Identification of a fungal cytochrome P450 with steroid two-step ordered selective hydroxylation characteristics in Colletotrichum lini

Microbial hydroxylation reaction has greatly enriched the number of steroids and created many meaningful new compounds. The dihydroxylation of dehydroepiandrosterone (DHEA) by filamentous fungi produces an important product 3β,7α,15α-trihydroxy-5-androstene-17-one (7α,15α-diOH-DHEA), which can be used as a key intermediate for the synthesis of contraceptive drospirenone. The introduction of microbial hydroxylation reaction reduces the traditional chemical synthesis process by 4 steps and greatly improves the productivity and economic efficiency. Colletotrichum lini is an industrial strain producing 7α,15α-diOH-DHEA, but the related cytochrome P450 that plays hydroxylation effect has not yet been discovered. In this work, a combination of quantitative proteomics, qRT-PCR, and functional expression in Pichia pastoris was used to identify highly induced steroid hydroxylase from Colletotrichum lini ST-1. A novel fungal cytochrome P450 monooxygenase CYP68JX was identified. The biotransformation in recombinant yeast confirmed that the cytochrome P450 has steroid C7α and C15α hydroxylase activities. The hydroxylation of DHEA by CYP68JX is an ordered reaction, proceeding from the C7 to the C15 site of the steroidal nucleus. The cloning and identification of the CYP68JX gene provide useful information for deepening the understanding regarding the structural basis of its regional and stereoselectivity.