Fangzhi Deng,Zhenru Zhou,Zhen Du,Mohamed Mohany,Qunyue Wu,Weiyang Liang,Lei Zhang,Shan Li
Murideoxycholic acid (MDCA), as a significant secondary bile acid derived from the metabolism of α/β-muricholic acid in rodents, is an important component in maintaining the bile acid homeostasis. However, the biosynthesis of MDCA remains a challenging task. Here, we present the development of cytochrome P450 monooxygenase CYP102A1 (P450 BM3) from Bacillus megaterium , employing semi-rational protein engineering technique. Following three rounds of mutagenesis, a triple variant (T260G/G328A/L82V) has been discovered that proficiently catalyzes the 6β-hydroxylation of lithocholic acid (LCA), thereby generating MDCA with an impressive 8.5-fold increase in yield compared to the template P450 BM3 mutant. The MDCA selectivity has been also promoted from 62.0% to 96.3%. This biocatalyst introduces a novel approach for the biosynthesis of MDCA from LCA. Furthermore, molecular docking and dynamics simulations have been employed to unravel the molecular mechanisms underlying the enhanced LCA conversion and MDCA selectivity.
Peng Shuaiying,Chu Zhongmei,Lu Jianfeng,Li Dongxiao,Wang Yonghong,Yang Shengli,Zhang Yi
The self-sufficient cytochrome P450 BM3 mutant (A74G/F87V/D168H/L188Q) can serve as a biocatalyst for whole-cell catalysis process of indigo. Nevertheless, the bioconversion yield of indigo is generally low under normal cultivation conditions (37 °C, 250 rpm). In this study, a recombinant E. coli BL21(DE3) strain was constructed to co-express the P450 BM3 mutant gene and GroEL/ES genes to investigate whether GroEL/ES can promote the indigo bioconversion yield in E. coli . The results revealed that the GroEL/ES system could significantly increase the indigo bioconversion yield, and the indigo bioconversion yield of the strain co-expressing P450 BM3 mutant and GroEL/ES was about 21-fold that of the strain only expressing the P450 BM3 mutant. In addition, the P450 BM3 enzyme content and in vitro indigo bioconversion yield were determined to explore the underlying mechanism for the improvement of indigo bioconversion yield. The results revealed that GroEL/ES did not increase indigo bioconversion yield by increasing the content of P450 BM3 enzyme and its enzymatic transformation efficiency. Moreover, GroEL/ES could improve the intracellular nicotinamide adenine dinucleotide phosphate (NADPH)/NADP + ratio. Given that NADPH is an important coenzyme in the catalytic process of indigo, the underlying mechanism for the improvement of indigo bioconversion yield is probably related to an increase in the intracellular NADPH/NADP + ratio.
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