Isolation Of Endophytic Bacteria With Antibacterial Active From Dysosma Versipellis And The Antibacterial Substances Analysis From The Endophytic Bacterium

In the face of microbial resistance,especially infections caused by Staphylococcus aureus,there is an urgent need to find new antimicrobial agents.Endophytes are microorganisms symbiotic in plants,whose special growth environment makes them have unique physiological and metabolic mechanisms,and can produce the same or similar compounds as the host plants.At the same time,endophytes have the characteristics of diverse species,short growth cycle and controllable metabolism,which can realize industrial production through fermentation.Dysosma versipellis are Berberis,its rhizome used in medicine,has the effect of clearing heat and detoxifying,promoting blood circulation and dispersing blood stasis,and has good antibacterial activity.In this paper,the endophytic bacteria with antibacterial activity were isolated from Dysosma versipellis,and the antibacterial substances were isolated and purified on this basis.In this paper,a total of 23 endophytic bacteria（including 12 endophytic bacteria and 11endophytic fungi）were isolated from the roots,stems and leaves of Dysosma versipellis.Among the 12 strains of endophytic bacteria,only DS6 and DS3 had antibacterial activity against Staphylococcus aureus.DS3 strain had better antibacterial activity than DS6 strain,and DS3 strain not only had strong antibacterial activity against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus and Bacillus subtilis.It is also effective against a variety of plant pathogens such as Verticillium dahliae,Fusarium acuminatum,Botrytis cinerea and Curvu Laria lunata also has antibacterial activity.Therefore,DS3 strain was selected for subsequent analysis.DS3 strain was identified as Actinacidiphila bryophytorum by 16S rDNA sequencing combined with whole-genome ANI and dDDH analyses.In addition,strain DS3 had the ability to produce catalase,protease,cellulase and decompose sulfur-containing compounds to produce hydrogen sulfide.Sequencing analysis of DS3 genome using Nanopore Prometh ION and Illumina Nova Seq6000 sequencing platforms showed that DS3 strain was composed of a linear chromosome of 8,265,668 bp,no plasmid,and GC content was 72.47%.DS3 contained 6tRNA,18 rRNA and 33 sRNA genes.Anti-SMASH online analysis of secondary metabolic Biosynthetic gene clusters（BGCs）in DS3 showed that:the strain contains 31 secondary metabolic BGCs,including PKS（Polyketide synthetases）,NRPS（Nonribosomal peptide synthetases）,Lanthipeptide,Siderophore and Terpene;six of them synthesize BGCs of known antibiotic classes and seven of unknown types.Single factor and orthogonal analysis were used to optimize the culture conditions of strain DS3,so as to improve the antibacterial activity of strain fermentation broth.The optimized culture conditions were as follows:3%（v/v）inoculation amount was added to seed liquid,yeast extract concentration was 0.2%（w/v）,starch concentration was 1%（w/v）, sodium chloride concentration was 0.15%（w/v）,fermentation liquid volumel was 0.56%was56%（v/v）,fermentation time was 6d,culture temperature was 26℃,pH was 6.0.After optimization,the plate antibacterial zone diameter of fermentation liquid was increased from19.69 mm to 22.03 mm.At the same time,the bacteriostatic substances in the fermentation broth of strain DS3 had thermal stability,acid-base stability and UV stability.DS3 fermentation liquor was extracted by ethyl acetate and concentrated by rotary evaporation to obtain the crude extract of the metabolites.The crude extract was separated and purified by silica gel chromatography,high performance liquid chromatography,semi-preparative high performance liquid chromatography and other separation methods.A total of 4 compounds with antibacterial activity were isolated,among which compound 1 was chromatography-pure.The molecular weight of compound 1 was 736.23.By nuclear magnetic resonance carbon（13C-NMR）and hydrogen（1H-NMR）analysis,the structure of the compound 1 was identified as dioxamycin(C₃₈H₄₀O₁₅).The minimum inhibitory concentrations of compound 1 against Staphylococcus aureus,methicillin-resistant Staphylococcus aureus,and Bacillus subtilis were 2μg/mL,2μg/mL,and 4μg/mL,respectively.