verA Gene is Involved in the Step to Make the Xanthone Structure of Demethylsterigmatocystin in Aflatoxin Biosynthesis

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.

Fungal secondary metabolite analysis applied to Cultural Heritage: the case of a contaminated library in Venice

The secondary metabolite production of several fungal strains of Aspergillus creber, Aspergillus jensenii, Aspergillus penicillioides, Aspergillus protuberus, Aspergillus vitricola, Cladosporium cladosporioides, Eurotium chevalieri, Eurotium halophilicum, Penicillium brevicompactum and Penicillium chrysogenum were characterised by liquid chromatography tamdem mass spectometry. All fungi were isolated from both air and book covers as well as from settled dust from a contaminated library in Venice (Italy). For A. creber and A. jensenii, we identified sterigmatocystin, methoxysterigmatocystin, versicolorin A and related precursors/side metabolites from the biosynthetic pathways. Deoxybrevianamid E, neoechinulin A, pseurotin A and D, and rugulusovin were principally detected from the strains of E. halophilicum, an emerging fungal species implicated in book contaminations in specific indoor niches. The analysis of settled dust showed a wide range of toxic or bioactive fungal metabolites. Forty-five different metabolites were identified in different concentrations; in particular, high amounts of asperglaucide, alamethicin, andrastin A, terrecyclic acid and neoechinulin A were detected. Also one bacterial metabolite, chloramphenicole was detected. This study increases the knowledge about metabolite production of several fungal species, as well as on the indoor presence of fungi that are not detected by aerobiological sampling. These results emphasise how routine dusting operations are necessary and essential in order to prevent further microbiological developments in library environments.

The Aflatoxin Biosynthesis Cluster Gene, aflX, Encodes an Oxidoreductase Involved in Conversion of Versicolorin A to Demethylsterigmatocystin

ABSTRACT Biosynthesis of the toxic and carcinogenic aflatoxins by the fungus Aspergillus flavus is a complicated process involving more that 27 enzymes and regulatory factors encoded by a clustered group of genes. Previous studies found that three enzymes, encoded by verA, ver-1, and aflY, are required for conversion of versicolorin A (VA), to demethylsterigmatocystin. We now show that a fourth enzyme, encoded by the previously uncharacterized gene, aflX (ordB), is also required for this conversion. A homolog of this gene, stcQ, is present in the A. nidulans sterigmatocystin (ST) biosynthesis cluster. Disruption of aflX in Aspergillus flavus gave transformants that accumulated ∼4-fold more VA and fourfold less aflatoxin than the untransformed strain. Southern and Northern blot analyses confirmed that aflX was the only gene disrupted in these transformants. Feeding ST or O-methylsterigmatocystin, but not VA or earlier precursor metabolites, restored normal levels of AF production. The protein encoded by aflX is predicted to have domains typical of an NADH-dependent oxidoreductase. It has 27% amino acid identity to a protein encoded by the aflatoxin cluster gene, aflO (avfA). Some of domains in the protein are similar to those of epoxide hydrolases.