Construction Of Steroid Hydroxylase Expression Platferm Based On Aspergillus Niger

Steroid hormone drugs are widely used for anti-inflammatory, anti-tumor and cardiovascular disease treatment and reproductive control. The structure of steroid compounds is very complex and theirs physiological and pharmacological activities depend on the substituents at specific sites on the steroid nucleus. Hydroxylation of steroid compounds is one of the key technologies for the preparation of steroid hormones and key intermediates. Because it is difficult to introduce hydroxyl group at specific sites of steroid nuclei by chemical synthesis, the steroid industry commonly uses microbial transformation,especially transformation by filamentous fungi such as Aspergillus ochraceus, Rhizopus nigricans and Absidia coerulea. etc to achieve regio- and stereoselective steroid hydroxylation. However, microbial steroid hydroxylation reaction often suffers from low concentrations of substrate feeding, low conversion rates and insufficient reaction specificity. Due to the poor understanding of the genetic background and lack of genetic tools for most of the filamentous fungal strains used in the steroid industry, it is formidable to rationally engineer efficient and robust production strains for large-scale steroid hydroxylation.In order to make full use of the industrially relevant steroid hydroxylase genes identified from the various production strains, this thesis mainly explored the construction of a heterologous expression host for steroid hydroxylase genes, with the aim to lay the foundation for engineering thr next generation of steroid conversion strains. Aspergillus niger, with a clear genetic background, availability of multiple genetic tools for targeted gene manipulation and an GRAS status, is an ideal host for expressing steroid hydroxylase genes. In this study, A. niger TCCC41650 was selected as the starting strain for host strain construction. To make it suitable for heterologous expression of fungal steroid hydroxylase genes, the endogenous CYP65 gene encoding a 16?-hydroxylase which is highly induced by the androst-4-ene-3,17-dione (4-AD),was knocked out,resulting in the creation of a fungal steroid hydroxylase gene expression platform. In addition, to assess the utility of the constructed steroid hydroxylase expression platform, experiments were conducted to investigate the scope of substrates that can be used for hydroxyl functionaliztion. The results showed that the engineered host strain exhibited no biotransformation activity on the commonly used steroid substrates such as 4-AD, D-ethylgonendione and 16,17a-epoxyprogesterone, indicating that the engineered platform can be applied to the heterologous expression of fungal hydroxylase genes to construct highly efficient production strains. Finally, to verify the performance of the engineered expression platform,The establishment of the steroid hydroxylase expression platform will facilitate the design and construction of highly efficient, and robust production strains for steroid biotransformation.