View source for Engineering a mevalonate pathway in Escherichia coli for production of terpenoids

'''Ana Kapraljević'''

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

== INTRODUCTION ==

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

=== Artemisnin ===

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

=== Biology of terpenoids ===

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

== METABOLIC ENGINEERING FOR PRODUCTION OD TERPENOIDS ==

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

=== Microbal host ===

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

=== Operons ===

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

== EXPERIMENT ==

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

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

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

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

=== Amorpadiene synthesis from mevalonate ===

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

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

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

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

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


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

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

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

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

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

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

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