Heterologous expression of genes from the proposed aspyridone biosynthetic cluster from Aspergillus nidulans in the host Aspergillus oryzae led to the production of eight different compounds in addition to aspyridone A 1, one of the previously observed products. The pathway genes were incapable of producing aspyridone B 2, the previously accepted final product of the pathway. Expression of restricted sets of genes in addition to the core polyketide synthase – nonribosomal peptide synthetase (PKS-NRPS) genes apdA and apdC revealed: that apdE encodes a cytochrome P450 enzyme with ring-expanding and unprecedented dephenylation activity; that apdB encodes an N-hydroxylase, an activity not previously suspected; that the productivity of ApdA and ApdC proteins appears to be significantly enhanced in the presence of the downstream ApdE oxidase; and no obvious chemical roles for ApdD and ApdG. Furthermore, the ApdC enoyl reductase appears to operate with different stereoselectivity in different PKS cycles. All of these features illustrate the inherent diversity of compounds potentially produced by the apd pathway and the high utility of a whole pathway expression strategy for investigating and revealing new biosynthetic chemistry in fungi.

A novel polyketide synthase nonribosomal peptide synthetase (PKS-NRPS) gene cluster was isolated from Beauveria bassiania 992.05. The cluster encodes the enzymes responsible for the biosynthesis of the new 2-pyridone desmethylbassianin (DMB). DMB is structurally related to tenellin from B. bassiana 110.25 but it differs in chain length and degree of methylation. Despite these programming differences the 20 kb DMB biosynthetic gene cluster has 90% sequence identity to the tenellin gene cluster. Silencing of the PKS-NRPS gene, dmbS, resulted in total loss of DMB production. Co-expression of dmbS in Aspergillus oryzae with its cognate trans-acting enoyl reductase gene, dmbC, produced predesmethylbassianin A, the first isolable precursor in the biosynthetic pathway. Expression of dmbS with the tenellin trans-acting enoyl reductase gene, tenC, also resulted in the production of predesmethylbassianin A. Co-expression of tenS, the tenellin PKS-NRPS, with dmbC produced pretenellin A. These results show that the tenS and dmbS encoded PKS-NRPS contains the programme for polyketide biosynthesis, while the trans-acting ERs appear to control the fidelity of the programme. Expression of a hybrid synthetase in which the PKS of the tenellin synthetase was fused to the NRPS from DMBS produced prototenellins A to C, indicating that the NRPS does not act as a selecting gatekeeper to affect the PKS programme.

Adaptation to a changing environment is essential for the survival and propagation of sessile organisms, such as plants or fungi. Filamentous fungi commonly respond to a worsening of their growth conditions by differentiation of asexually or sexually produced spores. The formation of these specialized cell types is, however, also triggered as part of the general life cycle by hyphal age or density. Spores typically serve for dispersal and, therefore, translocation but can also act as resting states to endure times of scarcity. Eukaryotic differentiation in response to environmental and self-derived signals is commonly mediated by three-tiered mitogen-activated protein (MAP) kinase signaling cascades. Here, we report that the MAP kinase Fus3 of the black mold Aspergillus niger (AngFus3) and its upstream kinase AngSte7 control vegetative spore formation and secondary metabolism. Mutants lacking these kinases are defective in conidium induction in response to hyphal density but are fully competent in starvation-induced sporulation, indicating that conidiation in A. niger is triggered by various independent signals. In addition, the mutants exhibit an altered profile of volatile metabolites and secrete dark pigments into the growth medium, suggesting a dysregulation of the secondary metabolism. By assigning the AngFus3 MAP kinase pathway to the transduction of a potentially self-derived trigger, this work contributes to the unraveling of the intricate signaling networks controlling fungal differentiation. Moreover, our data further support earlier observations that differentiation and secondary metabolism are tightly linked in filamentous fungi.