Metabolic and Cell Surface Engineering to Produce Isobutanol from Lignocellulosic Biomass in Yeast Saccharomyces cerevisiae

The production of higher alcohols including C3-C5 alcohols in engineered bacteria has received significant attention, because they can be used as fuels, fuel additives, or commodity chemicals. The budding yeast, Saccharomyces cerevisiae, has considerable potential as a producer of higher alcohols because of its capacity to naturally fabricate fusel alcohols, in addition to its robustness and tolerance to low pH. However, because its natural productivity is not significant, we considered a strategy of genetic engineering to increase production of higher alcohols. For example, the branched-chain higher alcohol isobutanol, which is involved in valine biosynthesis, offers higher octane values than their straight-chain counterparts with equivalent carbon numbers and can be used with current infrastructure in addition to possessing almost the same capabilities (energy density, hygroscopicity and octane number) as gasoline. However, its natural productivity in yeast is not significant. In this study, we show the engineering strategies of metabolic pathway and cell surface display to improve the isobutanol production in S. cerevisiae. To relieve the cofactor imbalance, we tested activation of the transhydrogenase-like shunt comprising pyruvate carboxylase (Pyc2), malate dehydrogenase (Mdh2) and malic enzyme (Mae1), the bypass for converting NADH to NADPH. As another approach, we tested the multiple gene knockout, which is related to the pyruvate metabolism, to improve the isobutanol production. Finally, we displayed the cellulose enzymes (endoglucanase and β-glucosidase) on the yeast cell surface and successfully produced isobutanol from cellobiose and β-glucan.

This work was supported by Special Coordination Funds for Promoting Science and Technology, Creation of Innovation Centers for Advanced Interdisciplinary Research Areas (Innovative Bioproduction Kobe; iBioK), MEXT, Japan, and in part by Industrial Technology Research Grant Program in 2011 from New Energy and Industrial Technology Development Organization (NEDO) of Japan.