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Molecular cloning and functional characterization of two cytochromes P450 CYP619 involved in biosynthesis of patulin in Aspergillus clavatus

INRA

ABSTRACT

Patulin is an acetate-derived tetraketide mycotoxin produced by several fungal species, especially Aspergillus, Penicillium and Byssochlamys species. The health risks due to patulin consumption by humans lead many countries to regulate it in human food. Previous studies have shown the involvement of cytochrome P450 monooxygenases into hydroxylation of two precursors of patulin, m-cresol and m-hydrobenzylalcohol. In the present study, two cytochrome P450 genes were identified in the genome sequence of Aspergillus clavatus, a patulin producing species. Both mRNAs were strongly co-expressed during the patulin production. CYP619C2 encoded by the first gene displays 529 aa, while the second cytochrome CYP 619C3 consists of 524 aa. The coding sequences were used to perform the heterologous expression of functional enzymes in Saccharomyces cerevisiae. The bioconversion assays showed that CYP619C3 catalysed the hydroxylation of m-cresol to yield m-hydroxybenzyl alcohol. CYP619C2 catalysed the hydroxylation of m-hydroxybenzyl alcohol and m-cresol in gentisyl alcohol and 2.5 dihydroxytoluene (toluquinol), respectively. Except this last compound, all enzymes products are known precursors of patulin. Taken together, these data suggests strongly the involvement of CYP 619C2 and CYP 619C3 in the biosynthesis of patulin. The CYP 619C2 and CYP 619C3 are located near to two others genes involved in patulin biosynthesis, namely 6-methylsalicylic acid synthase (6msas) and isoepoxydon dehydrogenase genes (idh). The present data associated with an analysis of the sequence of A. clavatus suggest the presence of a cluster of genes involved in the patulin biosynthesis consisting of 15 genes.

¹ E-mail: opuel{at}toulouse.inra.fr