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- Title
Production of 14α-Hydroxy Progesterone Using a Steroidal Hydroxylase from Cochliobolus lunatus Expressed in Escherichia coli.
- Authors
Chang, Yaowen; Liu, Han; Tian, Wei; Chang, Zunxue
- Abstract
Steroids with hydroxylation at C14 are drawing increased attention because of their diverse biological activities and applications. P-450lun from Cochliobolus lunatus is the first fungal cytochrome P450 reported to have 14α-hydroxylase activity. Studies have shown that P-450lun catalyzes the hydroxylation of progesterone (PROG) at C14α with low regiospecificity and activity. To improve its regiospecificity and activity for PROG, truncated forms of P-450lun and its cognate redox partner CPRlun were functionally co-expressed in Escherichia coli. Then, a semi-rational protein engineering approach was applied to P-450lun, resulting in a double-site mutant E109A/F297W with enhanced 14α-position selectivity for PROG compared with the wild-type P-450lun (97% vs. 28%). Protein structure analysis revealed that the F297W substitution can hinder the binding pose for 11β-hydroxylation product formation. Finally, whole-cell catalysis was optimized, and the final titer of 14α-OH-PROG reached 16.0 mg/L. This is the first report where a fungal 14α-hydroxylase was functionally expressed in Escherichia coli. The steroid hydroxylation system obtained in this study can serve as a basis for the synthesis of 14α-hydroxylated PROG and the rapid evolution of eukaryotic cytochrome P-450lun.
- Subjects
ESCHERICHIA coli; PROGESTERONE; CYTOCHROME P-450; PROTEIN engineering; PROTEIN structure
- Publication
Catalysts (2073-4344), 2024, Vol 14, Issue 4, p247
- ISSN
2073-4344
- Publication type
Article
- DOI
10.3390/catal14040247