Cloning And Indentification Of Penicillium Raistrickii Steroid 15α-hydrolyase Gene

Steroid hormones are an important class of drugs prescribed for treating a variety of diseases including rheumatoid arthritis,asthma,antitoxin,cancers as well as for contraception.C15 α,13-methy-estr-4-ene-3,17-dione is a key intermediate for synthesis of gestodene.Currently on an industrial scale,filamentous fungus Penicillium raistrickii is the favored microorganism for specific introduction of a hydroxyl group at the C15 position,but its low patching of materials limit the efficiency of industrial production.Previous studies show that P.raistrickii C15 α steroid hydroxylase belongs to the P450 superfamily,but C15 α-hydroxylase encoding gene remains unknown,and therefore genetic engineering approach cannot be applied to improve the biotransformation efficiency of currently used industrial strains.To clone and characterize the C15 α-hydroxylase in P.raistrickii,a series of experiments were conducted as follows:(1)Investigation of the ability of steroid substrates to induce the expression of C15α-hydroxylase activity in P.raistrickii and the results showed that C15 α-hydroxylase gene was induced at the transcription level;(2)based on the expression profiles of the P450 genes revealed by transcriptome sequencing,15 candidate C15α-hydroxylase genes from the 61 P450 genes predicted was amplified using qRT-PCR to examine expression differences under the substrate-induced and non-induced conditions.Two candidates genes 12820 and 61832 exhibiting high induction by the steroid substrate were identified;(3)Taking advantage of the established yeast platform for functional screening of CYP gene,expression of 6183 and 12820 genes in Saccharomyces cerevisiae,and 12820 in Pichia expression vectors were constructed and the corresponding recombinant strains were obtained;(4)steroid transformation experiments showed that recombinant yeast of 6183 was incapable of transforming 13-methy-estr-4-ene-3,17-dione,while the S.cerevisiae and Pichia pastoris recombinant strains of the 12820 gene were able to convert 13-methy-estr-4-ene-3,17-dione to C15 α,13-methy-estr-4-ene-3,17-dione,suggesting that 12820 is the 15α-hydroxylase gene;PRH is 1865bp long,contains 5 introns and 6 exons,the ORF is 1563bp which encodes 520 amino acids;(5)to determine whether there is additional C15α-hydroxylase gene in P.raistrickii,a targeted deletion construct of the 12820 gene was prepared and the protoplast transformation method established.Cloning and identification of the15α-hydroxylase gene in P.raistrickii will not only provide a solid foundation for elucidation of the detailed mechanism of steroid C15 αhydroxylation but also provide the valuable gene resource essential for construction of the next generation of industrial strains for efficient of steroid C15α-hydroxylation.