Funalenone

Aspergillus section Nigri is an important group of species for food and medical mycology, and biotechnology. The Aspergillus niger 'aggregate' represents its most complicated taxonomic subgroup containing eight morphologically indistinguishable taxa: A. niger, Aspergillus tubingensis, Aspergillus acidus, Aspergillus brasiliensis, Aspergillus costaricaensis, Aspergillus lacticoffeatus, Aspergillus piperis, and Aspergillus vadensis. Aspergillus awamori, first described by Nakazawa, has been compared taxonomically with other black aspergilli and recently it has been treated as a synonym of A. niger. Phylogenetic analyses of sequences generated from portions of three genes coding for the proteins β-tubulin (benA), calmodulin (CaM), and the translation elongation factor-1 alpha (TEF-1α) of a population of A. niger strains isolated from grapes in Europe revealed the presence of a cryptic phylogenetic species within this population, A. awamori. Morphological, physiological, ecological and chemical data overlap occurred between A. niger and the cryptic A. awamori, however the splitting of these two species was also supported by AFLP analysis of the full genome. Isolates in both phylospecies can produce the mycotoxins ochratoxin A and fumonisin B₂, and they also share the production of pyranonigrin A, tensidol B, funalenone, malformins, and naphtho-γ-pyrones. In addition, sequence analysis of four putative A. awamori strains from Japan, used in the koji industrial fermentation, revealed that none of these strains belong to the A. awamori phylospecies.

A characteristic hallmark of Aspergillus niger is the formation of black conidiospores. We have identified four loci involved in spore pigmentation of A. niger by using a combined genomic and classical complementation approach. First, we characterized a newly isolated color mutant, colA, which lacked pigmentation resulting in white or colorless conidia. Pigmentation of the colA mutant was restored by a gene (An12g03950) which encodes a putative 4'phosphopantetheinyl transferase protein (PptA). 4'Phosphopantetheinyl transferase activity is required for the activation of Polyketide Synthases (PKSs) and/or Non-Ribosomal Peptide Synthases (NRPSs). The loci whose mutation resulted in fawn, olive, and brown color phenotypes were identified by complementation. The fawn phenotype was complemented by a PKS protein (FwnA, An09g05730), the ovlA mutant by An14g05350 (OlvA) and the brnA mutant by An14g05370 (BrnA), the respective homologs of alb1/pksP, ayg1 and abr1 in A. fumigatus. Targeted disruption of the pptA, fwnA, olvA and brnA genes confirmed the complementation results. Disruption of the pptA gene abolished synthesis of all polyketides and non-ribosomal peptides, while the naphtho-γ-pyrone subclass of polyketides were specifically dependent on fwnA, and funalenone on fwnA, olvA and brnA. Thus, secondary metabolite profiling of the color mutants revealed a close relationship between polyketide synthesis and conidial pigmentation in A. niger.

Epigenetic modification is an important regulatory mechanism in the biosynthesis of secondary metabolites in Aspergillus species, which have been considered to be the treasure trove of new bioactive secondary metabolites. In this study, we reported that deletion of the epigenetic regulator gcnE, a histone acetyltransferase in the SAGA/ADA complex, resulted in the production of 12 polyketide secondary metabolites in A. niger FGSC A1279, which was previously not known to produce toxins or secondary metabolites. Chemical workup and structural elucidation by 1D/2D NMR and high resolution electrospray ionization mass (HR-ESIMS) yielded the novel compound nigerpyrone (1) and five known compounds: carbonarone A (2), pestalamide A (3), funalenone (4), aurasperone E (5), and aurasperone A (6). Based on chemical information and the literature, the biosynthetic gene clusters of funalenone (4), aurasperone E (5), and aurasperone A (6) were located on chromosomes of A. niger FGSC A1279. This study found that inactivation of GcnE activated the production of secondary metabolites in A. niger. The biosynthetic pathway for nigerpyrone and its derivatives was identified and characterized via gene knockout and complementation experiments. A biosynthetic model of this group of pyran-based fungal metabolites was proposed.