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Background  Isobutanol is an important target for biorefinery research as a next-generation biofuel and a building block for commodity chemical production. Metabolically engineered microbial strains to produce isobutanol have been successfully developed by introducing the Ehrlich pathway into bacterial hosts. Isobutanol-producing baker’s yeast ( Saccharomyces cerevisiae ) strains have been developed following the strategy with respect to its advantageous characteristics for cost-effective isobutanol production. However, the isobutanol yields and titers attained by the developed strains need to be further improved through engineering of  S. cerevisiae  metabolism.    Results  Two strategies including eliminating competing pathways and resolving the cofactor imbalance were applied to improve isobutanol production in  S. cerevisiae . Isobutanol production levels were increased in strains lacking genes encoding members of the pyruvate dehydrogenase complex such as  LPD1 , indicating that the pyruvate supply for isobutanol biosynthesis is competing with acetyl-CoA biosynthesis in mitochondria. Isobutanol production was increased by overexpression of enzymes responsible for transhydrogenase-like shunts such as pyruvate carboxylase, malate dehydrogenase, and malic enzyme. The integration of a single gene deletion  lpd1 Δ and the activation of the transhydrogenase-like shunt further increased isobutanol levels. In a batch fermentation test at the 50-mL scale from 100 g/L glucose using the two integrated strains, the isobutanol titer reached 1.62 ± 0.11 g/L and 1.61 ± 0.03 g/L at 24 h after the start of fermentation, which corresponds to the yield at 0.016 ± 0.001 g/g glucose consumed and 0.016 ± 0.0003 g/g glucose consumed, respectively.    Conclusions  These results demonstrate that downregulation of competing pathways and metabolic functions for resolving the cofactor imbalance are promising strategies to construct  S. cerevisiae  strains that effectively produce isobutanol.

Increased isobutanol production in Saccharomyces cerevisiae by eliminating competing pathways and resolving cofactor imbalance