Functional Gene Mining Of Three High-Salt Environmental Actinomvcetes And Preliminary Studies On Heterologous Expression Of 7 Gene Clusters Of Strain YIM 96748

As the secondary metabolites of microorganisms,natural products have complex and changeable structures and have antibacterial,anti-tumor and immunosuppressive activities,which play an important role in the fields of industry,agriculture,food and medical treatment.At present,the repetition rate of traditional fermentation separation is high and the development speed of new antibiotics are slow.To solve such problems,it is necessary to constantly update the microbial resource library and use efficient methods to obtain new metabolites.Actinomycetes in high salt environment are adapted to the special environment of high salt concentration.They have abundant gene cluster types and unique metabolic pathways and may produce a large number of new compouds with biological activit,which have a great prospect for the development of drugs and new antibiotics.The mining of functional genes and heterologous expression can find invisible or silent gene clusters,and efficiently obtain metabolites through heterologous expression methods,which can increase the utilization value of potential gene clusters.The experimental strains YIM 98790,YIM 98757^T and YIM S01182 were isolated from salt marsh samples of high-salt lakes in Xinjiang.Through polyphasic classification studies,the strains YIM 98757^T and YIM 98790 were determined to be new species of Holoechinothrix and Streptomyces,respectively,and YIM S01182 was a new genus of Nocardiopsaceae family.Use antiSMASH analysis tools to scan and analyze the genomes of YIM 98790,YIM98757^T,and YIM S01182.A total of 39 potential gene clusters were mined,including PKS,NRPS,saccharide,fatty?acid,terpene etc 12 single types and 11 types of heterozygous.This article mainly introduces two types of natural products,PKS and NRPS.Among them,antibiotic compouds such as Dynemicin A,Neocarzinostatin and Uncialamycin have important applications in the fields of medicine,industry,agriculture and animal husbandry.The experimental strain YIM 96748 is a halophilic actinomycete with siderophore activity in laboratory.Based on the establishment of the YIM 96748 genome library,this study performed heterologous expression of 7 potential gene clusters.First,prepare probes and screen from the library by colony in situ hybridization to obtain 7 single clones of gene clusters.Then,the 7 target fragments were first introduced into E.coli ET12567/p UZ8002 by electrotransformation,and then the target fragments were introduced into 4 commonly used heterologous expression host bacteria by conjugation transfer method.Finally,a total of 27 engineered strains except S.albus-cluster 26-b were obtained.And successfully verified 26 key genes in 13 engineered strains,including S.albus-cluster 3,S.albus-cluster 4 and S.albus-cluster 27,which were completely verified.Two engineering strains of S.albus-cluster3 and S.albus-cluster 4were fermented and cultured on 15 media.It was found that S.albus-cluster 3 appeared a series of new absorption peaks in medium 2,9,10 and 15,and it was speculated that the gene cluster was expressed.The No.9 medium was selected to expand the fermentation of S.albus-cluster 3,29 g of crude fermentation extract was obtained.Through the prediction and analysis of the gene clusters of experimental strains YIM98790,YIM 98757^T and YIM S01182,it has an important guiding role for the in-depth study of the subsequent metabolite acquisition and heterologous expression of the strain.The study on heterologous expression of 7 gene clusters of strain YIM 96748 shows that it is feasible to import gene clusters into host bacteria to obtain metabolites,which has important guiding significance for heterologous expression of potential gene clusters,and can promote the full development and utilization of potential gene clusters of halophilic actinomycetes.