Study On The Regulation Of ArgR In Daptomycin Biosynthesis In Streptomyces Roseosporus

Background: Daptomycin is a kind of new cyclic lipopeptide antibiotic and was reputed super antibiotic,due to its antibacterial activity against gram-positive pathogens including Staphylococcus aureus?MRSA?,penicillin-resistant Streptococcus pneumonia?PRSP?and vancomycin-resistant Enterococci?VRE?.Daptomycin is a small part of cyclic lipopeptide antibiotic produced by Non ribosomal peptide synthetase?NRPSs?in S.roseosporus NRRL 15998.Its low yield can't meet the clinical needs,but the importence in clinical medicine,promoting us to explore the function of S.roseosporus.In order to improve the production of daptomycin,several approaches have been made,promoting us to explore the mechanism of daptomycin biosynthesis by the method of metabolic engineering and synthetic biology.It has confirmed that the arginine repressor Arg R is a transcription factor regulating arginine biosynthesis universally exist in many bacterial genomes.Arg R could regulate other genes' expression about certain metabolic pathway in secondary metabolism.Arg R is the regulator of arginine biosynthesis genes in Streptomyces species.This kind of soil-dwelling could produce so many secondary metabolites which could use arginine or arginine-related molecules as precursors,regulating secondary metabolism and antibiotic biosynthesis in many Streptomyces.Arg R participate in a variety of metabolic processes in S.clavuligeerus,such as prune and clavulanic acid metabolism and biosynthesis of lincomycin.Arg R could control gene transcription and pigment biosynthesis in S.coelicolor.Orn was critical intermediate product in arginine biosynthesis.It is an important scientific issue whether Arg R could regulate daptomycin biosynthesis.These all pave the way for us to explore the function of argR in S.roseosporus.And pave the way for us to explore argR's universality in relation to secondary metabolism and to explore the mechanism of argR in the production of antibiotic,improving the production of daptomycin by the method of genetic engineering in S.roeosporus.Objective: We found that the expression of Arg R and Arg J were down-regulated,while Arg G was up-regulated in daptomycin producing strains through comparative proteome sequencing.This study focus on exploring the function of argR and the effect of argR on development and differentiation and secondary metabolism.We explore the regulation of argR in daptomycin biosynthesis in Streptomyces roseosporus.This study provides beneficial basis to analysis argR's function,mechanism and the way how to affect daptomycin production in S.roseosporus.Methods: 1.This study analysis the genome sequence of S.roseosporus NRRL 15998 by the method of bio information,confirming the function of argR,which could encode arginine repressor Arg R.To predict the conserve domain and to found homologous protein of Arg R,we compared Arg R with other reference strains using Clustal X and EScript.2.To explore the function of argR in S.roseosporus,we constructed disruption strains,complement strains and overexpression strains of argR by the method of PCR-targeting based on fosmid library,which we constructed early.3.In order to detect the function of argR in morphological development and differentiation,we choose different medium and AS-I medium with different salt ions to observe the growth of mycelium and spores production between wild type and mutant strains.To test whether argR could affect development and differentiation in S.roseosporus.4.In this study,q RT-PCR was used to detect the effect of argR on the cluster of arginine biosynthesis.And RT-PCR was used to detect whether argR could activate silence gene expression exists in S.roseosporus.5.To confirm the function of argR in antibiotic biosynthesis,we have attempted some alternative strategies.We ferment wild type,complement and overexpression strains,taking Micrococcus uterus as indictors to test biological activity.Then we conducted growth curve and yield curve.Furthermore,q RT-PCR was used to detect the regulation of argR on daptomycin in S.roseosporus..6.Arg R was expressed and purfied by constructing p ET23b-Arg R in E.coli BL21.Electrophoretic mobility shift assays?EMSA?was used to detect the regulation of Arg R on key genes in relation to daptomycin biosynthesis.Results: 1.We confirmed that argR exists in S.roseosporus genome?SSGG₀0667?and constructed argRDM,argRDMC and argROE strains successfully.2.The growth phenotypes of WT,argRDM and argROE on AS-I agar and in F10 A have no difference.WT and argRDM could produce spores on AS-I agar with 0.25 M Ca2+ and the production of spores of argRDM was 170 fold than that of WT.But argROE lose the ability to pruduce spores and to confirm the results by SEM.3.Disruption mutants of argR decreased antibiotic production by 21%,and the overexpression mutants of argR could increase its production by 1.6 times.Meanwhile,we detected the production of amino acids between WT and argRDM.4.We confirmed that argR could reduce the expression of dpt E,atr A,dpt R2 and dpt R3,decreasing daptomycin production.The expression of dpt E,atr A,dpt R2,dpt R3 was decreased separately by 26%?21.36%?73.28%,27.12% at 48 h,51.15%?75.45%?77.88%,29.82% at 72 h,65.87%?25.09%?15.07%,18.75% at 96 h.Otherwise,when knockout out argR could enhance arg CJBDR and arg GHs' expression about 60 folds and 100 folds,which exits in arginine biosynthesis genes cluster.5.Construction of protein expression vector p ET23b-Arg R and purified the object protein Arg R,which concentration was 8mg/ml.6.In this study,Electrophoretic mobility shift assays?EMSA?experiment was conducted to confirm Arg R could bind to the promoter area of dpt E,atr A,dpt R2,dpt R3,arg CJBDR and arg GH.7.Disruption mutants of argR failed to activate certain silence gene clusters in S.roseosporus.8.The content of Ser in bacteria and fermentation were decreased by 39.72% and 13.1%,and the content of Arg were increased by 77.44% and 180.43%.Conclusion: There is no obvious influence of argR on development and differentiation,but affecting the production of secondary metabolic antibiotic in S.roseosporus.argR could affect daptomycin production indirectly by regulating dpt E,atr A,dpt R2 and dpt R3,which are key genes associate with daptomycin biosynthesis.When distupted argR could decrease the production of Ser,affecting daptomycin biosynthesis.We first explore the function of argR in S.roseosporus.This paper lay foundations for further study on argR in S.roseosporus and the transformation of metabolic engineering of S.roseosporus.