Jae Woo Han

Publications (2)2.36 Total impact

• Article: Identification of the biosynthetic gene cluster for the antibiotic polyketide L-155,175 in Streptomyces hygroscopicus.

  Eun Young Kim, Jae Woo Han, Jee Yeon Lee, Beom Seok Kim
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  ABSTRACT: 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.
  Folia Microbiologica 06/2012; 57(6):543-50. · 0.68 Impact Factor
• Article: Site-directed modification of the adenylation domain of the fusaricidin nonribosomal peptide synthetase for enhanced production of fusaricidin analogs.

  Jae Woo Han, Eun Young Kim, Jung Min Lee, Yun Sung Kim, Eunjung Bang, Beom Seok Kim
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  ABSTRACT: Fusaricidins produced by Paenibacillus polymyxa DBB1709 are lipopeptide antibiotics active against fungi and Gram-positive bacteria. The cyclic hexapeptide structures of fusaricidins are synthesized by fusaricidin synthetase, a non-ribosomal peptide synthetase. The adenylation domain of the third module (FusA-A3) can recruit L: -Tyr, L: -Val, L: -Ile, L: -allo-Ile, or L: -Phe, which diversifies the fusaricidin structures. Since the L: -Phe-incorporated fusaricidin analog (LI-F07) exhibits more potent antimicrobial activity than other analogs, we modified a specificity-conferring sequence in the substrate binding pocket of FusA-A3 to direct the enhanced production of LI-F07. Base on comparison to the adenylation domain of gramicidin S synthetase 1 and tyrocidine synthetase 1, both of which mainly activate L: -Phe, six mutant strains with altered FusA-A3 were generated using site-directed mutagenesis. M3 (I239W, I299V), M5 (I299V, G322A, V330I), and M6 (S239W, I299V, G322A, V330I) mutants produced significantly more LI-F07 than the wild-type strain.
  Biotechnology Letters 03/2012; 34(7):1327-34. · 1.68 Impact Factor