Engineering a 7β-hydroxylase from fusarium longipes for enhancement of ursodeoxycholic acid production

Ursodeoxycholic acid (UDCA), a steroidal medication, is extensively used in the treatment of liver and gallbladder diseases. The cytochrome P450 enzyme (P450) with 7β-hydroxylation capability has attracted widespread attention owing to its essential role in biotransformation of biologically toxic lithocholic acid (LCA) into UDCA. However, the 7β-hydroxylases reported previously are confronted with severe challenges, including low catalytic efficiency and poor stereoselectivity, which limit the efficient biotransformation of LCA into UDCA. In this study, we discovered a novel 7β-hydroxylase (P450_FL) from Fusarium longipes through phylogenetic analysis with the capacity to catalytically hydroxylate LCA generating UDCA. A customized strategy based on active-site saturation mutagenesis/iterative combination mutagenesis and computer-aided design were employed to engineer P450_FL. The superior mutant M4 (T116N/V364I/L488I/E268K) was obtained, which exhibited a UDCA yield of 94.2 % with 97.1 % 7β-selectivity, an 8.2-fold increase in catalytic activity compared to wild-type P450_FL. The high titer of UDCA (3.9 g/L) was achieved through fed-batch cascade reactions, which was the highest UDCA yield catalyzed by engineered P450 enzyme. Molecular dynamics simulations revealed that the heightened catalytic activity of mutant M4 could be attributed to strengthened enzyme-substrate hydrogen bonding and hydrophobic interactions, as well as the optimization of electrostatic regulation. Overall, this work combining gene mining with further mutagenesis design could provide valuable insights for the engineering design of P450 and the green biosynthesis of UDCA in pharmaceutical industry.