Genome sequencing projects revealed massive cryptic gene clusters encoding the undiscovered secondary metabolites in Streptomyces. To investigate the metabolic products of silent gene clusters in Streptomyces chattano...Genome sequencing projects revealed massive cryptic gene clusters encoding the undiscovered secondary metabolites in Streptomyces. To investigate the metabolic products of silent gene clusters in Streptomyces chattanoogensis L10(CGMCC 2644), we used site-directed mutagenesis to generate ten mutants with point mutations in the highly conserved region of rpsL(encoding the ribosomal protein S12) or rpoB(encoding the RNA polymerase β-subunit). Among them, L10/RpoB(H437 Y) accumulated a dark pigment on a yeast extract-malt extract-glucose(YMG) plate. This was absent in the wild type. After further investigation, a novel angucycline antibiotic named anthrachamycin was isolated and determined using nuclear magnetic resonance(NMR) spectroscopic techniques. Quantitative real-time polymerase chain reaction(qRT-PCR) analysis and electrophoretic mobility shift assay(EMSA) were performed to investigate the mechanism underlying the activation effect on the anthrachamycin biosynthetic gene cluster. This work indicated that the rpoB-specific missense H437 Y mutation had activated anthrachamycin biosynthesis in S. chattanoogensis L10. This may be helpful in the investigation of the pleiotropic regulation system in Streptomyces.展开更多
基金Project supported by the National Natural Science Foundation of China(Nos.31520103901 and 3173002)
文摘Genome sequencing projects revealed massive cryptic gene clusters encoding the undiscovered secondary metabolites in Streptomyces. To investigate the metabolic products of silent gene clusters in Streptomyces chattanoogensis L10(CGMCC 2644), we used site-directed mutagenesis to generate ten mutants with point mutations in the highly conserved region of rpsL(encoding the ribosomal protein S12) or rpoB(encoding the RNA polymerase β-subunit). Among them, L10/RpoB(H437 Y) accumulated a dark pigment on a yeast extract-malt extract-glucose(YMG) plate. This was absent in the wild type. After further investigation, a novel angucycline antibiotic named anthrachamycin was isolated and determined using nuclear magnetic resonance(NMR) spectroscopic techniques. Quantitative real-time polymerase chain reaction(qRT-PCR) analysis and electrophoretic mobility shift assay(EMSA) were performed to investigate the mechanism underlying the activation effect on the anthrachamycin biosynthetic gene cluster. This work indicated that the rpoB-specific missense H437 Y mutation had activated anthrachamycin biosynthesis in S. chattanoogensis L10. This may be helpful in the investigation of the pleiotropic regulation system in Streptomyces.
