title: Aspergillus oryzae CsyB catalyzes the condensation of two β-ketoacyl-CoAs to form 3-acetyl-4-hydroxy-6-alkyl-α-pyrone.
abstract: The type III polyketide synthases from fungi produce a variety of secondary metabolites including pyrones, resorcinols, and resorcylic acids. We previously reported that CsyB from Aspergillus oryzae forms α-pyrone csypyrone B compounds when expressed in A. oryzae. Feeding experiments of labeled acetates indicated that a fatty acyl starter is involved in the reaction catalyzed by CsyB. Here we report the in vivo and in vitro reconstitution analysis of CsyB. When CsyB was expressed in Escherichia coli, we observed the production of 3-acetyl-4-hydroxy-α-pyrones with saturated or unsaturated straight aliphatic chains of C9-C17 in length at the 6 position. Subsequent in vitro analysis using recombinant CsyB revealed that CsyB could accept butyryl-CoA as a starter substrate and malonyl-CoA and acetoacetyl-CoA as extender substrates to form 3-acetyl-4-hydroxy-6-propyl-α-pyrone. CsyB also afforded dehydroacetic acid from two molecules of acetoacetyl-CoA. Furthermore, synthetic N-acetylcysteamine thioester of β-ketohexanoic acid was converted to 3-butanoyl-4-hydroxy-6-propyl-α-pyrone by CsyB. These results therefore confirmed that CsyB catalyzed the synthesis of β-ketoacyl-CoA from the reaction of the starter fatty acyl CoA thioesters with malonyl-CoA as the extender through decarboxylative condensation and further coupling with acetoacetyl-CoA to form 3-acetyl-4-hydroxy-6-alkyl-α-pyrone. CsyB is the first type III polyketide synthase that synthesizes 3-acetyl-4-hydroxy-6-alkyl-α-pyrone by catalyzed the coupling of two β-ketoacyl-CoAs.
authors.affiliation: From the School of Pharmacy, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba, Iwate 028-3694, Japan and.
authors.forename: Makoto
authors.initials: M
authors.lastname: Hashimoto
authors.affiliation: From the School of Pharmacy, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba, Iwate 028-3694, Japan and.
authors.forename: Tsukasa
authors.initials: T
authors.lastname: Koen
authors.affiliation: From the School of Pharmacy, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba, Iwate 028-3694, Japan and.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Takahashi
authors.affiliation: From the School of Pharmacy, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba, Iwate 028-3694, Japan and.
authors.forename: Chihiro
authors.initials: C
authors.lastname: Suda
authors.affiliation: the Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Kitamoto
authors.affiliation: From the School of Pharmacy, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba, Iwate 028-3694, Japan and ifujii@iwate-med.ac.jp.
authors.forename: Isao
authors.initials: I
authors.lastname: Fujii
mesh_terms: D000214:Acyl Coenzyme A / Q000737:chemistry / Q000378:metabolism; D001236:Aspergillus oryzae / Q000201:enzymology* / Q000235:genetics; D002384:Catalysis; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal; D007700:Kinetics; D008954:Models, Biological; D015394:Molecular Structure; D048630:Polyketide Synthases / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D011753:Pyrones / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D000214:Acyl Coenzyme A; D005656:Fungal Proteins; D011753:Pyrones; D011994:Recombinant Proteins; D048630:Polyketide Synthases
keywords: Aspergillus; Enzyme Catalysis; Enzyme Mechanism; Fatty Acid Metabolism; Natural Product Biosynthesis; Polyketide; Type III Polyketide Synthase; α-Pyrone