Liang Ziqi, Zhou Qiao, Li Yicheng, Liu Xiaoqing, Shen Yiwen, Tian Jian, Wang Xiaolu, Qin Xing, Wang Yuan, Luo Huiying, Yang Xiaojun, Yao Bin, Tu Tao
College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, China.
State Key Laboratory of Animal Nutrition and Feeding, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
J Agric Food Chem. 2025 Apr 30;73(17):10378-10388. doi: 10.1021/acs.jafc.4c12356. Epub 2025 Mar 20.
Cytochrome P450 enzymes (P450s) are promising candidates for the biosynthesis of 25-hydroxyvitamin D (25(OH)VD). 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)VD in 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.
细胞色素P450酶（P450s）是25-羟基维生素D（25(OH)VD）生物合成的有前景的候选者。然而，它们的工业应用受到诸如稳定性低、催化效率低和解偶联反应等挑战的限制。构建自给自足的P450s为解决这些限制提供了一种策略性解决方案，但需要对连接子进行优化以调节结构域间的构象动力学。在本研究中，我们将全细胞生物催化剂筛选与反应条件的系统优化相结合，包括共溶剂、细胞浓度和质粒选择，以提高催化性能。在优化条件下，血红素结构域Vdh-K1实现了91.6%的转化效率，随后被选用于嵌合酶组装。通过采用局部能量受挫分析来评估蛋白质的灵活性和别构动力学，我们鉴定出具有高度受挫连接子的嵌合P450变体。最优变体VK1-CYP116B46-L21表现出改善的热稳定性、催化活性和偶联效率，在全细胞催化中实现了4.89 mM（1.96 g/L）25(OH)VD的产量——这是迄今为止报道的最高产量。这项工作强调了计算受挫分析在优化多结构域酶的连接子动力学方面的实用性，并建立了一个可扩展、具有成本效益的框架，以推进用于高价值化合物工业生物合成的P450s系统。
