ABSTRACT
The afsRS(cla) global regulatory genes from Streptomyces clavuligerus activate the production of two antibiotics in Streptomyces lividans. In this study, we gained an increase of 38% in the production of natamycin (3.56 g/L) in an industrial strain Streptomyces gilvosporeus TZ1401 through the integration of pHL851 that bears the afsRS(cla) global regulatory genes into its genome. We discovered by quantitive real-time reverse transcription PCR (qRT-PCR) that the expression of 6 genes of the natamycin biosynthetic gene cluster were improved from 1.9 to 2.7 times. This suggests that afsRS(cla) improve the production of natamycin through increased transcription. This study provides a good example for applying afsRS(cla) in high yield breeding of industrial antibiotic producers.
Subject(s)
Anti-Bacterial Agents , Genes, Regulator , Industrial Microbiology , Multigene Family , Natamycin , Streptomyces , GeneticsABSTRACT
Tunicamycin, a potent reversible translocase I inhibitor, is produced by several Actinomycetes species. The tunicamycin structure is highly unusual, and contains an 11-carbon dialdose sugar and an α, β-1″,11'-glycosidic linkage. Here we report the identification of a gene cluster essential for tunicamycin biosynthesis by high-throughput heterologous expression (HHE) strategy combined with a bioassay. Introduction of the genes into heterologous non-producing Streptomyces hosts results in production of tunicamycin by these strains, demonstrating the role of the genes for the biosynthesis of tunicamycins. Gene disruption experiments coupled with bioinformatic analysis revealed that the tunicamycin gene cluster is minimally composed of 12 genes (tunA-tunL). Amongst these is a putative radical SAM enzyme (Tun B) with a potentially unique role in biosynthetic carbon-carbon bond formation. Hence, a seven-step novel pathway is proposed for tunicamycin biosynthesis. Moreover, two gene clusters for the potential biosynthesis of tunicamycin-like antibiotics were also identified in Streptomyces clavuligerus ATCC 27064 and Actinosynnema mirums DSM 43827. These data provide clarification of the novel mechanisms for tunicamycin biosynthesis, and for the generation of new-designer tunicamycin analogs with selective/enhanced bioactivity via combinatorial biosynthesis strategies.
Subject(s)
Actinobacteria , Genetics , Base Sequence , Biological Assay , Carbohydrate Sequence , Carbohydrates , Genetics , Cloning, Molecular , Gene Deletion , Gene Library , High-Throughput Screening Assays , Molecular Sequence Data , Multigene Family , Recombinant Proteins , Genetics , Sequence Analysis, DNA , Streptomyces , Genetics , Tunicamycin , Chemistry , GeneticsABSTRACT
The flanking fragments of the whiE(a) gene cluster was PCR amplified, cloned and used to construct the gene replacement plasmid pHL643. pHL643 was conjugated into Streptomyces avermitilis NRRL8165 followed by screening for double crossover event, yielding three apramycin resistance and thiostrepton sensitive isolates named ZJ1, ZJ2 and ZJ3, which were deficient in biosynthesis of the grey spore pigment. The whiE(a) gene replacement of these isolates was confirmed by Southern hybridization. Fermentation of the mutant strains in shaking flasks and HPLC analyses showed that the production of avermectins increased by 47% compared with that of the wild type, indicating that the spore pigment biosynthesis competes with the avermectins biosynthetic pathway.