Dataset Information

13C-metabolic flux analysis of ethanol-assimilating Saccharomyces cerevisiae for S-adenosyl-L-methionine production.

ABSTRACT: BACKGROUNDSaccharomyces cerevisiae is a host for the industrial production of S-adenosyl-L-methionine (SAM), which has been widely used in pharmaceutical and nutritional supplement industries. It has been reported that the intracellular SAM content in S. cerevisiae can be improved by the addition of ethanol during cultivation. However, the metabolic state in ethanol-assimilating S. cerevisiae remains unclear. In this study, 13C-metabolic flux analysis (13C-MFA) was conducted to investigate the metabolic regulation responsible for the high SAM production from ethanol.RESULTSThe comparison between the metabolic flux distributions of central carbon metabolism showed that the metabolic flux levels of the tricarboxylic acid cycle and glyoxylate shunt in the ethanol culture were significantly higher than that of glucose. Estimates of the ATP balance from the 13C-MFA data suggested that larger amounts of excess ATP was produced from ethanol via increased oxidative phosphorylation. The finding was confirmed by the intracellular ATP level under ethanol-assimilating condition being similarly higher than glucose.CONCLUSIONSThese results suggest that the enhanced ATP regeneration due to ethanol assimilation was critical for the high SAM accumulation.

SUBMITTER: Hayakawa K

PROVIDER: S-EPMC5977476 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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<sup>13</sup>C-metabolic flux analysis of ethanol-assimilating Saccharomyces cerevisiae for S-adenosyl-L-methionine production.

Hayakawa Kenshi K Matsuda Fumio F Shimizu Hiroshi H

Microbial cell factories 20180531 1

<h4>Background</h4>Saccharomyces cerevisiae is a host for the industrial production of S-adenosyl-L-methionine (SAM), which has been widely used in pharmaceutical and nutritional supplement industries. It has been reported that the intracellular SAM content in S. cerevisiae can be improved by the addition of ethanol during cultivation. However, the metabolic state in ethanol-assimilating S. cerevisiae remains unclear. In this study, <sup>13</sup>C-metabolic flux analysis (<sup>13</sup>C-MFA) was ...[more]

PMID: 29855316

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Project description:BackgroundThe yeast Saccharomyces cerevisiae is a promising host cell for producing a wide range of chemicals. However, attempts to metabolically engineer Crabtree-positive S. cerevisiae invariably face a common issue: how to reduce dominant ethanol production. Here, we propose a yeast metabolic engineering strategy for decreasing ethanol subgeneration involving tugging the carbon flux at an important hub branching point (e.g., pyruvate). Tugging flux at a central glycolytic overflow metabolism point arising from high glycolytic activity may substantially increase higher alcohol production in S. cerevisiae. We validated this possibility by testing 2,3-butanediol (2,3-BDO) production, which is routed via pyruvate as the important hub compound.ResultsBy searching for high-activity acetolactate synthase (ALS) enzymes that catalyze the important first-step reaction in 2,3-BDO biosynthesis, and tuning several fermentation conditions, we demonstrated that a stronger pyruvate pulling effect (tugging of pyruvate carbon flux) is very effective for increasing 2,3-BDO production and reducing ethanol subgeneration by S. cerevisiae. To further confirm the validity of the pyruvate carbon flux tugging strategy, we constructed an evolved pyruvate decarboxylase (PDC)-deficient yeast (PDCΔ) strain that lacked three isozymes of PDC. In parallel with re-sequencing to identify genomic mutations, liquid chromatography-tandem mass spectrometry analysis of intermediate metabolites revealed significant accumulation of pyruvate and NADH in the evolved PDCΔ strain. Harnessing the high-activity ALS and additional downstream enzymes in the evolved PDCΔ strain resulted in a high yield of 2,3-BDO (a maximum of 0.41 g g-1 glucose consumed) and no ethanol subgeneration, thereby confirming the utility of our strategy. Using this engineered strain, we demonstrated a high 2,3-BDO titer (81.0 g L-1) in a fed-batch fermentation using a high concentration of glucose as the sole carbon source.ConclusionsWe demonstrated that the pyruvate carbon flux tugging strategy is very effective for increasing 2,3-BDO production and decreasing ethanol subgeneration in Crabtree-positive S. cerevisiae. High activity of the common first-step enzyme for the conversion of pyruvate, which links to both the TCA cycle and amino acid biosynthesis, is likely important for the production of various chemicals by S. cerevisiae.

Dataset Information

13C-metabolic flux analysis of ethanol-assimilating Saccharomyces cerevisiae for S-adenosyl-L-methionine production.

Publications

<sup>13</sup>C-metabolic flux analysis of ethanol-assimilating Saccharomyces cerevisiae for S-adenosyl-L-methionine production.

Similar Datasets

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