Production of (R)-citramalate by engineered Saccharomyces cerevisiae

The budding yeast, Saccharomyces cerevisiae, , has a high tolerance to organic acids and alcohols, and thus grows well under toxic concentrations of various compounds in the culture medium, potentially allowing for highly efficient compound production. (R)-citramalate R )-citramalate is a raw material for methyl methacrylate and can be used as a metabolic intermediate in the biosynthesis of higher alcohols. (R)-citramalate R )-citramalate is synthesized from pyruvate and acetyl-CoA. Unlike Escherichia coli, , S. cerevisiae has organelles, and its intracellular metabolites are compartmentalized, preventing full use of intracellular acetyl-CoA. Therefore, in this study, to increase the amount of cytosolic acetyl-CoA for highly efficient production of (R)-citramalate, R )-citramalate, we inhibited the transport of cytosolic acetyl-CoA and pyruvate to the mitochondria. We also constructed a heterologous pathway to supply cytosolic acetyl-CoA. Additionally, we attempted to export (R)-citramalate R )-citramalate from cells by expressing a heterologous dicarboxylate transporter gene. We evaluated the effects of these approaches on (R)-citramalate R )-citramalate production and constructed a final strain by combining these positive approaches. The resulting strain produced 16.5 mM (R)- R )- citramalate in batch culture flasks. This is the first report of (R)-citramalate R )-citramalate production by recombinant S. cerevisiae, , and the (R)-citramalate R )-citramalate production by recombinant yeast achieved in this study was the highest reported to date.