| Micromonospora is a rare actinomycete that produces the most types and amounts of antibiotics except Streptomyces.It has excellent metabolic potential and can be used as a high-quality strain resource for antibiotic exploitation.In this study,31 strains of Micromonospora(belonging to 14 species)were identified from 547 strains of actinomycetes isolated from the sediment of the Tarim River.The taxonomic status of Micromonospora was determined through 16 S r RNA sequencing and phylogenetic tree construction.At the same time,(1)Detecting the key enzyme genes involved in the synthesis of Macrolides antibiotics and Ansamycins in the strain.(2)Four media were used for small-batch fermentation.The fermentation products were extracted and concentrated with 80% methanol.Using Escherichia coli,Staphylococcus aureus,Candida albicans and Erwinia amylovory as targets,the antibacterial activity of crude extracts was determined by the filter paper method.(3)The fermentation products of the strains were detected by Perform high-performance liquid chromatography(HPLC)detection.Preliminary exploration of metabolic potential of 31 strains of Micromonospora.It was found that 24 Micromonospora strains had the PKS-I gene,15 strains had the AHBA gene;There were 6 strains with antibacterial activities against Escherichia coli,4 strains against Staphylococcus aureus,9 strains against Candida albicans,and 21 strains against Erwinia amylovory,respectively;20 strains had both key enzyme genes for antibiotic synthesis and antibacterial activity.M.fluminis TRM99188,M.chalcea TRM99160,M.oryzae TRM99089,and M.purpureochromogenes TRM99166 showed antibacterial activity against two or more pathogens,which could be used for further exploration of secondary metabolites.The genome of four strains of Micromonospora was sequenced.The total length of the strain TRM99188 was 6842402 bp,(G+C)% was 73.33%,which was composed of 109 contigs and encoded 5961 genes.A total of 2405 protein-coding genes have been annotated in the KEGG database,accounting for 40.87% of the total protein sequence.According to the prediction of antiSMASH,the TRM99188 genome carries 33 gene clusters related to the synthesis of secondary metabolites,of which 10 gene clusters are related to antibiotic biosynthesis.The total length of the genome of strain TRM99089 is 7339319 bp,(G+C)% is73.80%,which is composed of 156 contigs and encodes 6350 genes.A total of 2519 protein-coding genes have been annotated in the KEGG database,accounting for 40.11% of the total protein sequence.It carries45 gene clusters related to the synthesis of secondary metabolites,of which 18 gene clusters are related to antibiotic biosynthesis.The total length of the strain TRM99160 genome is 6613541 bp,(G+C)% is73.01%,consisting of 73 contigs,encoding 6038 genes.A total of 2461 protein-coding genes have been annotated in the KEGG database,accounting for 41.23% of the total protein sequence,carrying 23 gene clusters related to the synthesis of secondary metabolites,7 of which are related to antibiotic biosynthesis.The total length of the genome of strain TRM99166 is 6964521 bp,(G+C)% is 72.61%,and consists of 157 contigs,encoding 6496 genes in total.A total of 2387 protein-coding genes have been annotated in the KEGG database,accounting for 37.19% of the total protein sequence.It carries 15 gene clusters related to the synthesis of secondary metabolites,including 4 gene clusters related to antibiotic biosynthesis.Based on the results of genomic analysis,the No.1 medium was selected for the mass fermentation of M.fluminis TRM99188.The fermentation products were separated into bacterial extract and fermentation broth extract,and the fermentation products were tested for bioactivity,TLC and HPLC.The results showed that the fermentation broth crude extract and bacterial crude extract had antagonistic activity against four target bacteria,respectively.HPLC detection showed that the number of substance absorption peaks in the HPLC spectrum of the fermentation broth extract was the highest,and the fermentation products mainly existed in the fermentation broth.HSQC-TOCSY detection was performed on the fermentation broth extract of strain TRM99188,and it was found that it has multiple signals such as polyketone region,amino sugar region,dilute region,sugar region,aromatic ring region,etc.Silica gel column chromatography and gel column chromatography for separation and purification,and NMR was used for structural identification.Four compounds were isolated: indole-3-carboxaldehyde,Genistein,Daidzein and Formononetin. |
