New insight into the ochratoxin A biosynthetic pathway through deletion of a nonribosomal peptide synthetase gene in Aspergillus carbonarius

Abstract

Ochratoxin A (OTA), a mycotoxin produced by Aspergillus and Penicillium species, is composed of a dihydroisocoumarin ring linked to phenylalanine, and its biosynthetic pathway has not yet been completely elucidated. Most of the knowledge regarding the genetic and enzymatic aspects of OTA biosynthesis has been elucidated in Penicillium species. In Aspergillus species, only pks genes involved in the initial steps of the pathway have been partially characterized. In our study, the inactivation of a gene encoding a nonribosomal peptide synthetase (NRPS) in OTA-producing A. carbonarius ITEM 5010 has eliminated the ability of this fungus to produce OTA. This is the first report on the involvement of an nrps gene product in OTA biosynthetic pathway in an Aspergillus species. The absence of OTA and ochratoxin α, the isocoumaric derivative of OTA, and the concomitant increase of ochratoxin β, the dechloro analog of ochratoxin α, were observed in the liquid culture of transformed strain. The data provide the first evidence that the enzymatic step adding phenylalanine to polyketide dihydroisocoumarin precedes the chlorination step to form OTA in A. carbonarius and that ochratoxin α is a product of hydrolysis of OTA, giving an interesting new insight into the biosynthetic pathway of the toxin.

Fig 1

Scheme showing all the different hypotheses of the OTA biosynthesis pathway according to the literature data available so far (27, 29). *, hypothetical intermediary compounds.

Fig 2

(A) Schematic representation of the AcOTAnrps gene deletion construct. (B) RT-PCR profile of AcOTAnrps gene expression in the wild-type, ΔkusA, and ΔAcOTAnrps strains grown on a medium permissive for OTA. β-Tubulin was used as a control. (C) Map of the AcOTAnrps locus in parental and ΔAcOTAnrps strains of A. carbonarius and Southern blot hybridizations from genomic DNA digested with BamHI and PstI. The restriction sites BamHI and PstI are indicated with B and P, respectively.

Fig 3

HPLC-FLD chromatograms of OTA and OTα. (A) Culture medium of wild-type A. carbonarius; (B) culture medium of ΔkusA mutant KB1039; (C) culture medium of ΔAcOTAnrps mutant KB1041 purified by IMA column before HPLC-FLD analysis; (D) OTA and OTα standards. Retention times: OTA, 10.35 min; OTα, 8.41 min.

Fig 4

Metabolites supposed to be involved in OTA biosynthesis and identified by HPLC-HRMS in cultures of ΔkusA (KB1039) and ΔAcOTAnrps (KB1041) strains incubated in triplicate for 7 days. *, not detected. Values are means ± standard errors. Different letters indicate statistical differences within pairs of data (P < 0.005).

Fig 5

Kinetics of OTA degradation and OTα formation by the ΔAcOTAnrps (KB1041) mutant during 25 days of incubation in minimal medium spiked with 3 μM OTA. The concentration of OTA remained unchanged in control medium over the entire duration of the experiments. Values are means ± standard errors.

Fig 6

New schematic representation of the last steps of the OTA biosynthetic pathway as proposed in this work.