Genome Mining Of Four Rare Actinomycete Strains For Discovery Of Novel Natural Products

With the development of genome sequencing technologies,researchers have found that rare actinomycetes contain abundant mysterious secondary metabolic pathways,revealing their potential to produce novel natural products.However,there are few studies on genomic mining of rare actinomycetes.This project aims at targeted discovery of novel secondary metabolites in rare actinomycetes.The outcomes of this project may provide new leads for further drug development.Previously,a number of 3-amino-5-hydroxybenzoic acid(AHBA)synthase gene positive rare actinomycetes were obtained by PCR screening.By bioinformatics analysis of the genome of these strains,we identified two novel pentaketide-type ansamycin biosynthetic gene clusters,mas and vas,in Micromonospora sp.HK160111 and Verrucosispora sp.NS0172,respectively,and an atypical PKS/NRPS hybrid ansamycin biosynthetic gene cluster in 14 Micromonospora sp.strains.In order to identify the compounds encoded by the above gene clusters,we established the genetic manipulation system for these AHBA synthase gene positive strains,and evaluated the activities of the promoters in these strains.First of all,we decided to activate the two novel 'pentaketide-type ansamycin biosynthetic gene clusters.For Micromonospora sp.HK160111,the mas gene cluster was activated by constitutively expression of the positive regulator gene mas13,and nine novel ansamycin microansamycin A-I were successfully isolated and identification through large scale fermentation.Using the same strategy,the vas gene cluster in Verrucosispora sp.NS0172 was also activated by constitutively expression of the vas12 gene,and two precursors were obtained from the mutant.For the atypical PKS/NRPS hybrid ansamycin biosynthetic gene cluster,we first deleted the biosynthetic essential gene in the target gene cluster in Micromonospora sp.FXYA29 and Micromonospora sp.FXY120.HPLC analysis of the metabolites of the mutant strains compared with the corresponding wild type indicated that the target gene cluster in these two strains are probably silent.Through constitutively expression of two LAL family regulator genes,one intermediate compound was isolated from the mutant,which indicated that partial of the target gene cluster was activated.In order to get the final products encoded by this kind of gene cluster,we adopt another approach.In brief,the fragment containing the promoter region of the first PKS gene was cloned upstream of the black pigment reporter gene,then the plasmid was integrated into the wild type strain to generate indicator strain,which will be cultivated on 22 different medium to identify the black pigment produced media.Now,we have identified that Micromonospora sp.FXY120 may produce the target compound in HI medium,which need to be further verified.In addition,bioinformatics analysis of the genome of Micromonospora sp.FXY120 revealed that it contains many novel secondary metabolic pathways.Through constitutive expression of positive regulator genes and promoter engineering,we obtained four mutants,the metabolites of which contain extra products compared with the wild type.The products remain to be isolated.The above results indicated that combining the traditional natural product discovery methods and genome-based mining strategies will facilitate the isolation of novel natural products in rare actinomycetes.It will set the foundation for further structure modifications and yield improvement.Overall,our work provided a good example for mining novel natural products in other resources.