High-throughput Screening And Metabolic Analysis Of FK506-producing Streptomyces Tsukubaensis

FK506,a widely used immunosuppressive agent to treat organ transplant rejection and autoimmune diseases,has an important applicable value.In this study,with the help of atmospheric and room temperature plasma(ARTP)and genome shuffling(GS)of Streptomyces tsukubaensis No.9993,a massive FK506-producing strain was obtained by high-throughput screening.Furthermore,gene engineering and transcriptional level analysis were performed to explore the regulatory mechanisms of FK506 biosynthesis,in order to improve the yield.Three stable and high-yielding strains were obtained.The S.tsukubaensis R3-C4 strain was the most productive,resulting in a concentration of 335 μg/mL(of FK506),2.6 times than that of the original wild-type strain.For the FK506 biosynthesis,extra alanine,tryptophan and shikimic acid indicated a positive contribution by shikimic acid pathway to the synthesis of FK506 in S.tsukubaensis.Further analyses using the precursors corresponding to different synthesis pathways of methylmalonyl-CoA(valine,sodium propionate and sodium malonate)suggested the up-regulation of FK506 synthesis in S.tsukubaensis at different levels.Among them,sodium propionate at 0.2 g/L had the most obvious promoting effect,which could increase the FK506 production by 21.7%.This result support the influence of the nessesary precursor methylmalonyl-CoA on the synthesis of FK506,and the delivery of methylmalonyl-CoA by PCC pathway is a key in increasing the yield of FK506 in S.tsukubaensis No.9993.The conclusions above were also evidenced by that overexpression of prpE(propionyl-CoA synthase gene)and pccB(propionyl-CoA carboxylase gene)in the wild-type strain increased FK506 production by 10.8%and 18.9%,respectively.Another experiment on the overexpression of dahp(DAHP synthase gene)in mutant YB-2809 led to an increase in the yield of FK506 by 24.0%,which was 53.3%higher than that of the wild-type strain.Exogenous 4%(v/v)DMSO could induce efflux of FK506,reduced feedback inhibition of secondary metabolites and increased FK506 production by 27.9%to 429 μg/mL,which was 3.3 times than that of the wild-type S.tsukubaensis.Analyses of the differences in morphology,fermentation characteristics and specific gene expression levels between S.tsukubaensis R3-C4 and the wild-type strain revealed that R3-C4 strain had stunted spore differentiation,thick mycelia and more red pigment,which were highly possibly related to the expression of bldD and cdgB.In addition,the expression levels of fkbO,fkbP,dahp,pccB and prpE all showed up-regulation at diverse degrees compared to the wild-type S.tsukubaensis.