Efficient C25-Hydroxylation of Vitamin D3 Utilizing an Artificial Self-Sufficient Whole-Cell Cytochrome P450 Biocatalyst

文献类型： 外文期刊

第一作者： Liang, Ziqi

作者： Liang, Ziqi;Zhou, Qiao;Li, Yicheng;Yang, Xiaojun;Liang, Ziqi;Zhou, Qiao;Li, Yicheng;Liu, Xiaoqing;Shen, Yiwen;Tian, Jian;Wang, Xiaolu;Qin, Xing;Wang, Yuan;Luo, Huiying;Yao, Bin;Tu, Tao

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关键词： 25-hydroxyvitamin D-3; P450s; self-sufficientP450s; local energetic frustration; linker engineering

期刊名称：JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY （ 影响因子：6.2； 五年影响因子：6.4 ）

ISSN： 0021-8561

年卷期： 2025 年 73 卷 17 期

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收录情况： SCI

摘要： Cytochrome P450 enzymes (P450s) are promising candidates for the biosynthesis of 25-hydroxyvitamin D-3 (25(OH)VD3). However, their industrial application is limited by challenges, such as low stability, inefficient catalysis, and uncoupling reactions. The construction of self-sufficient P450s offers a strategic solution to these limitations, but requires linker optimization to regulate interdomain conformational dynamics. In this study, we integrated whole-cell biocatalyst screening with systematic optimization of reaction conditions, including cosolvents, cell concentrations, and plasmid selection, to enhance catalytic performance. Under optimized conditions, the heme domain Vdh-K1 achieved a 91.6% conversion efficiency and was subsequently selected for chimeric enzyme assembly. By employing local energetic frustration analysis to evaluate protein flexibility and allosteric dynamics, we identified chimeric P450 variants with highly frustrated linkers. The optimal variant, VK1-CYP116B46-L21, exhibited improved thermostability, catalytic activity, and coupling efficiency, achieving a yield of 4.89 mM (1.96 g/L) 25(OH)VD3 in Escherichia coli whole-cell catalysis-the highest reported yield to date. This work underscores the utility of computational frustration analysis in refining linker dynamics for multidomain enzymes and establishes a scalable, cost-effective framework to advance P450s systems for industrial biosynthesis of high-value compounds.

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