Construction Of Recombinant Pichia Pastoris With High Efficiency Steroid Strain Of 15α-Hydroxylation And High Density Fermentation

15α-OH-D-ethylgonendione is a key intermediate in the synthesis of highly effective contraceptive pregnandienone.The production of 15α-OH-D-ethylgonendione using filamentous fungus Penicillium raistrickii has disadvantages in terms of substrate feeding（2‰）,conversion efficiency and wastewater treatment pressure.In order to improve the loading of substrate D-ethylgonendione and establish a more efficient and green transformation system,Pichia pichia（GS115）was used as the expression host to construct an efficient D-ethylgonendione 15α-hydroxylation reaction system.The research work is as follows:（1）15α-hydroxylase gene PRH of P.raistrickii was heterologously expressed in P.pastoris.In order to further improve substrate conversion and feeding concentrations,glucose 6-phosphate dehydrogenase gene ZWF1 from S.cerevisiae and gene PRH were co-expressed in P.pastoris.The transformation efficienciens of recombinant P.pastoris Z15134 and 15134 were compared by shaking flasks and 5 L fermentation tank transformation experiments.（2）The high-efficient recombinant strain was selected for high-density fermentation and growth conditions and substrate feeding of the strain were monitored using a 5 L fermentation tank.（3）The transformation products from 5 L fermentation tank were analyzed by HPLC.Transcriptome sequencing analysis was performed for the strain between transformation conditions of high density fermentation and shaking flasks.The results showed that:（1）the D-ethylgonendione transformation experiment of Z15134 and 15134 in shaking flasks showed that the substrate transformation time of Z15134 was shortened by 12 h at the substrate feeding of 1‰.（2）The substrate transformation efficiency of the recombinant P.pastoris Z15134was significantly higher than the control of 15134 at substrate feeding of 1‰in a 5 L fermentation tank.（3）The more efficient recombinant strain Z15134 further improved substrate D-ethylgonendione transformation efficiency using a 5 L fermentation tank with fermentation time of 196 h,substrate feeding of 10‰,product concentration of 5.79±0.18g/L and OD₆₀₀ of 276.8±1.39.（4）Transcriptomic sequencing was performed for Z15134 between transformation conditions of high density fermentation and shaking flasks.GO and KEGG pathway enrichment analysis of differentially expressed genes were performed,and the results of transcriptome sequencing were verified by q RT-PCR.In this thesis,a very efficient recombinant P.pastoris strain Z15134 was obtained by co-overexpressing P.raistrickii 15α-hydroxylase and S.cerevisiae glucose-6-phosphate dehydrogenase in P.pastoris,and an efficient production process of15α-OH-D-ethylgonendione was successfully established by using high density fermentation,which increased the substrate feeding by 400%.