The Develpoment Of A Rapid Screening Method For Strains Of Efficient Steroid Hydroxylation And The Construction Of A Selection Maker Of Absidia Coerulea

Steroid C11?-hydroxylation catalyzed by cells of certain filamentous fungi is essential for industrial production of some steroid drugs and key intermediates.Commonly used industrial strains for steroid C11?-hydroxylation is Absidia coerulea.However,in hydrocortisone biotechnological production,the side products always appear in the complete transformation products.In this thesis,we attempt to develop a rapid and simple method for determination of hydrocortisone biotransformation rates in order to breeding Absidia coerulea of high specific biotransformation ability via mutation breeding technology.In addition,a uracil auxotrophs of Absidia coerulea was constructed as a selection marker,it will also facilitate the elucidation of the function of steroid hydrolases,thus paving the way for rational design of next generation highly efficient industrial strains.In this study,concentrated sulphuric acid was employed for determination of hydrocortisone biotransformation rate,which can be monitored via dual wavelength analysis.A rapid and simple method for determination of hydrocortisone biotransformation rates was developed by combining the well-known Lambert Beer law and the 3D data modeling method.And the validation results,which were consistent with HPLC method,showed that the method could be used for high-throughput system.This study has a good application prospect in breeding Absidia coerulea of high specific biotransformation ability.Then the optimal conditions for micro-cells cultivation was determined by orthogonal test.Combined with the fast method for determination of HC biotransformation rate,we have successfully established a platform for high-throughput screening of strains.Thirdly,the mutants library based on atmospheric and room temperature plasma mutation was conducted.The high HC-yield strain was finally obtained through the first screening with high-throughput screening platform and the second screening with shake flask fermentation.The HC biotransformation rate of which increased by 8%,respectively,compared to the original strain on the same culture conditions.Based on the expression profiles of the pyrG gene encoding Orotidine-5'-monphosphate Decarboxylase revealed by transcriptome sequencing,one candidates genes pyrG-1 exhibiting high identity to Absidia coerulea from the 3 genes predicted was identified.Taking advantage of the established yeast platform for functional screening of pyrG gene,pyrG-1 in Cryptococcus neoformans expression vectors were constructed and the corresponding recombinant strains were obtained,suggesting that pyrG-1 is the target pyrG gene;To establish the protoplast transformation method,the conditions for the spore protoplasts preparation and regeneration in Absidia coerulea were studied.After the targeted deletion construct of the pyrG gene was prepared,a uracil auxotrophs of Absidia coerulea wasn' t finally obtained uesing the gene knockout technology.The high-throughput screening platform provide a solid foundation for a rapid screening system of industrial strains for efficient of steroid hydroxylation.In addition,the selection marker of pyrG gene,provide the valuable resource essential for the elucidation of the function of steroid hydrolases and the construction of the next generation of industrial strains for efficient of steroid hydroxylation.