Elucidation Of The Bleomycin Biosynthesis Activation And Transcriptor Mechanisms For The Construction Of High-producing Bleomycin Strain

As a broad-spectrum glycopeptide antibiotic,bleomycin is synthesized by S.verticillus.Now,the researches about bleomycin mainly focus on its analogues,and none is in the studies of regulatory mechanism.In the bleomycin synthesis,the high nutrition requirements and the more by-products lead to the low yield of bleomycin.To improve the bleomycin production,the global and specific transcriptional regulatory factors were studied during bleomycin synthesis,which were used for high-producing bleomycin strains construction,the detailed results are as follows:The N-acetylglucosamine metabolism can release the inhibitory effect of Das R on Glc NAc metabolism,sugar transport,antibiotic synthesis and development.Glc NAc addition significantly increased the bleomycin yield to 22.91±3.36 and11.44±2.39 mg/L in semi-synthetic liquid medium(M-ISP4)without bleomycin-synthesis ability,and inhibited the spore maturation in solid M-ISP4 medium.To verify Das R directly controls the synthesis of bleomycin,the N-acetylglucosamine metabolism-related genes in S.verticillus were sequenced.Bioinformatics analysis found that the promoter regions of das ABCD and nag KA-nag B respectively harbored a Das R binding sequence(dre site),but no potential dre site in the bleomycin gene cluster.The gel electrophoresis migration analysis(EMSA)shows that Das R could bind to the promoter of das A,nag K and nag B,and RT-q PCR showed that the expression level of nag regulon was increased 2 times and those of das ABCD were not changed under Glc NAc induction,indicating that N-acetylglucosamine metabolism relieved the inhibitory effect of Das R on the nag regulon but had no effect on das ABCD;Except the increased expression level of blm R-blm T,the expression of other genes in the bleomycin gene cluster did not change obviously under N-acetylglucosamine induction,and Das R was unable to bind to the promoter region of the operon blm R-blm T from EMSA.All indicated that Das R inhibit N-acetylglucosamine metabolic genes transcription but not directly control the bleomycin gene cluster expression.The increased bleomycin yield induced by N-acetylglucosamine may be directly derived from the expression change of blm R-blm T.The bleomycin production was significantly increased in the Blm R knockout mutant and recovered in Blm R complementary strain,indicating that Blm R is a pathway-specific negative regulator in bleomycin synthesis.The changes of the transcription level of the bleomycin gene cluster between the Blm R knockout mutant and the wild strain indicated that Blm R could only act as a self-regulatory factor and affect the expression of the gene blm T.However,the increased expression level of the blm R-blm T under N-acetylglucosamine induction indicates that the inhibition by Blm R may be released by the allosteric regulation and leads to the increased bleomycin production.Similar to other members in the Ars R/Smt B family regulator,Blm R bind to a12-2-12 bp DNA sequence.The amino acid sequence alignment showed that Blm R did not contain the metal binding site and redox sensing site.Also,the EMSA assay identified that the metal ions,bleomycin,N-acetylglucosamine,and N-acetylglucosamine 6-phosphate could not act as ligands to mediate the dissociation of the Blm R-DNA complex.To elucidate the allosteric regulation mechanism of the Blm R,the Blm R-DNA complex model was constructed by homology modeling and molecular dynamics simulation.The hydrogen bond interaction network indicated that Gln51,Arg14 and Arg58 could promote the recognition of Blm R to the DNA major groove,while Arg66 could maintain the stability between the w-HTH motif and the minor groove of DNA.In the absence of N-acetylglucosamine,both the yields of bleomycin A2 and B2 produced by DBlm R in M-ISP4 were lower than those in mutant strains(DBlm R and OBlm T)and wild strains under N-acetylglucosamine induction,which indicated that the effect of N-acetylglucosamine metabolism on bleomycin synthesis was not limited to the release of inhibition of Blm R,and involved in the regulation of intracellular metabolic flow as well.The overexpression of the mannose metabolism genes man A and man B could promote the synthesis of bleomycin.Combination with N-acetylglucosamine induction,the blm T,man A and man B over-expressing strain OBlm T/Man AB was constructed,the final yields of bleomycin A2 and B2 in the semi-synthetic medium reached 52.16±1.93 and 30.02±3.04 mg/L,which were 2.27 and 1.62 times than those of wild-type strain,respectively.