title: Heterologous gene expression and functional analysis of a type III polyketide synthase from Aspergillus niger NRRL 328.
abstract: Type III polyketide synthases (PKSs) catalyze the formation of pyrone- and resorcinol-types aromatic polyketides. The genomic analysis of the filamentous fungus Aspergillus niger NRRL 328 revealed that this strain has a putative gene (chr_8_2: 2978617-2979847) encoding a type III PKS, although its functions are unknown. In this study, for functional analysis of this putative type III PKS designated as An-CsyA, cloning and heterologous expression of the An-CsyA gene (An-csyA) in Escherichia coli were performed. Recombinant His-tagged An-CsyA was successfully expressed in E. coli BL21 (DE3), purified by Ni(2+)-affinity chromatography, and used for in vitro assay. Tests on the substrate specificity of the His-tagged An-CsyA with myriad acyl-CoAs as starter substrates and malonyl-CoA as extender substrate showed that His-tagged An-CsyA accepted fatty acyl-CoAs (C2-C14) and produced triketide pyrones (C2-C14), tetraketide pyrones (C2-C10), and pentaketide resorcinols (C10-C14). Furthermore, acetoacetyl-CoA, malonyl-CoA, isobutyryl-CoA, and benzoyl-CoA were also accepted as starter substrates, and both of triketide pyrones and tetraketide pyrones were produced. It is noteworthy that the His-tagged An-CsyA produced polyketides from malonyl-CoA as starter and extender substrates and produced tetraketide pyrones from short-chain fatty acyl-CoAs as starter substrates. Therefore, this is the first report showing the functional properties of An-CsyA different from those of other fungal type III PKSs.
authors.affiliation: Department of Applied Chemistry, Faculty of Science and Engineering, Waseda Universrity, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo 169-8555, Japan. Electronic address: kkohtaro@waseda.jp.
authors.forename: Kohtaro
authors.initials: K
authors.lastname: Kirimura
authors.affiliation: Department of Applied Chemistry, Faculty of Science and Engineering, Waseda Universrity, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo 169-8555, Japan.
authors.forename: Shotaro
authors.initials: S
authors.lastname: Watanabe
authors.affiliation: Department of Applied Chemistry, Faculty of Science and Engineering, Waseda Universrity, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo 169-8555, Japan.
authors.forename: Keiichi
authors.initials: K
authors.lastname: Kobayashi
mesh_terms: D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000502:physiology; D048630:Polyketide Synthases / Q000737:chemistry* / Q000502:physiology*; D011753:Pyrones / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012118:Resorcinols / Q000378:metabolism*
chemical_list: D011753:Pyrones; D011994:Recombinant Proteins; D012118:Resorcinols; D048630:Polyketide Synthases; C031389:resorcinol
keywords: Aspergillus niger NRRL 328; Pyrone-type polyketide; Resorcinol-type polyketide; Triacetic acid lactone; Type III polyketide synthase