Identification of the biosynthetic gene cluster for the antibiotic polyketide L-155,175 in Streptomyces hygroscopicus.
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.
Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, 5 Anam-dong, Seongbuk-gu, Seoul, Republic of Korea.
Eun Young
EY
Kim
Jae Woo
JW
Han
Jee Yeon
JY
Lee
Beom Seok
BS
Kim
D000890:Anti-Infective Agents / Q000378:metabolism*; D053898:Biosynthetic Pathways / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D055786:Gene Knockout Techniques; D015723:Gene Library; D018942:Macrolides / Q000378:metabolism*; D008969:Molecular Sequence Data; D005810:Multigene Family; D016366:Open Reading Frames; D061065:Polyketides / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism*
D000890:Anti-Infective Agents; D004269:DNA, Bacterial; D018942:Macrolides; D061065:Polyketides; C068817:hygrolidin K2