Study On Biological Characteristics And Metabolites Of Actinomyces 2F-1

Actinomycetes are a class of microbial resources that can produce abundant active secondary metabolites,which can inhibit or kill fungi,bacteria and insects.Using actinomycetes and their metabolites to develop highly efficient,low toxicity,residue-free and pollution-free biological agents has been a focus.In order to acquire new antimicrobial active substances and provide new resources for the developing of green biological agents,characteristics and metabolites of Actinomyces 2F-1 were determined.The results are as follows:1.The antimicrobial spectrum and growth characteristics of the strain 2F-1 were determined.Results showed that this antagonistic strain 2F-1 had a wide antimicrobial spectrum,was an obligate aerobic microorganism with high demand for oxygen,grows well at pH 4.0-10.0,and can adapt to osmotic pressure in a wide range from 0.5 g/L to 15 g/L of NaCl.When the strain 2F-1 had been irradiated under the UV lamp 30 cm away for 60 min,its growth was significantly inhibited,and this result means that strain 2F-1 is sensitive to UV light.2.Order to isolate and identify the secondary metabolites of stain 2F-1,chromatography of the crude extracts from its ferment liquid was performed by Sephadex G-50 and Sephadex G-15 gel column,and the effluents corresponding to the absorption peaks were collected to determine the antagonistic activity against Fusarium oxysporum f.sp.lycopersici.The effluents with antagonistic activity were frozen and concentrated.Analysis of LC-MS combined with NIST database indicated that the antagonistic subsatnces might be anthraquinones or amines.3.Results of whole genome sequencing of Actinomyces 2F-1 showed that genome total length of strain 2F-1 was 7590656 bp with 72.3%GC.It contained 7258 protein-coding genes（CDs）,88 tRNA genes and 21 rRNA genes.General function prediction indicated that most of the genes were involved in the metabolism of carbohydrates and amino acids.Using antiSMASH software,24 clusters of secondary metabolite synthesis genes including three polyketide（PKS）types,five non-ribosomal peptide（NRPS）types,six terpene,three heterozygous types,three siderophore,melanin,RiPP-like,one lanthipeptide-class-i and one lanthipeptide-class-iv were predicted.Three of them showed 100%similarity to gene clusters encoding known compounds（JBIR-126,alkylresorcinol,venezuelin）.Sixteen gene clusters had the similarity between 3%and 90%to known gene clusters,and the remaining 5 gene clusters could not be matched.4.Wild-type strain 2F-1 was mutated by UV,and four mutant strains with weakened antagonistic activity against E.oxysporun f.sp.lycopersici were obtained.Among these mutant strains,UV80S26 lost its antagonistic activity completely.By whole genome resequencing of UV80S26 mutant,18 missense mutated genes were located.Among them,glgEl,acsA₄,pip₃ and parB genes were involved in the synthesis of secondary metabolites of Actinomyces.5.The metabolites of wild-type strain 2F-1 and its mutant strain UV80S26 were analyzed based on high-resolution mass spectrometry（HRMS）detection technique,combined with HMDB public database for substance annotation.At the same time,combined the multivariate statistical analysis of the VIP value（VIP≥1）of OPLS-DA and the univariate statistical analysis of the P value（P<0.05）of t-test to screen the differential metabolites between different strains were performed.43 down-regulated differential metabolites were obtained,including organic acids and their derivatives,lipids and lipid-like molecules,benzene types,organic heterocycles,organic nitrogen,organic oxygen,alkaloid and its derivatives,and unknown compounds.All down-regulated metabolites were analyzed one by one,and it was found that Amphotericin b,a polyene macrolide antifungal substance which only 2.7%of that in the control,had a good inhibitory effect on F.oxysporum f.sp.lycopersici.Therefore,we think that Amphotericin b,a polyketide compound in strain 2F-1,may play the antagonistic function against pathogens.6.The genetic transformation system of Actinobacteria 2F-1 was established,and the GFP gene was constructed into overexpression vector pSET152.The recombinant plasmid pSET152-GFP was obtained and transformed into E.coli 12567.The positive transformants were conjugational transferred with strain 2F-1 to obtain the transformants that could stably express GFP.The correlation between the missense mutation gene and the antibioticproducing ability of strain 2F-1 will be further studied in the future.