Approaches and Recent Developments for the Commercial Production of Semi-synthetic Artemisinin.

The antimalarial drug artemisinin is a natural product produced by the plant Artemisia annua. Extracts of A. annua have been used in Chinese herbal medicine for over two millennia. Following the re-discovery of A. annua extract as an effective antimalarial, and the isolation and structural elucidation of artemisinin as the active agent, it was recommended as the first-line treatment for uncomplicated malaria in combination with another effective antimalarial drug (Artemisinin Combination Therapy) by the World Health Organization (WHO) in 2002. Following the WHO recommendation, the availability and price of artemisinin fluctuated greatly, ranging from supply shortfalls in some years to oversupply in others. To alleviate these supply and price issues, a second source of artemisinin was sought, resulting in an effort to produce artemisinic acid, a late-stage chemical precursor of artemisinin, by yeast fermentation, followed by chemical conversion to artemisinin (i.e., semi-synthesis). Engineering to enable production of artemisinic acid in yeast relied on the discovery of A. annua genes encoding artemisinic acid biosynthetic enzymes, and synthetic biology to engineer yeast metabolism. The progress of this effort, which resulted in semi-synthetic artemisinin entering commercial production in 2013, is reviewed with an emphasis on recent publications and opportunities for further development. Aspects of both the biology of artemisinin production in A. annua, and yeast strain engineering are discussed, as are recent developments in the chemical conversion of artemisinic acid to artemisinin.

Developing fermentative terpenoid production for commercial usage.

Terpenoids comprise a large (>55000) family of compounds, very few of which have been used commercially due to low and economically unpractical production in their native hosts (generally plants and microorganisms). Two examples of natural terpenoid production are described (rubber and astaxanthin), but the advent of metabolic engineering has allowed the development of fermentative production processes using heterologous microorganisms. The two biochemical pathways responsible for terpenoid production are described, along with manipulations that enable production of terpenoids at economically viable levels. Finally, this article reviews some terpenoids that are currently in commercial production or development, ranging from semisynthetic production of the antimalarial drug artemisinin, through fragrance molecules, to commodity chemicals such as isoprene and ß-farnesene.

Semi-synthetic artemisinin: a model for the use of synthetic biology in pharmaceutical development.

Recent developments in synthetic biology, combined with continued progress in systems biology and metabolic engineering, have enabled the engineering of microorganisms to produce heterologous molecules in a manner that was previously unfeasible. The successful synthesis and recent entry of semi-synthetic artemisinin into commercial production is the first demonstration of the potential of synthetic biology for the development and production of pharmaceutical agents. In this Review, we describe the metabolic engineering and synthetic biology approaches that were used to develop this important antimalarial drug precursor. This not only demonstrates the incredible potential of the available technologies but also illuminates how lessons learned from this work could be applied to the production of other pharmaceutical agents.

De novo sequencing, assembly and analysis of the genome of the laboratory strain Saccharomyces cerevisiae CEN.PK113-7D, a model for modern industrial biotechnology.

Saccharomyces cerevisiae CEN.PK 113-7D is widely used for metabolic engineering and systems biology research in industry and academia. We sequenced, assembled, annotated and analyzed its genome. Single-nucleotide variations (SNV), insertions/deletions (indels) and differences in genome organization compared to the reference strain S. cerevisiae S288C were analyzed. In addition to a few large deletions and duplications, nearly 3000 indels were identified in the CEN.PK113-7D genome relative to S288C. These differences were overrepresented in genes whose functions are related to transcriptional regulation and chromatin remodelling. Some of these variations were caused by unstable tandem repeats, suggesting an innate evolvability of the corresponding genes. Besides a previously characterized mutation in adenylate cyclase, the CEN.PK113-7D genome sequence revealed a significant enrichment of non-synonymous mutations in genes encoding for components of the cAMP signalling pathway. Some phenotypic characteristics of the CEN.PK113-7D strains were explained by the presence of additional specific metabolic genes relative to S288C. In particular, the presence of the BIO1 and BIO6 genes correlated with a biotin prototrophy of CEN.PK113-7D. Furthermore, the copy number, chromosomal location and sequences of the MAL loci were resolved. The assembled sequence reveals that CEN.PK113-7D has a mosaic genome that combines characteristics of laboratory strains and wild-industrial strains.

Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin.

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.

Induction of multiple pleiotropic drug resistance genes in yeast engineered to produce an increased level of anti-malarial drug precursor, artemisinic acid.

BACKGROUND: Due to the global occurrence of multi-drug-resistant malarial parasites (Plasmodium falciparum), the anti-malarial drug most effective against malaria is artemisinin, a natural product (sesquiterpene lactone endoperoxide) extracted from sweet wormwood (Artemisia annua). However, artemisinin is in short supply and unaffordable to most malaria patients. Artemisinin can be semi-synthesized from its precursor artemisinic acid, which can be synthesized from simple sugars using microorganisms genetically engineered with genes from A. annua. In order to develop an industrially competent yeast strain, detailed analyses of microbial physiology and development of gene expression strategies are required. RESULTS: Three plant genes coding for amorphadiene synthase, amorphadiene oxidase (AMO or CYP71AV1), and cytochrome P450 reductase, which in concert divert carbon flux from farnesyl diphosphate to artemisinic acid, were expressed from a single plasmid. The artemisinic acid production in the engineered yeast reached 250 microg mL(-1) in shake-flask cultures and 1 g L(-1) in bio-reactors with the use of Leu2d selection marker and appropriate medium formulation. When plasmid stability was measured, the yeast strain synthesizing amorphadiene alone maintained the plasmid in 84% of the cells, whereas the yeast strain synthesizing artemisinic acid showed poor plasmid stability. Inactivation of AMO by a point-mutation restored the high plasmid stability, indicating that the low plasmid stability is not caused by production of the AMO protein but by artemisinic acid synthesis or accumulation. Semi-quantitative reverse-transcriptase (RT)-PCR and quantitative real time-PCR consistently showed that pleiotropic drug resistance (PDR) genes, belonging to the family of ATP-Binding Cassette (ABC) transporter, were massively induced in the yeast strain producing artemisinic acid, relative to the yeast strain producing the hydrocarbon amorphadiene alone. Global transcriptional analysis by yeast microarray further demonstrated that the induction of drug-resistant genes such as ABC transporters and major facilitator superfamily (MSF) genes is the primary cellular stress-response; in addition, oxidative and osmotic stress responses were observed in the engineered yeast. CONCLUSION: The data presented here suggest that the engineered yeast producing artemisinic acid suffers oxidative and drug-associated stresses. The use of plant-derived transporters and optimizing AMO activity may improve the yield of artemisinic acid production in the engineered yeast.

Balancing a heterologous mevalonate pathway for improved isoprenoid production in Escherichia coli.

Engineering biosynthetic pathways in microbes for the production of complex chemicals and pharmaceuticals is an attractive alternative to chemical synthesis. However, in transferring large pathways to alternate hosts and manipulating expression levels, the native regulation of carbon flux through the pathway may be lost leading to imbalances in the pathways. Previously, Escherichia coli was engineered to produce large quantities of isoprenoids by creating a mevalonate-based isopentenyl pyrophosphate biosynthetic pathway [Martin, V.J., Pitera, D.J., Withers, S.T., Newman, J.D., Keasling, J.D., 2003. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat. Biotechnol. 21, 796-802]. The strain produces high levels of isoprenoids, but upon further investigation we discovered that the accumulation of pathway intermediates limited flux and that high-level expression of the mevalonate pathway enzymes inhibited cell growth. Gene titration studies and metabolite profiling using liquid chromatography-mass spectrometry linked the growth inhibition phenotype with the accumulation of the pathway intermediate 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA). Such an accumulation implies that the activity of HMG-CoA reductase was insufficient to balance flux in the engineered pathway. By modulating HMG-CoA reductase production, we eliminated the pathway bottleneck and increased mevalonate production. These results demonstrate that balancing carbon flux through the heterologous pathway is a key determinant in optimizing isoprenoid biosynthesis in microbial hosts.

The global transcriptional response of Bacillus subtilis to peroxide stress is coordinated by three transcription factors.

Bacillus subtilis exhibits a complex adaptive response to low levels of peroxides. We used global transcriptional profiling to monitor the magnitude and kinetics of changes in the mRNA population after exposure to either hydrogen peroxide (H(2)O(2)) or tert-butyl peroxide (t-buOOH). The peroxide stimulons could be largely accounted for by three regulons controlled by the PerR, sigma(B), and OhrR transcription factors. Three members of the PerR regulon (katA, mrgA, and zosA) were strongly induced by H(2)O(2) and weakly induced by t-buOOH. The remaining members of the PerR regulon were only modestly up-regulated by peroxide treatment. Overall, the magnitude of peroxide induction of PerR regulon genes corresponded well with the extent of derepression in a perR mutant strain. The sigma(B) regulon was activated by 58 micro M H(2)O(2) but not by 8 micro M H(2)O(2) and was strongly activated by either t-buOOH or, in a control experiment, tert-butyl alcohol. Apart from the sigma(B) regulon there was a single gene, ohrA, that was strongly and rapidly induced by t-buOOH exposure. This gene, controlled by the peroxide-sensing repressor OhrR, was not induced by any of the other conditions tested.

Defining the Bacillus subtilis sigma(W) regulon: a comparative analysis of promoter consensus search, run-off transcription/macroarray analysis (ROMA), and transcriptional profiling approaches.

The Bacillus subtilis extracytoplasmic function (ECF) sigma factor sigma(W) controls a large regulon that is strongly induced by alkali shock. To define the physiological role of sigma(W) we have sought to identify the complete set of genes under sigma(W) control. Previously, we described a promoter consensus search procedure to identify sigma(W) controlled genes. Herein, we introduce a novel method to identify additional target promoters: run-off transcription followed by macroarray analysis (ROMA). We compare the resulting list of targets with those identified in conventional transcriptional profiling studies and using the consensus search approach. While transcriptional profiling identifies genes that are strongly dependent on sigma(W) for in vivo expression, some sigma(W)-dependent promoters are not detected due to the masking effects of other promoter elements, overlapping recognition with other ECF sigma factors, or both. Taken together, the consensus search, ROMA, and transcriptional profiling approaches establish a minimum of 30 promoter sites (controlling approximately 60 genes) as direct targets for activation by sigma(W). Significantly, no single approach identifies more than approximately 80% of the regulon so defined. We therefore suggest that a combination of two or more complementary approaches be employed in studies seeking to achieve maximal coverage when defining bacterial regulons. Our results indicate that sigma(W) controls genes that protect the cell against agents that impair cell wall biosynthesis but fail to reveal any connection to operons likely to function in adaptation to alkaline growth conditions. This is consistent with the observation that a sigW mutant is unaffected in its ability to survive alkali shock. We conclude that in B. subtilis sudden imposition of alkali stress activates the sigma(W) stress response, perhaps by impairing the ability of the cell wall biosynthetic machinery to function.