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dc.contributor.authorXu, Guoqiang
dc.contributor.authorShi, Xiangliu
dc.contributor.authorGao, Yuhao
dc.contributor.authorWang, Jiyue
dc.contributor.authorCheng, Hui
dc.contributor.authorLiu, Yang
dc.contributor.authorChen, Yuanyuan
dc.contributor.authorLi, Jiayu
dc.contributor.authorXu, Xiaopeng
dc.contributor.authorZha, Jian
dc.contributor.authorXia, Ke
dc.contributor.authorLinhardt, Robert J.
dc.contributor.authorZhang, Xiaomei
dc.contributor.authorShi, Jinsong
dc.contributor.authorKoffas, Mattheos A.G.
dc.contributor.authorXu, Zhenghong
dc.date2022
dc.date.accessioned2022-06-27T15:36:07Z
dc.date.available2022-06-27T15:36:07Z
dc.date.issued2022-01-01
dc.identifier.citationSemi-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.
dc.identifier.issn1873295X
dc.identifier.issn1369703X
dc.identifier.urihttps://doi.org/10.1016/j.bej.2021.108238
dc.identifier.urihttps://hdl.handle.net/20.500.13015/5437
dc.descriptionBiochemical Engineering Journal, 177, 108238
dc.descriptionNote : 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.
dc.description.abstractDicarboxylic 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.
dc.description.sponsorshipNational Natural Science Foundation of China
dc.description.urihttps://login.libproxy.rpi.edu/login?url=https://doi.org/10.1016/j.bej.2021.108238
dc.languageen_US
dc.language.isoENG
dc.relation.ispartofThe Linhardt Research Labs Online Collection
dc.relation.ispartofRensselaer Polytechnic Institute, Troy, NY
dc.relation.ispartofBiochemical Engineering Journal
dc.relation.urihttps://harc.rpi.edu/
dc.subjectBiology
dc.subjectChemistry and chemical biology
dc.subjectChemical and biological engineering
dc.subjectBiomedical engineering
dc.titleSemi-rational evolution of pyruvate carboxylase from Rhizopus oryzae for elevated fumaric acid synthesis in Saccharomyces cerevisiae
dc.typeArticle
dcterms.accessRightshttps://login.libproxy.rpi.edu/login?url=https://doi.org/10.1016/j.bej.2021.108238
dcterms.isPartOfJournal
dcterms.isVersionOfhttps://doi.org/10.1016/j.bej.2021.108238
dc.rights.holderIn 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/
dc.creator.identifierhttps://orcid.org/0000-0003-2219-5833
dc.relation.departmentThe Linhardt Research Labs.
dc.relation.departmentThe Shirley Ann Jackson, Ph.D. Center for Biotechnology and Interdisciplinary Studies (CBIS)
rpi.description.volume177


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