Production of the antimalarial drug precursor artemisinic acid in engineered yeast

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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.

Contents

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

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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

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