Abstract
In the biosynthesis of aflatoxin, verA, ver-1, ordB, and hypA genes of the aflatoxin gene cluster are involved in the pathway from versicolorin A (VA) to demethylsterigmatocystin (DMST). We herein isolated each disruptant of these four genes to determine their functions in more detail. Disruptants of ver-1, ordB, and hypA genes commonly accumulated VA in their mycelia. In contrast, the verA gene disruptant accumulated a novel yellow fluorescent substance (which we named HAMA) in the mycelia as well as culture medium. Feeding HAMA to the other disruptants commonly caused the production of aflatoxins B1 (AFB1) and G1 (AFG1). These results indicate that HAMA pigment is a novel aflatoxin precursor which is involved at a certain step after those of ver-1, ordB, and hypA genes between VA and DMST. HAMA was found to be an unstable substance to easily convert to DMST and sterigmatin. A liquid chromatography-mass spectrometry (LC-MS) analysis showed that the molecular mass of HAMA was 374, and HAMA gave two close major peaks in the LC chromatogram in some LC conditions. We suggest that these peaks correspond to the two conformers of HAMA; one of them would be selectively bound on the substrate binding site of VerA enzyme and then converted to DMST. VerA enzyme may work as a key enzyme in the creation of the xanthone structure of DMST from HAMA.
Highlights
Aflatoxins (AFs) are highly toxic, carcinogenic, teratogenic, and mutagenic secondary metabolites produced primarily by certain strains of Aspergillus flavus and Aspergillus parasiticus [1]
Preparation of verA Disruptant verA gene of A. parasiticus SYS4 was disrupted via the double-crossover strategy by using the linear DNA fragments of pVERA-DD, which was constructed with the flanking sequences of verA and disruption vector (Figure 2A)
When ptrA gene was used as a probe, beside the band that was suspected to correspond to the putative homologous nmt-1 gene in the A. parasiticus genome [39], specific ptrA hybridization signals of 2.06 kb for EcoRI digestion were detected only in the A. parasiticus genome [39], specific ptrA hybridization signals of 2.06 kb for EcoRI digestion were detected only in the genomic DNA of verA disruptant (Figure 2C, Right)
Summary
Aflatoxins (AFs) are highly toxic, carcinogenic, teratogenic, and mutagenic secondary metabolites produced primarily by certain strains of Aspergillus flavus and Aspergillus parasiticus [1]. Other strains (A. nomius, A. pseudotamarii, A. bombycis and more) have been reported to produce AFs, and aflatoxigenic fungi were shown to be widely distributed mainly in tropical and subtropical regions [2,3,4,5,6]. The AF contamination of crops such as corn, cotton, and nuts has serious effects on the health of animals and humans, and it causes economic problems due to crop losses [7,8,9]. Aflatoxigenic fungi can potentially produce eight types of AF: the four major AFs, i.e., aflatoxins. B1 (AFB1 ), B2 (AFB2 ), G1 (AFG1 ), and G2 (AFG2 ) and the four minor AFs, aflatoxins M1 (AFM1 ), Int. J.
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