| Steroids are an important class of drugs prescribed for treating a variety of diseases including rheumatoid arthritis,asthma,antitoxin,cancers as well as for contraception.Hydrocortisone(HC)is mainly be used to cure collagenous diseases,such as rheumatic fever,rheumatic arthritis,lupus erythematosus,anaphylactic disease and addison's disease.Meanwhile,HC is also used in emergency treatment of coma,shock and serious infection.Currently,filamentous fungus Absidia coerulea is applied to produce HC using cortexolone-21 acetate(RSA)as substrate on an industrial scale in China.However,low inventory of substrate and weak specificity of hydroxylation limit the efficiency of industrial production.Steroid 11?-hydroxylase of Absidia coerulea belongs to the cytochrome P450(CYP)family,and it's coding gene still remains unknown until now.Therefore,genetic engineering approach cannot be used to improve the biotransformation efficiency of current industrial strainsTo clone and characterize the 11?-hydroxylase gene of Absidiacoerulea,a series of experiments were conducted as follows:(1)An induction assay was carried out to prove that the steroid hydroxylation activity of Absidia coerulea is induced by substrate RSA.(2)Total RNA of Absidia coerulea with RSA induction was extracted and sequenced,15 CYP genes were found out from the data of transcriptome sequencing.(3)qRT-PCR was applied to identify the expression of CYP genes of Absidia coerulea in transcriptional level.The expression of CYP-7?CYP-8 and CYP-15 is obviously up-regulate with RSA induction compare to non-induction.(4)The recombinant saccharymyces cerevisiae of CYP-7 and CYP-8 were constructed.The recombinant yeast of CYP-7 is able to transform RSA to HC.(5)CYP-7 was named as CYP5311B2 according to nelson's nomenclature.CYP5311B2 contains 7 introns and 8 exons and encodes 527 amino acids. |
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