| 3β,7α,15α-trihydroxy-5-androsten-17-one(7α,15α-diOH-DHEA) is an important drug intermediate in the synthesis of drospirenone, which can be produced by introducing two hydroxyls in C7 and C15 of DHEA through bioconversion. Drospirenone is the major composition of Yasmin, the new generation of female oral contraceptives. However, the product yield of 7α,15α-diOH-DHEA is low in manufacturing production because of the low substrate concentration and poor conversion efficiency in the bioconversion of DHEA. Previous studies showed that adding proper organic solvent into the steroid bioconversion system could improve the conversion rate. The research was focused on the bioconversion of DHEA into 7α,15α-diOH-DHEA by Colletotrichum lini ST-1. To enhance the production of 7α,15α-diOH-DHEA, the effects of common organic solvents on dihydroxylation of DHEA was studied. And the mechanism of the optimum organic solvent-ethylene glycol was investigated through comparative proteomic technology. The main results are as follows:(1) The effects of sixteen common organic solvents on dihydroxylation of DHEA by C.lini ST-1 were investigated. The results showed the alcohols with more than three carbon atom had asverse effect on the bioconvension. Moreover, the production of 7α,15α-diOH-DHEA could be significantly enhanced by addition of ethylene glycol. With 0.5%(v/v) ethylene glycol, the yield of 7α,15α-diOH-DHEA was increased to 80.1%, at the substrate DHEA concentration of 10 g·L-1, which was 1.5-fold higher than that of control.(2) The bioconversion conditions with ethylene glycol were optimized. With the addition of 0.5% ethylene glycol into the transformation medium, the conversion efficiency in the bioconversion of DHEA was dramatically improved. Under the optimal condtion, the concentration of DHEA was successfully increased to 12 g·L-1, and the conversion rate of DHEA was reached to 90.7%. Meanwhile, the yield of 7α,15α-diOH-DHEA was maintained at 79.7%, which was 2-fold higher than that of control. The production of by-product was obviously decreased.(3) The mechanism of ethylene glycol on the bioconversion of DHEA by C.lini ST-1 was preliminary studied. The concentrations of ethylene glycol and its metabolic products were tested. The results showed during the metabolism of ethylene glycol by C.lini ST-1, the pH of the transformation system was stabilizesd and coenzyme NADPH was generated, which could provide adequate reducing power for the bioconversion. Based on the results of 2-DE analysis, the expressions of cytochrome P450 and 6-phosphaogluconate dehydrogenase were up-regulated, which suggested that those two enzymes played critical roles in the dihydroxylation of DHEA. Moreover, qPCR was used to evaluate the transcriptional level of the key enzymes. The results of qPCR were consistent with the analysis of 2-DE. Those above results demonstrated that the addition of ethylene glycol could not only stabilize the pH, but also increase the content of coenzyme NADPH, 6-phosphaogluconate dehydrogenase and cytochrome P450, which were helpful of the dihydroxylation of DHEA in C. lini ST-1. |
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