| 3?,7?,15?-Trihydroxy-5-androsten-17-one(7?,15?-diOH-DHEA)is a key drug intermediate in the synthesis of drospirenone which is the major composition of the novel oral contraceptive Yasmin.Because Yasmin is the first global sales volume,the commercial production of 7?,15?-diOH-DHEA has recently become a research focus.7?,15?-diOH-DHEA can be synthesized from dehydroepiandrosterone(DHEA)through microbial dihydroxylation at the position of C7 and C15 by Colletotrichum lini ST which was screened and preserved in our laboratory.However,the low substrate concentration,poor catalytic activity and conversion efficiency greatly limited the industrial scale production.Therefore,significant works were focused on breeding new microbial strains with high conversion efficiency and 7?,15?-diOH-DHEA yield by genome shuffling.And the mechanism for the enhancement of hydroxylation ability was investigated.Subsequently,a cytochrome P450 catalyzing the dihydroxylation of DHEA was obtained and successfully expressed for the first time.The main results are as follows:(1)The recombinants with high 7?,15?-diOH-DHEA yield were obtained.A high-yield strain C.lini ST-y9 with ethanol tolarence was screened by ARTP.Another high-yield strain C.lini ST-0317 with DHEA tolarence was preserved in our laboratory.C.lini ST-y9 and C.lini ST-0317 were subjected to recursive protoplast fusion.After three rounds of genome shuffling,three recombinants with high 7?,15?-diOH-DHEA yield were obtained.(2)The properties of the recombinants were evaluated and the mechanism for the enhancement of hydroxylation ability was preliminary explored.Among the three recombinants,C.lini ST-F307 had high genetic stability and the 7?,15?-di OH-DHEA molar yield reached 54.8% from 10 g·L-1DHEA,which was 26.7% higher than that of the initial C.lini ST strain(28.1%).Through conditions optimization,the 7?,15?-diOH-DHEA molar yield was raised to 69.9% from 12 g/L DHEA with 1.5% ethanol as co-solvent.Because the hydroxylation of steroid is mainly catalyzed by cytochrome P450 system,the P450 enzyme activity of different C.lini strains(the initial strain C.lini ST,the parental strains C.lini ST-0317,C.lini ST-y9 and the recombinant C.lini ST-F307)were studied.C.lini ST-F307 showed highest P450 enzyme activity(45.2 U·mg-1).Furthermore,the gene transcript levels of partial cytochrome P450 s were also analyzed and that of C.lini ST-F307 were significantly improved compared with other strains.It was found that the improvement of P450 gene transcript levels and enzyme activity led to the production enhancement of C.lini ST-F307.(3)A cytochrome P450 from C.lini ST-F307 was obtained and its hydroxylation function was verified.According to the above results,the gene transcript level of CYP3 located in the microsomes in C.lini ST-F307 exhibited the highest.Therefore,it was presumed that CYP3 might be a key enzyme for DHEA dihydroxylation.The complete geneof CYP3 was then amplified.After further analysis,this CYP3 protein belonged to the CYP68 J subfamily and was named CYP68 JX.In addition,CYP68 JX was successfully expressed in Pichia pastoris GS115 and the whole cell transformation was carried out.The7?,15?-diOH-DHEA molar yield of GS115-pPIC3.5K-cyp68 jx was 31.8% from 2 g·L-1DHEA,which was 2.8-fold that of GS115-pPIC3.5K(11.3%).The results showed that CYP68 JX was a target enzyme catalyzing the dihydroxylation of DHEA. |
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