Study On The Technology Of 4-OH-CsA Overproduction By Exogenous Soybean Oil Addition

4-OH-CsA,a region-specific hydroxylation product of cyclosporine A(CsA),which retains the anti-HIV/HCV and hair growth-stimulating activities while avoiding the immunosuppressive effect of CsA.However,the low conversion rate and poor understanding of N.dietziae seriously restrict its clinical application.In this study,a stable genetic performance strain was obtained using the compound mutation of ultraviolet-LiCl with transformation efficiency increased by 25 %,reaching 180.3 mg/L,but did not meet the requirements of the enterprise(300 mg/L).In order to further improve the fermentation yield,we carried out a large number of screening optimization experiments.We first found that soybean oil can enhance the production of 4-OH-CsA.Then,the production of 4-OH-CsA reached 280.6 mg/L when supplementing 0.1 %(w/v)soybean oil into the production medium,which was improved by 55.6 % compared with the initial strain.We used comparative proteomics and metabolomics to analyze the strengthen mechanism of soybean oil on 4-OH-CsA overproduction.Based on two-dimensional electrophoresis and MALDI-TOF/TOF-MS analysis,95 differentially expressed proteins were identified and mainly involved in central carbon metabolism,genetic information processing and energy metabolism,especially the redox process,indicating that soybean oil can promote cell growth,and strengthen the redox metabolism,which provide sufficient oxygen substrate to the hydroxylation of CsA.However,there is no significant differences in lipid metabolism were ditected there.Considering that the soybean oil could possibly participate in some specific metabolic processes of N.dietziae,a further metabolomic research based on GC-MS was conducted to investigate the intracellular metabolites discrepancies under soybean oil condition.Results indicated that soybean oil had a great effect on amino acid metabolism and tricarboxylic acid cycle but not fatty acid metabolism.Subsequently,the major hydrolysates of soybean oil and some other plant oils were fed into the production medium,suggesting that soybean oil could promote intracellular oxygen transfer and cell biosynthesis by changing fermentation characteristics.In order to further improve the catalytic activity of high abundance protein in proteomics research,we further add metal cofactors in soybean oil fermentation system,which validates the above conclusions and further increase the yield of 4-OH-CsA to 327.5 mg/L.