(752e) Deciphering the Elusive Role of Branched-Chain Amino Acid Transaminases in the Production of Branched-Chain Higher Alcohols in Saccharomyces cerevisiae | AIChE

(752e) Deciphering the Elusive Role of Branched-Chain Amino Acid Transaminases in the Production of Branched-Chain Higher Alcohols in Saccharomyces cerevisiae

Authors 

Avalos, J. L. - Presenter, Princeton University
The yeast Saccharomyces cerevisiae is an attractive host for the production of branched-chain higher alcohols (BCHAs), including isobutanol, isopentanol, and 2-methyl-1-butanol, which are promising advanced biofuels. Yeast is known to naturally produce these alcohols as degradation products of valine, leucine, and isoleucine, respectively. Metabolic engineering of BCHA biosynthesis in yeast is complicated by the separation of the upstream (branched-chain amino acid biosynthesis) and downstream (Ehrlich degradation) biosynthetic pathways between the mitochondria and the cytosol, respectively. Moreover, each of these compartments contains distinct branched-chain amino acid transaminases (BCATs), encoded by BAT1 (the mitochondrial BCAT) and BAT2 (the cytosolic BCAT). Previous studies examining the effects of deleting or overexpressing BAT1 and/or BAT2 have yielded inconsistent results, which have obscured the role of these key enzymes and further complicated our ability to engineer optimal BCHA biosynthetic pathways in yeast. In this work, we resolve these previous conflicting results and conclusively elucidate the roles of BCATs in the biosynthesis of BCHAs. We achieve this by taking a rational and combinatorial approach to metabolic engineering and pathway compartmentalization. With this new knowledge, and by manipulating transamination activity alone, we were able to increase isobutanol production by more than 15-fold over wild type strains. Further addressing the intracellular transport bottleneck due to transamination activity in different compartments, we obtained an additional doubling of BCHA production. This study answers the longstanding question of the role of transamination activity in BCHA production in yeast, and develops valuable strains for future optimization of these advanced biofuels.