Role Of The Two-component System AfsQ1/Q2 In Regulation Of The Yellow-pigmented Coelimycin P2 Biosynthesis By Streptomyces Coelicolor

Streptomyces have abundant regulatory mechanisms.Because of the important role of Streptomyces in the production of antibiotics,it is of great importance to make a research on the regulatory mechanism of secondary metabolism,especially of the antibiotic production.In this study,we mainly make an insight into the role of the two-component system Afs Q1/Q2 in regulation of the yellow-pigmented coelimycin P2 biosynthesis by Streptomyces coelicolor.We previously demonstrated that in Streptomyces coelicolor,the two-component system(TCS)Afs Q1/Q2 activates the production of a yellow colored coelimycin P2(also named as y CPK)under the condition of minimal medium(MM)supplemented with 75 m M glutamate(Glu),and the response reg?lator Afs Q1 co?ld specifically bind to the intergenic region between two coelimycin P2 biosynthetic genes,cpk A and cpk D.In this study,a more in-depth investigation was performed to elucidate the molecular mechanism underlying the role of Afs Q1/Q2 in regulation of coelimycin P2 biosynthesis.To avoid the interference of ACT?RED for us to observe the production of coelimycin P2,we knock out the pathway-specific regulatory gene act II-ORF4?red D in Streptomyces coelicolor wide type M145,which results in the original strain SBJ512 without the ability of the production of ACT and RED.Firstly,we study the effect of different concentration of Glu on the production of coelimycin P2,we found that SBJ512 produce the most coelimycin P2 under the condition of 75 m M Glu Then under the condition of minimal medium(MM)supplemented with 75 m M glutamate(Glu),we found the deletion of afs Q1/Q2 result nearly the total loss of coelimycin P2 production,and through transcription analysis,we found that deletion of afs Q1/Q2 resulted in markedly decrease expression of the whole coelimycin P2 biosynthetic gene cluster,Electrophoretic mobility shift assays(EMSAs)revealed that Afs Q1 only bound to the target site identified previously,but not to any other promoters in the coelimycin P2 biosynthetic gene cluster.Base substitutions of the Afs Q1-binding motif between cpk A and cpk D only resulted in the drastically reduced transcription of the cpk A/B/C operon(encoding three giant,type I polyketide synthases)and has little effects on the expression of other genes in the gene cluster.These res?lts clearly suggested that in addition to the direct role of Afs Q1/Q2 in regulation of coelimycin production,which is mediated via the coelimycin P2 biosynthetic genes,other unknown mechanisms are also involved.