which would in PCR reaction amplify even cytochrome P450 monooxygenases from different species, in our case from’’ A. annua’’.

At the same time total RNA from trichome enriched cells of ‘’A.anua’’ was extracted. Using total RNA, cDNA pool was prepared.

Researchers were hoping to find enzymes from P450 family that existed and were transcribed and translated in ‘’A. annua’’, because there was a strong possibility , that one of those enzymes was the enzyme they were looking for.

Retrieved cDNA pool and synthesized degenerative primers were used in PCR reaction. Result of this pcr reaction was the isolation of several unique P450 fragments from an ‘’A. annua’’ trichome-enriched complementary DNA pool.

Comparing DNA sequences of those fragments single ‘’A. annua’’ P450 gene fragment was spotted. It had 85–88% identity at the amino-acid level to ESTs of unknown function from both sunflower and lettuce. Sequence identity of this ‘’A. annua’’ P450 fragment to other P450 fragments outside the Asteraceae family was much lower. This P450 gene was therefore a suitable candidate for a cytochrome P450 monooxygenase (CYP71AV1)/amorpha-4,11-diene oxidase (AMO) enzyme.

With DNA sequence, isolation of open reading frame of 495 amino acids of CYP71AV1 enzyme from ‘’A. annua’’ was possible.

As previously stated CYP71AV1 as all cytochrome P450 enzyme needs its native redoxs partner. They used ‘’A. annua’’ cytochrome P450 oxidoreductase (CPR) as a redox partner.

===Rewriting the genome of ordinary ‘’Saccharomyces cerevisiae’’ to encourage it to make artemisinic acid===

With missing genes discovered Ro and coworkers could start developing synthetic yeast strain which could produce atremisinic acid. Since yeast is eukaryotic microbe problems such as lack of heterologous gene expression and nonfunctional membrane – bound proteins were excluded.

What they needed to do was:

1. To engineer the farnesyl pyrophosphate (FPP) biosynthetic pathway to increase FPP production and decrease its use for sterols

2. To introduce the amorphadiene synthase gene (ADS) from ‘’A. annua’’ into the high FPP producer to convert FPP to amorphadiene, and

3. To clone a novel cytochrome P450 that performs a three-step oxidation of amorphadiene to artemisinic acid from’’ A. annua’’ and expressing it in the amorphadiene producer

First step was to upregulate the expression of some genes encoding for enzymes present in mevalonate pathway and to downregulate enzyme ERG9, which catalyzes conversion of FPP into squalene (encodes squalene synthase,which catalyses the competing reaction of joining two farnesyl diphosphate moieties to form squalene (Paddon et al. 2013)). Thereby total concentration of FPP in the cell would increase providing more substrate (FPP) for amorphadiene synthase to convert FPP to amorphadiene.

Using Yeast Integrating plasmids - These plasmids lack an ORI and must be integrated directly into the host chromosome via homologous recombination – Ro and coworkers inserted following genes under strong GAL1 promoters into yeast genome one by one. They used standard lithium acetate method. After every successful transformation three to ten colonies from each transformation were tested for the highest amorphadiene producing transformation.

After transfection with plasmid harboring amorphadiene synthase gene (ADS) from ‘’A. annua’’, changes in mevalonate pathway were needed.

tHMGR:
Firstly they added gene coding tHMGR enzyme. Overexpression of a truncated, soluble form of 3 - hydroy – 3 - methylglutaryl - coenzyme A reductase (tHMGR) – enzyme catalyzing conversion of HMG-CoA into mevalonate, improved amorphadiene production approximately fivefold.

upc2-1:
After that upc2-1, asemi-dominant mutant allele that enhances the activity of UPC2 was added. UPC2 is a global transcription factor regulating the biosynthesis of sterols in ‘’S. cerevisiae’’ (Davies et al. 2005).

ERG9:
To downregulate enzyme ERG9, which catalyzes conversion of FPP into squalene (the first step after FPP in the sterol biosynthetic pathway), its promoter was changed from its original promotor into Pmet3, a methionine-represible promoter. This increased amorphadiene production an additional twofold.

tHMGR:
Additionally they inserted another copy of tHMGR gene into the chromosome. This insertion increased amorphadiene
production by 50% .

ERG20:
Finally they put ERG20, gene encoding FPP synthase, under strong GAL1 promotor.

Result of all these insertions and modifications was strain called EPY224 which was able to produce 153 mg per litre of amorphadiene.

Of course, both previously mentioned; CYP71AV1 and its cytochrome P450 oxidoreductase (CPR) as a redox partner, were still needed to be placed between yeast genes. Vector with CPR gene and CYP71AV1 gene under the control of galactose-inducible promoters was inserted via transformation into transgenic yeast strain EPY224.

To detect whether desired substance (artemisinic acid) was produced with synthetic yeast strain, yeast coulture medium and cell pellet were investigated via gas chromatography followed by mass spectrophotometer to detect newly synthesized substances. Artemisinic acid from ‘’A. annua’’ was used as reference for retention time. Results showed, that more than 95% of this novel compound was associated with the cell pellet. Authors claim, that easy removal of artemisinic acid by alkaline buffer confirms the idea that artemisinic acid is efficiently transported out of yeast cells but remains bound to the cell surface when it is protonated under acidic culture conditions.

Using single silica gel column chromatographic separation, artemisinic acid was purified. NMR spectra also confirmed that artemisinic acid produced by yeast strain matches artemisinic acid extracted from ‘’A. annua’’.

== Artemisinic acid production in yeast today==
Even though results in 2006 were very promising some problems still remained unsolved. Besides, in attempts to scale up the process new issues emerged. One of the biggest was low viability of synthetic yeast strains due to rapid accumulation of artemisinic acid and poor pairing of cytochrome P450 and their reductases, which resulted in formation of reactive oxygen species.

In 2013 Paddon and coworkers publish an article where a significantly improved, re-engineered yeast strain that is able to produce much more artemisinic acid is described. There are three enzymes added: CYB5, ADH1 and ALDH1, all of them are involved in the oxidation of amorphadiene to artemisinic acid in ‘’A. annua’’ plants. Also MET3 promoter is replaced with the copper-regulated CTR3 promoter, enabling restriction of ERG9 expression by addition of the inexpensive repressor CuSO4 to the medium rather than the more expensive methionine (Paddon et al. 2013) .

Commercial production of semisynthetic artemisinin is now underway at Sanofi's site in Garessio, Italy. The innovative industrial process to produce semisynthetic artemisinin consists in the production of artemisinic acid through fermentation—which is performed by Huvepharma, in Bulgaria—followed by a synthetic transformation of the artemisinic acid into artemisinin via photochemistry, which will be performed at Sanofi’s Garessio site. The production goal is set at 35 tons for 2013. It is expected to increase to 50-60 tons per year in 2014, supplying approximately 1/3 of the global annual need for artemisinin (Pantjushenko. 2013) .

==References==
Adams MD, Kelley JM, Gocayne JD, et al. (1991). "Complementary DNA sequencing: expressed sequence tags and human genome project". Science 252 (5013): 1651–6. doi:10.1126/science.2047873.

Akhila A, Thakur RS, Popli SP (1987) Biosynthesis of artemisininin Artemisia annua. Phytochemistry 26:1927–1930

Bertea, C. M. et al. (2005) Identification of intermediates and enzymes involved in the early steps of artemisinin biosynthesis in Artemisia annua. Planta Med. 71, 40–-47.

Boumeester HJ, Wallaart TE, Janssen MH, van Loo B,Jansen BJ, Posthumus MA, Schmidt CO, de Kraker JW, Knig WA, Franssen MC (1999) Amorpha-4,11-diene synthase catalyze the first probable step in artemisinin biosynthesis. Phytochemistry 52:843–854

Brock Biology of Microorganisms (11th Edition) (2006) by Michael T. Madigan, John M. Martinko, David Stahl, David P. Clark
Connolly JD, Hill RA (1991) Dictionary of terpenoids. Vol 1, Mono- and Sesquiterpenoids. Chapman and Hall, London

Covello PS, Teoh KH, Polichuk DR, Reed DW, Nowak G (2007) Functional genomics and the biosynthesis of artemisinin. Phytochemistry 68:1864–1871

Davies B S J, Wang H S, Rine J (2005) Dual activators of the sterol biosynthetic pathway of Saccharomyces cerevisiae: Similar activation/regulatory domains but different response mechanisms. Mol. Cell. Biol. 25, 7375–-7385

Foley M, Tilley L (1998) Quinoline antimalarials:Mechanisms of action and resistance and prospects for new agents. Pharmacol Ther 79:55–87

Layne SP (2006) "Principles of Infectious Disease Epidemiology" (PDF). EPI 220. UCLA Department of Epidemiology. Archived from the original on 2006-02-20. Retrieved 2007-06-15
Liu JM, Ni MY, Fan JF, Tu YY, Wu ZH, Wu YL, Chou WS (1979) Structure and reaction of arteannuin. Acta Chim Sin 37:129–143
Pantjushenko E (2013) "Sanofi and PATH announce the launch of large-scale production of semisynthetic artemisinin against malaria". PATH.
Sigel R, Sigel A, Sigel H(2007). The Ubiquitous Roles of Cytochrome P450 Proteins: Metal Ions in Life Sciences. New York: Wiley.

Warhurst D. (2001) New developments: Chloroquine-resistance in Plasmodium falciparum. Drug Resistance Updates 4:141–144

Zeng Q, Qiu F, Yuan L. (2008) Production of artemisinin by genetically-modified microbes. Biotechnol Lett. 30:581–592.
Paddon CJ, Westfall PJ, Pitera DJ, Benjamin K, Fisher K, McPhee D, Leavell MD, Tai A, Main A, Eng D, Polichuk DR, Teoh KH, Reed DW, Treynor T, Lenihan J, Fleck M, Bajad S, Dang G, Dengrove D, Diola D, Dorin G, Ellens KW, Fickes S, Galazzo J, Gaucher SP, Geistlinger T, Henry R, Hepp M, Horning T, Iqbal T, Jiang H, Kizer L, Lieu B, Melis D, Moss N, Regentin R, Secrest S, Tsuruta H, Vazquez R, Westblade LF, Xu L, Yu M, Zhang Y, Zhao L, Lievense J, Covello PS, Keasling JD, Reiling KK, Renninger NS, Newman JD (2013) High-level semi-synthetic production of the potent antimalarial artemisinin. Nature; 496(7446):528-32

Production of the antimalarial drug precursor artemisinic acid in engineered yeast

2015-01-20T04:29:32Z

Živa Mersetič: /* artemisinic acid production in yeast today */

This coursework is my attempt to paraphrase and explain in simple words the article of Ro DK, Paradise EM, Ouellet M, Fisher KJ, Newman KL, Ndungu JM, Ho KA, Eachus RA, Ham TS, Kirby J, Chang MC, Withers ST, Shiba Y, Sarpong R, Keasling JD with the title Production of the antimalarial drug precursor artemisinic acid in engineered yeast. The article was published in 2006 in Nature and it is one of the most cited articles in history os synthetic biology.
The article is one of many articles contributing to the realization of one of the biggest practical application of synthetic biology. The authors describe how they managed to prepare a yeast strain ‘’Saccharomyces cerevisiae’’, also known as Baker’s yeast, which was able to produce artemisinic acid. The production of anti-malarial drugs Artemisinic acid can be afterwards chemically transformed into artemisinin, which is today the first line treatment for malaria worldwide.
I divided this coursework in three parts. In the first part some aspects of malaria as disease and history of treatment are described. Second part is trying to explain the work behind the article. The third part is highlighting what happened after happy ending of the article in 2006, in other words, how things went on until today.
==Malaria and treatment==
=== Malaria===
Malaria is an emerging epidemic disease, occurring in warmer parts of the world. Because of its occurrence in tropical and subtropical areas where a lot of moisture is present its name is derived from Italian words for “bad air” [2]. It is endemic in a broad band around the equator in America, Asia and Africa. Most of deaths (85–90%) are misled in Sub-Saharan Africa (Layne SP. 2006).
It is caused by four species of sporozoa, but mostly by ‘’Plasmodium vivax ‘’and ‘’Plasmidium falciparum’’. This parasite performs part if its life cycle in human and part of it in the mosquito called ‘’Anopheles’’. Female mosquitoes of genus ‘’Anopheles’’ transmit protozoan ‘’Plasmodium falciparum’’ from person to person. The life cycle of ‘’Plasmidium falciparum’’ is complex. When mosquito injects saliva together with ‘’Plasmidium falciparum’’ sporozoites into a human host, those small, elongated cells travel through the bloodstream to the liver, where they convert into larger cells called schizont. Those cells divide into many small cells called merozoites, which enter bloodstream and infects erythrocytes. In red blood cells merozites grow, divide and exit cell by cell lysis. This asexual reproduction cycle takes approximately 48 hours. During this 48-hour period, malaria specific syndromes occur, such as chills, followed by fever up to 40◦C when ‘’Plasmidium falciparum’’ cells are released from cells. Because of the loss of red blood cells, malaria generally causes anemia.
Not all protozoal cells liberated from red blood cells are able to infect other erythrocytes. The protozoan cells, that cannot infect red blood cells are named gametocytes and are infective only for mosquito. When another mosquito feeds with infected blood gametocytes enter its digestive tract. In mosquito sexual production occurs and zygote is formed, which forms number of sporozoites. Some of these reach the salivary gland of the mosquito, and are injected into next human reservoir by the bite of mosquito (Madigan et al. 2006).

Picture of Plasmodium life cycle: http://www.uni-tuebingen.de/modeling/Mod_Malaria_Cycle_en.html

===Treatment===
Until now no antimalarial vaccine had been developed. Consequently all the treatment of malaria relies upon antilamarial drugs. First drug against malaria was quinine. It was extracted from the bark of the Cinchona tree in 1820 and was the only drug used for malaria treatment until 1930s (Foley et al. 1998) . Due to the occurrence of resistance of plasmodium species against quinine, search of new active pharmaceutical substances against malaria started. In the context of those researches chloroquinine and other synthetic guinoline antimalarials such as mefloquine were developed (Liu et al. 1979) . Unfortunately, plasmodium species developed resistance against those drugs, too (Warhurst 2001) .

In China another antimalarial drug was discovered in glandular trichomes on leaves and floral buds of plant called sweet wormwood or ‘’Artemisia annua’’. It was called artemisinin. Molecules of artemisinin contain a peroxide (O-O) group. In the presence of iron from damaged blood cells, the peroxide group is assumed to generate reactive free radicals which could destroy the DNA of the plasmodium (Liu et al. 1979) .
Today World Health Organisation recommends artemisinin-based combination therapies.
== Production of the antimalarial drug precursor artemisinic acid in engineered yeast==

===Mevalonate pathway and artemisinin synthesis pathway===

Even though artemisinin can be extracted from ‘’A. annua’’ plants, this way of obtaining the drug faces multiple obstacles. One of the major obstacle in direct extraction from plants lies in dependence of artemisinin assay in plant tissue upon natural environmental variability. Contamination by other plant trepenes, can cause problems in steps of purification (Zeng et al. 2008) .
In order to avoid those problems, which in effect cause major fluctuations in artemisinin price and thereby make it unaffordable for third world patients, Ro and coworkers published an article describing production of artemisinic acid in yeast ‘’Saccharomyces cerevisiae’’. Their idea was to rewrite the genome of ordinary brewer’s yeast to encourage it to make artemisinic acid. ‘’Saccharomyces cerevisiae’’ in an eukaryotic microorganism and can be easily grown in large bioreactors in industrial environment, thus making artemisinin production and especially purification cheaper.
Artemisinin has been categorized as a terpenoid or isoprenoid (Connolly et al. 1991) .

Ro and coworkers knew, that artemisinin biosynthesis pathway consists of two stages.

In the first stage linear isoprene precursors, such as GPP ( geranyl pyrophosphate ) later converted into FPP (farnesyl pyrophosphate) are synthesized from Acetyl-CoA via mevalonate pathway.

This mevalonate pathway is present in all organisms including ‘’Saccharomices cerevisiae’’. Here should be mentioned that in ‘’Saccharomices cerevisiae’’ mevalonate pathway which starts from Acetyl-CoA and goes through intermediates such as HMG-CoA, mevalonate, IPP, GPP and FPP continues into synthesis of Squalene, which is then used for the synthesis of ergosterol (Covello et al. 2008) . Ergosterol is found in cell membranes of fungi and protozoa where stabilizes the membrane an makes it less flexible (Madigan et al. 2006).

In the second stage cyclic trepenes are synthesized from linear isoprene precursors ( GPP, FPP ). In case of artemisinic acid synthesis cyclic trepene is amorpha – 4, 11 diene from ‘’A. annua’’. This intermediate is in next steps transformed into artemisinic acid.
Schematic representation of the mevalonate pathway in ‘’S. cerevisiae’’ and its crossroad with atremisinic acid synthesis pathway can be seen following this link: http://www.nature.com/nature/journal/v440/n7086/fig_tab/nature04640_F1.html#figure-title

The involvement of mevalonate pathway in artemisinin biosynthesis has been already proven (Akhila et al. 1987). It was also known, that amorpha – 4, 11 diene, the first intermediate in the second stage of artemisinin is cyclized FPP by enzyme amorpha – 4, 11 diene synthase (ADS) (Bouwmeester et al. 1999). Gene encoding this enzyme was already known.
However, the gene and amino acid sequence of the next enzyme in pathway was still unknown. This at the time unknown enzyme should be able to catalyze reaction of oxidation of amorpha – 4, 11 diene into artemisinic acid via artemisinic aldehyde and artemisinic alcohol intermediates.
Ro and coworkers made great breakthrough and identified a cytochrome P450 monooxygenase (CYP71AV1)/
amorpha-4,11-diene oxidase (AMO) for the oxidation of amorpha - 4,11-diene.

=== Isolating genes encoding enzymes responsible for oxidizing amorphadiene to artemisinic acid in ‘’A. annua ‘’===

Previous research demonstrated, that enzyme catalyzing the first regiospecific hydroxylation of amorphadiene in A. annua belongs to group on enzymes called cytochrome P450 monooxygenase (P450) (Bertea et al. 2005).

Cytochrome P450 enzymes (CYPs) are, in general, oxidase enzymes. The most common reaction catalyzed by those enzymes is an insertion of one atom of oxygen into the aliphatic position of an organic substrate while the other oxygen atom is reduced to water. Most CYPs require a protein partner to deliver one or more electrons (Siegel et al. 2007).

In order to find this specific enzyme they collected P450-expressed-sequence tags (ESTs) belonging to sunflower and lettuce from the Asteraceae EST-database (http://www.cgpdb.ucdavis.edu).
EST is abbreviation for an expressed sequence tag. They are short sub-sequences of a cDNA sequence. ESTs have a relatively low quality of sequence. Their length is limited by current technology to approximately 500 to 800 nucleotides. They may be used to identify gene transcripts, to unravel new genes or to determine gene sequences. They also represent portions of expressed genes (Adams et al. 1991).

With known amino acid sequences of cytochrome P450 monooxygenases from sunflowers and lettuce (CYP71 subfamily), they designed degenerated primers, which would in PCR reaction amplify even cytochrome P450 monooxygenases from different species, in our case from’’ A. annua’’.

At the same time total RNA from trichome enriched cells of ‘’A.anua’’ was extracted. Using total RNA, cDNA pool was prepared.

Researchers were hoping to find enzymes from P450 family that existed and were transcribed and translated in ‘’A. annua’’, because there was a strong possibility , that one of those enzymes was the enzyme they were looking for.

Retrieved cDNA pool and synthesized degenerative primers were used in PCR reaction. Result of this pcr reaction was the isolation of several unique P450 fragments from an ‘’A. annua’’ trichome-enriched complementary DNA pool.

Comparing DNA sequences of those fragments single ‘’A. annua’’ P450 gene fragment was spotted. It had 85–88% identity at the amino-acid level to ESTs of unknown function from both sunflower and lettuce. Sequence identity of this ‘’A. annua’’ P450 fragment to other P450 fragments outside the Asteraceae family was much lower. This P450 gene was therefore a suitable candidate for a cytochrome P450 monooxygenase (CYP71AV1)/amorpha-4,11-diene oxidase (AMO) enzyme.

With DNA sequence, isolation of open reading frame of 495 amino acids of CYP71AV1 enzyme from ‘’A. annua’’ was possible.

As previously stated CYP71AV1 as all cytochrome P450 enzyme needs its native redoxs partner. They used ‘’A. annua’’ cytochrome P450 oxidoreductase (CPR) as a redox partner.

===Rewriting the genome of ordinary ‘’Saccharomyces cerevisiae’’ to encourage it to make artemisinic acid===

With missing genes discovered Ro and coworkers could start developing synthetic yeast strain which could produce atremisinic acid. Since yeast is eukaryotic microbe problems such as lack of heterologous gene expression and nonfunctional membrane – bound proteins were excluded.

What they needed to do was:

1. To engineer the farnesyl pyrophosphate (FPP) biosynthetic pathway to increase FPP production and decrease its use for sterols

2. To introduce the amorphadiene synthase gene (ADS) from ‘’A. annua’’ into the high FPP producer to convert FPP to amorphadiene, and

3. To clone a novel cytochrome P450 that performs a three-step oxidation of amorphadiene to artemisinic acid from’’ A. annua’’ and expressing it in the amorphadiene producer

First step was to upregulate the expression of some genes encoding for enzymes present in mevalonate pathway and to downregulate enzyme ERG9, which catalyzes conversion of FPP into squalene (encodes squalene synthase,which catalyses the competing reaction of joining two farnesyl diphosphate moieties to form squalene (Paddon et al. 2013)). Thereby total concentration of FPP in the cell would increase providing more substrate (FPP) for amorphadiene synthase to convert FPP to amorphadiene.
Using Yeast Integrating plasmids - These plasmids lack an ORI and must be integrated directly into the host chromosome via homologous recombination – Ro and coworkers inserted following genes under strong GAL1 promoters into yeast genome one by one. They used standard lithium acetate method. After every successful transformation three to ten colonies from each transformation were tested for the highest amorphadiene producing transformation.

After transfection with plasmid harboring amorphadiene synthase gene (ADS) from ‘’A. annua’’, changes in mevalonate pathway were needed.

Firstly they added gene coding tHMGR enzyme. Overexpression of a truncated, soluble form of 3 - hydroy – 3 - methylglutaryl - coenzyme A reductase (tHMGR) – enzyme catalyzing conversion of HMG-CoA into mevalonate, improved amorphadiene production approximately fivefold.

After that upc2-1, asemi-dominant mutant allele that enhances the activity of UPC2 was added. UPC2 is a global transcription factor regulating the biosynthesis of sterols in ‘’S. cerevisiae’’ (Davies et al. 2005).

To downregulate enzyme ERG9, which catalyzes conversion of FPP into squalene (the first step after FPP in the sterol biosynthetic pathway), its promoter was changed from its original promotor into Pmet3, a methionine-represible promoter. This increased amorphadiene production an additional twofold.

Additionally they inserted another copy of tHMGR gene into the chromosome. This insertion increased amorphadiene
production by 50% .

Finally they put ERG20, gene encoding FPP synthase, under strong GAL1 promotor.

Result of all these insertions and modifications was strain called EPY224 which was able to produce 153 mg per litre of amorphadiene.

Of course, both previously mentioned CYP71AV1 and its cytochrome P450 oxidoreductase (CPR) as a redox partner were still needed to be placed between yeast genes. vector with CPR gene and CYP71AV1 gene under the control of galactose-inducible promoters was inserted via transformation into transgenic yeast strain EPY224.

To detect whether desired substance (artemisinic acid) was produced with synthetic yeast strain, yeast coulture medium and cell pellet were investigated via gas chromatography followed by mass spectrophotometer to detect newly synthesized substances. Artemisinic acid from ‘’A. annua’’ was used as reference for retention time. Results showed, that more than 95% of this novel compound was associated with the cell pellet. Authors claim, that easy removal of artemisinic acid by alkaline buffer confirms the idea that artemisinic acid is efficiently transported out of yeast cells but remains bound to the cell surface when it is protonated under acidic culture conditions.

Using single silica gel column chromatographic separation, artemisinic acid was purified. NMR spectra also confirmed that artemisinic acid produced by yeast strain matches artemisinic acid extracted from ‘’A. annua’’.
== Artemisinic acid production in yeast today==
Even though results in 2006 were very promising some problems still remained unsolved. Besides, in attempts to scale up the process new issues emerged. One of the biggest was low viability of synthetic yeast strains due to rapid accumulation of artemisinic acid and poor pairing of cytochrome P450 and their reductases, which resulted in formation of reactive oxygen species.

In 2013 Paddon and coworkers publish an article where a significantly improved, re-engineered yeast strain that is able to produce much more artemisinic acid is described. There are three enzymes added: CYB5, ADH1 and ALDH1, all of them are involved in the oxidation of amorphadiene to artemisinic acid in ‘’A. annua’’ plants. Also MET3 promoter is replaced with the copper-regulated CTR3 promoter, enabling restriction of ERG9 expression by addition of the inexpensive repressor CuSO4 to the medium rather than the more expensive methionine (Paddon et al. 2013) .

Commercial production of semisynthetic artemisinin is now underway at Sanofi's site in Garessio, Italy. The innovative industrial process to produce semisynthetic artemisinin consists in the production of artemisinic acid through fermentation—which is performed by Huvepharma, in Bulgaria—followed by a synthetic transformation of the artemisinic acid into artemisinin via photochemistry, which will be performed at Sanofi’s Garessio site. The production goal is set at 35 tons for 2013. It is expected to increase to 50-60 tons per year in 2014, supplying approximately 1/3 of the global annual need for artemisinin (Pantjushenko. 2013) .

==References==
Adams MD, Kelley JM, Gocayne JD, et al. (1991). "Complementary DNA sequencing: expressed sequence tags and human genome project". Science 252 (5013): 1651–6. doi:10.1126/science.2047873.

Akhila A, Thakur RS, Popli SP (1987) Biosynthesis of artemisininin Artemisia annua. Phytochemistry 26:1927–1930

Bertea, C. M. et al. (2005) Identification of intermediates and enzymes involved in the early steps of artemisinin biosynthesis in Artemisia annua. Planta Med. 71, 40–-47.

Boumeester HJ, Wallaart TE, Janssen MH, van Loo B,Jansen BJ, Posthumus MA, Schmidt CO, de Kraker JW, Knig WA, Franssen MC (1999) Amorpha-4,11-diene synthase catalyze the first probable step in artemisinin biosynthesis. Phytochemistry 52:843–854

Brock Biology of Microorganisms (11th Edition) (2006) by Michael T. Madigan, John M. Martinko, David Stahl, David P. Clark
Connolly JD, Hill RA (1991) Dictionary of terpenoids. Vol 1, Mono- and Sesquiterpenoids. Chapman and Hall, London

Covello PS, Teoh KH, Polichuk DR, Reed DW, Nowak G (2007) Functional genomics and the biosynthesis of artemisinin. Phytochemistry 68:1864–1871

Davies B S J, Wang H S, Rine J (2005) Dual activators of the sterol biosynthetic pathway of Saccharomyces cerevisiae: Similar activation/regulatory domains but different response mechanisms. Mol. Cell. Biol. 25, 7375–-7385

Foley M, Tilley L (1998) Quinoline antimalarials:Mechanisms of action and resistance and prospects for new agents. Pharmacol Ther 79:55–87

Layne SP (2006) "Principles of Infectious Disease Epidemiology" (PDF). EPI 220. UCLA Department of Epidemiology. Archived from the original on 2006-02-20. Retrieved 2007-06-15
Liu JM, Ni MY, Fan JF, Tu YY, Wu ZH, Wu YL, Chou WS (1979) Structure and reaction of arteannuin. Acta Chim Sin 37:129–143
Pantjushenko E (2013) "Sanofi and PATH announce the launch of large-scale production of semisynthetic artemisinin against malaria". PATH.
Sigel R, Sigel A, Sigel H(2007). The Ubiquitous Roles of Cytochrome P450 Proteins: Metal Ions in Life Sciences. New York: Wiley.

Warhurst D. (2001) New developments: Chloroquine-resistance in Plasmodium falciparum. Drug Resistance Updates 4:141–144

Zeng Q, Qiu F, Yuan L. (2008) Production of artemisinin by genetically-modified microbes. Biotechnol Lett. 30:581–592.
Paddon CJ, Westfall PJ, Pitera DJ, Benjamin K, Fisher K, McPhee D, Leavell MD, Tai A, Main A, Eng D, Polichuk DR, Teoh KH, Reed DW, Treynor T, Lenihan J, Fleck M, Bajad S, Dang G, Dengrove D, Diola D, Dorin G, Ellens KW, Fickes S, Galazzo J, Gaucher SP, Geistlinger T, Henry R, Hepp M, Horning T, Iqbal T, Jiang H, Kizer L, Lieu B, Melis D, Moss N, Regentin R, Secrest S, Tsuruta H, Vazquez R, Westblade LF, Xu L, Yu M, Zhang Y, Zhao L, Lievense J, Covello PS, Keasling JD, Reiling KK, Renninger NS, Newman JD (2013) High-level semi-synthetic production of the potent antimalarial artemisinin. Nature; 496(7446):528-32

Production of the antimalarial drug precursor artemisinic acid in engineered yeast

2015-01-20T04:28:12Z

Živa Mersetič: /* References */

This coursework is my attempt to paraphrase and explain in simple words the article of Ro DK, Paradise EM, Ouellet M, Fisher KJ, Newman KL, Ndungu JM, Ho KA, Eachus RA, Ham TS, Kirby J, Chang MC, Withers ST, Shiba Y, Sarpong R, Keasling JD with the title Production of the antimalarial drug precursor artemisinic acid in engineered yeast. The article was published in 2006 in Nature and it is one of the most cited articles in history os synthetic biology.
The article is one of many articles contributing to the realization of one of the biggest practical application of synthetic biology. The authors describe how they managed to prepare a yeast strain ‘’Saccharomyces cerevisiae’’, also known as Baker’s yeast, which was able to produce artemisinic acid. The production of anti-malarial drugs Artemisinic acid can be afterwards chemically transformed into artemisinin, which is today the first line treatment for malaria worldwide.
I divided this coursework in three parts. In the first part some aspects of malaria as disease and history of treatment are described. Second part is trying to explain the work behind the article. The third part is highlighting what happened after happy ending of the article in 2006, in other words, how things went on until today.
==Malaria and treatment==
=== Malaria===
Malaria is an emerging epidemic disease, occurring in warmer parts of the world. Because of its occurrence in tropical and subtropical areas where a lot of moisture is present its name is derived from Italian words for “bad air” [2]. It is endemic in a broad band around the equator in America, Asia and Africa. Most of deaths (85–90%) are misled in Sub-Saharan Africa (Layne SP. 2006).
It is caused by four species of sporozoa, but mostly by ‘’Plasmodium vivax ‘’and ‘’Plasmidium falciparum’’. This parasite performs part if its life cycle in human and part of it in the mosquito called ‘’Anopheles’’. Female mosquitoes of genus ‘’Anopheles’’ transmit protozoan ‘’Plasmodium falciparum’’ from person to person. The life cycle of ‘’Plasmidium falciparum’’ is complex. When mosquito injects saliva together with ‘’Plasmidium falciparum’’ sporozoites into a human host, those small, elongated cells travel through the bloodstream to the liver, where they convert into larger cells called schizont. Those cells divide into many small cells called merozoites, which enter bloodstream and infects erythrocytes. In red blood cells merozites grow, divide and exit cell by cell lysis. This asexual reproduction cycle takes approximately 48 hours. During this 48-hour period, malaria specific syndromes occur, such as chills, followed by fever up to 40◦C when ‘’Plasmidium falciparum’’ cells are released from cells. Because of the loss of red blood cells, malaria generally causes anemia.
Not all protozoal cells liberated from red blood cells are able to infect other erythrocytes. The protozoan cells, that cannot infect red blood cells are named gametocytes and are infective only for mosquito. When another mosquito feeds with infected blood gametocytes enter its digestive tract. In mosquito sexual production occurs and zygote is formed, which forms number of sporozoites. Some of these reach the salivary gland of the mosquito, and are injected into next human reservoir by the bite of mosquito (Madigan et al. 2006).

Picture of Plasmodium life cycle: http://www.uni-tuebingen.de/modeling/Mod_Malaria_Cycle_en.html

===Treatment===
Until now no antimalarial vaccine had been developed. Consequently all the treatment of malaria relies upon antilamarial drugs. First drug against malaria was quinine. It was extracted from the bark of the Cinchona tree in 1820 and was the only drug used for malaria treatment until 1930s (Foley et al. 1998) . Due to the occurrence of resistance of plasmodium species against quinine, search of new active pharmaceutical substances against malaria started. In the context of those researches chloroquinine and other synthetic guinoline antimalarials such as mefloquine were developed (Liu et al. 1979) . Unfortunately, plasmodium species developed resistance against those drugs, too (Warhurst 2001) .

In China another antimalarial drug was discovered in glandular trichomes on leaves and floral buds of plant called sweet wormwood or ‘’Artemisia annua’’. It was called artemisinin. Molecules of artemisinin contain a peroxide (O-O) group. In the presence of iron from damaged blood cells, the peroxide group is assumed to generate reactive free radicals which could destroy the DNA of the plasmodium (Liu et al. 1979) .
Today World Health Organisation recommends artemisinin-based combination therapies.
== Production of the antimalarial drug precursor artemisinic acid in engineered yeast==

===Mevalonate pathway and artemisinin synthesis pathway===

Even though artemisinin can be extracted from ‘’A. annua’’ plants, this way of obtaining the drug faces multiple obstacles. One of the major obstacle in direct extraction from plants lies in dependence of artemisinin assay in plant tissue upon natural environmental variability. Contamination by other plant trepenes, can cause problems in steps of purification (Zeng et al. 2008) .
In order to avoid those problems, which in effect cause major fluctuations in artemisinin price and thereby make it unaffordable for third world patients, Ro and coworkers published an article describing production of artemisinic acid in yeast ‘’Saccharomyces cerevisiae’’. Their idea was to rewrite the genome of ordinary brewer’s yeast to encourage it to make artemisinic acid. ‘’Saccharomyces cerevisiae’’ in an eukaryotic microorganism and can be easily grown in large bioreactors in industrial environment, thus making artemisinin production and especially purification cheaper.
Artemisinin has been categorized as a terpenoid or isoprenoid (Connolly et al. 1991) .

Ro and coworkers knew, that artemisinin biosynthesis pathway consists of two stages.

In the first stage linear isoprene precursors, such as GPP ( geranyl pyrophosphate ) later converted into FPP (farnesyl pyrophosphate) are synthesized from Acetyl-CoA via mevalonate pathway.

This mevalonate pathway is present in all organisms including ‘’Saccharomices cerevisiae’’. Here should be mentioned that in ‘’Saccharomices cerevisiae’’ mevalonate pathway which starts from Acetyl-CoA and goes through intermediates such as HMG-CoA, mevalonate, IPP, GPP and FPP continues into synthesis of Squalene, which is then used for the synthesis of ergosterol (Covello et al. 2008) . Ergosterol is found in cell membranes of fungi and protozoa where stabilizes the membrane an makes it less flexible (Madigan et al. 2006).

In the second stage cyclic trepenes are synthesized from linear isoprene precursors ( GPP, FPP ). In case of artemisinic acid synthesis cyclic trepene is amorpha – 4, 11 diene from ‘’A. annua’’. This intermediate is in next steps transformed into artemisinic acid.
Schematic representation of the mevalonate pathway in ‘’S. cerevisiae’’ and its crossroad with atremisinic acid synthesis pathway can be seen following this link: http://www.nature.com/nature/journal/v440/n7086/fig_tab/nature04640_F1.html#figure-title

The involvement of mevalonate pathway in artemisinin biosynthesis has been already proven (Akhila et al. 1987). It was also known, that amorpha – 4, 11 diene, the first intermediate in the second stage of artemisinin is cyclized FPP by enzyme amorpha – 4, 11 diene synthase (ADS) (Bouwmeester et al. 1999). Gene encoding this enzyme was already known.
However, the gene and amino acid sequence of the next enzyme in pathway was still unknown. This at the time unknown enzyme should be able to catalyze reaction of oxidation of amorpha – 4, 11 diene into artemisinic acid via artemisinic aldehyde and artemisinic alcohol intermediates.
Ro and coworkers made great breakthrough and identified a cytochrome P450 monooxygenase (CYP71AV1)/
amorpha-4,11-diene oxidase (AMO) for the oxidation of amorpha - 4,11-diene.

=== Isolating genes encoding enzymes responsible for oxidizing amorphadiene to artemisinic acid in ‘’A. annua ‘’===

Previous research demonstrated, that enzyme catalyzing the first regiospecific hydroxylation of amorphadiene in A. annua belongs to group on enzymes called cytochrome P450 monooxygenase (P450) (Bertea et al. 2005).

Cytochrome P450 enzymes (CYPs) are, in general, oxidase enzymes. The most common reaction catalyzed by those enzymes is an insertion of one atom of oxygen into the aliphatic position of an organic substrate while the other oxygen atom is reduced to water. Most CYPs require a protein partner to deliver one or more electrons (Siegel et al. 2007).

In order to find this specific enzyme they collected P450-expressed-sequence tags (ESTs) belonging to sunflower and lettuce from the Asteraceae EST-database (http://www.cgpdb.ucdavis.edu).
EST is abbreviation for an expressed sequence tag. They are short sub-sequences of a cDNA sequence. ESTs have a relatively low quality of sequence. Their length is limited by current technology to approximately 500 to 800 nucleotides. They may be used to identify gene transcripts, to unravel new genes or to determine gene sequences. They also represent portions of expressed genes (Adams et al. 1991).

With known amino acid sequences of cytochrome P450 monooxygenases from sunflowers and lettuce (CYP71 subfamily), they designed degenerated primers, which would in PCR reaction amplify even cytochrome P450 monooxygenases from different species, in our case from’’ A. annua’’.

At the same time total RNA from trichome enriched cells of ‘’A.anua’’ was extracted. Using total RNA, cDNA pool was prepared.

Researchers were hoping to find enzymes from P450 family that existed and were transcribed and translated in ‘’A. annua’’, because there was a strong possibility , that one of those enzymes was the enzyme they were looking for.

Retrieved cDNA pool and synthesized degenerative primers were used in PCR reaction. Result of this pcr reaction was the isolation of several unique P450 fragments from an ‘’A. annua’’ trichome-enriched complementary DNA pool.

Comparing DNA sequences of those fragments single ‘’A. annua’’ P450 gene fragment was spotted. It had 85–88% identity at the amino-acid level to ESTs of unknown function from both sunflower and lettuce. Sequence identity of this ‘’A. annua’’ P450 fragment to other P450 fragments outside the Asteraceae family was much lower. This P450 gene was therefore a suitable candidate for a cytochrome P450 monooxygenase (CYP71AV1)/amorpha-4,11-diene oxidase (AMO) enzyme.

With DNA sequence, isolation of open reading frame of 495 amino acids of CYP71AV1 enzyme from ‘’A. annua’’ was possible.

As previously stated CYP71AV1 as all cytochrome P450 enzyme needs its native redoxs partner. They used ‘’A. annua’’ cytochrome P450 oxidoreductase (CPR) as a redox partner.

===Rewriting the genome of ordinary ‘’Saccharomyces cerevisiae’’ to encourage it to make artemisinic acid===

With missing genes discovered Ro and coworkers could start developing synthetic yeast strain which could produce atremisinic acid. Since yeast is eukaryotic microbe problems such as lack of heterologous gene expression and nonfunctional membrane – bound proteins were excluded.

What they needed to do was:

1. To engineer the farnesyl pyrophosphate (FPP) biosynthetic pathway to increase FPP production and decrease its use for sterols

2. To introduce the amorphadiene synthase gene (ADS) from ‘’A. annua’’ into the high FPP producer to convert FPP to amorphadiene, and

3. To clone a novel cytochrome P450 that performs a three-step oxidation of amorphadiene to artemisinic acid from’’ A. annua’’ and expressing it in the amorphadiene producer

First step was to upregulate the expression of some genes encoding for enzymes present in mevalonate pathway and to downregulate enzyme ERG9, which catalyzes conversion of FPP into squalene (encodes squalene synthase,which catalyses the competing reaction of joining two farnesyl diphosphate moieties to form squalene (Paddon et al. 2013)). Thereby total concentration of FPP in the cell would increase providing more substrate (FPP) for amorphadiene synthase to convert FPP to amorphadiene.
Using Yeast Integrating plasmids - These plasmids lack an ORI and must be integrated directly into the host chromosome via homologous recombination – Ro and coworkers inserted following genes under strong GAL1 promoters into yeast genome one by one. They used standard lithium acetate method. After every successful transformation three to ten colonies from each transformation were tested for the highest amorphadiene producing transformation.

After transfection with plasmid harboring amorphadiene synthase gene (ADS) from ‘’A. annua’’, changes in mevalonate pathway were needed.

Firstly they added gene coding tHMGR enzyme. Overexpression of a truncated, soluble form of 3 - hydroy – 3 - methylglutaryl - coenzyme A reductase (tHMGR) – enzyme catalyzing conversion of HMG-CoA into mevalonate, improved amorphadiene production approximately fivefold.

After that upc2-1, asemi-dominant mutant allele that enhances the activity of UPC2 was added. UPC2 is a global transcription factor regulating the biosynthesis of sterols in ‘’S. cerevisiae’’ (Davies et al. 2005).

To downregulate enzyme ERG9, which catalyzes conversion of FPP into squalene (the first step after FPP in the sterol biosynthetic pathway), its promoter was changed from its original promotor into Pmet3, a methionine-represible promoter. This increased amorphadiene production an additional twofold.

Additionally they inserted another copy of tHMGR gene into the chromosome. This insertion increased amorphadiene
production by 50% .

Finally they put ERG20, gene encoding FPP synthase, under strong GAL1 promotor.

Result of all these insertions and modifications was strain called EPY224 which was able to produce 153 mg per litre of amorphadiene.

Of course, both previously mentioned CYP71AV1 and its cytochrome P450 oxidoreductase (CPR) as a redox partner were still needed to be placed between yeast genes. vector with CPR gene and CYP71AV1 gene under the control of galactose-inducible promoters was inserted via transformation into transgenic yeast strain EPY224.

To detect whether desired substance (artemisinic acid) was produced with synthetic yeast strain, yeast coulture medium and cell pellet were investigated via gas chromatography followed by mass spectrophotometer to detect newly synthesized substances. Artemisinic acid from ‘’A. annua’’ was used as reference for retention time. Results showed, that more than 95% of this novel compound was associated with the cell pellet. Authors claim, that easy removal of artemisinic acid by alkaline buffer confirms the idea that artemisinic acid is efficiently transported out of yeast cells but remains bound to the cell surface when it is protonated under acidic culture conditions.

Using single silica gel column chromatographic separation, artemisinic acid was purified. NMR spectra also confirmed that artemisinic acid produced by yeast strain matches artemisinic acid extracted from ‘’A. annua’’.
== artemisinic acid production in yeast today==
Even though results in 2006 were very promising some problems still remained unsolved. Besides, in attempts to scale up the process new issues emerged. One of the biggest was low viability of synthetic yeast strains due to rapid accumulation of artemisinic acid and poor pairing of cytochrome P450 and their reductases, which resulted in formation of reactive oxygen species.
In 2013 Paddon and coworkers publish an article where a significantly improved, re-engineered yeast strain that is able to produce much more artemisinic acid is described.
There are three enzymes added: CYB5, ADH1 and ALDH1, all of them are involved in the oxidation of amorphadiene to artemisinic acid in ‘’A. annua’’ plants

Also MET3 promoter is replaced with the copper-regulated CTR3 promoter, enabling restriction of ERG9 expression by addition of the inexpensive repressor CuSO4 to the medium rather than the more expensive methionine (Paddon et al. 2013) .
Commercial production of semisynthetic artemisinin is now underway at Sanofi's site in Garessio, Italy. The innovative industrial process to produce semisynthetic artemisinin consists in the production of artemisinic acid through fermentation—which is performed by Huvepharma, in Bulgaria—followed by a synthetic transformation of the artemisinic acid into artemisinin via photochemistry, which will be performed at Sanofi’s Garessio site. The production goal is set at 35 tons for 2013. It is expected to increase to 50-60 tons per year in 2014, supplying approximately 1/3 of the global annual need for artemisinin (Pantjushenko. 2013) .

==References==
Adams MD, Kelley JM, Gocayne JD, et al. (1991). "Complementary DNA sequencing: expressed sequence tags and human genome project". Science 252 (5013): 1651–6. doi:10.1126/science.2047873.

Akhila A, Thakur RS, Popli SP (1987) Biosynthesis of artemisininin Artemisia annua. Phytochemistry 26:1927–1930

Bertea, C. M. et al. (2005) Identification of intermediates and enzymes involved in the early steps of artemisinin biosynthesis in Artemisia annua. Planta Med. 71, 40–-47.

Boumeester HJ, Wallaart TE, Janssen MH, van Loo B,Jansen BJ, Posthumus MA, Schmidt CO, de Kraker JW, Knig WA, Franssen MC (1999) Amorpha-4,11-diene synthase catalyze the first probable step in artemisinin biosynthesis. Phytochemistry 52:843–854

Brock Biology of Microorganisms (11th Edition) (2006) by Michael T. Madigan, John M. Martinko, David Stahl, David P. Clark
Connolly JD, Hill RA (1991) Dictionary of terpenoids. Vol 1, Mono- and Sesquiterpenoids. Chapman and Hall, London

Covello PS, Teoh KH, Polichuk DR, Reed DW, Nowak G (2007) Functional genomics and the biosynthesis of artemisinin. Phytochemistry 68:1864–1871

Davies B S J, Wang H S, Rine J (2005) Dual activators of the sterol biosynthetic pathway of Saccharomyces cerevisiae: Similar activation/regulatory domains but different response mechanisms. Mol. Cell. Biol. 25, 7375–-7385

Foley M, Tilley L (1998) Quinoline antimalarials:Mechanisms of action and resistance and prospects for new agents. Pharmacol Ther 79:55–87

Layne SP (2006) "Principles of Infectious Disease Epidemiology" (PDF). EPI 220. UCLA Department of Epidemiology. Archived from the original on 2006-02-20. Retrieved 2007-06-15
Liu JM, Ni MY, Fan JF, Tu YY, Wu ZH, Wu YL, Chou WS (1979) Structure and reaction of arteannuin. Acta Chim Sin 37:129–143
Pantjushenko E (2013) "Sanofi and PATH announce the launch of large-scale production of semisynthetic artemisinin against malaria". PATH.
Sigel R, Sigel A, Sigel H(2007). The Ubiquitous Roles of Cytochrome P450 Proteins: Metal Ions in Life Sciences. New York: Wiley.

Warhurst D. (2001) New developments: Chloroquine-resistance in Plasmodium falciparum. Drug Resistance Updates 4:141–144

Zeng Q, Qiu F, Yuan L. (2008) Production of artemisinin by genetically-modified microbes. Biotechnol Lett. 30:581–592.
Paddon CJ, Westfall PJ, Pitera DJ, Benjamin K, Fisher K, McPhee D, Leavell MD, Tai A, Main A, Eng D, Polichuk DR, Teoh KH, Reed DW, Treynor T, Lenihan J, Fleck M, Bajad S, Dang G, Dengrove D, Diola D, Dorin G, Ellens KW, Fickes S, Galazzo J, Gaucher SP, Geistlinger T, Henry R, Hepp M, Horning T, Iqbal T, Jiang H, Kizer L, Lieu B, Melis D, Moss N, Regentin R, Secrest S, Tsuruta H, Vazquez R, Westblade LF, Xu L, Yu M, Zhang Y, Zhao L, Lievense J, Covello PS, Keasling JD, Reiling KK, Renninger NS, Newman JD (2013) High-level semi-synthetic production of the potent antimalarial artemisinin. Nature; 496(7446):528-32

Production of the antimalarial drug precursor artemisinic acid in engineered yeast

2015-01-20T04:27:41Z

Živa Mersetič: /* References */

This coursework is my attempt to paraphrase and explain in simple words the article of Ro DK, Paradise EM, Ouellet M, Fisher KJ, Newman KL, Ndungu JM, Ho KA, Eachus RA, Ham TS, Kirby J, Chang MC, Withers ST, Shiba Y, Sarpong R, Keasling JD with the title Production of the antimalarial drug precursor artemisinic acid in engineered yeast. The article was published in 2006 in Nature and it is one of the most cited articles in history os synthetic biology.
The article is one of many articles contributing to the realization of one of the biggest practical application of synthetic biology. The authors describe how they managed to prepare a yeast strain ‘’Saccharomyces cerevisiae’’, also known as Baker’s yeast, which was able to produce artemisinic acid. The production of anti-malarial drugs Artemisinic acid can be afterwards chemically transformed into artemisinin, which is today the first line treatment for malaria worldwide.
I divided this coursework in three parts. In the first part some aspects of malaria as disease and history of treatment are described. Second part is trying to explain the work behind the article. The third part is highlighting what happened after happy ending of the article in 2006, in other words, how things went on until today.
==Malaria and treatment==
=== Malaria===
Malaria is an emerging epidemic disease, occurring in warmer parts of the world. Because of its occurrence in tropical and subtropical areas where a lot of moisture is present its name is derived from Italian words for “bad air” [2]. It is endemic in a broad band around the equator in America, Asia and Africa. Most of deaths (85–90%) are misled in Sub-Saharan Africa (Layne SP. 2006).
It is caused by four species of sporozoa, but mostly by ‘’Plasmodium vivax ‘’and ‘’Plasmidium falciparum’’. This parasite performs part if its life cycle in human and part of it in the mosquito called ‘’Anopheles’’. Female mosquitoes of genus ‘’Anopheles’’ transmit protozoan ‘’Plasmodium falciparum’’ from person to person. The life cycle of ‘’Plasmidium falciparum’’ is complex. When mosquito injects saliva together with ‘’Plasmidium falciparum’’ sporozoites into a human host, those small, elongated cells travel through the bloodstream to the liver, where they convert into larger cells called schizont. Those cells divide into many small cells called merozoites, which enter bloodstream and infects erythrocytes. In red blood cells merozites grow, divide and exit cell by cell lysis. This asexual reproduction cycle takes approximately 48 hours. During this 48-hour period, malaria specific syndromes occur, such as chills, followed by fever up to 40◦C when ‘’Plasmidium falciparum’’ cells are released from cells. Because of the loss of red blood cells, malaria generally causes anemia.
Not all protozoal cells liberated from red blood cells are able to infect other erythrocytes. The protozoan cells, that cannot infect red blood cells are named gametocytes and are infective only for mosquito. When another mosquito feeds with infected blood gametocytes enter its digestive tract. In mosquito sexual production occurs and zygote is formed, which forms number of sporozoites. Some of these reach the salivary gland of the mosquito, and are injected into next human reservoir by the bite of mosquito (Madigan et al. 2006).

Picture of Plasmodium life cycle: http://www.uni-tuebingen.de/modeling/Mod_Malaria_Cycle_en.html

===Treatment===
Until now no antimalarial vaccine had been developed. Consequently all the treatment of malaria relies upon antilamarial drugs. First drug against malaria was quinine. It was extracted from the bark of the Cinchona tree in 1820 and was the only drug used for malaria treatment until 1930s (Foley et al. 1998) . Due to the occurrence of resistance of plasmodium species against quinine, search of new active pharmaceutical substances against malaria started. In the context of those researches chloroquinine and other synthetic guinoline antimalarials such as mefloquine were developed (Liu et al. 1979) . Unfortunately, plasmodium species developed resistance against those drugs, too (Warhurst 2001) .

In China another antimalarial drug was discovered in glandular trichomes on leaves and floral buds of plant called sweet wormwood or ‘’Artemisia annua’’. It was called artemisinin. Molecules of artemisinin contain a peroxide (O-O) group. In the presence of iron from damaged blood cells, the peroxide group is assumed to generate reactive free radicals which could destroy the DNA of the plasmodium (Liu et al. 1979) .
Today World Health Organisation recommends artemisinin-based combination therapies.
== Production of the antimalarial drug precursor artemisinic acid in engineered yeast==

===Mevalonate pathway and artemisinin synthesis pathway===

Even though artemisinin can be extracted from ‘’A. annua’’ plants, this way of obtaining the drug faces multiple obstacles. One of the major obstacle in direct extraction from plants lies in dependence of artemisinin assay in plant tissue upon natural environmental variability. Contamination by other plant trepenes, can cause problems in steps of purification (Zeng et al. 2008) .
In order to avoid those problems, which in effect cause major fluctuations in artemisinin price and thereby make it unaffordable for third world patients, Ro and coworkers published an article describing production of artemisinic acid in yeast ‘’Saccharomyces cerevisiae’’. Their idea was to rewrite the genome of ordinary brewer’s yeast to encourage it to make artemisinic acid. ‘’Saccharomyces cerevisiae’’ in an eukaryotic microorganism and can be easily grown in large bioreactors in industrial environment, thus making artemisinin production and especially purification cheaper.
Artemisinin has been categorized as a terpenoid or isoprenoid (Connolly et al. 1991) .

Ro and coworkers knew, that artemisinin biosynthesis pathway consists of two stages.

In the first stage linear isoprene precursors, such as GPP ( geranyl pyrophosphate ) later converted into FPP (farnesyl pyrophosphate) are synthesized from Acetyl-CoA via mevalonate pathway.

This mevalonate pathway is present in all organisms including ‘’Saccharomices cerevisiae’’. Here should be mentioned that in ‘’Saccharomices cerevisiae’’ mevalonate pathway which starts from Acetyl-CoA and goes through intermediates such as HMG-CoA, mevalonate, IPP, GPP and FPP continues into synthesis of Squalene, which is then used for the synthesis of ergosterol (Covello et al. 2008) . Ergosterol is found in cell membranes of fungi and protozoa where stabilizes the membrane an makes it less flexible (Madigan et al. 2006).

In the second stage cyclic trepenes are synthesized from linear isoprene precursors ( GPP, FPP ). In case of artemisinic acid synthesis cyclic trepene is amorpha – 4, 11 diene from ‘’A. annua’’. This intermediate is in next steps transformed into artemisinic acid.
Schematic representation of the mevalonate pathway in ‘’S. cerevisiae’’ and its crossroad with atremisinic acid synthesis pathway can be seen following this link: http://www.nature.com/nature/journal/v440/n7086/fig_tab/nature04640_F1.html#figure-title

The involvement of mevalonate pathway in artemisinin biosynthesis has been already proven (Akhila et al. 1987). It was also known, that amorpha – 4, 11 diene, the first intermediate in the second stage of artemisinin is cyclized FPP by enzyme amorpha – 4, 11 diene synthase (ADS) (Bouwmeester et al. 1999). Gene encoding this enzyme was already known.
However, the gene and amino acid sequence of the next enzyme in pathway was still unknown. This at the time unknown enzyme should be able to catalyze reaction of oxidation of amorpha – 4, 11 diene into artemisinic acid via artemisinic aldehyde and artemisinic alcohol intermediates.
Ro and coworkers made great breakthrough and identified a cytochrome P450 monooxygenase (CYP71AV1)/
amorpha-4,11-diene oxidase (AMO) for the oxidation of amorpha - 4,11-diene.

=== Isolating genes encoding enzymes responsible for oxidizing amorphadiene to artemisinic acid in ‘’A. annua ‘’===

Previous research demonstrated, that enzyme catalyzing the first regiospecific hydroxylation of amorphadiene in A. annua belongs to group on enzymes called cytochrome P450 monooxygenase (P450) (Bertea et al. 2005).

Cytochrome P450 enzymes (CYPs) are, in general, oxidase enzymes. The most common reaction catalyzed by those enzymes is an insertion of one atom of oxygen into the aliphatic position of an organic substrate while the other oxygen atom is reduced to water. Most CYPs require a protein partner to deliver one or more electrons (Siegel et al. 2007).

In order to find this specific enzyme they collected P450-expressed-sequence tags (ESTs) belonging to sunflower and lettuce from the Asteraceae EST-database (http://www.cgpdb.ucdavis.edu).
EST is abbreviation for an expressed sequence tag. They are short sub-sequences of a cDNA sequence. ESTs have a relatively low quality of sequence. Their length is limited by current technology to approximately 500 to 800 nucleotides. They may be used to identify gene transcripts, to unravel new genes or to determine gene sequences. They also represent portions of expressed genes (Adams et al. 1991).

With known amino acid sequences of cytochrome P450 monooxygenases from sunflowers and lettuce (CYP71 subfamily), they designed degenerated primers, which would in PCR reaction amplify even cytochrome P450 monooxygenases from different species, in our case from’’ A. annua’’.

At the same time total RNA from trichome enriched cells of ‘’A.anua’’ was extracted. Using total RNA, cDNA pool was prepared.

Researchers were hoping to find enzymes from P450 family that existed and were transcribed and translated in ‘’A. annua’’, because there was a strong possibility , that one of those enzymes was the enzyme they were looking for.

Retrieved cDNA pool and synthesized degenerative primers were used in PCR reaction. Result of this pcr reaction was the isolation of several unique P450 fragments from an ‘’A. annua’’ trichome-enriched complementary DNA pool.

Comparing DNA sequences of those fragments single ‘’A. annua’’ P450 gene fragment was spotted. It had 85–88% identity at the amino-acid level to ESTs of unknown function from both sunflower and lettuce. Sequence identity of this ‘’A. annua’’ P450 fragment to other P450 fragments outside the Asteraceae family was much lower. This P450 gene was therefore a suitable candidate for a cytochrome P450 monooxygenase (CYP71AV1)/amorpha-4,11-diene oxidase (AMO) enzyme.

With DNA sequence, isolation of open reading frame of 495 amino acids of CYP71AV1 enzyme from ‘’A. annua’’ was possible.

As previously stated CYP71AV1 as all cytochrome P450 enzyme needs its native redoxs partner. They used ‘’A. annua’’ cytochrome P450 oxidoreductase (CPR) as a redox partner.

===Rewriting the genome of ordinary ‘’Saccharomyces cerevisiae’’ to encourage it to make artemisinic acid===

With missing genes discovered Ro and coworkers could start developing synthetic yeast strain which could produce atremisinic acid. Since yeast is eukaryotic microbe problems such as lack of heterologous gene expression and nonfunctional membrane – bound proteins were excluded.

What they needed to do was:

1. To engineer the farnesyl pyrophosphate (FPP) biosynthetic pathway to increase FPP production and decrease its use for sterols

2. To introduce the amorphadiene synthase gene (ADS) from ‘’A. annua’’ into the high FPP producer to convert FPP to amorphadiene, and

3. To clone a novel cytochrome P450 that performs a three-step oxidation of amorphadiene to artemisinic acid from’’ A. annua’’ and expressing it in the amorphadiene producer

First step was to upregulate the expression of some genes encoding for enzymes present in mevalonate pathway and to downregulate enzyme ERG9, which catalyzes conversion of FPP into squalene (encodes squalene synthase,which catalyses the competing reaction of joining two farnesyl diphosphate moieties to form squalene (Paddon et al. 2013)). Thereby total concentration of FPP in the cell would increase providing more substrate (FPP) for amorphadiene synthase to convert FPP to amorphadiene.
Using Yeast Integrating plasmids - These plasmids lack an ORI and must be integrated directly into the host chromosome via homologous recombination – Ro and coworkers inserted following genes under strong GAL1 promoters into yeast genome one by one. They used standard lithium acetate method. After every successful transformation three to ten colonies from each transformation were tested for the highest amorphadiene producing transformation.

After transfection with plasmid harboring amorphadiene synthase gene (ADS) from ‘’A. annua’’, changes in mevalonate pathway were needed.

Firstly they added gene coding tHMGR enzyme. Overexpression of a truncated, soluble form of 3 - hydroy – 3 - methylglutaryl - coenzyme A reductase (tHMGR) – enzyme catalyzing conversion of HMG-CoA into mevalonate, improved amorphadiene production approximately fivefold.

After that upc2-1, asemi-dominant mutant allele that enhances the activity of UPC2 was added. UPC2 is a global transcription factor regulating the biosynthesis of sterols in ‘’S. cerevisiae’’ (Davies et al. 2005).

To downregulate enzyme ERG9, which catalyzes conversion of FPP into squalene (the first step after FPP in the sterol biosynthetic pathway), its promoter was changed from its original promotor into Pmet3, a methionine-represible promoter. This increased amorphadiene production an additional twofold.

Additionally they inserted another copy of tHMGR gene into the chromosome. This insertion increased amorphadiene
production by 50% .

Finally they put ERG20, gene encoding FPP synthase, under strong GAL1 promotor.

Result of all these insertions and modifications was strain called EPY224 which was able to produce 153 mg per litre of amorphadiene.

Of course, both previously mentioned CYP71AV1 and its cytochrome P450 oxidoreductase (CPR) as a redox partner were still needed to be placed between yeast genes. vector with CPR gene and CYP71AV1 gene under the control of galactose-inducible promoters was inserted via transformation into transgenic yeast strain EPY224.

To detect whether desired substance (artemisinic acid) was produced with synthetic yeast strain, yeast coulture medium and cell pellet were investigated via gas chromatography followed by mass spectrophotometer to detect newly synthesized substances. Artemisinic acid from ‘’A. annua’’ was used as reference for retention time. Results showed, that more than 95% of this novel compound was associated with the cell pellet. Authors claim, that easy removal of artemisinic acid by alkaline buffer confirms the idea that artemisinic acid is efficiently transported out of yeast cells but remains bound to the cell surface when it is protonated under acidic culture conditions.

Using single silica gel column chromatographic separation, artemisinic acid was purified. NMR spectra also confirmed that artemisinic acid produced by yeast strain matches artemisinic acid extracted from ‘’A. annua’’.
== artemisinic acid production in yeast today==
Even though results in 2006 were very promising some problems still remained unsolved. Besides, in attempts to scale up the process new issues emerged. One of the biggest was low viability of synthetic yeast strains due to rapid accumulation of artemisinic acid and poor pairing of cytochrome P450 and their reductases, which resulted in formation of reactive oxygen species.
In 2013 Paddon and coworkers publish an article where a significantly improved, re-engineered yeast strain that is able to produce much more artemisinic acid is described.
There are three enzymes added: CYB5, ADH1 and ALDH1, all of them are involved in the oxidation of amorphadiene to artemisinic acid in ‘’A. annua’’ plants

Also MET3 promoter is replaced with the copper-regulated CTR3 promoter, enabling restriction of ERG9 expression by addition of the inexpensive repressor CuSO4 to the medium rather than the more expensive methionine (Paddon et al. 2013) .
Commercial production of semisynthetic artemisinin is now underway at Sanofi's site in Garessio, Italy. The innovative industrial process to produce semisynthetic artemisinin consists in the production of artemisinic acid through fermentation—which is performed by Huvepharma, in Bulgaria—followed by a synthetic transformation of the artemisinic acid into artemisinin via photochemistry, which will be performed at Sanofi’s Garessio site. The production goal is set at 35 tons for 2013. It is expected to increase to 50-60 tons per year in 2014, supplying approximately 1/3 of the global annual need for artemisinin (Pantjushenko. 2013) .

==References==
Adams MD, Kelley JM, Gocayne JD, et al. (1991). "Complementary DNA sequencing: expressed sequence tags and human genome project". Science 252 (5013): 1651–6. doi:10.1126/science.2047873.

Akhila A, Thakur RS, Popli SP (1987) Biosynthesis of artemisininin Artemisia annua. Phytochemistry 26:1927–1930

Bertea, C. M. et al. (2005) Identification of intermediates and enzymes involved in the early steps of artemisinin biosynthesis in Artemisia annua. Planta Med. 71, 40–-47.

Boumeester HJ, Wallaart TE, Janssen MH, van Loo B,Jansen BJ, Posthumus MA, Schmidt CO, de Kraker JW, Knig WA, Franssen MC (1999) Amorpha-4,11-diene synthase catalyze the first probable step in artemisinin biosynthesis. Phytochemistry 52:843–854

Brock Biology of Microorganisms (11th Edition) (2006) by Michael T. Madigan, John M. Martinko, David Stahl, David P. Clark
Connolly JD, Hill RA (1991) Dictionary of terpenoids. Vol 1, Mono- and Sesquiterpenoids. Chapman and Hall, London

Covello PS, Teoh KH, Polichuk DR, Reed DW, Nowak G (2007) Functional genomics and the biosynthesis of artemisinin. Phytochemistry 68:1864–1871

Davies B S J, Wang H S, Rine J (2005) Dual activators of the sterol biosynthetic pathway of Saccharomyces cerevisiae: Similar activation/regulatory domains but different response mechanisms. Mol. Cell. Biol. 25, 7375–-7385

Foley M, Tilley L (1998) Quinoline antimalarials:Mechanisms of action and resistance and prospects for new agents. Pharmacol Ther 79:55–87

Layne SP (2006) "Principles of Infectious Disease Epidemiology" (PDF). EPI 220. UCLA Department of Epidemiology. Archived from the original on 2006-02-20. Retrieved 2007-06-15
Liu JM, Ni MY, Fan JF, Tu YY, Wu ZH, Wu YL, Chou WS (1979) Structure and reaction of arteannuin. Acta Chim Sin 37:129–143
Pantjushenko E (2013) "Sanofi and PATH announce the launch of large-scale production of semisynthetic artemisinin against malaria". PATH.
Sigel R, Sigel A, Sigel H(2007). The Ubiquitous Roles of Cytochrome P450 Proteins: Metal Ions in Life Sciences. New York: Wiley. ISBN 0-470-01672-8.

Warhurst D. (2001) New developments: Chloroquine-resistance in Plasmodium falciparum. Drug Resistance Updates 4:141–144

Zeng Q, Qiu F, Yuan L. (2008) Production of artemisinin by genetically-modified microbes. Biotechnol Lett. 30:581–592.
Paddon CJ, Westfall PJ, Pitera DJ, Benjamin K, Fisher K, McPhee D, Leavell MD, Tai A, Main A, Eng D, Polichuk DR, Teoh KH, Reed DW, Treynor T, Lenihan J, Fleck M, Bajad S, Dang G, Dengrove D, Diola D, Dorin G, Ellens KW, Fickes S, Galazzo J, Gaucher SP, Geistlinger T, Henry R, Hepp M, Horning T, Iqbal T, Jiang H, Kizer L, Lieu B, Melis D, Moss N, Regentin R, Secrest S, Tsuruta H, Vazquez R, Westblade LF, Xu L, Yu M, Zhang Y, Zhao L, Lievense J, Covello PS, Keasling JD, Reiling KK, Renninger NS, Newman JD (2013) High-level semi-synthetic production of the potent antimalarial artemisinin. Nature; 496(7446):528-32

Production of the antimalarial drug precursor artemisinic acid in engineered yeast

2015-01-20T04:26:53Z

Živa Mersetič:

This coursework is my attempt to paraphrase and explain in simple words the article of Ro DK, Paradise EM, Ouellet M, Fisher KJ, Newman KL, Ndungu JM, Ho KA, Eachus RA, Ham TS, Kirby J, Chang MC, Withers ST, Shiba Y, Sarpong R, Keasling JD with the title Production of the antimalarial drug precursor artemisinic acid in engineered yeast. The article was published in 2006 in Nature and it is one of the most cited articles in history os synthetic biology.
The article is one of many articles contributing to the realization of one of the biggest practical application of synthetic biology. The authors describe how they managed to prepare a yeast strain ‘’Saccharomyces cerevisiae’’, also known as Baker’s yeast, which was able to produce artemisinic acid. The production of anti-malarial drugs Artemisinic acid can be afterwards chemically transformed into artemisinin, which is today the first line treatment for malaria worldwide.
I divided this coursework in three parts. In the first part some aspects of malaria as disease and history of treatment are described. Second part is trying to explain the work behind the article. The third part is highlighting what happened after happy ending of the article in 2006, in other words, how things went on until today.
==Malaria and treatment==
=== Malaria===
Malaria is an emerging epidemic disease, occurring in warmer parts of the world. Because of its occurrence in tropical and subtropical areas where a lot of moisture is present its name is derived from Italian words for “bad air” [2]. It is endemic in a broad band around the equator in America, Asia and Africa. Most of deaths (85–90%) are misled in Sub-Saharan Africa (Layne SP. 2006).
It is caused by four species of sporozoa, but mostly by ‘’Plasmodium vivax ‘’and ‘’Plasmidium falciparum’’. This parasite performs part if its life cycle in human and part of it in the mosquito called ‘’Anopheles’’. Female mosquitoes of genus ‘’Anopheles’’ transmit protozoan ‘’Plasmodium falciparum’’ from person to person. The life cycle of ‘’Plasmidium falciparum’’ is complex. When mosquito injects saliva together with ‘’Plasmidium falciparum’’ sporozoites into a human host, those small, elongated cells travel through the bloodstream to the liver, where they convert into larger cells called schizont. Those cells divide into many small cells called merozoites, which enter bloodstream and infects erythrocytes. In red blood cells merozites grow, divide and exit cell by cell lysis. This asexual reproduction cycle takes approximately 48 hours. During this 48-hour period, malaria specific syndromes occur, such as chills, followed by fever up to 40◦C when ‘’Plasmidium falciparum’’ cells are released from cells. Because of the loss of red blood cells, malaria generally causes anemia.
Not all protozoal cells liberated from red blood cells are able to infect other erythrocytes. The protozoan cells, that cannot infect red blood cells are named gametocytes and are infective only for mosquito. When another mosquito feeds with infected blood gametocytes enter its digestive tract. In mosquito sexual production occurs and zygote is formed, which forms number of sporozoites. Some of these reach the salivary gland of the mosquito, and are injected into next human reservoir by the bite of mosquito (Madigan et al. 2006).

Picture of Plasmodium life cycle: http://www.uni-tuebingen.de/modeling/Mod_Malaria_Cycle_en.html

===Treatment===
Until now no antimalarial vaccine had been developed. Consequently all the treatment of malaria relies upon antilamarial drugs. First drug against malaria was quinine. It was extracted from the bark of the Cinchona tree in 1820 and was the only drug used for malaria treatment until 1930s (Foley et al. 1998) . Due to the occurrence of resistance of plasmodium species against quinine, search of new active pharmaceutical substances against malaria started. In the context of those researches chloroquinine and other synthetic guinoline antimalarials such as mefloquine were developed (Liu et al. 1979) . Unfortunately, plasmodium species developed resistance against those drugs, too (Warhurst 2001) .

In China another antimalarial drug was discovered in glandular trichomes on leaves and floral buds of plant called sweet wormwood or ‘’Artemisia annua’’. It was called artemisinin. Molecules of artemisinin contain a peroxide (O-O) group. In the presence of iron from damaged blood cells, the peroxide group is assumed to generate reactive free radicals which could destroy the DNA of the plasmodium (Liu et al. 1979) .
Today World Health Organisation recommends artemisinin-based combination therapies.
== Production of the antimalarial drug precursor artemisinic acid in engineered yeast==

===Mevalonate pathway and artemisinin synthesis pathway===

Even though artemisinin can be extracted from ‘’A. annua’’ plants, this way of obtaining the drug faces multiple obstacles. One of the major obstacle in direct extraction from plants lies in dependence of artemisinin assay in plant tissue upon natural environmental variability. Contamination by other plant trepenes, can cause problems in steps of purification (Zeng et al. 2008) .
In order to avoid those problems, which in effect cause major fluctuations in artemisinin price and thereby make it unaffordable for third world patients, Ro and coworkers published an article describing production of artemisinic acid in yeast ‘’Saccharomyces cerevisiae’’. Their idea was to rewrite the genome of ordinary brewer’s yeast to encourage it to make artemisinic acid. ‘’Saccharomyces cerevisiae’’ in an eukaryotic microorganism and can be easily grown in large bioreactors in industrial environment, thus making artemisinin production and especially purification cheaper.
Artemisinin has been categorized as a terpenoid or isoprenoid (Connolly et al. 1991) .

Ro and coworkers knew, that artemisinin biosynthesis pathway consists of two stages.

In the first stage linear isoprene precursors, such as GPP ( geranyl pyrophosphate ) later converted into FPP (farnesyl pyrophosphate) are synthesized from Acetyl-CoA via mevalonate pathway.

This mevalonate pathway is present in all organisms including ‘’Saccharomices cerevisiae’’. Here should be mentioned that in ‘’Saccharomices cerevisiae’’ mevalonate pathway which starts from Acetyl-CoA and goes through intermediates such as HMG-CoA, mevalonate, IPP, GPP and FPP continues into synthesis of Squalene, which is then used for the synthesis of ergosterol (Covello et al. 2008) . Ergosterol is found in cell membranes of fungi and protozoa where stabilizes the membrane an makes it less flexible (Madigan et al. 2006).

In the second stage cyclic trepenes are synthesized from linear isoprene precursors ( GPP, FPP ). In case of artemisinic acid synthesis cyclic trepene is amorpha – 4, 11 diene from ‘’A. annua’’. This intermediate is in next steps transformed into artemisinic acid.
Schematic representation of the mevalonate pathway in ‘’S. cerevisiae’’ and its crossroad with atremisinic acid synthesis pathway can be seen following this link: http://www.nature.com/nature/journal/v440/n7086/fig_tab/nature04640_F1.html#figure-title

The involvement of mevalonate pathway in artemisinin biosynthesis has been already proven (Akhila et al. 1987). It was also known, that amorpha – 4, 11 diene, the first intermediate in the second stage of artemisinin is cyclized FPP by enzyme amorpha – 4, 11 diene synthase (ADS) (Bouwmeester et al. 1999). Gene encoding this enzyme was already known.
However, the gene and amino acid sequence of the next enzyme in pathway was still unknown. This at the time unknown enzyme should be able to catalyze reaction of oxidation of amorpha – 4, 11 diene into artemisinic acid via artemisinic aldehyde and artemisinic alcohol intermediates.
Ro and coworkers made great breakthrough and identified a cytochrome P450 monooxygenase (CYP71AV1)/
amorpha-4,11-diene oxidase (AMO) for the oxidation of amorpha - 4,11-diene.

=== Isolating genes encoding enzymes responsible for oxidizing amorphadiene to artemisinic acid in ‘’A. annua ‘’===

Previous research demonstrated, that enzyme catalyzing the first regiospecific hydroxylation of amorphadiene in A. annua belongs to group on enzymes called cytochrome P450 monooxygenase (P450) (Bertea et al. 2005).

Cytochrome P450 enzymes (CYPs) are, in general, oxidase enzymes. The most common reaction catalyzed by those enzymes is an insertion of one atom of oxygen into the aliphatic position of an organic substrate while the other oxygen atom is reduced to water. Most CYPs require a protein partner to deliver one or more electrons (Siegel et al. 2007).

In order to find this specific enzyme they collected P450-expressed-sequence tags (ESTs) belonging to sunflower and lettuce from the Asteraceae EST-database (http://www.cgpdb.ucdavis.edu).
EST is abbreviation for an expressed sequence tag. They are short sub-sequences of a cDNA sequence. ESTs have a relatively low quality of sequence. Their length is limited by current technology to approximately 500 to 800 nucleotides. They may be used to identify gene transcripts, to unravel new genes or to determine gene sequences. They also represent portions of expressed genes (Adams et al. 1991).

With known amino acid sequences of cytochrome P450 monooxygenases from sunflowers and lettuce (CYP71 subfamily), they designed degenerated primers, which would in PCR reaction amplify even cytochrome P450 monooxygenases from different species, in our case from’’ A. annua’’.

At the same time total RNA from trichome enriched cells of ‘’A.anua’’ was extracted. Using total RNA, cDNA pool was prepared.

Researchers were hoping to find enzymes from P450 family that existed and were transcribed and translated in ‘’A. annua’’, because there was a strong possibility , that one of those enzymes was the enzyme they were looking for.

Retrieved cDNA pool and synthesized degenerative primers were used in PCR reaction. Result of this pcr reaction was the isolation of several unique P450 fragments from an ‘’A. annua’’ trichome-enriched complementary DNA pool.

Comparing DNA sequences of those fragments single ‘’A. annua’’ P450 gene fragment was spotted. It had 85–88% identity at the amino-acid level to ESTs of unknown function from both sunflower and lettuce. Sequence identity of this ‘’A. annua’’ P450 fragment to other P450 fragments outside the Asteraceae family was much lower. This P450 gene was therefore a suitable candidate for a cytochrome P450 monooxygenase (CYP71AV1)/amorpha-4,11-diene oxidase (AMO) enzyme.

With DNA sequence, isolation of open reading frame of 495 amino acids of CYP71AV1 enzyme from ‘’A. annua’’ was possible.

As previously stated CYP71AV1 as all cytochrome P450 enzyme needs its native redoxs partner. They used ‘’A. annua’’ cytochrome P450 oxidoreductase (CPR) as a redox partner.

===Rewriting the genome of ordinary ‘’Saccharomyces cerevisiae’’ to encourage it to make artemisinic acid===

With missing genes discovered Ro and coworkers could start developing synthetic yeast strain which could produce atremisinic acid. Since yeast is eukaryotic microbe problems such as lack of heterologous gene expression and nonfunctional membrane – bound proteins were excluded.

What they needed to do was:

1. To engineer the farnesyl pyrophosphate (FPP) biosynthetic pathway to increase FPP production and decrease its use for sterols

2. To introduce the amorphadiene synthase gene (ADS) from ‘’A. annua’’ into the high FPP producer to convert FPP to amorphadiene, and

3. To clone a novel cytochrome P450 that performs a three-step oxidation of amorphadiene to artemisinic acid from’’ A. annua’’ and expressing it in the amorphadiene producer

First step was to upregulate the expression of some genes encoding for enzymes present in mevalonate pathway and to downregulate enzyme ERG9, which catalyzes conversion of FPP into squalene (encodes squalene synthase,which catalyses the competing reaction of joining two farnesyl diphosphate moieties to form squalene (Paddon et al. 2013)). Thereby total concentration of FPP in the cell would increase providing more substrate (FPP) for amorphadiene synthase to convert FPP to amorphadiene.
Using Yeast Integrating plasmids - These plasmids lack an ORI and must be integrated directly into the host chromosome via homologous recombination – Ro and coworkers inserted following genes under strong GAL1 promoters into yeast genome one by one. They used standard lithium acetate method. After every successful transformation three to ten colonies from each transformation were tested for the highest amorphadiene producing transformation.

After transfection with plasmid harboring amorphadiene synthase gene (ADS) from ‘’A. annua’’, changes in mevalonate pathway were needed.

Firstly they added gene coding tHMGR enzyme. Overexpression of a truncated, soluble form of 3 - hydroy – 3 - methylglutaryl - coenzyme A reductase (tHMGR) – enzyme catalyzing conversion of HMG-CoA into mevalonate, improved amorphadiene production approximately fivefold.

After that upc2-1, asemi-dominant mutant allele that enhances the activity of UPC2 was added. UPC2 is a global transcription factor regulating the biosynthesis of sterols in ‘’S. cerevisiae’’ (Davies et al. 2005).

To downregulate enzyme ERG9, which catalyzes conversion of FPP into squalene (the first step after FPP in the sterol biosynthetic pathway), its promoter was changed from its original promotor into Pmet3, a methionine-represible promoter. This increased amorphadiene production an additional twofold.

Additionally they inserted another copy of tHMGR gene into the chromosome. This insertion increased amorphadiene
production by 50% .

Finally they put ERG20, gene encoding FPP synthase, under strong GAL1 promotor.

Result of all these insertions and modifications was strain called EPY224 which was able to produce 153 mg per litre of amorphadiene.

Of course, both previously mentioned CYP71AV1 and its cytochrome P450 oxidoreductase (CPR) as a redox partner were still needed to be placed between yeast genes. vector with CPR gene and CYP71AV1 gene under the control of galactose-inducible promoters was inserted via transformation into transgenic yeast strain EPY224.

To detect whether desired substance (artemisinic acid) was produced with synthetic yeast strain, yeast coulture medium and cell pellet were investigated via gas chromatography followed by mass spectrophotometer to detect newly synthesized substances. Artemisinic acid from ‘’A. annua’’ was used as reference for retention time. Results showed, that more than 95% of this novel compound was associated with the cell pellet. Authors claim, that easy removal of artemisinic acid by alkaline buffer confirms the idea that artemisinic acid is efficiently transported out of yeast cells but remains bound to the cell surface when it is protonated under acidic culture conditions.

Using single silica gel column chromatographic separation, artemisinic acid was purified. NMR spectra also confirmed that artemisinic acid produced by yeast strain matches artemisinic acid extracted from ‘’A. annua’’.
== artemisinic acid production in yeast today==
Even though results in 2006 were very promising some problems still remained unsolved. Besides, in attempts to scale up the process new issues emerged. One of the biggest was low viability of synthetic yeast strains due to rapid accumulation of artemisinic acid and poor pairing of cytochrome P450 and their reductases, which resulted in formation of reactive oxygen species.
In 2013 Paddon and coworkers publish an article where a significantly improved, re-engineered yeast strain that is able to produce much more artemisinic acid is described.
There are three enzymes added: CYB5, ADH1 and ALDH1, all of them are involved in the oxidation of amorphadiene to artemisinic acid in ‘’A. annua’’ plants

Also MET3 promoter is replaced with the copper-regulated CTR3 promoter, enabling restriction of ERG9 expression by addition of the inexpensive repressor CuSO4 to the medium rather than the more expensive methionine (Paddon et al. 2013) .
Commercial production of semisynthetic artemisinin is now underway at Sanofi's site in Garessio, Italy. The innovative industrial process to produce semisynthetic artemisinin consists in the production of artemisinic acid through fermentation—which is performed by Huvepharma, in Bulgaria—followed by a synthetic transformation of the artemisinic acid into artemisinin via photochemistry, which will be performed at Sanofi’s Garessio site. The production goal is set at 35 tons for 2013. It is expected to increase to 50-60 tons per year in 2014, supplying approximately 1/3 of the global annual need for artemisinin (Pantjushenko. 2013) .

==References==
Adams MD, Kelley JM, Gocayne JD, et al. (1991). "Complementary DNA sequencing: expressed sequence tags and human genome project". Science 252 (5013): 1651–6. doi:10.1126/science.2047873. PMID 2047873.

Akhila A, Thakur RS, Popli SP (1987) Biosynthesis of artemisininin Artemisia annua. Phytochemistry 26:1927–1930

Bertea, C. M. et al. (2005) Identification of intermediates and enzymes involved in the early steps of artemisinin biosynthesis in Artemisia annua. Planta Med. 71, 40–-47.

Boumeester HJ, Wallaart TE, Janssen MH, van Loo B,Jansen BJ, Posthumus MA, Schmidt CO, de Kraker JW, Knig WA, Franssen MC (1999) Amorpha-4,11-diene synthase catalyze the first probable step in artemisinin biosynthesis. Phytochemistry 52:843–854

Brock Biology of Microorganisms (11th Edition) (2006) by Michael T. Madigan, John M. Martinko, David Stahl, David P. Clark
Connolly JD, Hill RA (1991) Dictionary of terpenoids. Vol 1, Mono- and Sesquiterpenoids. Chapman and Hall, London

Covello PS, Teoh KH, Polichuk DR, Reed DW, Nowak G (2007) Functional genomics and the biosynthesis of artemisinin. Phytochemistry 68:1864–1871

Davies B S J, Wang H S, Rine J (2005) Dual activators of the sterol biosynthetic pathway of Saccharomyces cerevisiae: Similar activation/regulatory domains but different response mechanisms. Mol. Cell. Biol. 25, 7375–-7385

Foley M, Tilley L (1998) Quinoline antimalarials:Mechanisms of action and resistance and prospects for new agents. Pharmacol Ther 79:55–87

Layne SP (2006) "Principles of Infectious Disease Epidemiology" (PDF). EPI 220. UCLA Department of Epidemiology. Archived from the original on 2006-02-20. Retrieved 2007-06-15
Liu JM, Ni MY, Fan JF, Tu YY, Wu ZH, Wu YL, Chou WS (1979) Structure and reaction of arteannuin. Acta Chim Sin 37:129–143
Pantjushenko E (2013) "Sanofi and PATH announce the launch of large-scale production of semisynthetic artemisinin against malaria". PATH.
Sigel R, Sigel A, Sigel H(2007). The Ubiquitous Roles of Cytochrome P450 Proteins: Metal Ions in Life Sciences. New York: Wiley. ISBN 0-470-01672-8.

Warhurst D. (2001) New developments: Chloroquine-resistance in Plasmodium falciparum. Drug Resistance Updates 4:141–144

Zeng Q, Qiu F, Yuan L. (2008) Production of artemisinin by genetically-modified microbes. Biotechnol Lett. 30:581–592.
Paddon CJ, Westfall PJ, Pitera DJ, Benjamin K, Fisher K, McPhee D, Leavell MD, Tai A, Main A, Eng D, Polichuk DR, Teoh KH, Reed DW, Treynor T, Lenihan J, Fleck M, Bajad S, Dang G, Dengrove D, Diola D, Dorin G, Ellens KW, Fickes S, Galazzo J, Gaucher SP, Geistlinger T, Henry R, Hepp M, Horning T, Iqbal T, Jiang H, Kizer L, Lieu B, Melis D, Moss N, Regentin R, Secrest S, Tsuruta H, Vazquez R, Westblade LF, Xu L, Yu M, Zhang Y, Zhao L, Lievense J, Covello PS, Keasling JD, Reiling KK, Renninger NS, Newman JD (2013) High-level semi-synthetic production of the potent antimalarial artemisinin. Nature; 496(7446):528-32

Production of the antimalarial drug precursor artemisinic acid in engineered yeast

2015-01-20T02:33:47Z

Živa Mersetič: Removing all content from page

Production of the antimalarial drug precursor artemisinic acid in engineered yeast

2015-01-20T00:33:58Z

Živa Mersetič: /* Production of the antimalarial drug precursor artemisinic acid in engineered yeast */

Production of the antimalarial drug precursor artemisinic acid in engineered yeast

2015-01-19T21:05:47Z

Živa Mersetič:

Production of the antimalarial drug precursor artemisinic acid in engineered yeast

2015-01-19T20:55:44Z

Živa Mersetič: New page: =Production of the antimalarial drug precursor artemisinic acid in engineered yeast= This coursework is my attempt to paraphrase and explain in simple words the article of Ro DK, Paradise...

=Production of the antimalarial drug precursor artemisinic acid in engineered yeast=

This coursework is my attempt to paraphrase and explain in simple words the article of Ro DK, Paradise EM, Ouellet M, Fisher KJ, Newman KL, Ndungu JM, Ho KA, Eachus RA, Ham TS, Kirby J, Chang MC, Withers ST, Shiba Y, Sarpong R, Keasling JD with the title Production of the antimalarial drug precursor artemisinic acid in engineered yeast. The article was published in 2006 in Nature and it is one of the most cited articles in history.
The authors describe how they managed to prepare a yeast strain Saccharomyces cerevisiae, also known as Baker’s yeast, which was able to produce artemisinic acid. Artemisinic acid can be afterwards chemically transformed into artemisinin, which is today the first line treatment for malaria worldwide.
I divided this coursework in three parts. In the first part some aspects of malaria as disease and history of treatment are described. Second part is trying to explain the work behind the article. The third part is highlighting what happened after happy ending of the article in 2006, in other words, how things went on until today.
==Part one:==
=== MALARIA===
Malaria is an emerging epidemic disease, occurring in warmer parts of the world. Because of its occurrence in tropical and subtropical areas where a lot of moisture is present its name is derived from Italian words for “bad air” [2]. It is endemic in a broad band around the equator in America, Asia and Africa. Most of deaths (85–90%) are misled in Sub-Saharan Africa [1].
It is caused by four species of sporozoa, but mostly by Plasmodium vivax and Plasmidium falciparum. This parasite performs part if its life cycle in human and part of it in the mosquito called Anopheles. Female mosquitoes of genus Anopheles transmit protozoan Plasmodium falciparum from person to person. The life cycle of Plasmidium falciparum is complex. When mosquito injects saliva together with Plasmidium falciparum sporozoites into a human host, those small, elongated cells travel through the bloodstream to the liver, where they convert into larger cells called schizont. Those cells divide into many small cells called merozoites, which enter bloodstream and ifect erythrocytes. In red blood cells merozites grow, divide and exit cell by cell lysis. This asexual reproductin cycle takes approximately 48 hours. During this 48-hour period, malaria specific sindroms occur, such as chills, followed by fever up to 40◦C when Plasmidium falciparum cells are released from cells. Because of the loss of red blood cells, malaria generally causes anemia.
Not all protozoal cells liberated from red blood cells are able to infect other erithrocytes. The protozoal cells, that cannot infect red blood cells are named gametocytes and are infective only for mosquito.When another mosquito feeds with infected blood gametocytes enter its digestive tract. In mosquito sexual production occurs and zigote is formed, which forms number of sporozoites. Some of these reach the salivary gland of the mosquito, and are injected into next human reservoir by the bite of mosquito [2].
Picture of Plasmodium life cycle: http://www.uni-tuebingen.de/modeling/Mod_Malaria_Cycle_en.html
TREATMENT
Until now no antimalarial vaccine had been developed. Consequently all the treatment of malaria relies upon antilamarial drugs. First drug against malaria was quinine. It was extracted from the bark of the Cinchona tree in 1820 and was the only drug used for malaria treatment until 1930s [3]. Due to the occurrence of resistance of plasmodium species against quinine, search of new active pharmaceutical substances against malaria started. In the context of those researches chloroquinine and other synthetic guinoline antimalarials such as mefloquine were developed[3]. Unfortunately, plasmodium species developed resistance against those drugs, too [4].

In China another antimalarial drug was discovered in glandular trichomes on leaves and floral buds of plant called sweet wormwood or Artemisia annua. It was called artemisinin. Molecules of artemisinin contain a peroxide (O-O) group. In the presence of iron from damaged blood cells, the peroxide group is assumed to generate reactive free radicals which could destroy the DNA of the plasmodium[5].
Today World Health Organisation recommends artemisinin-based combination therapies.
Part two:
Even though artemisinin can be exctacted from A. annua plants, this way of obtaining the drug faces multiple obstacles. One of the major obstacle in direct extraction from plants lies in dependence of artemisinin assay in plant tissue upon natural environmental variability. Contamination by other plant trepenes, can cause problems in steps of purification [6].
In order to avoid those problems, which in effect cause major fluctuations in artemisinin price and thereby making it unaffordable for third world patients, Ro and coworkers published an article describing productin of artemisinic acid in yeast Saccharomyces cerevisiae. Saccharomyces cerevisiae in an eucariotic microorganism and can be easily grown in large bioreactors in industrial environment, thus making artemisinin production and especially purification cheaper.
Artemisinin has been categorized as a terpenoid or isoprenoid [7].

Ro and coworkers knew, that artmisinin biosynthesis pathway consists of two stages.

In the first stage linear isoprene precursors, such as GPP ( geranyl pyrophosphate ) later converted into FPP (farnesyl pyrophosphate) are synthesized from Acetyl-CoA via mevalonate pathway.

This mevalonate pathway is present in all organisms including Saccharomices cerevisiae. We should mention that in Saccharomices cerevisiae mevalonate pathway which starts from Acetyl-CoA and goes through intermediates such as HMG-CoA, Mevalonate, IPP, GPP and FPP continues into synthesis of Squalene, which is then used for the synthesis of Ergosterol [8]. Ergosterol is found in cell membranes of fungi and protozoa where stabilizes the membrane an makes it less flexible [2].

In the second stage cyclic trepenes are synthesized from linear isoprene precursors ( GPP, FPP ). In case of artemisinic acid synthesis cyclic trepene is Amorpha – 4, 11 diene from A. annua. This intermediate is in next steps transformed into Artemisinic acid.

Malaria patients can be treated with highly effective Artemisinin-based Combination Therapies (ACTs), but cultivating and extracting artemisinin, which comes from the Chinese Sweet Wormwood plant, is expensive and time consuming. Lack of access to this vital compound prevents millions of people in the developing world from receiving critical ACTs.
http://amyris.com/products/artemisinin/
Semi-synthetic artemisinin has been in the pipeline since 2006, when Jay Keasling’s group at the Lawrence Berkeley National Laboratory in California, US, reported rewriting the genome of ordinary brewer’s yeast to encourage it to make artemisinic acid.1 But piecing together a practical route for making the drug precursor in yeast and then transforming it into the finished product has proved tricky. It has remained cheaper and more straightforward to extract the drug from its natural source.
EST
Shivashankar H. Nagaraj, Robin B. Gasser, Shoba Ranganathan, A hitchhiker's guide to expressed sequence tag (EST) analysis Brief Bioinform (2007) 8 (1): 6-21 first published online May 23, 2006 doi:10.1093/bib/bbl015

Yeast Integrating plasmids (YIp): These plasmids lack an ORI and must be integrated directly into the host chromosome via homologous recombination.

SB students resources

2015-01-19T20:51:26Z

Živa Mersetič:

===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 (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

Predstavitev na površini bakterij

2013-10-20T10:57:06Z

Živa Mersetič:

'''Predstavitev hipervariabilnih domen težkih verig protiteles kamele na površini po Gramu pozitivnih bakterij'''

V članku so tehniko predstavitve na površini celic Gram-pozitivnih bakterij Staphylococcus carnosus primerjali s tehniko predstavitve na fagih. Na površini bakterije so kot prvič predstavili knjižnico fragmentov protiteles imenovanih nanotelesca, ki so pravzaprav variabilne domene težkih verig protiteles kamele. V tej raziskavi so bila ta nanotelesca usmerjena proti zelenemu fluorescirajočemu proteinu GFP (antigen). Cilj raziskave je bil odgovor na vprašanje, ali lahko predstavitev knjižnjice nanotelesc na površini bakterij konkurira oziroma dopolnjuje predstavitev na površini fagov.
Predstavitev na površini celic se bistveno razlikuje od predstavitve na površini fagov v načinu prebiranja oz. selekcije klonov. Selekcija fagov z vezavnimi molekulami (ki se vežejo na antigen) na svoji površini pri predstavitvi na fagih poteka po načinu afinitetne kromatografije, saj imobilizirani antigeni zadržijo delce z vezavnimi molekulami, kar kasneje eluiramo. Ker so celice bistveno večje od virusov, nam to omogoča detekcijo s pomočjo pretočne citometrije in ločevanje na podlagi fluorescence. Za sortiranje celic, ki imajo na svoji površini izražene rekombinantne proteine so avtorji uporabili tehniko FACS - Flow cytometry and Fluorescence-activated cell sorting. FACS lahko celice poleg analiziranja in štetja še sortira glede na njihove lastnosti. Sortiranje poteka glede na količino in vrsto fluorescence, ki ga posamezna celica odda. Po tem ko gredo celice mimo vira svetlobe, pretočni citometer tok suspenzije spremeni v posamezne kapljice. Vsaka kapljica vsebuje celico. Glede na valovno dolžino in količino oddane fluorescenčne svetlobe določene celice kapljica s to celico v električnem polju prejme določen naboj. Glede na naboj so nato celice fizično ločene.
Prvi del eksperimenta je bila predstavitev na površini fagov. V ta namen so najprej sprožili imunski odziv kameljih protiteles proti GFP proteinom in izolirali protitelesa. Sledilo je pripravljanje fagov, ki so nanotelesca proti GFP predstavljali na svoji površini. Selekcija fagov z nanotelesci je potekala z vezavo fagov na imobiliziran GFP in kasnejšo elucijo. Sledilo je sekvenciranje genov za nanotelesca in preverjanje nanotelesc s tehniko ELISA.
Preverjanje s tehniko ELISA temelji na preverjanju topnih protiteles, ki jih producirajo fagi, ki smo jih pred tem selekcionirali kot tiste, ki na svoji površini predstavljajo protitelesa, ki se vežejo na želeni antigen. Dejstvo, da poleg nanotelesc ujetih v membrano, nastajajo tudi topna nanotelesca, je posledica tega, da zaradi doseganja monovalentnega izražanja nanotelesc na površini, med gen za nanotelesce in gen III. vstavijo stop kodon, zaradi česar nastajajo tudi manjše količine topnih nanotelesc.
Drugi del eksperimenta je predstavljala predstavitev na površini bakterijskih celic. V ta namen so gene za nanotelesca vstavili v vektor za predstavitev na površini bakterijskih celic pSCZ1.
Tehnologija predstavitve na površini po Gramu pozitivnih bakterij temelji na tem, da želeni gen vstavimo za gen za domeno stafilokoknega proteina A, ki je usidrana v celično steno.
Celice se nato ločujejo s pomočjo pretočne citometrije – metodo FACS. V primeru predstavitve na površini celic, celic ne inkubiramo skupaj z imobiliziranim antigenom za nanotelesca, ampak s topnimi GFP proteini v raztopini, skupaj s topnimi fluorescentno označenimi reporterskimi proteini (HSA), ki se vežejo na ABP protein. Tiste celice, ki torej vežejo GFP in HSA in fluorescirajo nad nekim pragom, pretočni citometer loči od ostalih, pa tudi med seboj. Tudi tukaj korak selekcije izvajamo večkrat, saj celice z domnevno izraženimi nanotelesci še enkrat namnožimo, še enkrat zmešamo z GFP in HSA proteinom ter ločujemo z metodo FACS. Sledi sekvenciranje in ponovno preverjanje s pretočno citometrijo.
Podatki iz tega članka so pokazali, da sta obe tehniki nekoliko pristranski in da se predstavljena nanotelesca nekoliko razlikujejo glede na to ali so bila izražena na fagu ali na celični površini. Kaj je razlog teh razlik še ni znano, zato avtorji zagovarjajo hkratno uporabo obeh tehnik, saj bi na ta način iz določene genske knjižnice lahko izolirali karseda veliko število različnih nanotelesc, oziroma v drugih raziskavah drugih vezavnih proteinov, ki se vežejo na določen antigen.