Search

Saccharomyces cerevisiae is engineered to produce high concentrations of artemisinic acid, a precursor of the artemisinin used in combination therapies for malaria treatment; an efficient and practical chemical process to convert artemisinic acid to artemisinin is also developed. Artemisinin-based combination therapies are the treatment of choice for uncomplicated Plasmodium falciparum malaria, but the supply of plant-derived artemisinin can sometimes be unreliable, causing shortages and high prices. This manuscript describes a viable industrial process for the production of semisynthetic artemisinin, with the potential to help stabilize artemisinin supply. The process uses Saccharomyces cerevisiae yeast engineered to produce high yields of artemisinic acid, a precursor of artemisinin. The authors have also developed an efficient and scalable chemical process to convert artemisinic acid to artemisinin. In 2010 there were more than 200 million cases of malaria, and at least 655,000 deaths1. The World Health Organization has recommended artemisinin-based combination therapies (ACTs) for the treatment of uncomplicated malaria caused by the parasite Plasmodium falciparum. Artemisinin is a sesquiterpene endoperoxide with potent antimalarial properties, produced by the plant Artemisia annua. However, the supply of plant-derived artemisinin is unstable, resulting in shortages and price fluctuations, complicating production planning by ACT manufacturers2. A stable source of affordable artemisinin is required. Here we use synthetic biology to develop strains of Saccharomyces cerevisiae (baker’s yeast) for high-yielding biological production of artemisinic acid, a precursor of artemisinin. Previous attempts to produce commercially relevant concentrations of artemisinic acid were unsuccessful, allowing production of only 1.6 grams per litre of artemisinic acid3. Here we demonstrate the complete biosynthetic pathway, including the discovery of a plant dehydrogenase and a second cytochrome that provide an efficient biosynthetic route to artemisinic acid, with fermentation titres of 25 grams per litre of artemisinic acid. Furthermore, we have developed a practical, efficient and scalable chemical process for the conversion of artemisinic acid to artemisinin using a chemical source of singlet oxygen, thus avoiding the need for specialized photochemical equipment. The strains and processes described here form the basis of a viable industrial process for the production of semi-synthetic artemisinin to stabilize the supply of artemisinin for derivatization into active pharmaceutical ingredients (for example, artesunate) for incorporation into ACTs. Because all intellectual property rights have been provided free of charge, this technology has the potential to increase provision of first-line antimalarial treatments to the developing world at a reduced average annual price.

Artemisinin-based combination therapies (ACTs) are currently unaffordable for many of the people who need them most. A major cost component of ACTs is the plant-derived artemisinin. A fermentation process for a precursor to artemisinin might provide a viable second source to stabilize the artemisinin supply and therefore reduce price. The heterologous production of artemisinic acid, an artemisinin precursor, by Saccharomyces cerevisiae was improved 25-fold from a 100 mg/L flask process to a 2.5 g/L process in bioreactors. A defined medium fed-batch process with galactose as the carbon source and inducer was developed, with titers of 1.3 g/L. In this strain ERG9 was controlled with promoter Pmet3 so that methionine repressed the sterol biosynthesis pathway and increased precursor availability for artemisinic acid biosynthesis. Addition of methionine to the process increased artemisinic acid titers to 1.8 g/L. A dissolved oxygen-stat algorithm was developed, which simultaneously controlled the agitation and feed pump. This improved process control and increased titers to 2.5 g/L.

SummaryGeldanamycin, a polyketide natural product, is of significant interest for development of new anticancer drugs that target the protein chaperone Hsp90. While the chemically reactive groups of geldanamycin have been exploited to make a number of synthetic analogs, including 17-allylamino-17-demethoxy geldanamycin (17-AAG), currently in clinical evaluation, the “inert” groups of the molecule remain unexplored for structure-activity relationships. We have used genetic engineering of the geldanamycin polyketide synthase (GdmPKS) gene cluster in Streptomyces hygroscopicus to modify geldanamycin at such positions. Substitutions of acyltransferase domains were made in six of the seven GdmPKS modules. Four of these led to production of 2-desmethyl, 6-desmethoxy, 8-desmethyl, and 14-desmethyl derivatives, including one analog with a four-fold enhanced affinity for Hsp90. The genetic tools developed for geldanamycin gene manipulation will be useful for engineering additional analogs that aid the development of this chemotherapeutic agent.

Substitution of racemic diketide thioesters for optically pure compounds in precursor-fed fermentations was investigated. These substrates were shown to be as effective as optically pure materials in diketide-fed fermentation processes for producing analogs of 6-deoxyerythronolide B. However, since half of the racemic mixture is not utilizable for polyketide biosynthesis, higher total levels of diketide are required. Toxicity to cells was evident at high diketide concentrations. In fermenters, exhaust gas analysis was used to indicate the optimal time for diketide addition. While both enantiomers were shown to disappear from cultures at similar rates, the presence of unincorporated enantiomer had minimal effect on polyketide production within a range of feed concentrations. Use of racemic diketide thioesters was successful and dramatically reduced the cost of the fermentation process. © 2002 Society of Chemical Industry

Recently, the epothilone polyketide synthase (PKS) was successfully introduced into a heterologous production host for the large-scale production of epothilone D. We have found that at least three other epothilones can also be produced as the major fermentation product of this recombinant strain by supplementation of specific substrates to the production media. Addition of acetate or propionate to the media results in modulation of the epothilone D:C ratio, whereas addition of l-serine with either acetate or propionate yields epothilone H1 or H2 as the major product. This strategy permits production of at least four novel epothilones by culturing a single host with a genetically modified epothilone PKS in various media. Journal of Industrial Microbiology & Biotechnology (2002) 28, 17–20 DOI: 10.1038/sj/jim/7000209

FK520, also called ascomycin, is an immunosuppressive agent produced by Streptomyces hygroscopicus. Engineering the polyketide synthase genes of the parent strain generated novel FK520 analogs with the potential for improved in vivo stability. By replacing the acyl transferase (AT) domain in the polyketide synthase module 8 with an AT specific for methylmalonyl CoA (the rapamycin AT 3), the strain produced 13-desmethoxy-13-methyl-FK520 (13dmmFK520). Process development and scale-up studies of this recombinant S. hygroscopicus strain producing 13dmmFK520 are described here. Production kinetics and compound stability in fermentation broth were significantly different compared to the native FK520. Fermentation of the new strain resulted in the synthesis of a contaminating substance that co-purified with the 13dmmFK520. To optimize 13dmmFK520 production and to facilitate purification, growth parameters and media development were examined. Although a medium was identified that increased product titers by ca. 300%, the ratio of impurity to product was doubled. Lower dissolved oxygen (20% compared to 50% and 80%) increased titers by 20% with no appreciable effect on the concentration of impurity. Increasing the fermentation pH from 6.0 to 6.5 did not change the 13dmmFK520 titer, but reduced the impurity-to-product ratio by approximately 450%. Journal of Industrial Microbiology & Biotechnology (2002) 28, 12–16 DOI: 10.1038/sj/jim/7000208

Malaria, caused by Plasmodium sp , results in almost one million deaths and over 200 million new infections annually. The World Health Organization has recommended that artemisinin-based combination therapies be used for treatment of malaria. Artemisinin is a sesquiterpene lactone isolated from the plant Artemisia annua . However, the supply and price of artemisinin fluctuate greatly, and an alternative production method would be valuable to increase availability. We describe progress toward the goal of developing a supply of semisynthetic artemisinin based on production of the artemisinin precursor amorpha-4,11-diene by fermentation from engineered Saccharomyces cerevisiae , and its chemical conversion to dihydroartemisinic acid, which can be subsequently converted to artemisinin. Previous efforts to produce artemisinin precursors used S. cerevisiae S288C overexpressing selected genes of the mevalonate pathway [Ro et al. (2006) Nature 440:940–943]. We have now overexpressed every enzyme of the mevalonate pathway to ERG20 in S. cerevisiae CEN.PK2, and compared production to CEN.PK2 engineered identically to the previously engineered S288C strain. Overexpressing every enzyme of the mevalonate pathway doubled artemisinic acid production, however, amorpha-4,11-diene production was 10-fold higher than artemisinic acid. We therefore focused on amorpha-4,11-diene production. Development of fermentation processes for the reengineered CEN.PK2 amorpha-4,11-diene strain led to production of > 40 g/L product. A chemical process was developed to convert amorpha-4,11-diene to dihydroartemisinic acid, which could subsequently be converted to artemisinin. The strains and procedures described represent a complete process for production of semisynthetic artemisinin.

Artemisinin is a sesquiterpene lactone endoperoxide with potent antimalarial properties, recommended by the World Health Organization for the treatment of malaria in artemisinin combination therapies (ACTs). It is extracted from the plant Artemisia annua, but its supplies are limited and its price is volatile. In order to increase supply and stabilize the price of artemisinin, a semisynthesis has been developed, whereby an artemisinin precursor (amorpha-4,11-diene) is produced in microbes and the isolated precursor converted chemically to artemisinin. Escherichia coli has been engineered to produce amorpha-4,11-diene by the expression of a heterologous mevalonate pathway along with amorpha-4,11-diene synthase (ADS) from A. annua. Development of the E. coli platform to increase production of amorpha-4,11-diene from 24 mg/L to >25 g/L is described. ADS has also been expressed in the yeast model system Saccharomyces cerevisiae which, following manipulation of the mevalonate pathway, produced 150 mg/L of amorpha-4,11-diene. The cDNAs encoding the cytochrome P450 that oxidizes amorpha-4,11-diene to artemisinic acid, CYP71AV1, and its cognate reductase were isolated from A. annua and expressed in amorpha-4,11-diene-producing E. coli and yeast, leading to the production of >1 g/L artemisinic acid from both organisms. A route for the chemical conversion of artemisinic acid to artemisinin is described. Production of semisynthetic artemisinin may lead to the development of a second source of the drug for incorporation into ACTs.

BackgroundArtemisinin derivatives are the key active ingredients in Artemisinin combination therapies (ACTs), the most effective therapies available for treatment of malaria. Because the raw material is extracted from plants with long growing seasons, artemisinin is often in short supply, and fermentation would be an attractive alternative production method to supplement the plant source. Previous work showed that high levels of amorpha-4,11-diene, an artemisinin precursor, can be made in Escherichia coli using a heterologous mevalonate pathway derived from yeast (Saccharomyces cerevisiae), though the reconstructed mevalonate pathway was limited at a particular enzymatic step.Methodology/ principal findingsBy combining improvements in the heterologous mevalonate pathway with a superior fermentation process, commercially relevant titers were achieved in fed-batch fermentations. Yeast genes for HMG-CoA synthase and HMG-CoA reductase (the second and third enzymes in the pathway) were replaced with equivalent genes from Staphylococcus aureus, more than doubling production. Amorpha-4,11-diene titers were further increased by optimizing nitrogen delivery in the fermentation process. Successful cultivation of the improved strain under carbon and nitrogen restriction consistently yielded 90 g/L dry cell weight and an average titer of 27.4 g/L amorpha-4,11-diene.Conclusions/ significanceProduction of >25 g/L amorpha-4,11-diene by fermentation followed by chemical conversion to artemisinin may allow for development of a process to provide an alternative source of artemisinin to be incorporated into ACTs.

Precursor-directed biosynthesis was used to produce different triketide lactones (R-TKLs) in a fermentation process. Plasmids expressing engineered versions of the first subunit of 6-deoxyerythronolide B synthase (DEBS1) fused to the terminal DEBS thioesterase (TE) were introduced into three different Streptomyces strains. The DEBS1 protein fused to TE had either an inactivated ketosynthase domain (KS1 degrees ) or a partial DEBS1 lacking module 1 but containing module 2 (M2+TE). Different synthetic precursors were examined for their effect on R-TKL production. An overproducing strain of S. coelicolor expressing the M2+TE protein was found to be best for production of R-TKLs. Racemic precursors were as effective as enantiomerically pure precursors in the fermentation process. The R group on the precursor significantly affected titer (propyl >> chloromethyl > vinyl). The R-TKLs were unstable in fermentation broth at pH 6-8. A two-phase fermentation with a pH shift was implemented to stabilize the products. The fermentation pH initially was controlled at optimal values for cell growth (pH 6.5) and then shifted to 5.5 during production. This doubled peak titers and stabilized the product. Finally, the concentration of synthetic precursor in the fermentation was optimized to improve production. A maximum titer of 500 mg/L 5-chloromethyl-TKL was obtained using 3.5 g/L precursor.

The heterologous production of epothilone D in Myxococcus xanthus was improved by 140-fold from an initial titer of 0.16 mg/L with the incorporation of an adsorber resin, the identification of a suitable carbon source, and the implementation of a fed-batch process. To reduce the degradation of epothilone D in the basal medium, XAD-16 (20 g/L) was added to stabilize the secreted product. This greatly facilitated its recovery and enhanced the yield by three-fold. The potential of using oils as a carbon source for cell growth and product formation was also evaluated. From a screen of various oils, methyl oleate was shown to have the greatest impact. At the optimal concentration of 7 mL/L in a batch process, the maximum cell density was increased from 0.4 g dry cell weight (DCW)/L to 2 g DCW/L. Product yield, however, depended on the presence of trace elements in the production medium. With an exogenous supplement of trace metals to the basal medium, the peak epothilone D titer was enhanced eight-fold. This finding demonstrates the significant role of metal ions in cell metabolism and in epothilone biosynthesis. To further increase the product yield, a continuous fed-batch process was used to promote a higher cell density and to maintain an extended production period. The optimized fed-batch cultures consistently yielded a cell density of 7 g DCW/L and an average production titer of 23 mg/L.

A fermentation process employing precursor-directed biosynthesis is being developed for the manufacture of 6-deoxyerythronolide B (6-dEB) analogues. Through a plasmid-based system in Streptomyces coelicolor, 6-dEB synthesis is catalyzed by 6-dEB synthase (DEBS). 6-dEB synthesis is abolished by inactivation of the ketosynthase (KS) 1 domain of DEBS but can be restored by providing synthetic activated diketides. Because of its inherent catalytic flexibility, the KS1-deficient DEBS is capable of utilizing unnatural diketides to form various 13-substituted 6-dEBs. Here we characterize process variables associated with diketide feeding in shake-flask experiments. 13-R-6-dEB production was found to depend strongly on diketide feed concentrations, on the growth phase of cultures at feeding time, and on the R-group present in the diketide moiety. In all cases a major portion of the fed diketides was degraded by the cells.