Semi-rational evolution of pyruvate carboxylase from Rhizopus oryzae for elevated fumaric acid synthesis in Saccharomyces cerevisiae
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Authors
Xu, Guoqiang
Shi, Xiangliu
Gao, Yuhao
Wang, Jiyue
Cheng, Hui
Liu, Yang
Chen, Yuanyuan
Li, Jiayu
Xu, Xiaopeng
Zha, Jian
Issue Date
2022-01-01
Type
Article
Language
ENG
Keywords
Biology , Chemistry and chemical biology , Chemical and biological engineering , Biomedical engineering
Alternative Title
Abstract
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.
Description
Biochemical Engineering Journal, 177, 108238
Note : if this item contains full text it may be a preprint, author manuscript, or a Gold OA copy that permits redistribution with a license such as CC BY. The final version is available through the publisher’s platform.
Note : if this item contains full text it may be a preprint, author manuscript, or a Gold OA copy that permits redistribution with a license such as CC BY. The final version is available through the publisher’s platform.
Full Citation
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.
Publisher
Terms of Use
Journal
Volume
Issue
PubMed ID
DOI
ISSN
1873295X
1369703X
1369703X