Feng Xisong, Gao Mengjian, Wei Wanqing, Song Wei, Wen Jian, Wang Hongyu, Hu Guipeng, Li Xiaomin, Gao Cong, Wu Jing
School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, China.
School of Biotechnology and Key Laboratory of Industrial Biotechnology of Ministry of Education, Jiangnan University, Wuxi 214122, China.
J Agric Food Chem. 2025 Apr 30;73(17):10439-10448. doi: 10.1021/acs.jafc.5c01010. Epub 2025 Apr 16.
25-hydroxyvitamin D (25(OH)VD) is an important daily nutritional supplement, and direct enzymatic C25 hydroxylation of vitamin D (VD) to 25(OH)VD is a green and sustainable method. However, the low catalytic activity and electron transfer efficiency of redox partners of cytochrome P450 limited its production. Here, structure-guided semirational design of CYP109E1 led to the mutant CYP109E1, which showed a 2-fold increase in 25(OH)VD production compared to the wild-type. Furthermore, the novel reductase domain BmRD was first employed to construct a fusion protein with CYP109E1. Notably, truncating only two amino acids at the N-terminus of BmRD generated a fusion protein Chimera-2, resulting in a 38.5% increase in 25(OH)VD production. Under optimized conditions, the engineered strain 03-2 produced 491.3 mg/L 25(OH)VD with a conversion of 49.0% and a space-time yield of 61.4 mg/(L·h). This work demonstrates the modification and optimization potential of P450 and lays a theoretical foundation for the industrialization of the 25(OH)VD biosynthesis.
25-羟基维生素D（25(OH)VD）是一种重要的日常营养补充剂，维生素D（VD）直接酶促C25羟基化生成25(OH)VD是一种绿色可持续的方法。然而，细胞色素P450氧化还原伙伴的低催化活性和电子转移效率限制了其产量。在此，通过对CYP109E1进行结构导向的半理性设计得到突变体CYP109E1，与野生型相比，其25(OH)VD产量提高了2倍。此外，首次采用新型还原酶结构域BmRD与CYP109E1构建融合蛋白。值得注意的是，仅在BmRD的N端截短两个氨基酸就产生了融合蛋白Chimera-2，使25(OH)VD产量提高了38.5%。在优化条件下，工程菌株03-2产生了491.3 mg/L的25(OH)VD，转化率为49.0%，时空产率为61.4 mg/(L·h)。这项工作展示了P450的修饰和优化潜力，为25(OH)VD生物合成的工业化奠定了理论基础。
