Study On Secondary Metabolites Of TRM 15522 Based On Genome Mining Technology

Microbial secondary metabolites represented by antibiotics have been an important breakthrough in the development of many new drugs for many years.They have a wide variety,rich activity,and a wide range of applications.Strtomyces alarensis TRM 15522 is a strain isolated from the soil of continuous cropping cotton fields by Laboratory of Microbiology and Genetic Resources,Tarim University.It is a new species of actinomycete has good inhibitory activity against Fusarium oxysporum,Verticillium dahliae,Staphylococcus aureus,Candida albicans and other agricultural pathogens.In order to study the secondary metabolites of TRM 15522 in depth,this study through the genome mining technology conducted whole-genome sequencing and analysis of TRM 15522,systematically excavated its secondary metabolites and related gene clusters.The main results are as follows:1.TRM 15522 whole genome analysis.COG analysis of the whole genome sequence of TRM 15522 showed that its protein functions are mainly concentrated in transcription,carbohydrate transport and metabolism,amino acid transport and metabolism,and signal transduction mechanisms.KEGG analysis shows that metabolism-related systems in the metabolic pathway account for a relatively high proportion.The anti SMASH analysis showed that its genome carries a complete gene cluster that synthesizes PKS,NRPS,terpenes,bacteriocins,peptides,siderophores,indole and other active metabolites.2.Induce TRM 15522 product changes through ribosome engineering technology.9 antibiotics commonly used in ribosome engineering,include gentamicin,neomycin,lincomycin,fusidic acid,rifamycin,chloramphenicol,kanamycin,paromocin and streptomycin were used to induce TRM 15522,and the product changes were determined by detecting the difference peaks.The analysis showed that Fus,Rif,Lin and Kan all induced TRM 15522 to produce new chromatographic peaks,but the peak time was different,which may be different products;Fus,Str,Lin,Kan,Par and Gent induced the compound peak area at 4.0 min increased significantly,among which the Gent induction effect was the best,reaching more than 10 times;Fus and Str induced the compound peak area at 8.2 min increased significantly,and the Fus induction effect was the best,reaching more than 2 times;Fus,Str,Lin,Chlo,Neo,Kan,and Par induced the compound peak area at 11.4 min increased significantly,and the induction effect is in the order of Par>Fus>Lin>Str>Neo>Chlo>Kan,the induced yield of Par increases up to 7 times.Kan,which has the worst induction effect,also has a 2-fold increase.3.Construction of gene cluster overexpression vector driven by kas Op strong promoter.Using in situ activation method and homologous double exchange technology,two vectors that containing the 5'-end upstream and downstream homology arms of the Cluster 27 and Cluster 38 core operons and Kan+kas Op fragments were designed and constructed respectively.Intended to activate the expression of gene clusters.4.Constructed a TRM 15522 genome library and selected active metabolite gene clusters for heterologous expression.The ligation of the TRM 15522 genomic DNA recovered by partial restriction digestion with the Cosmid vector and the packaging of the ligation product by the phage packaging protein resulted in constructed the Cosmid library of TRM 15522.Taking Cluster 11,Cluster 12 and Cluster 25 as the target gene clusters,from which Cosmids containing relatively complete three gene clusters were screened respectively,and the three Cosmids were recombined by the PCR-targeting method and integrated into the heterologous expression host strain S.albus genome.After fermenting the recombinant strains,the fermented products were detected by HPLC,and it was found that the heterologous expression strains of Cluster 12 and Cluster 25 both had differential peaks.To sum up,this study is based on the genome mining technology to study the secondary metabolites of TRM 15522,and initially reveals the biosynthetic potential of TRM 15522,which provides guiding ideas and technical support for the gene cluster analysis and natural product mining of other similar strains in the future.