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GSF2 deletion increases lactic acid production by alleviating glucose repression in Saccharomyces cerevisiae.

ABSTRACT: Improving lactic acid (LA) tolerance is important for cost-effective microbial production of LA under acidic fermentation conditions. Previously, we generated LA-tolerant D-LA-producing S. cerevisiae strain JHY5310 by laboratory adaptive evolution of JHY5210. In this study, we performed whole genome sequencing of JHY5310, identifying four loss-of-function mutations in GSF2, SYN8, STM1, and SIF2 genes, which are responsible for the LA tolerance of JHY5310. Among the mutations, a nonsense mutation in GSF2 was identified as the major contributor to the improved LA tolerance and LA production in JHY5310. Deletion of GSF2 in the parental strain JHY5210 significantly improved glucose uptake and D-LA production levels, while derepressing glucose-repressed genes including genes involved in the respiratory pathway. Therefore, more efficient generation of ATP and NAD⁺ via respiration might rescue the growth defects of the LA-producing strain, where ATP depletion through extensive export of lactate and proton is one of major reasons for the impaired growth. Accordingly, alleviation of glucose repression by deleting MIG1 or HXK2 in JHY5210 also improved D-LA production. GSF2 deletion could be applied to various bioprocesses where increasing biomass yield or respiratory flux is desirable.

SUBMITTER: Baek SH

PROVIDER: S-EPMC5052599 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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GSF2 deletion increases lactic acid production by alleviating glucose repression in Saccharomyces cerevisiae.

Baek Seung-Ho SH Kwon Eunice Y EY Kim Seon-Young SY Hahn Ji-Sook JS

Scientific reports 20161006

Improving lactic acid (LA) tolerance is important for cost-effective microbial production of LA under acidic fermentation conditions. Previously, we generated LA-tolerant D-LA-producing S. cerevisiae strain JHY5310 by laboratory adaptive evolution of JHY5210. In this study, we performed whole genome sequencing of JHY5310, identifying four loss-of-function mutations in GSF2, SYN8, STM1, and SIF2 genes, which are responsible for the LA tolerance of JHY5310. Among the mutations, a nonsense mutation ...[more]

PMID: 27708428

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Project description:l-Lactic acid (LA) is a three-carbon hydroxycarboxylic acid with extensive applications in food, cosmetic, agricultural, pharmaceutical, and bioplastic industries. However, microbial LA production is limited by its intrinsic inefficiency of cellular metabolism. Here, pathway engineering was used to rewire the biosynthetic pathway for LA production in Saccharomyces cerevisiae by screening heterologous l-lactate dehydrogenase, reducing ethanol accumulation, and introducing a bacterial acetyl coenzyme A (acetyl-CoA) synthesis pathway. To improve its intrinsic efficiency of LA export, transporter engineering was conducted by screening the monocarboxylate transporters and then strengthening the capacity of LA export, leading to LA production up to 51.4 g/L. To further enhance its intrinsic efficiency of acid tolerance, adaptive evolution was adopted by cultivating yeast cells with a gradual increase in LA levels during 12 serial subcultures, resulting in a 17.5% increase in LA production to 60.4 g/L. Finally, the engineered strain S.c-NO.2-100 was able to produce 121.5 g/L LA, with a yield of up to 0.81 g/g in a 5-L batch bioreactor. The strategy described here provides a guide for developing efficient cell factories for the production of the other industrially useful organic acids. IMPORTANCE Saccharomyces cerevisiae is one of the most widely engineered cell factories for the production of organic acids. However, microbial production of l-lactic acid is limited by its intrinsic inefficiency of cellular metabolism in S. cerevisiae. Here, the transmission efficiency of the biosynthetic pathway was improved by pathway optimization to increase l-lactic acid production. Then, the synthetic ability for l-lactic acid was further enhanced by adaptive evolution to improve acid tolerance of S. cerevisiae. Based on these strategies, the final engineered S. cerevisiae strain achieved high efficiency of l-lactic acid production. These findings provide new insight into improving the intrinsic efficiency of cellular metabolism and will help to construct superior industrial yeast strains for high-level production of other organic acids.

Project description:BackgroundSaccharomyces cerevisiae, engineered for L-lactic acid production from glucose and xylose, is a promising production host for lignocellulose-to-lactic acid processes. However, the two principal engineering strategies-pyruvate-to-lactic acid conversion with and without disruption of the competing pyruvate-to-ethanol pathway-have not yet resulted in strains that combine high lactic acid yields (YLA) and productivities (QLA) on both sugar substrates. Limitations seemingly arise from a dependency on the carbon source and the aeration conditions, but the underlying effects are poorly understood. We have recently presented two xylose-to-lactic acid converting strains, IBB14LA1 and IBB14LA1_5, which have the L-lactic acid dehydrogenase from Plasmodium falciparum (pfLDH) integrated at the pdc1 (pyruvate decarboxylase) locus. IBB14LA1_5 additionally has its pdc5 gene knocked out. In this study, the influence of carbon source and oxygen on YLA and QLA in IBB14LA1 and IBB14LA1_5 was investigated.ResultsIn anaerobic fermentation IBB14LA1 showed a higher YLA on xylose (0.27 g g Xyl-1 ) than on glucose (0.18 g g Glc-1 ). The ethanol yields (YEtOH, 0.15 g g Xyl-1 and 0.32 g g Glc-1 ) followed an opposite trend. In IBB14LA1_5, the effect of the carbon source on YLA was less pronounced (~?0.80 g g Xyl-1 , and 0.67 g g Glc-1 ). Supply of oxygen accelerated glucose conversions significantly in IBB14LA1 (QLA from 0.38 to 0.81 g L-1 h-1) and IBB14LA1_5 (QLA from 0.05 to 1.77 g L-1 h-1) at constant YLA (IBB14LA1 ~?0.18 g g Glc-1 ; IBB14LA1_5 ~?0.68 g g Glc-1 ). In aerobic xylose conversions, however, lactic acid production ceased completely in IBB14LA1 and decreased drastically in IBB14LA1_5 (YLA aerobic???0.25 g g Xyl-1 and anaerobic?~?0.80 g g Xyl-1 ) at similar QLA (~?0.04 g L-1 h-1). Switching from aerobic to microaerophilic conditions (pO2?~?2%) prevented lactic acid metabolization, observed for fully aerobic conditions, and increased QLA and YLA up to 0.11 g L-1 h-1 and 0.38 g g Xyl-1 , respectively. The pfLDH and PDC activities in IBB14LA1 were measured and shown to change drastically dependent on carbon source and oxygen.ConclusionEvidence from conversion time courses together with results of activity measurements for pfLDH and PDC show that in IBB14LA1 the distribution of fluxes at the pyruvate branching point is carbon source and oxygen dependent. Comparison of the performance of strain IBB14LA1 and IBB14LA1_5 in conversions under different aeration conditions (aerobic, anaerobic, and microaerophilic) further suggest that xylose, unlike glucose, does not repress the respiratory response in both strains. This study proposes new genetic engineering targets for rendering genetically engineering S. cerevisiae better suited for lactic acid biorefineries.

Dataset Information

GSF2 deletion increases lactic acid production by alleviating glucose repression in Saccharomyces cerevisiae.

Publications

GSF2 deletion increases lactic acid production by alleviating glucose repression in Saccharomyces cerevisiae.

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