The Regulatory Mechanisms Of Daptomycin Biosynthesis In High-quality And Efficiency

Daptomycin,a novel cyclic lipopetide antibiotic,is isolated from cultures of Streptomyces roseosporus.It performs broad-spectrum antimicrobial activity against Gram-positive pathogens in vitro,and is deemed as the substitute of vancomycin.According to the research of daptomycin,its structure,modification,biosynthetic pathway,antibacterial mechanism and physicochemical property have been studied thoroughly.However,little is reported about the regulatory mechanism of daptomycin biosynthesis and A21978C1-3 analogs producing in fermentation,which influence the final quality of daptomycin.In this study,we focus on the regulatory cascade and pleiotropic regulatory mechanism in the process of daptomycin biosynthesis,and the regulatory mechanism of branched-chain fatty acid precursors for A21978C1-3 analogs production in industrial producer S.roseosporus L30.Here,we establish a new Himar1-based random mutagenesis strategy and a DNA-affinity system for screening pleiotropic regulators in vivo and in vitro,respectively.Two important pleiotropic regulators PhaR and AtrA,involving in regulating daptomycin biosynthetic gene cluster(dpt)transcriptional levels,were isolated successfully.PhaR is a negative regulator of dpt cluster.It binds directly to dptEp,which induces the transcription of a large operon containing the core genes from dptE to dptH in dpt cluster,to suppress the promoter activity and daptomycin production.The daptomycin yield of phaR gene deletion mutant is 42.5%higher than that of WT in fed-batch fermentation,up to 926 mg/L.Moreover,PhaR shows positively autoregulation and feedback regulation,its inhibition to downstream genes is gradually relieved as the fermentation proceed.AtrA,a TetR family transcriptional regulator,is conserved in Streptomyces.It positively regulates daptomycin biosynthesis by binding to dptEp directly.Another pleiotropic regulator AdpA,a highly conserved protein in A-factor signaling pathway of S.roseosporus,increases the expression of atrA by specifically binding to atrAp,advancing the daptomycin production indirectly.Meanwhile,ArpA,the A-factor receptor protein in the A-factor signaling pathway,inhibits the transcription of adpA gene.According to the mechanism,we knocked out arpA gene,and promoted daptomycin production by about 2.5 fold.What’s more,PhaR,AtrA,AdpA and ArpA are all contributed to morphological development in S.roseosporus.Our works have also examined the biosynthetic process of A21978C1-3 branched-chain fatty acid precursors and their regulatory mechanisms.There are two allele clusters,bkdA1B1C1/bkdA2B2C2,encoding branched-chain a-keto acid dehydrogenase complex(BCDH complex)in the genome of S.roseosporus,and a regulatory gene bkdR locating upstream of bkdA2B2C2.We reveal that BkdR positively regulates the expression of bkdA2B2C2 and the biosynthesis of daptomycin,but it does not influence the transcriptional levels of dpt cluster.BkdR is negatively autoregulated.The deletion of bkdA2B2C2 would only partly eliminate the A21978C1-3 analogs,while the absence of bkdA1B1C1 has a very significant effect.The results suggest that bkdA1B1C1 gene cluster occupies the dominant position in the catabolism of branched-chain amino acids and bkdA2B2C2 is an assistant.Furthermore we constructed a new strain to eliminate the biosynthesis of A21978C1-3 by deleting both bkdA1B1C1 and bkdA2B2C2 clusters,and achieved high-quality production of daptomycin.In this study,our works reveal the regulatory mechanisms of daptomycin biosynthesis,establish the signaling pathway from extracellular stimulation of the potential A-factor to daptomycin gene cluster expression and construct the high producing strains to achieve efficient production of daptomycin in S.roseosporus.Meanwhile,we expound the regulatory mechanism of branched-chain fatty acid precursors for A21978C1-3 analogs production,and eliminate them in fermentation by genetic modification achieving daptomycin production in high-quality.