Research On Intestinal Bacterial Diversity Of Eight Species Of Fish In Beibu Gulf And Preliminary Exploration On Heterologous Expression Technology

Intestinal microbes are an important resource bank for developing cultivable microbial resources and mining new biosynthetic gene clusters and bioactive substances.This study is focused on the pure cultured bacteria,the bacterial diversity and strain application potential of eight species of marine fish in the Beibu Gulf of Guangxi were studied.In addition,a detailed bacterial taxonomic study was conducted on a potential new taxa isolated from Asian elephant feces.Finally,the heterologous expression of the bacterial secondary metabolite biosynthetic gene cluster was initially explored.Firstly,we isolated and cultured the intestinal bacteria of 8 species marine fish from Guangxi Beibu Gulf and analyzed their diversity.A total of 228 strains bacteria were isolated by using 5 types of isolation media.Among them,96 strains actinomycetes were distributed in10 orders,12 families,and 21 genera;132 strains non-actinomycetes were distributed in 3 phyla,4 classes,6 orders,9 families and 13 genera.When R2 A was used as isolation medium,it had good separation effect.Secondly,228 bacteria were eduplicated,and the potential application value of the isolates was evaluated in three aspects: potential new taxon,antibiotic biosynthesis gene and antipathogen activity.The 16 S r RNA similarity was lower than 98.7% as the threshold for species identification,a total of 13 potential new taxa were isolated,which distributed in 9 genera.The number of positive detection of Malacidin,Lugdunin,Teixobactin,Leinamycin,Glutarimide,Salinomycin,Diterpenes,PKS-II and Clavulanic acid were 1,0,2,12,8,25,10,11 and 9,respectively,distributed in 24 genera.The number of strains with activity against Gibberella saubinetii,Fusarium coeruleum,Botrytis cinerea,Pseudomonas aeruginosa,Bacillus subtilis,Staphylococcus aureus,Mycobacterium tuberculosis,Escherichia coli,Candida albicans and Aspergillus niger were 9,11,11,3,14,16,14,12,15 and 14,respectively,distributed in 12 genera.Thirdly,through polyphasic taxonomic studies,strain YIM 103518 was identified as a novel species of genus Acinetobacter,named Acinetobacter faecale sp.nov..Finally,six biosynthetic gene clusters of secondary metabolites were heterologously expressed.The heterogenously expressed mutant strains of the 6 gene clusters were successfully constructed by conjugational transfer.The corresponding metabolites were not detected by using various fermentation media,further research and exploration were needed.The research suggests that gut microorganisms are not only the source of new culturable microbial species,but also have the potential to produce a variety of bioactive metabolites.What's more,gut microorganisms can also provide a large number of biosynthetic gene clusters for the study of synthetic biology.