Identification And Regulation Of Antmicrobial Substances Produced By Streptomyces Fia Vogriseus NJ-4

Over the years,people have urgent need for new antibiotics for clinical application as the appearance of more and more antibiotic-resistant bacteria such as methicillin-resistant Staphylococcus aureus(MRSA),vancomycin-resistant Enterococci(VRE)and penicillin-resistant Streptococcus pneumoniae(PRSP),2/3 Antibiotics currently in use are produced by actinomycetes.The purpose of this work was(1)to isolate and screen antagonistic actinomycetes from the soil sample;(2)to identify the isolate strain by physiological and biochemical characteristics,16S rDNA sequence homology analysis;(3)to purify and characterize the antimicrobial compounds from Streptomyces flavogriseus NJ-4;(4)to optimize the fermentation condition to improve the yield of actinomycin D;(5)to research the mechanism of the regulation of antimicrobial substances synthesis.The results are described as follows:1L Screening and identification of the isolate of actinomycetes with wide-spectrum antimicrobial activity.About 260 actinomycetes were isolated from samples from different soil samples collected from different three regions of China.Among them,8 strains were found to have a broad-spectrum and high antimicrobial activity using the agar block and shaking flask method.Finally,the strain NJ-4 was determined for further research because of its broad-spectrum antibacterial and antifungal activities.Based on the morphological and cultural characteristics,the physiological and biochemical properties,strain NJ-4 was belonging to the genus of Streptomyces flavogriseus.From the 16S rDNA sequence analysis(GenBank accession NO.KM102731),strain NJ-4 has 99%similarity with that of Streptomyces flavogriseus ATCC33331.Phylogenetic analysis based on 16S rDNA gene sequence analysis revealed that strain NJ-4 formed a distinct phylogenetic cluster w:ith Streptomyces flavogriseus in the phylogenetic tree.Based on all above,strain NJ-4 was identified as Streptomyces flavogriseus.2.Purification and characterization of antimicrobial compounds from the culture supernatant of Streptomyces flavogriseus NJ-4.The fermentation broth of 5.flavogriseus NJ-4 was Gause’s synthetic medium,antimicrobial compounds were extracted from the fermentation broth of S.flavogriseus NJ-4 using ethyl acetate,the organic fractions were dried and concentrated under reduced pressure,the residue was then dissolved in methanol and loaded onto a Sephadex LH-20 column.Elution was carried out with 80%methanol,two antimicrobial activity fractions were obtained.The first antimicrobial activity fraction was further purified through RP-HPLC,three antimicrobial activity peaks were obtained.The molecules of the three antimicrobial compounds were detected by liquid chromatography-tandem mass spectrometry,the first antagonistic compound with m/z 1269 was identical to actinomycin X0β,the second antagonistic compound with m/z 1271 was identical to actinomycin X0β,the third antagonistic compound with m/z 1269 was identical to actinomycin D.The second antimicrobial activity fraction with m/z 214 was identical to holomycin.The two antagonistic compounds’(m/z 1271,m/z 214)structures were elucidated by UV,IR and NMR,the structures of the two antagonistic compounds were confirmed to be actinomycin D and holomycin.Actinomycin D and holomycin were active against all tested bacteria and cancer cells including Staphylococcus aureus,Pseudomonas aeruginosa.,Bacillus cereus,Micrococcus luteus,Pseudomonas fluorescens,Escherichia coli,Salmonella typhimurium and Salmonella enteritidis;lung cancer A549 cells,gastric cancer BGC-823 cells and HepG2 cells.Actinomycin D was also active against all tested filamentous fungi including Aspergillus niger,Aspergillus oryzae,Fusarium solani,Fusarium graminearum and Rhizopus stolonifer.Holomycin was only active against Fusarium solani,Fusarium graminearum,but no active against Aspergillus niger,Aspergillus oryzae and Rhizopus stolonifer.3.Optimization of fermentation conditions for improved holomycin production by S.flavogriseus NJ-4.The medium screening experiment indicated that S.flavogriseus NJ-4 could obtain high yield of holomycin in Gause’s Corn Flour medium.The further Plackett-Burman design was undertaken to screen the key factors of medium components and fermentation conditions rapidly from the related 11 factors.By analyzing the statistical regression and the prediction profiler,the concentrations of corn flour,KNO3 and FeSO4 were the key factors significantly affected the production of holomycin.According to the results of Plackett-Burman design,a 3 factor with 3 level BBD design were applied to optimize the holomycin production.The regression equation model for prediction was established,by analysis of the 3D plots and their corresponding plots,the optimum values of the concentrations of corn flour,KNO3 and FeSO4 for obtaining the most production of holomycin were 21.5 g/L,1.3 g/L,0 g/L,0.016 mg/L,respectively.The optimized fermentation conditions allowed actinomycin D production increased to 31.27 mg/L.4.The regulation of holomycin synthesis by peptone of S.flavogriseus NJ-4.We found that in the production of holomycin by S.flavogriseus NJ-4 the amount of peptone in Gause’s Synthetic medium increased to 0.3%could completely inhibite the synthesis of holomycin,the effect of peptone on the expression of holomycin biosynthesis genes was determine by real-time fluorescence quantitative PCR.The results showed that holomycin biosynthesis genes were significantly down-regulated by adding peptone to the medium.The synthesis of holomycin was affected by the redox potential of the medium.The effect of the amount of peptone adding to Gause’s Synthetic medium on redox potential of the medium was detected using redox potential meter.The results show that the redox potential of the medium decreased significantly with the amount of peptone increased.The synthesis of holomycin was inhibited with the decreased of the redox potential of the medium by S.flavogriseus NJ-4.The form of coenzyme was effected by the trend of redox potential.The redox potential of Gause’s Synthetic medium was decreased with the adding of peptone.The ratio of NADH and NADP/NADPH was decreased when the redox potential of Gause’s Synthetic medium decreased.This indicate that the content of oxidized coenzyme NAD and NADP was decreased,the content of reduced coenzyme NADH and NADPH was increased.5.Research the mechanism of the regulation of holomycin synthesis by the method of metabonomics.The differences metabolites of S.flavogriseus NJ-4 were determined using the gas chromatography-mass spectrometry.Characteristic metabolites were identified using the principal component analysis(PCA)and orthogonal partial least squares discriminant analysis(OPLS-DA)multi-dimensional statistical analysiss.The results showed that metabolites of ribitol,succinic acid,phosphate,N-Acetyl-beta-D-mannosamine,D-glyceric acid,lactulose were upregulated;levoglucosan,sulfate,threonine,and proline,were downregulated.By the analysis of metabolic pathway,we know that the levels of ATP and sulfuric acid regulate the synthesis of holomycin of S.flavogriseus NJ-4.