Study On 1-Dehydrogenation Of 21-Acetoxy-4-Pregnene-11β,17α-Diol-3,20-Dione By Nocardia Simplex And The Identification Of Key Enzymes

Prednisolone is used in allergic and autoimmune inflammatory diseases and collagen diseases.It is widely used in clinic and is a key intermediate in the production of steroids such as budesonide and methylprednisolone.The market demand is huge.At present,the biological conversion of prednisolone has the problems of expensive substrate,long reaction period and low conversion efficiency.Therefore,in this paper,the low-cost hydrocortisone acetate(HCA)is used as a substrate,an efficient biotransformation process for the production of prednisolone based on HCA biotransformation based on cyclodextrin recycling technology was established through the technologies of optimal strains,cosolvent selection and resting cell transformation.Finally,through bioinformatics analysis,gene heterologous expression and other means,the key KsdD enzymes that play the function of 1-dehydrogenation in Nocardia simplex were preliminarily studied,and the research results provide basic data and theoretical basis for the construction of strains for the efficient production of prednisolone through strain modification in the later period.In this paper,the strains of 1-dehydrogenation stored in the laboratory were used as biocatalysts to screen the most suitable strains for HCA biotransformation.The results showed that the Arthrobacter simplex had no ability to transform HCA,and the Nocardia simplex showed better results.The product was confirmed to be prednisolone by thin layer chromatography(TLC)and nuclear magnetic resonance(NMR)analyses.A process for producing prednisolone was established through Nocardia simplex growth cell transformation.Under the conversion system of 4%ethanol as a co-solvent,the product generation rate reached 91.6%when 10 g/L HCA was converted for 96h.In the small-scale experiment of a 2L fermentor,with a conversion system of 0.75 filling factor,5%inoculation volume,and 4%ethanol as a co-solvent,the conversion rate of 10 g/L HCA for 96 h reached 95.8%.A process for producing prednisolone was established through Nocardia simplex resting cell transformation.Under the transformation system of 10 g/L hydroxypropyl-β-cyclodextrin(HP-β-CD)as a cosolvent,the product generation rate reached 86.3%when 10 g/L HCA was converted for 32h,which was 66.7%shorter than that when transformed with growing cells.The resting cell transformation process was further optimized,and the optimal transformation conditions for the conversion of HCA to prednisolone were determined:at 8 h of fermentation culture,0.25 g/L HC was added for induction,10 g/L HP-β-CD was used for solubilization,and the product generation rate reached 97.1%when 20 g/L HCA was converted for 32h.At the same time,the recycling process of HP-β-CD in HCA biotransformation was established.After HP-β-CD was recycled for 7 times,the product generation rate could still reach more than 90%.A preliminary study was conducted on the key enzymes that play the function of 1-dehydrogenation on this strain.Through heterologous expression means,the six target genes screened from the genome sequencing results were expressed in E.coli BL21(DE3).The results of enzyme activity and whole bacteria transformation showed that only the gene annotated 1＿1592 showed the catalytic activity of 1-dehydrogenation.Using gene overexpression strategy,we conducted a preliminary exploration on the construction of ksdD from Nocardia simplex overexpression strain using E.coli-Arthrobacter sp.shuttle plasmid pART2.In the end,the expected overexpression strains were not obtained,and the reasons need to be further studied.