Dicarboxylic acids are widely used organic acids in food, pharmaceutical and chemical industries. Pyruvate carboxylase (PYC) plays a pivotal role in the production of dicarboxylic acids by microbial fermentation. In this study, we constructed a synthetic pathway of fumaric acid by deleting FUM1 in Saccharomyces cerevisiae cells lacking both PDC1 and ADH1 to reduce the carbon flow toward ethanol. Furthermore, with overexpression of the heterologous Rhizopus oryzae pyruvate carboxylase (RoPYC) gene in S. cerevisiae cells lacking PDC1, ADH1, and FUM1, the fermentation reached a fumaric acid titer of 226.0 ± 2.2 mg/L. Based on this model, we demonstrated that each of the three mutations (N315F[Asn→Phe], R485P[Arg→Pro], and N1078F[Asn→Phe]) or codon optimization of RoPYC significantly increased fumaric acid production. In a flask, a maximum fumaric acid titer of 465.5 ± 6.5 mg/L was achieved by combining the R485P mutation and codon optimization of RoPYC. Overall, this study is the first to apply site-saturation mutagenesis and codon optimization of the PYC gene to improve fumaric acid production, suggesting that PYC mutant is a good starting point to further enhance of fumaric acid production.

Improvement of fumaric acid biosynthesis in engineered Saccharomyces cerevisiae by site-directed mutagenesis and codon optimization

254th National Meeting (2017)

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