The fungal kingdom has provided advances in our ability to identify biosynthetic gene clusters (BGCs) and to examine how gene composition of BGCs evolves across species and genera. However, little is known about the evolution of specific BGC regulators that mediate how BGCs produce secondary metabolites (SMs). A bioinformatics search for conservation of the Aspergillus fumigatus xanthocillin BGC revealed an evolutionary trail of xan-like BGCs across Eurotiales species. Although the critical regulatory and enzymatic genes were conserved in Penicillium expansum, overexpression (OE) of the conserved xan BGC transcription factor (TF) gene, PexanC, failed to activate the putative xan BGC transcription or xanthocillin production in P. expansum, in contrast to the role of AfXanC in A. fumigatus. Surprisingly, OE::PexanC was instead found to promote citrinin synthesis in P. expansum via trans induction of the cit pathway-specific TF, ctnA, as determined by cit BGC expression and chemical profiling of ctnA deletion and OE::PexanC single and double mutants. OE::AfxanC results in significant increases of xan gene expression and metabolite synthesis in A. fumigatus but had no effect on either xanthocillin or citrinin production in P. expansum. Bioinformatics and promoter mutation analysis led to the identification of an AfXanC binding site, 5'-AGTCAGCA-3', in promoter regions of the A. fumigatus xan BGC genes. This motif was not in the ctnA promoter, suggesting a different binding site of PeXanC. A compilation of a bioinformatics examination of XanC orthologs and the presence/absence of the 5'-AGTCAGCA-3' binding motif in xan BGCs in multiple Aspergillus and Penicillium spp. supports an evolutionary divergence of XanC regulatory targets that we speculate reflects an exaptation event in the Eurotiales. IMPORTANCE Fungal secondary metabolites (SMs) are an important source of pharmaceuticals on one hand and toxins on the other. Efforts to identify the biosynthetic gene clusters (BGCs) that synthesize SMs have yielded significant insights into how variation in the genes that compose BGCs may impact subsequent metabolite production within and between species. However, the role of regulatory genes in BGC activation is less well understood. Our finding that the bZIP transcription factor XanC, located in the xanthocillin BGC of both Aspergillus fumigatus and Penicillium expansum, has functionally diverged to regulate different BGCs in these two species emphasizes that the diversification of BGC regulatory elements may sometimes occur through exaptation, which is the co-option of a gene that evolved for one function to a novel function. Furthermore, this work suggests that the loss/gain of transcription factor binding site targets may be an important mediator in the evolution of secondary-metabolism regulatory elements.

Transcription Factor Repurposing Offers Insights into Evolution of Biosynthetic Gene Cluster Regulation.

Transcriptomic analysis of inhibition by eugenol of ochratoxin A biosynthesis and growth of Aspergillus carbonarius

Unveiling ochratoxin a controlling and biodetoxification molecular mechanisms: Opportunities to secure foodstuffs from OTA contamination.

The ochratoxin A (OTA) biosynthetic gene cluster includes a bZIP transcription factor (TF) gene (OTAbzip) that has been identified in different fungal species. However, most previous studies identified the OTAbzip gene in ochratoxigenic fungi using bioinformatics methods, while few studies focused on deleting the gene, let alone overexpressing it, to characterize the function of the OTAbZIP TF. Here, we characterized the AnOTAbZIP TF in an ochratoxigenic isolate of Aspergillus niger by deleting and overexpressing the AnOTAbzip gene and examining the role of AnOTAbZIP in morphological development, OTA biosynthesis, and pathogenicity. Chemical and gene expression analyses revealed that AnOTAbZIP positively regulates OTA biosynthesis, since the loss of OTA production and the downregulation of the OTA biosynthetic genes were observed in the ΔAnOTAbzip strain, compared with the wild-type (WT) and OE::AnOTAbzip strains. In terms of pathogenicity, the ΔAnOTAbzip strain produced a greater lesion on grape berries, especially with respect to the OE::AnOTAbzip strain, rather than WT. Finally, the ΔAnOTAbzip strain was also more tolerant to oxidative stress with respect to the OE::AnOTAbzip and WT strains in that order. These new findings improve our understanding of the AnOTAbZIP regulatory mechanism and help develop strategies to attenuate plant pathogenicity and reduce OTA biosynthesis of A. niger.

Deletion and Overexpression of the AnOTAbzip Gene, a Positive Regulator of Ochratoxin A Biosynthesis in Aspergillus niger.

Three stilbenes make difference to the antifungal effects on ochratoxin A and its precursor production of Aspergillus carbonarius.

Aspergillus carbonarius is the principal fungal species responsible for ochratoxin A (OTA) contamination of grapes and derived products in the main viticultural regions worldwide. In recent years, co-expressed genes representing a putative-OTA gene cluster were identified, and the deletion of a few of them allowed the partial elucidation of the biosynthetic pathway in the fungus. In the putative OTA-gene cluster is additionally present a bZIP transcription factor (AcOTAbZIP), and with this work, A. carbonarius ΔAcOTAbZIP strains were generated to study its functional role. According to phylogenetic analysis, the gene is conserved in the OTA-producing fungi. A Saccharomyces cerevisiae transcription factor binding motif (TFBM) homolog, associated with bZIP transcription factors was present in the A. carbonarius OTA-gene cluster no-coding regions. AcOTAbZIP deletion results in the loss of OTA and the intermediates OTB and OTβ. Additionally, in ΔAcOTAbZIP strains, a down-regulation of AcOTApks, AcOTAnrps, AcOTAp450, and AcOTAhal genes was observed compared to wild type (WT). These results provide evidence of the direct involvement of the AcOTAbZIP gene in the OTA biosynthetic pathway by regulating the involved genes. The loss of OTA biosynthesis ability does not affect fungal development as demonstrated by the comparison of ΔAcOTAbZIP strains and WT strains in terms of vegetative growth and asexual sporulation on three different media. Finally, no statistically significant differences in virulence were observed among ΔAcOTAbZIP strains and WT strains on artificially inoculated grape berries, demonstrating that OTA is not required by A. carbonarius for the pathogenicity process.

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Federica Garrapa

Papers

Functional Role of Aspergillus carbonarius AcOTAbZIP Gene, a bZIP Transcription Factor within the OTA Gene Cluster