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Evidence of the Involvement of a Cyclase Gene in the Biosynthesis of Ochratoxin A in Aspergillus carbonarius.

ABSTRACT: Ochratoxin A (OTA) is a well-known mycotoxin with wide distribution in food and feed. Fungal genome sequencing has great utility for identifying secondary metabolites gene clusters for known and novel compounds. A comparative analysis of the OTA-biosynthetic cluster in A. steynii, A. westerdijkiae, A. niger, A. carbonarius, and P. nordicum has revealed a high synteny in OTA cluster organization in five structural genes (otaA, otaB, ota, otaR1, and otaD). Moreover, a recent detailed comparative genome analysis of Aspergilli OTA producers led to the identification of a cyclase gene, otaY, located in the OTA cluster between the otaA and otaB genes, encoding for a predicted protein with high similarity to SnoaLs domain. These proteins have been shown to catalyze ring closure steps in the biosynthesis of polyketide antibiotics produced in Streptomyces. In the present study, we demonstrated an upregulation of the cyclase gene in A. carbonarius under OTA permissive conditions, consistent with the expression trends of the other OTA cluster genes and their role in OTA biosynthesis by complete gene deletion. Our results pointed out the involvement of a cyclase gene in OTA biosynthetic pathway for the first time. They represent a step forward in the understanding of the molecular basis of OTA biosynthesis in A. carbonarius.

SUBMITTER: Ferrara M

PROVIDER: S-EPMC8705981 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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Evidence of the Involvement of a Cyclase Gene in the Biosynthesis of Ochratoxin A in Aspergillus carbonarius.

Ferrara Massimo M Gallo Antonia A Cervini Carla C Gambacorta Lucia L Solfrizzo Michele M Baker Scott E SE Perrone Giancarlo G

Toxins 20211213 12

Ochratoxin A (OTA) is a well-known mycotoxin with wide distribution in food and feed. Fungal genome sequencing has great utility for identifying secondary metabolites gene clusters for known and novel compounds. A comparative analysis of the OTA-biosynthetic cluster in A. steynii, A. westerdijkiae, A. niger, A. carbonarius, and P. nordicum has revealed a high synteny in OTA cluster organization in five structural genes (otaA, otaB, ota, otaR1, and < ...[more]

PMID: 34941729

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Project description:UnlabelledAspergillus carbonarius is the main responsible fungus of ochratoxin A (OTA) contamination of grapes and derived products. To date, the biosynthetic mechanism of this mycotoxin has been partially elucidated. Availability of genome sequence of A. carbonarius has allowed the identification of a putative gene cluster involved in OTA biosynthesis. This region hosts the previously characterized AcOTAnrps and AcOTApks genes encoding two key enzymes of the biosynthetic pathway. At about 4,400 nucleotides downstream of these loci, a gene encoding a putative flavin dependent-halogenase came out from the annotation data. Its proximity to OTA biosynthetic genes and its sequence analysis have suggested a role in the biosynthesis of OTA, directed to the introduction of the chlorine atom in the C-5 position of the final molecular structure of this mycotoxin. The deduced protein sequence of the halogenase gene, we designated AcOTAhal, shows a high similarity to a halogenase that is located in the OTA cluster of A. niger The deletion of the halogenase gene completely eliminated the production of ochratoxin A in A. carbonarius and determined a significant increase of ochratoxin B, as confirmed by mass spectrometry analysis. Moreover, its expression profile was similar to the two biosynthetic genes previously identified, AcOTApks and AcOTAnrps, indicating a strong correlation of the AcOTAhal gene with the kinetics of OTA accumulation in A. carbonarius. Therefore, experimental evidence confirmed that the chlorination step which converts OTB in OTA represents the final stage of the biosynthetic pathway, supporting our earlier hypothesis on the order of enzymatic steps of OTA biosynthesis in A. carbonariusImportanceOchratoxin A is a potent mycotoxin classified as a possible carcinogen for humans, and Aspergillus carbonarius is the main agent responsible for OTA accumulation in grapes. We demonstrate here that a flavin-halogenase is implicated in the biosynthesis of OTA in A. carbonarius The encoding gene, AcOTAhal, is contiguous to biosynthetic genes that we have already described (nrps and pks), resulting as part of the biosynthetic cluster. The encoded protein is responsible of the introduction of chlorine atom in the final molecular structure and acts at the last step in the pathway. This study can be considered a continuation of an earlier study wherein we started to clarify the molecular basis of OTA biosynthesis in A. carbonarius, which has not been completely elucidated until now. This research represents an important step forward to a better understanding of the production mechanism, which will contribute to the development of improved control strategies to reduce the risk of OTA contamination in food products.

Dataset Information

Evidence of the Involvement of a Cyclase Gene in the Biosynthesis of Ochratoxin A in Aspergillus carbonarius.

Publications

Evidence of the Involvement of a Cyclase Gene in the Biosynthesis of Ochratoxin A in <i>Aspergillus carbonarius</i>.

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