Secondary Metabolites Research Of Actinomyces From Lichens Based On Genome Sequencing

Lichens are stable symbiotic organisms formed by fungi and cyanobacteria(or green algae).Lichens are widely distributed and can survive in a variety of extreme environments,they play an important role in the ecological environment by producing virtue of the metabolic products.In addition to symbiotic fungi and algae,there are a large number of bacteria in the lichens which provide nitrogen,phosphorus and growth hormone substances for fungi and algae in the lichens symbiosis system,even assist in photosynthesis and resistance to biotic stress,etc.Lichen bacteria contain a large number of actinomycetes which play an ecological protective role.However,there are few studies on the species diversity,special biological activities of lichens actinomycetes and their status and roles in lichens symbiotic ecosystems.Therefore,the laboratory carried out research on the metabolites of actinomycetes from lichens in order to clarify the biological status of lichen bacteria in the lichens symbiosis system.In this experiment,the whole-genome sequencing,functional annotation,activity analysis and product prediction were performed on the strain 9846 a from the lichens.According to the genome-wide shotgun strategy,the whole genome double-end sequencing of the rare actinomycetes strain 9846 a was carried out using the Illumina MiSeq platform Next-Generation Sequencing(NGS).The data of the strain 9846 a showed that the total number of Reads are 5,825,924,and the total number of bases are 172,494,754 bp,the bases with recognition accuracy above 99% account for81.63% of the total number of bases,and the GC content is 67.4%.Screening and evaluating the downtime data and filtering the low quality data,then recapitulate high-quality data for functional analysis and product prediction.The antibioticresistance gene and the antibiotic synthesis gene are simultaneously present in the strain 9846 a,at the same time,the CRISPRs palindromic sequence also exists.In the functional annotation,it was found that there are 4,125 protein-coding genes in the whole genome of strain 9846 a,and its gene products are mainly concentrated in biosynthesis processes,such as transportation and metabolism of carbohydrates and amino acids.In its metabolic pathway,63 genes related to the synthesis of antibacterial products were found,and 36 genes related to cell viability(cell replication and repair function),they were mapped to five metabolic pathways.The FASTA formatted file was uploaded to the antiSMASH platform to predict the secondary metabolites that can be produced by strain 9846 a.Through the alignment of homologous gene clusters,the main secondary metabolites that may be produced by strain 9846 a are natural carotenoids,branched fatty acids and alkyl resorcinol.On the basis of genome-wide analysis and product prediction,strain 9846 a was subjected to shaker fermentation using ISP2 liquid medium,co-fermentation 50 L,and the fermentation broth was extracted by steaming to obtain 5.9 g of crude extract.The antibacterial activity of the fermentation product was analyzed by double-layer plate method,and it was found to have an ideal inhibitory effect on Escherichia coli,Staphylococcus aureus and Bacillus subtilis.Then,using silica gel column chromatography,gel column chromatography,high-performance liquid phase preparation and countercurrent chromatography,the product was separated and purified to obtain 12 components.Nuclear magnetic resonance spectroscopy(NMR)and mass spectrometry(MS)were used to detect the alignment of the spectra,and the molecular formula and structural formula of the compound were inferred.The molecular and structural formulas of the compounds were deduced by comparison of NMR and MS spectra,six long-chain aliphatic hydrocarbons were identified,including alkanes and olefins.Long-chain aliphatic hydrocarbons can resist the adverse environment of the environment when the lichens system is subjected to external environmental stress such as high salt,low temperature and low oxygen stress,maintain the stability of the environment inside the lichens system.Long-chain aliphatic hydrocarbons can resist the adverse environment of the outside world andmaintain the stability of the internal environment of the lichens system,especially when the local clothing system is subjected to external environmental pressures such as high salt,low temperature and low oxygen.In this experiment,the whole genome sequencing and gene analysis of the rare actinomycetes strain 9846 a were carried out,the secondary metabolites were further isolated and identified and the activity analysis was provided,which provided a reference method for the development and utilization of natural products in lichen bacteria.In order to further clarify the status and role of lichen bacteria in the lichens symbiosis system,the research on lichen bacteria has positive significance.