title: Sequencing, physical organization and kinetic expression of the patulin biosynthetic gene cluster from Penicillium expansum.
abstract: Patulin is a polyketide-derived mycotoxin produced by numerous filamentous fungi. Among them, Penicillium expansum is by far the most problematic species. This fungus is a destructive phytopathogen capable of growing on fruit, provoking the blue mold decay of apples and producing significant amounts of patulin. The biosynthetic pathway of this mycotoxin is chemically well-characterized, but its genetic bases remain largely unknown with only few characterized genes in less economic relevant species. The present study consisted of the identification and positional organization of the patulin gene cluster in P. expansum strain NRRL 35695. Several amplification reactions were performed with degenerative primers that were designed based on sequences from the orthologous genes available in other species. An improved genome Walking approach was used in order to sequence the remaining adjacent genes of the cluster. RACE-PCR was also carried out from mRNAs to determine the start and stop codons of the coding sequences. The patulin gene cluster in P. expansum consists of 15 genes in the following order: patH, patG, patF, patE, patD, patC, patB, patA, patM, patN, patO, patL, patI, patJ, and patK. These genes share 60-70% of identity with orthologous genes grouped differently, within a putative patulin cluster described in a non-producing strain of Aspergillus clavatus. The kinetics of patulin cluster genes expression was studied under patulin-permissive conditions (natural apple-based medium) and patulin-restrictive conditions (Eagle's minimal essential medium), and demonstrated a significant association between gene expression and patulin production. In conclusion, the sequence of the patulin cluster in P. expansum constitutes a key step for a better understanding of the mechanisms leading to patulin production in this fungus. It will allow the role of each gene to be elucidated, and help to define strategies to reduce patulin production in apple-based products.
authors.affiliation: INRA, UMR 1331 Toxalim, Research Centre in Food Toxicology, 180 Chemin de Tournefeuille, F-31027 Toulouse Cedex, France; Université de Toulouse III, ENVT, INP, UMR 1331, Toxalim, F-31076, Toulouse, France; Université Saint-Joseph, Centre d'Analyses et de Recherches (Faculté des Sciences), Campus des Sciences et Technologies, Mar Roukos, Mkallès, P.O. Box 11-514 Riad El Solh, Beyrouth 1107 2050, Lebanon.
authors.forename: Joanna
authors.initials: J
authors.lastname: Tannous
authors.affiliation: INRA, UMR 1331 Toxalim, Research Centre in Food Toxicology, 180 Chemin de Tournefeuille, F-31027 Toulouse Cedex, France; Université de Toulouse III, ENVT, INP, UMR 1331, Toxalim, F-31076, Toulouse, France; Université Saint-Joseph, Centre d'Analyses et de Recherches (Faculté des Sciences), Campus des Sciences et Technologies, Mar Roukos, Mkallès, P.O. Box 11-514 Riad El Solh, Beyrouth 1107 2050, Lebanon.
authors.forename: Rhoda
authors.initials: R
authors.lastname: El Khoury
authors.affiliation: INRA, UMR 1331 Toxalim, Research Centre in Food Toxicology, 180 Chemin de Tournefeuille, F-31027 Toulouse Cedex, France; Université de Toulouse III, ENVT, INP, UMR 1331, Toxalim, F-31076, Toulouse, France.
authors.forename: Selma P
authors.initials: SP
authors.lastname: Snini
authors.affiliation: INRA, UMR 1331 Toxalim, Research Centre in Food Toxicology, 180 Chemin de Tournefeuille, F-31027 Toulouse Cedex, France; Université de Toulouse III, ENVT, INP, UMR 1331, Toxalim, F-31076, Toulouse, France.
authors.forename: Yannick
authors.initials: Y
authors.lastname: Lippi
authors.affiliation: Université Saint-Joseph, Centre d'Analyses et de Recherches (Faculté des Sciences), Campus des Sciences et Technologies, Mar Roukos, Mkallès, P.O. Box 11-514 Riad El Solh, Beyrouth 1107 2050, Lebanon.
authors.forename: André
authors.initials: A
authors.lastname: El Khoury
authors.affiliation: Laboratory of Microorganisms and Food Irradiation, Lebanese Atomic Energy Commission-CNRS, P.O. Box 11-8281, Riad El Solh, 1107 2260 Beirut, Lebanon.
authors.forename: Ali
authors.initials: A
authors.lastname: Atoui
authors.affiliation: Université Saint-Joseph, Centre d'Analyses et de Recherches (Faculté des Sciences), Campus des Sciences et Technologies, Mar Roukos, Mkallès, P.O. Box 11-514 Riad El Solh, Beyrouth 1107 2050, Lebanon.
authors.forename: Roger
authors.initials: R
authors.lastname: Lteif
authors.affiliation: INRA, UMR 1331 Toxalim, Research Centre in Food Toxicology, 180 Chemin de Tournefeuille, F-31027 Toulouse Cedex, France; Université de Toulouse III, ENVT, INP, UMR 1331, Toxalim, F-31076, Toulouse, France.
authors.forename: Isabelle P
authors.initials: IP
authors.lastname: Oswald
authors.affiliation: INRA, UMR 1331 Toxalim, Research Centre in Food Toxicology, 180 Chemin de Tournefeuille, F-31027 Toulouse Cedex, France; Université de Toulouse III, ENVT, INP, UMR 1331, Toxalim, F-31076, Toulouse, France. Electronic address: opuel@toulouse.inra.fr.
authors.forename: Olivier
authors.initials: O
authors.lastname: Puel
mesh_terms: D001483:Base Sequence; D003062:Codon; D017931:DNA Primers / Q000737:chemistry; D005638:Fruit / Q000382:microbiology; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D027845:Malus / Q000382:microbiology; D058977:Molecular Sequence Annotation; D008969:Molecular Sequence Data; D005810:Multigene Family; D010365:Patulin / Q000096:biosynthesis / Q000235:genetics*; D010407:Penicillium / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D012689:Sequence Homology, Nucleic Acid
chemical_list: D003062:Codon; D017931:DNA Primers; D010365:Patulin
keywords: Apples; Gene cluster; Gene expression; Mycotoxins; Patulin; Penicillium expansum