Production of (R)-citramalate by engineered .

The budding yeast, , 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. ()-citramalate is a raw material for methyl methacrylate and can be used as a metabolic intermediate in the biosynthesis of higher alcohols. ()-citramalate is synthesized from pyruvate and acetyl-CoA. Unlike , 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 ()-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 ()-citramalate from cells by expressing a heterologous dicarboxylate transporter gene. We evaluated the effects of these approaches on ()-citramalate production and constructed a final strain by combining these positive approaches. The resulting strain produced 16.5 mM ()-citramalate in batch culture flasks. This is the first report of ()-citramalate production by recombinant , and the ()-citramalate production by recombinant yeast achieved in this study was the highest reported to date.