title: An Aspergillus flavus secondary metabolic gene cluster containing a hybrid PKS-NRPS is necessary for synthesis of the 2-pyridones, leporins.
abstract: The genome of the filamentous fungus, Aspergillus flavus, has been shown to harbor as many as 56 putative secondary metabolic gene clusters including the one responsible for production of the toxic and carcinogenic, polyketide synthase (PKS)-derived aflatoxins. Except for the production of aflatoxins, cyclopiazonic acid and several other metabolites the capability for metabolite production of most of these putative clusters is unknown. We investigated the regulation of expression of the PKS-non-ribosomal peptide synthetase (NRPS) containing cluster 23 and determined that it produces homologs of the known 2-pyridone leporin A. Inactivation and overexpression of a cluster 23 gene encoding a putative Zn(2)-Cys(6) transcription factor (AFLA_066900, lepE) resulted in downregulation of nine and up-regulation of 8, respectively, of the fifteen SMURF-predicted cluster 23 genes thus allowing delineation of the cluster. Overexpression of lepE (OE::lepE) resulted in transformants displaying orange-red pigmented hyphae. Mass spectral analysis of A. flavus OE::lepE extracts identified the known 2-pyridone metabolite, leporin B, as well as the previously unreported dehydroxy-precursor, leporin C. We provide strong evidence that leporin B forms a unique trimeric complex with iron, not found previously for other 2-pyridones. This iron complex demonstrated antiinsectan and antifeedant properties similar to those previously found for leporin A. The OE::lepE strain showed reduced levels of conidia and sclerotia suggesting that unscheduled leporin production affects fungal developmental programs.
authors.affiliation: U.S. Dept. of Agriculture, Southern Regional Research Center, New Orleans, LA 70124, USA. Electronic address: jeff.cary@ars.usda.gov.
authors.forename: Jeffrey W
authors.initials: JW
authors.lastname: Cary
authors.affiliation: Laboratory of Food Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium.
authors.forename: Valdet
authors.initials: V
authors.lastname: Uka
authors.affiliation: Laboratory of Food Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Han
authors.affiliation: Dept. of Organic Chemistry, Faculty of Sciences, Ghent University, Krijgslaan 281, 9000 Ghent, Belgium.
authors.forename: Dieter
authors.initials: D
authors.lastname: Buyst
authors.affiliation: U.S. Dept. of Agriculture, Southern Regional Research Center, New Orleans, LA 70124, USA.
authors.forename: Pamela Y
authors.initials: PY
authors.lastname: Harris-Coward
authors.affiliation: U.S. Dept. of Agriculture, Southern Regional Research Center, New Orleans, LA 70124, USA.
authors.forename: Kenneth C
authors.initials: KC
authors.lastname: Ehrlich
authors.affiliation: U.S. Dept. of Agriculture, Southern Regional Research Center, New Orleans, LA 70124, USA.
authors.forename: Qijian
authors.initials: Q
authors.lastname: Wei
authors.affiliation: U.S. Dept. of Agriculture, Southern Regional Research Center, New Orleans, LA 70124, USA.
authors.forename: Deepak
authors.initials: D
authors.lastname: Bhatnagar
authors.affiliation: U.S. Dept. of Agriculture, National Center for Agricultural Utilization Research, Peoria, IL 61604, USA.
authors.forename: Patrick F
authors.initials: PF
authors.lastname: Dowd
authors.affiliation: Dept. of Biological Sciences, Northern Illinois University, DeKalb, IL 60115, USA.
authors.forename: Stacey L
authors.initials: SL
authors.lastname: Martens
authors.affiliation: Dept. of Biological Sciences, Northern Illinois University, DeKalb, IL 60115, USA.
authors.forename: Ana M
authors.initials: AM
authors.lastname: Calvo
authors.affiliation: Dept. of Organic Chemistry, Faculty of Sciences, Ghent University, Krijgslaan 281, 9000 Ghent, Belgium.
authors.forename: José C
authors.initials: JC
authors.lastname: Martins
authors.affiliation: Dept. of Veterinary Public Health and Food Safety, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium.
authors.forename: Lynn
authors.initials: L
authors.lastname: Vanhaecke
authors.affiliation: Lab of Microbiology, Faculty of Pharmaceutical Sciences, Ottergemsesteenweg 460, 9000 Ghent, Belgium.
authors.forename: Tom
authors.initials: T
authors.lastname: Coenye
authors.affiliation: Laboratory of Food Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium.
authors.forename: Sarah
authors.initials: S
authors.lastname: De Saeger
authors.affiliation: Laboratory of Food Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium.
authors.forename: José Diana
authors.initials: JD
authors.lastname: Di Mavungu
mesh_terms: D001231:Aspergillus flavus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D005810:Multigene Family; D010453:Peptide Synthases / Q000235:genetics / Q000378:metabolism*; D010860:Pigments, Biological / Q000032:analysis; D048630:Polyketide Synthases / Q000235:genetics / Q000378:metabolism*; D011728:Pyridones / Q000378:metabolism*; D064210:Secondary Metabolism
chemical_list: D010860:Pigments, Biological; D011728:Pyridones; D048630:Polyketide Synthases; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase
keywords: Aspergillus flavus; Gene cluster; High resolution mass spectrometry; Leporin; Natural products; Secondary metabolism