Study On Metabolic Mechanism Of FK506 Overproduction In Streptomyces Tsukubaensis By Adding Soybean Oil

FK506(tacrolimus)is a 23-membered polyketide macrolide produced by various Streptomyces species.It widely used to prevent rejection and treat the autoimmune diseases.FK506 has gradually replaced the utilization of cyclosporine since the FDA approval in 1994,and now it becomes one of the best-selling medicines for treating solid-organ transplantation in the world.Feeding soybean oil has great effect on the production of macrolides,especially for FK506.In this study,feeding soybean oil into the fermentation culture of S.tsukubaensis improved FK506 production by 88.8 %.However,it has not been reported from molecular mechanism in other literature on FK506 overproduction by soybean oil,this is the first time for us to in-depth analysis.In order to study the mechanism of overproduction,we combined comparative proteomics with metabolomics,comprehensive analysis three aspects of intracellular enzymes,metabolites and metabolic pathways.To decipher the overproduction mechanism,comparative proteomic and metabolomic analysis was carried out.More than 500 protein spots were isolated in this study.A total of 77 protein spots with differential expression.And 74 spots were present at higher levels in soybean oil-fed samples,while only three protein spots showed higher abundances under the control condition.MALDI-TOF/TOF-MS was used to identified and function annotations of these protein spots.And 66 intracellular metabolites were measured by GC-MS.18 key metabolites was related FK506 biosynthesis in PLS analysis.The analysis of proteome and metabolome indicated that feeding soybean oil as a supplementary carbon source could not only strengthen the FK506 precursor metabolism and energy metabolism,but also tune the pathways related to transcriptional regulation,translation and stress response,suggesting a better intracellular metabolic environment for the synthesis of FK506.Based on these analyses,twenty key metabolites and precursors of FK506 were supplemented into the soybean oil medium.Among them,lysine,citric acid,shikimic acid and malonic acid performed excellently for promoting the FK506 production and biomass.Especially,the addition of malonic acid achieved the highest FK506 production 165.05 ± 3.6 mg/L and the biomass reached 12.4 ± 0.4g/L.