Construction Of Steroid Hydroxylase Gene Expression Platform Of Neurospora Crassa

Microbial transformation of steroids is a critical process for the steroid industry,especially the employment of filamentous fungi for regio-and stereospecific hydroxylation of steroids to produce most of the key corticosteroid intermediates.However,the current industrial strains suffer from low conversion rate and high levels of by products.Recently our research group has cloned and identified several steroid hydroxylase genes from various filamentous fungal production strains.Given that tools for genetic manipulation in the production strains are not developed,it is difficult to use these hydroxylation genes for strain engineering in the short term.To take full advantage of the identified valuable hydroxylase genes,it is necessary to develop a highly effective heterologous expression system for fungal steroid hydroxylase gene.The fact that Neurospora crassa grows fast on simple nutrient medium and has well-established genetic background,availability of a variety of genetic tools and easy genetic transformation makes it an ideal host for expression of fungal steroid hydroxylase genes derived from other filamentous fungi.Thus the aim of this thesis research is to explore the feasibility of modifying N.crassa for hydroxylase gene expression.N.crassa has cytochrome P450 enzyme system capable of hydroxylating 4-AD,16a,17a-Epoxyprogesterone and D-Ethylgonendione at undesirable positions,thus it is necessary to identify and delete the target gene/genes responsible for the steroid hydroxylating activities to make it suitable for expressing heterologous fungal steroid hydroxylase.To identify the target cytochrome P450 gene,its steroid hydroxylating activity was shown to be highly induced by steroid substrates.The expression profile of the 42 cytochrome P450 genes under steroid induction conditions was determined by RT-PCR and the expression of 4 cytochrome P450 genes was detected only in induction conditions.The functional role of these four genes were investigated by construction of respective expression vectors and introduced into Pichia pastoris.Sterod transformation experiments showed that recombinant Pichia cells expressing gene NCU05001 exhibited strong steroid transformation activities.Further analysis byHPLC showed that the products from both recombinant Pichia cells and N.crassa are basically identical,indicating that NCU05001 is the major gene involved in steroid transformation activity in N.crassa.The cloning and identification of NCU05001 will facilitate the construction of N.crassa as a host for high levels of steroid hydroxylase gene expression.