Targeted Isolation Of Actinoplanes And The Mining Of Their Secondary Metabolites

Actinoplanes was the second largest genus of rare actinomycetes in Micromonosporaceae,which can be used to prepare teicoplanin and other anti-infective drugs.As an important medicinal microbiologic resource,the establishment of corresponding targeted separation methods and the mining of secondary metabolites were of great research significance.Established a targeted isolation method of Actinoplanes.By adding polyaspartic acid(PASP)to the soil to enrichActinoplanesinsitu,combinedwiththeseparationprocessof trapping-drying-dehydration-rehydration-centrifugation,then the selective separation medium was used to culture,and the single colonies with orange,wrinkled and earthy smell were selected to amplify and sequence the 16S r RNA gene of actinomycetes.By using this method,291 strains of actinomycetes were isolated,including 198 Actinoplanes,the rate of which was 68.04%,4 suspected new species of Actinoplanes were founded.A new potential Actinoplanes species of TRM66264-DLM was identified by polyphase classification.The strain was gram-positive,without aerial hyphae,and the sporangium was irregular spherical.The whole genome size of the strain was 10.19 MB,and the content of(G+C)%was 70.48%.The average nucleotide identity(ANI)of the strain and the most similar strain Actinoplanes bogorensis LIPI11-2-Ac043^Twas 83.39%,and the digital DNA hybridization(d DDH)value was 27%.The whole-cell sugars contained xylose,arabinose,glucose,and galactose.Meso-diaminopimelic acid was the major cell-wall peptidoglycan.The major polar lipids were disphosphatidylglycerol(DPG),phosphatidylglycerol(PG),phosphatidylethanolamine(PE),phosphotidylinositol(PI).The predominant menaquinones were identified as MK-9(H₄).The major cellular fatty acids(>5%)were iso-C_16:0,anteiso-C_15:0,iso-C_15:0,C_17:1?8c,anteiso-C_17:0,and C_17:0for strain TRM66264-DLM.Combined with physiological,biochemical,and chemical characteristics,the strain was identified as a new species of Actinoplanes polyasparticus TRM66264-DLM.Secondary metabolites of TRM66264-DLM were mined.By optimizing the fermentation process,the optimal seed medium was determined as the oat urea medium,and the optimal fermentation medium was the04 fermentation medium.The strain was fermented with 10 L under optimal fermentation conditions and separated by silicone chromatography and gel chromatography to obtain an active compound,which was identified as acridone by 1D&2D NMR,which had good antibacterial activity against E.coli(Escherichia coli)ATCC 25922,Candida albicans(Candida albicans)ATCC 64550,and Klebsiella pneumoniae(Klebsiella pneumonia)ATCC 10031.