Influence Of Seven Regulation Related Genes On Daptomycin Production

Daptomycin,a lipopeptide antibiotic,has its potential in vitro bactericidal activity against most clinically relevant strains of Gram-positive bacteria.Streptomyces sp.HCCB10043 can produce A21978C,which is the leading compound of daptomycin.Therefore,it has vital significance for genetic engineering to achieve high-production strain.Seven possible regulation related genes were selected as targeted genes in this study by transcriptome analysis,including tfpA?arpA?afpA?dgkA?mfpA?asnC and lfpA.These genes were deleted by homologous recombination in HCCB 10043.Then the strains with complementation and over-expression were used to confirm the function of these genes.Then the gene knockout were also carried out in high-producing mutant A9.1.Temperature-sensitive shuttle plasmid pKC1139 was used as knockout vector to construct recombinant plasmids.Then the recombinant plasmids were transferred into HCCB 10043 by conjugation and the mutants were verified by single-and double-exchange screening.?tfpA??arpA??afpA??dgkA??mfpA,?asnC and ?lfpA knockout mutants were obtained.The plasmid pLYW2 was used as complementary vector to construct recombinant plasmids.The plasmid pWMH3 containing oriT was used as over-expressed vector to construct recombinant plasmids.Then the recombinant plasmids were individually transformed into knockout mutants and HCCB 10043 by conjugation.The corresponding gene complementary mutants and over-expressed mutants were obtained.2.The production of A21978C in mutants was detected via HPLC.Compared with the original strain HCB10043,production of A21978C in the ?tfpA??mfpA??asnC and?lfpA mutants increased obviously.Therefore,tfpA?mfpA?asnC and lfpA genes exert a strong influence on A21978C production and are negative regulatory genes.While production of A21978C in the ?arpA??afpA??dgkA mutants decreased obviously.Therefore,arpA?afpA and dgkA genes exert a strong influence on A21978C production and are positive regulatory genes.However,no new compounds in mutants were detected via UPLC-MS.While we found that these genes also exerted a strong influence on arylomycin production.3.Six recombinant deleted plasmids were transformed into high-producing mutant A9 by conjugation and the mutants were verified by single-and double-exchange screening.Then the deleted mutants in A9 were obtained.While the over-expressed plasmid containing arpA gene was also transformed into A9 by conjunction and the over-expressed mutant was obtained.Then the mutants of higher production than A9 were achieved,including LYPLA141,LYPLA96,LYPLA243,LYPLA158 and LYPLAG260.So the regulatory function of those genes was further confirmed by the results.4.The influence of tfpA and mfpA regulatory genes on their upstream and downstream genes or A21978C biosynthetic gene cluster was studied by RT-PCR.Compared with the original strain HCCB10043,the different transcription levels in the deletion mutants indicated that dptM gene expression level were improved and there was no obvious relation between them and their upstream and downstream genes.Therefore,the transcription of tfpA,mfpA genes may relate to dptM gene involved in daptomycin export.While we found that aryA gene expression level also improved.So there was also some relation between the transcription of tfpA,mfpA genes and the arylomycin production.