Gene Cluster Mining Of Secondary Metabolite From Streptomyces Luozhongensis TRM49605 Based On Large Fragment Cloning Technology

Actinomycetes are rich in secondary metabolites,is an important source for the minging new drugs.Xinjiang's typical extreme saline-alkaline environment contains a large number of new species of actinomycetes,and may evolve unique secondary metabolites when adapting to extreme environments such as high salinity and alkalinity,therefore arouse the attention of researchers.In this study,a new Streptomyces species TRM49605 isolated from Lop Nur,a typical extreme saline-alkaline environment in Xinjiang was selected for gene cluster analysis,and three gene clusters were cloned and heterologously expressed by using Red/ET recombination technology.Through heterologous expression to determine the biological function of the biosynthetic gene cluster and expect to obtain secondary metabolites with novel structure and strong activity,at the same time,reserve more genetic resources for genetic modification and gene combination biology.(1)Analysis of secondary metabolic potential of Streptomyces luozhongensis In order to explore the secondary metabolic potential of Streptomyces luozhongensis TRM49605,whole genome sequencing and analysis was performed in this study.The results showed that genomic DNA of Streptomyces luozhongensis was 7 156 507 bp and 63 potential natural product biosynthetic gene clusters revealed by antiSMASH analysis.To probe the heterologous expression of different similarity gene clusters and assess the secondary metabolic potential of TRM49605,three representative gene clusters were selected for analysis,of which Cluster24 is a nucleoside antibiotic biosynthesis gene cluster,29 393 bp in length sonsists of 26 genes composition,with 92% similarity to the tunicamycin gene cluster;Cluster17 is17 066 bp in length and consists of 14 genes composition with 4% similar to the dutomycin biosynthesis gene cluster;the full length of Cluster36 is 44 395 bp,was composed of 42 genes and contains multiple valamycin-related genes,the similarity to the valinomycin gene cluster is 16%.The analysis results showthat Streptomyces luozhongensis has abundant secondary metabolic potential,which known biosynthetic gene clusters as well as those with unknown functions,and has the potential for deep mining.Genomic analysis and prediction of related gene clusters provide reference data for accurately assessing the secondary metabolic potential of Streptomyces luozhongensis,and at the same time provide a working basis for natural product mining in the post-genomic era.(2)Cloning and heterologous expression of gene clustersIn order to determine the biosynthetic potential of TRM49605 and the biological function of related gene clusters,Cluster17,Cluster24,and Cluster36 were cloned based on Red/ET system,and introduced the target gene cluster into S.coelicolor M145 by conjugal transfer to heterologous expression.Through HPLC detection analysis,the product of Cluster24,tunicamycin was obtained,consistent with the prediction;the expected products were not detected for the other 2 gene clusters,and no other characteristic peaks that distinguish them from the host bacteria were present.After replacing the heterologous host bacteria with S.lividans TK64 and fermentation medium,the target product and the difference from the heterologous host bacteria have not been found,the reason for analysis may be that the selected gene cluster is a defective gene cluster and does not have the ability to express a certain product.Streptomyces coelicolor::pTRM605-24-03,a recombinant bacterium carrying a tunicamycin gene cluster,was found to have significant antibacterial activity against Bacillus subtilis through the bioactivity experiments of tunicamycin heterologous expression products,which is a potential drug pilot compound,and is valuable for in-depth research.The results of this study indicate that gene clusters with higher similarity have a more complete biosynthetic enzyme system,and are more likely to obtain products by heterologous expression.In this study,based on gene cluster mining in the post-genomic era,initially revealed the biosynthetic potential of Streptomyces TRM49605,which will provide guidance and technical support for future gene cluster analysis and natural product mining.