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

Zobrazit více v PubMed

J Am Chem Soc. 2002 May 15;124(19):5268-9 PubMed

Microbiology (Reading). 2005 Oct;151(Pt 10):3161-3169 PubMed

Nat Prod Rep. 2001 Aug;18(4):380-416 PubMed

Proc Natl Acad Sci U S A. 2003 Feb 18;100(4):1541-6 PubMed

Angew Chem Int Ed Engl. 2009;48(26):4688-716 PubMed

Gene. 2000 Jun 13;251(1):81-90 PubMed

Proc Natl Acad Sci U S A. 1998 Apr 28;95(9):4853-8 PubMed

Nat Prod Rep. 2009 Jan;26(1):90-114 PubMed

Gene. 2000 Mar 7;245(1):203-11 PubMed

FEMS Microbiol Lett. 2003 Jan 28;218(2):223-30 PubMed

Gene. 2002 Feb 20;285(1-2):257-67 PubMed

Structure. 2010 Jan 13;18(1):94-105 PubMed

J Am Chem Soc. 2010 May 12;132(18):6402-11 PubMed

Antimicrob Agents Chemother. 2002 May;46(5):1174-82 PubMed

J Antibiot (Tokyo). 2004 Oct;57(10):655-61 PubMed

Curr Opin Chem Biol. 2012 Apr;16(1-2):117-23 PubMed

Biochemistry. 2001 Dec 25;40(51):15464-70 PubMed

J Antibiot (Tokyo). 1984 May;37(5):610-3 PubMed

J Antibiot (Tokyo). 1983 Dec;36(12):1783-6 PubMed

J Antibiot (Tokyo). 1984 Feb;37(2):110-7 PubMed

Arch Microbiol. 2005 May;183(4):277-85 PubMed

J Antibiot (Tokyo). 1985 Feb;38(2):161-8 PubMed

Nat Prod Rep. 1998 Jun;15(3):221-40 PubMed

Org Biomol Chem. 2011 Apr 7;9(7):2053-6 PubMed

Evolution of cyclizing 5-aminolevulinate synthases in the biosynthesis of actinomycete secondary metabolites: outcomes for genetic screening techniques

Zobrazit více v PubMed

GENBANK
HE648167