title: Establishing a new methodology for genome mining and biosynthesis of polyketides and peptides through yeast molecular genetics.
abstract: Fungal genome sequencing has revealed many genes coding for biosynthetic enzymes, including polyketide synthases and nonribosomal peptide synthetases. However, characterizing these enzymes and identifying the compounds they synthesize remains a challenge, whether the genes are expressed in their original hosts or in more tractable heterologous hosts, such as yeast. Here, we developed a streamlined method for isolating biosynthetic genes from fungal sources and producing bioactive molecules in an engineered Saccharomyces cerevisiae host strain. We used overlap extension PCR and yeast homologous recombination to clone desired fungal polyketide synthase or a nonribosomal peptide synthetase genes (5-20 kb) into a yeast expression vector quickly and efficiently. This approach was used successfully to clone five polyketide synthases and one nonribosomal peptide synthetase, from various fungal species. Subsequent detailed chemical characterizations of the resulting natural products identified six polyketide and two nonribosomal peptide products, one of which was a new compound. Our system should facilitate investigating uncharacterized fungal biosynthetic genes, identifying novel natural products, and rationally engineering biosynthetic pathways for the production of enzyme analogues possessing modified bioactivity.
authors.affiliation: Department of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, 422-8526, Japan.
authors.forename: Kan'ichiro
authors.initials: K
authors.lastname: Ishiuchi
authors.forename: Takehito
authors.initials: T
authors.lastname: Nakazawa
authors.forename: Takashi
authors.initials: T
authors.lastname: Ookuma
authors.forename: Satoru
authors.initials: S
authors.lastname: Sugimoto
authors.forename: Michio
authors.initials: M
authors.lastname: Sato
authors.forename: Yuta
authors.initials: Y
authors.lastname: Tsunematsu
authors.forename: Noriyasu
authors.initials: N
authors.lastname: Ishikawa
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Noguchi
authors.forename: Kinya
authors.initials: K
authors.lastname: Hotta
authors.forename: Hisao
authors.initials: H
authors.lastname: Moriya
authors.forename: Kenji
authors.initials: K
authors.lastname: Watanabe
mesh_terms: D053898:Biosynthetic Pathways; D005818:Genetic Engineering / Q000379:methods; D016681:Genome, Fungal; D015394:Molecular Structure; D010453:Peptide Synthases / Q000235:genetics* / Q000378:metabolism; D048630:Polyketide Synthases / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D048630:Polyketide Synthases; D010453:Peptide Synthases