Microbial communication has attracted notable attention as an indicator of microbial interactions that lead to marked alterations of secondary metabolites (SMs) in varied environments. However, the mechanisms responsible for SM regulation are not fully understood, especially in fungal-fungal interactions. Here, cocultivation of an endophytic fungus Epicoccum dendrobii with the model fungus Aspergillus nidulans and several other filamentous fungi triggered widespread alteration of SMs. Multiple silent biosynthetic gene clusters in A. nidulans were activated by transcriptome and metabolome analysis. Unprecedentedly, gene deletion and replacement proved that a partial loss-of-function VeA1 protein, but not VeA, was associated with the widespread SM changes in both A. nidulans and A. fumigatus during cocultivation. VeA1 regulation required the transcription factor SclB and the velvet complex members LaeA and VelB for producing aspernidines as representative formation of SMs in A. nidulans . This study provides new insights into the mechanism that trigger metabolic changes during fungal-fungal interactions.

The strategies of genetic dereplication and manipulation of epigenetic regulators to activate the cryptic gene clusters are effective to discover natural products with novel structure in filamentous fungi. In this study, a combination of genetic dereplication (deletion of pesthetic acid biosynthetic gene, PfptaA) and manipulation of epigenetic regulators (deletion of histone methyltransferase gene PfcclA and histone deacetylase gene PfhdaA) was developed in plant endophytic fungus Pestalotiopsis fici. The deletion of PfptaA with PfcclA and/or PfhdaA led to isolation of 1 novel compound, pestaloficiol X (1), as well as another 11 known compounds with obvious yield changes. The proposed biosynthesis pathway of pestaloficiol X was speculated using comparative analysis of homologous biosynthetic gene clusters. Moreover, phenotypic effects on the conidial development and response to oxidative stressors in the mutants were explored. Our results revealed that the new strain with deletion of PfcclA or PfhdaA in ΔPfptaA background host can neutralise the hyperformation of conidia in the PfptaA mutant, and that the ΔPfptaA ΔPfhdaA mutant was generally not sensitive to oxidative stressors as much as the ΔPfptaA ΔcclA mutant in comparison with the single mutant ΔPfptaA or the parental strains. This combinatorial approach can be applied to discover new natural products in filamentous fungi.

Nonribosomal peptide synthetase (NRPS)-like enzymes containing A-T-R domain architecture are also known as carboxylate reductases (CARs) for aldehyde generation. To identify new members of CARs, we established a virtual library containing 84 fungal CARs distributed in seven distinct clades by genome mining and phylogenetic analysis. Nine CARs, including PnlA from Pestalotiopsis fici and eight known CARs, were clustered in clade VI and proposed to catalyze the reduction of nonreducing polyketide synthase (NR-PKS)-derived aryl carboxylic acids. The recombinant protein PnlA was overproduced and purified to apparent homogeneity from Saccharomyces cerevisiae. In vitro enzyme assays of PnlA with 28 different benzoic acid derivatives (1–28) revealed the corresponding aldehyde formation in 14 cases (1–14). Comparison of conversion yields indicated the high preference of PnlA toward 3,5-dimethylorsellinic acid (DMOA, 4) and vanillic acid (10). A specificity-conferring code Q355 in PnlA was postulated by sequence alignment with the known CARs in clade VI. Our study provides an updated virtual library of fungal CAR enzymes and expands the biocatalytic selectivity of CARs.