Application of Functional Genomics to Pathway Optimization for Increased Isoprenoid Production

Kizer, Lance; Pitera, Douglas J.; Pfleger, Brian F.; Keasling, Jay D.

doi:10.1128/aem.02750-07

Cited by 161 publications

(130 citation statements)

References 64 publications

(55 reference statements)

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“…Development of a two-phase partitioning bioreactor allowed production of 0.5 g∕L of amorpha-4,11-diene (9). Analysis of transcriptional responses and pathway metabolites showed that 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase limited production, leading to perturbation of fatty acid biosynthesis and generalized membrane stress (10,11). Subsequent replacement of the S. cerevisiae HMG-CoA reductase and HMG-CoA synthase with equivalent enzymes from Staphylococcus aureus, and concomitant fermentation development, allowed production of 27 g∕L of amorpha-4,11-diene (12).…”

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confidence: 99%

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

Westfall

Pitera

Lenihan

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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627

448

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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.

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confidence: 99%

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

Westfall

Pitera

Lenihan

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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627

448

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“…These include lactate (van Maris et al 2004;Ishida et al 2006), malate (Zelle et al 2008), isoprenoids (Shiba et al 2007;Herrero et al 2008;Kizer et al 2008), glycerol (Geertman et al 2006;Cordier et al 2007), and ethanol (Alper et al 2006;Bro et al 2006), among others. Although nonfermentative by-products represent a class of biologically interesting and commercially attractive small molecules, efforts aimed at engineering microbes for increased production of these metabolites are comparatively infrequent.…”

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confidence: 99%

Systems-Level Engineering of Nonfermentative Metabolism in Yeast

Kennedy

Boyle²,

Waks

et al. 2009

Genetics

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We designed and experimentally validated an in silico gene deletion strategy for engineering endogenous one-carbon (C1) metabolism in yeast. We used constraint-based metabolic modeling and computer-aided gene knockout simulations to identify five genes (ALT2, FDH1, FDH2, FUM1, and ZWF1), which, when deleted in combination, predicted formic acid secretion in Saccharomyces cerevisiae under aerobic growth conditions. Once constructed, the quintuple mutant strain showed the predicted increase in formic acid secretion relative to a formate dehydrogenase mutant ( fdh1 fdh2), while formic acid secretion in wild-type yeast was undetectable. Gene expression and physiological data generated post hoc identified a retrograde response to mitochondrial deficiency, which was confirmed by showing Rtg1-dependent NADH accumulation in the engineered yeast strain. Formal pathway analysis combined with gene expression data suggested specific modes of regulation that govern C1 metabolic flux in yeast. Specifically, we identified coordinated transcriptional regulation of C1 pathway enzymes and a positive flux control coefficient for the branch point enzyme 3-phosphoglycerate dehydrogenase (PGDH).Together, these results demonstrate that constraint-based models can identify seemingly unrelated mutations, which interact at a systems level across subcellular compartments to modulate flux through nonfermentative metabolic pathways.

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“…It will not be surprising if systems-level analyses reveal many strategies that can be combined to optimize production. Many recent cell-wide studies have, in fact, focused on understanding the cellular physiology of production microbes exposed to industrial processing conditions [ 67], stress and starvation [ 65], and heterologous metabolic pathways with their potentially toxic intermediates [ 54] and are beginning to find use in engineering better hosts. While the majority of functional genomics studies to date have been targeted toward understanding cellular physiology, the expec-tation is that this information can now be used to deduce pathway bottlenecks and optimize cellular circuit design.…”

Section: Discussionmentioning

confidence: 99%

“…Imbalances in enzymatic activity can also result in the accumulation of toxic or inhibitory pathway intermediates, which may drastically reduce cellular growth as well as production levels. Func-tional genomics can be important in troubleshooting such issues [ 54]. As an example, the accumulation of 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) was found to be a bottleneck in the production of iso-prenoids in E. coli and was corrected by overexpression of tHMG1 [ 55] or by downregulating the synthesis of HMG-CoA [ 56].…”

Section: Applicability Of Functional Genomics To Metabolic Engineeringmentioning

confidence: 99%

Importance of systems biology in engineering microbes for biofuel production

Mukhopadhyay

Redding

Rutherford

et al. 2008

Current Opinion in Biotechnology

124

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Microorganisms have been rich sources for natural products, some of which have found use as fuels, commodity chemicals, specialty chemicals, polymers, and drugs, to name a few. The recent interest in production of transportation fuels from renewable resources has catalyzed numerous research endeavors that focus on developing microbial systems for production of such natural products. Eliminating bottlenecks in microbial metabolic pathways and alleviating the stresses due to production of these chemicals are crucial in the generation of robust and efficient production hosts. The use of systemslevel studies makes it possible to comprehensively understand the impact of pathway engineering within the context of the entire host metabolism, to diagnose stresses due to product synthesis, and provides the rationale to cost-effectively engineer optimal industrial microorganisms.

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Application of Functional Genomics to Pathway Optimization for Increased Isoprenoid Production

Cited by 161 publications

References 64 publications

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

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

Systems-Level Engineering of Nonfermentative Metabolism in Yeast

Importance of systems biology in engineering microbes for biofuel production

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