The 14α-hydroxysteroids have specific anti-gonadotropic and carcinolytic biological activities and can be produced by microbial biotransformation. The steroid 11β-/14α-hydroxylase P-450 from Cochliobolus lunatus is the only fungal cytochrome P450 enzyme identified to date with steroid C14 hydroxylation ability. Previous work has mainly revealed the 11β-hydroxylation activity of the P-450 towards cortexolone (RSS) substrate; however， the potential steroid 14α-hydroxylation activity of this enzyme， especially for androstenedione (AD) substrate， has not yet conducted in-depth testing. In this work， we further tested the steroid 14α-hydroxylation activity of the P-450 towards RSS and AD in the Sacchar... More
The 14α-hydroxysteroids have specific anti-gonadotropic and carcinolytic biological activities and can be produced by microbial biotransformation. The steroid 11β-/14α-hydroxylase P-450 from Cochliobolus lunatus is the only fungal cytochrome P450 enzyme identified to date with steroid C14 hydroxylation ability. Previous work has mainly revealed the 11β-hydroxylation activity of the P-450 towards cortexolone (RSS) substrate; however， the potential steroid 14α-hydroxylation activity of this enzyme， especially for androstenedione (AD) substrate， has not yet conducted in-depth testing. In this work， we further tested the steroid 14α-hydroxylation activity of the P-450 towards RSS and AD in the Saccharomyces cerevisiae system. We demonstrated that P-450 functions as the specific 14α-hydroxylase towards the AD substrate (regiospecificity > 99%); however， it showed a poor C14-hydroxylation regiospecificity (around 40%) for the RSS substrate. In addition， through transcriptome analysis combined with gene functional characterizations， we also identified and cloned the gene for the P-450-associated redox partner CPR. Finally， through codon optimization， knockout of genes for the side reactions related enzymes GCY1 and YPR1， and increasing copies of the P-450 and CPR， we developed a recombinant S. cerevisiae biocatalyst based on the C. lunatus steroid 14α-hydroxylation system to produce 14α-hydroxysteroids. Initial production of 14α-OH-AD (150 mg/L day productivity， 99% regioisomeric purity， and 60% w/w yield) and 14α-OH-RSS (64 mg/L day productivity， 40% regioisomeric purity， and 26% w/w yield) were separately achieved in shake flasks; these results represent the highest level of 14α-hydroxysteroid production in the current yeast system.
