Colletotrichum lini is used as an industrial stain for the dihydroxylation of steroid compound dehydroepiandrosterone (DHEA) to biosynthesize 3β,7α,15α-trihydroxy-5-androstene-17-one (7α,15α-diOH-DHEA), a key intermediate of the most popular oral contraceptive “Yasmin”. This work aimed to enhance 7α,15α-diOH-DHEA production in C. lini CGMCC 6051 through ethanol induction. With 0.6% (v/v) ethanol induction and 10 g/L DHEA concentration, the 7α,15α-diOH-DHEA molar yield reached 58.8%, which was increased by 67.5% than that of the control. iTRAQ-based quantitative proteomic analysis was applied to explore the probable molecular mechanism of C. lini response to ethanol induction. A total of 50 differential expressed proteins was affected by ethanol induction, and could be related to multiple metabolic pathways. Most of differently expressed proteins were functionally mapped into pathways of transport, steroids metabolism, or redox reaction. Other proteins for energy, transcription and translation, and carbohydrate metabolism might have important roles in the cellular response to ethanol induction. In addition, the levels of cytochrome P450 and NAD(P)H-cytochrome P450 reductase were remarkably higher under ethanol induction, and their functions on DHEA dihydroxylation were first proposed in C. lini. Our results provide critical clues in revealing the dihydroxylation mechanism and are important for efficient microbiological hydroxylation of steroidal compounds in the future.

Biological significance

iTRAQ strategy was first used to compare the proteomes of ethanol induction during the dihydroxylation reaction by Colletotrichum lini CGMCC 6051. The changes in protein provided a comprehensive overview of DHEA dihydroxylation in C. lini, including the proteins for steroids metabolism, redox reaction, transport, transcription and translation, energy and carbohydrate metabolism. Cytochrome P450, NADPH-cytochrome P450 reductase, and NADH-cytochrome b5 reductase were highlighted due to their outstanding contribution to DHEA dihydroxylation. The results help us understand the molecular mechanism underlying ethanol induction in C. lini and would guide strain engineering to further improve dihydroxylation efficiency.