Dataset Information

Engineering strategy of yeast metabolism for higher alcohol production.

ABSTRACT:

Background

While Saccharomyces cerevisiae is a promising host for cost-effective biorefinary processes due to its tolerance to various stresses during fermentation, the metabolically engineered S. cerevisiae strains exhibited rather limited production of higher alcohols than that of Escherichia coli. Since the structure of the central metabolism of S. cerevisiae is distinct from that of E. coli, there might be a problem in the structure of the central metabolism of S. cerevisiae. In this study, the potential production of higher alcohols by S. cerevisiae is compared to that of E. coli by employing metabolic simulation techniques. Based on the simulation results, novel metabolic engineering strategies for improving higher alcohol production by S. cerevisiae were investigated by in silico modifications of the metabolic models of S. cerevisiae.

Results

The metabolic simulations confirmed that the high production of butanols and propanols by the metabolically engineered E. coli strains is derived from the flexible behavior of their central metabolism. Reducing this flexibility by gene deletion is an effective strategy to restrict the metabolic states for producing target alcohols. In contrast, the lower yield using S. cerevisiae originates from the structurally limited flexibility of its central metabolism in which gene deletions severely reduced cell growth.

Conclusions

The metabolic simulation demonstrated that the poor productivity of S. cerevisiae was improved by the introduction of E. coli genes to compensate the structural difference. This suggested that gene supplementation is a promising strategy for the metabolic engineering of S. cerevisiae to produce higher alcohols which should be the next challenge for the synthetic bioengineering of S. cerevisiae for the efficient production of higher alcohols.

SUBMITTER: Matsuda F

PROVIDER: S-EPMC3184262 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Publications

Engineering strategy of yeast metabolism for higher alcohol production.

Matsuda Fumio F Furusawa Chikara C Kondo Takashi T Ishii Jun J Shimizu Hiroshi H Kondo Akihiko A

Microbial cell factories 20110908

<h4>Background</h4>While Saccharomyces cerevisiae is a promising host for cost-effective biorefinary processes due to its tolerance to various stresses during fermentation, the metabolically engineered S. cerevisiae strains exhibited rather limited production of higher alcohols than that of Escherichia coli. Since the structure of the central metabolism of S. cerevisiae is distinct from that of E. coli, there might be a problem in the structure of the central metabolism of S. cerevisiae. In this ...[more]

PMID: 21902829

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Project description:Branched-chain higher alcohols (BCHAs), or fusel alcohols, including isobutanol, isoamyl alcohol, and active amyl alcohol, are useful compounds in several industries. The yeast Saccharomyces cerevisiae can synthesize these compounds via the metabolic pathways of branched-chain amino acids (BCAAs). Branched-chain amino acid aminotransaminases (BCATs) are the key enzymes for BCHA production via the Ehrlich pathway of BCAAs. BCATs catalyze a bidirectional transamination reaction between branched-chain α-keto acids (BCKAs) and BCAAs. In S. cerevisiae, there are two BCAT isoforms, Bat1 and Bat2, which are encoded by the genes BAT1 and BAT2. Although many studies have shown the effects of deletion or overexpression of BAT1 and BAT2 on BCHA production, there have been no reports on the enhancement of BCHA production by functional variants of BCATs. Here, to improve BCHA productivity, we designed variants of Bat1 and Bat2 with altered enzyme activity by using in silico computational analysis: the Gly333Ser and Gly333Trp Bat1 and corresponding Gly316Ser and Gly316Trp Bat2 variants, respectively. When expressed in S. cerevisiae cells, most of these variants caused a growth defect in minimal medium. Interestingly, the Gly333Trp Bat1 and Gly316Ser Bat2 variants achieved 18.7-fold and 17.4-fold increases in isobutanol above that for the wild-type enzyme, respectively. The enzyme assay revealed that the catalytic activities of all four BCAT variants were lower than that of the wild-type enzyme. Our results indicate that the decreased BCAT activity enhanced BCHA production by reducing BCAA biosynthesis, which occurs via a pathway that directly competes with BCHA production. IMPORTANCE Recently, several studies have attempted to increase the production of branched-chain higher alcohols (BCHAs) in the yeast Saccharomyces cerevisiae. The key enzymes for BCHA biosynthesis in S. cerevisiae are the branched-chain amino acid aminotransaminases (BCATs) Bat1 and Bat2. Deletion or overexpression of the genes encoding BCATs has an impact on the production of BCHAs; however, amino acid substitution variants of Bat1 and Bat2 that could affect enzymatic properties-and ultimately BCHA productivity-have not been fully studied. By using in silico analysis, we designed variants of Bat1 and Bat2 and expressed them in yeast cells. We found that the engineered BCATs decreased catalytic activities and increased BCHA production. Our approach provides new insight into the functions of BCATs and will be useful in the future construction of enzymes optimized for high-level production of BCHAs.

Dataset Information

Engineering strategy of yeast metabolism for higher alcohol production.

Background

Results

Conclusions

Publications

Engineering strategy of yeast metabolism for higher alcohol production.

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