Exploring Natural Product Biosynthesis Gene Cluster In Streptomyces Netropsis DSM 40846 By Genome Mining

Streptomyces netropsis DSM 40846 has attracted extensive attention because it can produce a variety of antibiotics,including the polyketone antibiotic aureothin,the pyrrolamide antibiotics distamycin A and congocidine.Bioinformatics analysis found that there are still plentiful natural product biosynthesis gene clusters with unknown functions in the genome sequence,including PKS,NRPS and Ri PP gene clusters,suggesting its potential for genome mining.In this study,these potential natural product biosynthesis gene cluster in the genome of S.netropsis DSM 40846 were investigated by genome mining.40 potential natural product biosynthetic gene clusters were discovered from the genome of S.netropsis DSM 40846 by anti SMASH analysis,including 22 PKS,NRPS and Ri PP gene clusters which might produce bioactive natural products.The constructed genomic cosmid library of S.netropsis DSM 40846 combined with the genomic BAC library constructed earlier were screened by PCR.the positive clones containing 18 natural product biosynthetic gene clusters were selected out and introduced into the host strain S.lividans or S.albus by conjugation.5 gene clusters products were found by detecting the metabolites of heterologous expression strains with bioassay and TLC.The following was the research content of these 5 gene clusters.The biosynthetic gene cluster of distamycin A is separated in two different regions part1 and part2.Only co-expresseion of two parts in one host strain can result in the production of distamycin A.The positive clones 16H5 and 18H11 respectively containing two parts of the gene cluster were screened from the library.After changing the vector,part1 and part2 were introduced into the heterologous host S.albus for co-expression by two compatible vectors p MSB152 B and p WHM4 S,respectively.The heterologous expression strain capable of producing distamycin A was successfully obtained.The gene cluster cluster21-1 has high homology with the lassopeptide citrulassin B biosynthetic gene cluster,suggesting that it most likely produces citrulassin B.The positive clone 3B8 of cluster21-1 was introduced into the heterologous expression host strain S.albus,heterologous expression strain S.albus::3B8 had significant inhibitory effect on Staphylococcus aureus.The results of HPLC detection also indicated the production of heterologous expression products.The gene cluster cluster11-2 has high homology with the isofurannaphthoquinone biosynthesis gene cluster.Heterologous expression strains S.albus::1H9 and S.albus::3E1containing cluster11-2 produced special red-brown pigments which showed bright orange fluorescence under 365 nm ultraviolet light.TLC detected at least 3 distinct components,HPLC analysis further determined their characteristic UV absorption peaks,and LC-HRMS detected their mass-to-charge ratios and possible molecular formulas.Based on the structural characteristics of isofurannaphthoquinone compounds,the structures of these heterologous expression products were preliminarily speculated.The type Ⅰ polyketide synthase gene clusters cluster15-1 and cluster8-1 have low homology with known natural product biosynthetic gene clusters,indicating they have the potential to produce new polyketide.The putative heterologous expression products were found in the heterologous expression strains of these two gene clusters by TLC and HPLC analysis,and the mass-to-charge ratio of the products were further determined by LC-HRMS analysis.We analyzed the genome of S.netropsis DSM 40846 and found 40 natural product biosynthetic gene clusters,screened positive clones of 18 PKS,NRPS or Ri PP-like biosynthetic gene clusters from the genomic library,and confirmed 5 gene clusters produced heterologous expression products by analyzing metabolites,which provided the basis for the discovery and structural elucidation of novel natural products.