Metabolic Engineering of Yeast Central Metabolism for Higher Alcohol Production

Isobutanol is a target for biorefinery research as a next-generation biofuel and a building block for commodity chemical production. Metabolically engineered Saccharomyces cerevisiae strains to produce isobutanol have been developed by introducing the Ehrlich pathway with respect to its advantageous characteristics for cost-effective production. In this study, the central metabolism in S. cerevisiae was engineered to improve isobutanol production based on understanding S. cerevisiae metabolism by flux balance analysis (FBA) and absolute quantification of intermediate.

FBA using S. cerevisiae metabolic model pointed out that a redox imbalance caused by an activation of isobutanol biosynthesis could be relieved by introducing the Enter-Doudoroff (ED) pathway or pyridine nucleotide transhydrogenase. Metabolically engineered strains expressing Escherichia coli eda and edd genes in BY4742gnd1Δ showed a faster growth rate indicating a function of ED pathway in S. cerevisiae. An absolute quantification of the metabolite levels in BY4742pfk1Δ strain showed that a tight control of glucose-6-phoshate dehydrogenase reaction hampers a redirection of glycolytic flux into ED pathway, since levels of 6-phosphogluconate in BY4742pfk1Δ (0.08 mmol/g fw) was essentially identical with wild type strain. In order to implement a transhydrogenase function, metabolic shunts including pyruvate carboxylase, malate dehydrogenase, and malic enzyme were constructed in BY4741lpd1Δ strain. In a batch fermentation test from 100 g/L glucose using the engineered strains, the isobutanol titer reached 1.62 ± 0.11 g/L at 24 h after the start of fermentation, which corresponds to the yield at 0.016 ± 0.001 g/g glucose consumed. This work was supported by industrial technology research grant program in 2011 from NEDO of Japan.