Ettect Of Exogenous Propionyl Coa Metabolic Pathway Enhancement On Sterol Transformation Of Mycobacterium Fortuitum

The 9-hydroxyl-4-androsteroid-3,17-dione(9-OH-AD),as an important steroidal precursor substance,can synthesize a variety of steroidal drugs and plays an important role in the steroidal drug industry.In the process of 9-OH-AD production,occasional mycobacteria first metabolizes plant sterols to propionyl-Co A and acetyl-Co A,which causes an instantaneous increase in intracellular propionyl-Co A content,affecting the growth and transformation of strains.At present,most studies have focused on the three endogenous metabolic pathways reported in mycobacteria,whether the exogenous introduction of propionyl-Co A metabolic pathway can regulate propionyl-Co A metabolism,its effect on phytosterol transformation are not clear.Therefore,this study takes Mycobacterium fortuitum,the main production strain of 9-OH-AD,as the research object.Based on the previous cofactor regulation work of our research group,combined with classical methods such as genetic engineering and metabolic engineering and the overall regulation strategy of cofactor regulation.A new exogenous propionyl-Co A metabolic pathway is introduced to eliminate the impact of too high intracellular propionyl-Co A content on the product yield of engineering strains.It provides stable and excellent strains for the efficient production of 9-OH-AD,and also provides a new strategy for the construction of other plant sterol transformation engineering strains.Firstly,this paper studied the effect of propionyl-Co A:succinate-Co A transferase(scp C)from E.coli BL21 on the transformation reaction of Mycobacterium fortuitum,constructed the overexpression strain MFT-scp C,and detected the growth performance,9-OH-AD conversion rate and intracellular propionyl-Co A content of the strain.The results showed that the excessive propionyl-Co A in the cell was transformed into succinate-Co A,which relieved the toxicity to the strain and improved the cell viability.The conversion of9-OH-AD was improved.Secondly,the effects of type II NADH dehydrogenase(ndh)and9-hydroxylase(ksh)on phytosterol transformation were studied.The tandem expression strains MFT-scp C-ndh and MFT-scp C-ndh-ksh were constructed,and the transformation efficiency,intracellular NAD~+,NADH content and ATP content of the strain were detected.The results showed that the modified strain MFT-scp C-ndh solved the problem of intracellular cofactor imbalance in the process of phytosterol side chain degradation to a certain extent.The ratio of NAD~+/NADH and ATP content were increased,the dynamic balance of intracellular cofactors was maintained,and the inhibition of phytosterol conversion was reduced.At this time,the conversion rate was 66.4%after 84 h fermentation.However,the conversion rate difference between MFT-scp C-ndh-ksh and MFT-scp C-ndh was only 1.6%.Then the succinate-Co A transferase(Aar C)in Lactobacillus was studied,and the three gene tandem expression strain MFT-scp C-ndh-Aar C was constructed.All strains were fermented and transformed.The results showed that MFT-scp C-ndh-Aar C could convert a large amount of succinate-Co A accumulated in the metabolic process into acetyl-Co A and enter the TCA cycle,which improved the conversion rate of 9-OH-AD.When the strain was fermented for 84 h,the conversion rate of phytosterol could reach 76.7%,which was 13.6%higher than that of the original strain.The fermentation conditions such as substrate concentration,p H value of fermentation broth,ratio of substrate to cyclodextrin and substrate release time were optimized.The results showed that when the substrate concentration was 7 g/L,the optimum ratio of substrate to cyclodextrin was 1:2,and the optimum p H value of fermentation broth was 7.5,the molar conversion of recombinant strain MFT-scp C-ndh-Aar C 9-OH-AD reached87.2%,increased by 10.5%compared with that before optimization,and increased by37%compared with that of original strain when the substrate concentration was 5 g/L.