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3β,7α,15α-Trihydroxy-5-androsten-17-one (7α,15α-diOH-DHEA) is a key intermediate of the novel oral contraceptive Yasmin. It can be catalyzed from dehydroepiandrosterone (DHEA) through Colletotrichum lini. Improvement of 7α,15α-diOH-DHEA production was performed through recursive protoplast fusion of C. lini ST in a genome shuffling format. 7α,15α-diOH-DHEA yield of the best performing recombinant C. lini ST-F307 reached 6.08 g/L from 10 g/L DHEA, and this was 94.9% higher than that of the initial C. lini ST strain. Through optimized conditions, the 7α,15α-diOH-DHEA yield was increased to 9.32 g/L from 12 g/L DHEA, with 1.5% ethanol as cosolvent. This is the highest reported substrate concentration and 7α,15α-diOH-DHEA production with one-step substrate addition. Moreover, C. lini ST-F307 showed high P450 enzyme activity and gene transcript levels of several cytochrome P450s, and this might contribute to the enhancement of 7α,15α-diOH-DHEA production. Genome shuffling was an efficient approach to breed high-yield strains. [ABSTRACT FROM AUTHOR]

Dihydroxylation of dehydroepiandrosterone (DHEA) is an essential step in the synthesis of many important pharmaceutical intermediates. However, the solution to the problem of low biohydroxylation conversion in the biotransformation of DHEA has yet to be found. The effects of natural oils on the course of dihydroxylation of DHEA to 3β,7α,15α-trihydroxy-5-androsten-17-one (7α,15α-diOH-DHEA) were studied. With rapeseed oil (2 %, v/v) addition, the bioconversion efficiency was improved, and the 7α,15α-diOH-DHEA yield was increased by 40.8 % compared with that of the control at DHEA concentration of 8.0 g/L. Meantime, the ratio of 7α,15α-diOH-DHEA to 7α-OH-DHEA was also increased by 4.5 times in the rapeseed oil-containing system. To explain the mechanism underlying the increase of 7α,15α-diOH-DHEA yield, the effects of rapeseed oil on the pH of the bioconversion system, the cell growth and integrity of Gibberella intermedia CA3-1, as well as the membrane composition were systematically studied. The addition of rapeseed oil enhanced the substrate dispersion and maintained the pH of the system during bioconversion. Cells grew better with favorable integrity. The fatty acid profile of G. intermedia cells revealed that rapeseed oil changed the cell membrane composition and improved cell membrane permeability for lipophilic substrates. [ABSTRACT FROM AUTHOR]