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Metabolic engineering v.44, 2017년, pp.302 - 312   SCIE
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Uncovering the role of branched-chain amino acid transaminases in Saccharomyces cerevisiae isobutanol biosynthesis

Hammer, Sarah K. (Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ 08544, USA ) ; Avalos, José (Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ 08544, USA ) ; L. ;
  • 초록  

    Abstract Isobutanol and other branched-chain higher alcohols (BCHAs) are promising advanced biofuels derived from the degradation of branched-chain amino acids (BCAAs). The yeast Saccharomyces cerevisiae is a particularly attractive host for the production of BCHAs due to its high tolerance to alcohols and prevalent use in the bioethanol industry. Degradation of BCAAs begins with transamination reactions, catalyzed by branched-chain amino acid transaminases (BCATs) located in the mitochondria (Bat1p) and cytosol (Bat2p). However, the roles that these transaminases play in isobutanol production remain poorly understood and obscured by conflicting reports in the literature. In this work, we elucidate the influence of BCATs on isobutanol production in two genetic backgrounds (CEN.PK2-1C and BY4741). In the process, we uncover and characterize two competing isobutanol pathways, which can be manipulated by overexpressing or deleting BAT1 or BAT2 , and adding or removing valine from the fermentation media. We show that deletion of BAT1 alone increases isobutanol production by 14.2-fold over wild type strains in media lacking valine, and examine how interactions between valine and the regulatory protein Ilv6p affect isobutanol production. Compartmentalizing the five-gene isobutanol biosynthetic pathway in mitochondria of BAT1 deletion strains results in an additional 2.1-fold increase in isobutanol production in the absence of valine. While valine inhibits isobutanol production, it boosts 2-methyl-1-butanol production. This work clarifies the role of transamination activity in BCHA biosynthesis, and develops valuable strategies and strains for future optimization of isobutanol production. Highlights Two endogenous competing pathways for isobutanol production are identified. Deletion of BAT1 increases isobutanol production 14.2-fold in media lacking valine. Evidence for the role of Ilv6p in acetolactate synthase inhibition by valine. Intracellular ketoisovalerate transport is identified as a key bottleneck in biosynthesis. Evidence for valine selective inhibition of acetolactate synthase is provided.


  • 주제어

    Saccharomyces cerevisiae .   Branched-chain amino acid transaminases .   Isobutanol .   2-methyl-1-butanol .   Metabolic engineering .   Mitochondrial engineering.  

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