Preliminary Study On The Expression Of Halophilic Actinomyces YIM 96748 Gene Cluster

Amycolatopsis is a kind of microbial resource with great application prospects,and can produce a large number of biologically active secondary metabolites.At present,rifamycin,vancomycin,balimycin,chloramphenicol,telavancin,oritavancin,dethymicin and other important antibiotics have been excavated from Amycolatopsis,which has made enormous contributions to human health.With the widespread use of antibiotics,pathogenic microorganisms have evolved complex antibiotic resistance mechanisms,and drug-resistant pathogenic microorganisms continue to emerge,posing a serious threat to human health.The development of new antibiotics,especially those with new modes of action,is one of the necessary elements to avoid the catastrophic epidemic of drug-resistant pathogenic microorganisms in the future.Halophilic actinomycetes are a group of microorganisms that can tolerate high-salt environments.They need to produce corresponding physiological mechanisms in order to adapt to the high-saline environment.In the process of adapting to the environment,they may produce secondary metabolites different from other actinomycete species.Through research on halophilic actinomycetes,it is possible to discover new active metabolites and thus develop antibiotics with new modes of action.The experimental strain YIM 96748 was isolated from soil samples collected from Xinjiang Salt Lake.First,the growth conditions of the strains are studied.The analysis of16S r RNA phylogenetic tree,genome-wide phylogenetic tree,ANI value(88.97%)and DNA-DNA value(59.60%)of YIM 96748 and reference strain Amycolatopsis cihanbeyliensis BNT52~T showed that the YIM 96748 is a potentially new species of Amycolatopsis.YIM96748 genomic data,through the integrated use of anti SMASH,NP.Searcher,PRISM 3,Deep BGC,clusterfinder,ARTS and other genome mining tools,found that it has a 32 gene clusters with potentially diverse functions,and the potential to produce a variety of new active compounds.The experimental strain YIM 96748 was isolated from soil samples collected from Xinjiang Salt Lake.The results of YIM 96748 16S r RNA gene sequence comparisonshowed that Amycolatopsis cihanbeyliensis BNT52~T was the strain with the highest similarity,with a similarity of 98.96%,and the other strains'similarities were lower than97%.The DNA-DNA hybridization value and the ANI value of YIM 96748 and Amycolatopsis cihanbeyliensis BNT52T are 59.60%,88.97%respectively,indicating that YIM 96748 and Amycolatopsis cihanbeyliensis BNT52T are different species.Further polyphasic Taxonomy studies indicate that YIM 96748 is a potential new species of Amycolatopsis.Through the analysis of YIM 96748 genomic data,it was found that it has 32 gene clusters with potentially diverse functions and the potential ability to produce a variety of new active compounds.Preliminary results of activity screening show that YIM 96748has activity against Staphylococcus aureus and iron carrier compounds,which is of great research value.First,traditional fermentation studies were carried out on the YIM 96748 gene cluster.After media selection,fermentation was carried out using 1#medium.Finally,4.16g of crude extract was obtained,from which two known compounds were isolated,4-Methoxyphenylacetic acid and Ergosterol endoperoxide.The experimental results show that the halophilic actinomycete YIM 96748 adopts traditional fermentation and the functional gene cluster has little or no expression,which requires new attempts.Therefore,we conducted a preliminary exploration of heterologous expression.In order to clone potential gene clusters,a YIM 96748 genomic library containing1800 clones was first constructed.In the process of diversity sequencing and detection of cloned fragments of the genomic library,clones containing the complete fragment of gene cluster 18 were obtained.This gene cluster has the potential to synthesize new types of terpenoids,and it was used as the target gene cluster for heterologous expression.explore.The gene cluster 18 was successfully introduced into a heterologous expression engineering strain Streptomyces albus J1074.Ten medium were used for preliminary fermentation exploration of the expression products of engineering strains,and A3medium was selected for expansion fermentation to obtain metabolites of the target gene cluster.At present,5 samples that may contain the expression product of gene cluster 18have been obtained.This study on the expression of YIM 96748 gene clusters shows that it is feasible for halophilic actinomycetes to obtain expression products of functional gene clusters by heterologous expression methods.It has an important guiding significance for the subsequent mining of halophilic Actinomycetes'resources in the laboratory...