Identification of the biosynthetic gene cluster for the antibiotic polyketide L-155,175 in Streptomyces hygroscopicus
Language English Country United States Media print-electronic
Document type Journal Article, Research Support, Non-U.S. Gov't
- MeSH
- Anti-Infective Agents metabolism MeSH
- Biosynthetic Pathways genetics MeSH
- DNA, Bacterial chemistry genetics MeSH
- Gene Library MeSH
- Gene Knockout Techniques MeSH
- Macrolides metabolism MeSH
- Molecular Sequence Data MeSH
- Multigene Family * MeSH
- Open Reading Frames MeSH
- Polyketides metabolism MeSH
- Sequence Analysis, DNA MeSH
- Streptomyces genetics metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Anti-Infective Agents MeSH
- DNA, Bacterial MeSH
- hygrolidin K2 MeSH Browser
- Macrolides MeSH
- Polyketides MeSH
The antibiotic L-155,175, a potent antiparasitic and antifungal compound, has an unusual structure involving 16-membered macrolides that contain a tetrahydropyran ring connected through a three-carbon linker chain. To identify the biosynthetic gene cluster for L-155,175, a genomic DNA library of Streptomyces hygroscopicus ATCC31955 was constructed and screened with a degenerate primer set designed from a conserved region of the ketosynthase (KS) domain. Sequence analysis of a fosmid clone, pEY1D8 (34 kb), revealed multiple open reading frames (ORFs) encoding type I polyketide synthase (PKS). To determine whether the cloned genes are involved in L-155,175 biosynthesis, a deletion mutant (1D8m) was generated by homologous recombination, in which the gene encoding the KS domain was substituted with an apramycin-resistance gene by PCR-targeted Streptomyces gene replacement. LC-MS analysis showed that L-155,175 production was completely abolished in the 1D8m strain, thereby proving that the cloned gene is responsible for L-155,175 biosynthesis. The sequencing of two other fosmid clones (pEY8B10 and pEY1C9) harboring overlapping sequences from pEY1D8 revealed a 60-kb DNA segment encoding six ORFs for type I PKS harboring 12 modules. The domain organization of the PKS modules encoded by PKS exactly matched the structure of L-155,175. This is the first report on the gene cluster involved in the biosynthesis of L-155,175.
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