A fungal P450 enzyme from Fusarium equiseti HG18 with 7β-hydroxylase activity in biosynthesis of ursodeoxycholic acid

• Published in: The Journal of steroid biochemistry and molecular biology 2024-06, Vol.240, p.106507, Article 106507
• Main Authors: Zhou, Zhen-Ru, Liu, Fen, Li, Shan, Dong, Chang-Zhi, Zhang, Lei
• Format: Article
• Language: English
• Subjects: 7β-Hydroxylase; Cytochrome P-450 Enzyme System - chemistry; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; Fungal Proteins - chemistry; Fungal Proteins - genetics; Fungal Proteins - metabolism; Fusarium - enzymology; Fusarium - genetics; Fusarium - metabolism; Fusarium equiseti HG18; Hydroxylation; Life Sciences; Lithocholic acid; Lithocholic Acid - chemistry; Lithocholic Acid - metabolism; Molecular Docking Simulation; Mutagenesis, Site-Directed; Saccharomycetales; Substrate Specificity; Ursodeoxycholic acid; Ursodeoxycholic Acid - chemistry; Ursodeoxycholic Acid - metabolism
• ISSN: 0960-0760; 1879-1220; 1879-1220
• DOI: 10.1016/j.jsbmb.2024.106507

Cytochrome P450 enzyme with 7β-hydroxylation capacity has attracted widespread attentions due to the vital roles in the biosynthesis of ursodeoxycholic acid (UDCA), a naturally active molecule for the treatment of liver and gallbladder diseases. In this study, a novel P450 hydroxylase (P450FE) was screen out from Fusarium equiseti HG18 and identified by a combination of genome and transcriptome sequencing, as well as heterologous expression in Pichia pastoris. The biotransformation of lithocholic acid (LCA) by whole cells of recombinant Pichia pastoris further confirmed the C7β-hydroxylation with 5.2% UDCA yield. It was firstly identified a fungal P450 enzyme from Fusarium equiseti HG18 with the capacity to catalyze the LCA oxidation producing UDCA. The integration of homology modeling and molecular docking discovered the substrate binding to active pockets, and the key amino acids in active center were validated by site-directed mutagenesis, and revealed that Q112, V362 and L363 were the pivotal residues of P450FE in regulating the activity and selectivity of 7β-hydroxylation. Specifically, V362I mutation exhibited 2.6-fold higher levels of UDCA and higher stereospecificity than wild-type P450FE. This advance provided guidance for improving the catalytic efficiency and selectivity of P450FE in LCA hydroxylation, indicative of the great potential in green synthesis of UDCA from biologically toxic LCA. [Display omitted] •The strategy of combining genome and transcriptome sequencing effectively reduced the workload of traditional large-scale genome screening.•A novel cytochrome P450 enzyme (P450FE) with the 7β-hydroxylase capacity was discovered from Fusarium equiseti HG18.•Heterologous expression of P450FE was successfully achieved in Pichia pastoris.•Q112, V362 and L363 were the key amino acid residues regulated the 7β-hydroxylation activity and selectivity of P450FE.