Clonging And Identification Of Steroid C₁₁?-hyroxylase Gene From Aspergillus Niger

Steroid drugs are important class of drugs commonly used in the clinic.It is mainly prescribed for the treatment of rheumatoid arthritis,asthma,antitoxin,tumor and other diseases as well as for birth control.Structures of Steroid compounds are very complex and their activities depend on the functionalization at various positions of the core skeleton.The major hurdle of Chemical synthesis route to the synthesis of various steroid derivatives is its poor selectivity,especially it is extremely difficult to incorpate an oxygen atom at C₁₁? of the steroid nucleus.However,microbial transformation for steroid C₁₁? hydroxylation displays strong regio-and stereospecificity and produces less unwanted side products compared with chemical synthesis routes.Aspergillus niger can transform steroids,but its products has not yet been identified,hydroxylation activity and clear genetic basis are not clear,which greatly limiting the orientation transformation Aspergillus niger species.The major goal of this research is to clone and characterize the steroid hydroxylation gene in Aspergillus niger The transformation product was determined as 11?-hydroxy-16a,17a-epoxy progesterone based on analyses of TLC,HPLC,¹H and ¹³C spectroscopy.In addition,the 11?-hydroxylation activities of A.niger ATCC1015 on 16a,17a-epoxy progesterone were shown to be induced by substrate 16a,17a-epoxy progesterone.Given that fungal steroid hydroxylases are members of the cytochrome P450 family,we selected 57 CYP genes as potential candidates of the target hydroxylase gene?s?.Quantitative RT-PCR showed that expression of CYP5061 of 57 CYP genes was highly induced by 16?,17?-epoxy progesterone treatment,suggesting that CYP5061is a candidate steroid C₁₁?-hydroxy lation gene.Construction of CYP5061 gene expression vector and transformation into INVScI cells showed that Z-CYP5061 recombinant INVScI cells are capable of transforming 16a,17-a-Epoxyprogesterone.The transformation product was determined as 11?-hydroxy-16?,17?-epoxy progesterone based on analyses of TLC and HPLC,indicating that the gene CYP5061 is the steroid Cua hydroxylase gene in Aspergillus niger.We used the method of Agrobacterium to knockout the CYP5061 gene with the vector pPZP-CYP5061.The cloning and identification of CYP5061 gene in Aspergillus niger should facilitate the genetic engineering of next generation of industrial strains for steroid C₁₁? hydroxylation.