Semi-rational evolution of pyruvate carboxylase from Rhizopus oryzae for elevated fumaric acid synthesis in Saccharomyces cerevisiae

Xu, Guoqiang
Shi, Xiangliu
Gao, Yuhao
Wang, Jiyue
Cheng, Hui
Liu, Yang
Chen, Yuanyuan
Li, Jiayu
Xu, Xiaopeng
Zha, Jian
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Biology , Chemistry and chemical biology , Chemical and biological engineering , Biomedical engineering
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Semi-rational evolution of pyruvate carboxylase from Rhizopus oryzae for elevated fumaric acid synthesis in Saccharomyces cerevisiae, G. Xu, X. Shi, Y. Gao, J. Wang, H. Cheng, Y. Liu, Y. Chen, J. Li, X. Xu, J. Zha, K. Xia, R. J. Linhardt, X. Zhang, J. Shi, M.A.G. Koffas, Z. Xu, Biochemical Engineering Journal, 177, 108238, 2022.
Dicarboxylic acids are widely used in food, pharmaceutical, and chemical industries. Pyruvate carboxylase (PYC) plays a pivotal role in the production of dicarboxylic acids in microbial fermentation process. Our previous work showed that heterologous expression of pyruvate carboxylase (RoPYC) from Rhizopus oryzae resulted in an increase in fumaric acid titer to 226.0 ± 2.2 mg/L from 194.0 ± 4.0 mg/L in the S. cerevisiae pdc1adh1fum1 strain. However, PYC still remained the metabolic step limiting the production of target carboxylic acids. In this study, semi-rational evolution of pyruvate carboxylase by site-saturation mutagenesis combined with codon optimization was conducted to further improve fumaric acid synthesis. We demonstrated that each of three mutations (N315F, R485P and N1078F) or codon optimization of RoPYC significantly increased the production of fumaric acid. A maximal titer of 465.5 ± 6.5 mg/L was achieved in flasks by the strain expressing codon-optimized RoPYC mutant (R485P). Enzyme assays of these mutants showed higher PYC activities, while homology modeling indicated that the increased PYC activities could be attributed to the modulation of the allosteric domain and the biotin carboxylation domain. In addition, both calcium ion and carbon dioxide displayed positive effects on the fumaric acid production by this mutant. Overall, the strategy described here demonstrated an effective way for elevating PYC activity and further enhance the synthesis of dicarboxylic acids.
Biochemical Engineering Journal, 177, 108238
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The Linhardt Research Labs.
The Shirley Ann Jackson, Ph.D. Center for Biotechnology and Interdisciplinary Studies (CBIS)
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Rensselaer Polytechnic Institute, Troy, NY
Biochemical Engineering Journal