Production of (R)-citramalate by engineered Saccharomyces cerevisiae

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