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

    Author
    Xu, Guoqiang; Shi, Xiangliu; Gao, Yuhao; Wang, Jiyue; Cheng, Hui; Liu, Yang; Chen, Yuanyuan; Li, Jiayu; Xu, Xiaopeng; Zha, Jian; Xia, Ke; Linhardt, Robert J.; Zhang, Xiaomei; Shi, Jinsong; Koffas, Mattheos A.G.; Xu, Zhenghong
    ORCID
    https://orcid.org/0000-0003-2219-5833
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    Other Contributors
    Date Issued
    2022-01-01
    Subject
    Biology; Chemistry and chemical biology; Chemical and biological engineering; Biomedical engineering
    Degree
    Terms of Use
    In Copyright : this Item is protected by copyright and/or related rights. You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s). https://rightsstatements.org/page/InC/1.0/;
    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.
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    URI
    https://doi.org/10.1016/j.bej.2021.108238; https://hdl.handle.net/20.500.13015/5437
    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.
    Department
    The Linhardt Research Labs.; The Shirley Ann Jackson, Ph.D. Center for Biotechnology and Interdisciplinary Studies (CBIS);
    Relationships
    The Linhardt Research Labs Online Collection; Rensselaer Polytechnic Institute, Troy, NY; Biochemical Engineering Journal; https://harc.rpi.edu/;
    Access
    https://login.libproxy.rpi.edu/login?url=https://doi.org/10.1016/j.bej.2021.108238;
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