Sesquiterpenyl epoxy-cyclohexenoids (SECs) show impressive biological activities. However, the key pathways for SECs still remain unambiguous. Unexpectedly, 11 new SECs and derivatives with diverse oxidation patterns were isolated after the deletion of gene 274. A high accumulation of toluquinol and its new glycosides in mutant Δ276 and further isolation of the most crucial precursors farnesyl hydroquinone, farnesyl quinone, and three new derivatives from mutant Δ278 confirm that farnesylation at toluquinol is the key step for SECs.

Arthrobotrys oligospora is the first recognized nematode-trapping fungus and by far the most abundant in the environment. Our recent study revealed the polyketide synthase (PKS) gene AOL_s00215g283 in A. oligospora involved in the production of many secondary metabolites and the trap formation of the fungus. Here we report that the disruption of two genes in the upstream flanking region of the gene AOL_s00215g283, AOL_s00215g281 and AOL_s00215g282, which putatively encoded one amidohydrolase and one cytochrome P450 monooxygenase, respectively, both resulted in significant nematicidal activity of the cultural broths of the mutants and loss of morphological regulatory arthrosporols. Chemical investigation revealed the huge accumulation of 6-methylsalicylic acid in the cultural broth of the mutant ΔAOL_s00215g281 and the high production of m-cresol in the mutant ΔAOL_s00215g282, respectively. Further bioassay revealed that 6-methylsalicylic acid and m-cresol displayed significant nematicidal activity toward root-knot nematodes Meloidogyne incognita with IC90 values of 300 and 100 μg/mL, respectively. The mutant ΔAOL_s00215g282 displayed a more complex metabolite profile than the mutant ΔAOL_s00215g281, suggesting that m-cresol was a more versatile key precursor than 6-methylsalicylic acid. These findings not only demonstrated that the gene AOL_s00215g283 encodes the 6-methylsalicylic acid synthase and the gene AOL_s00215g281 encodes the decarboxylase for 6-methylsalicylic acid but also provided evidence for the potential functions of the precursors in fungal complex biosynthetic pathways and had more implications for the establishment of efficient fungal biocontrol agents.Keywords: Arthrobotrys oligospora; biosynthesis; gene; nematode-trapping fungus;

A group of morphology regulatory arthrosporol metabolites have been recently characterized from carnivorous fungus Arthrobotrys oligospora that can develop trapping networks to capture their prey. A combination of genetic manipulation and chemical analyses was applied to characterize the function of one polyketide synthase (PKS) gene AOL_s00215g283 in A. oligospora, which was putatively involved in the production of 6-methylsalicylic acid. High-performance liquid chromatography analysis showed that the disruption of the PKS gene not only led to the total loss of the arthrosporol A but also resulted in significant reduction in the production of secondary metabolites in the cultural broth of the mutant ΔAOL_s00215g283 strain. Interestingly, the mutant strain displayed significant increases in the trap formation and the nematicidal activity by 10 and 2 times, respectively, higher than the wild-type strain. These findings revealed a pathogenicity-related biosynthetic gene of this agriculturally important biological agent and have implications for establishment of efficient fungal biocontrol agents.

Nematophagous fungi are globally distributed soil fungi and well-known natural predators of soil-dwelling nematodes. Pochonia chlamydosporia can be found in diverse nematode-suppressive soils as a parasite of nematode eggs and is one of the most studied potential biological control agents of nematodes. However, little is known about the functions of small molecules in the process of infection of nematodes by this parasitic fungus or about small-molecule-mediated interactions between the pathogenic fungus and its host. Our recent study demonstrated that a P. chlamydosporia strain isolated from root knots of tobacco infected by the root-knot nematode Meloidogyne incognita produced a class of yellow pigment metabolite aurovertins, which induced the death of the free-living nematode Panagrellus redivevus. Here we report that nematicidal P. chlamydosporia strains obtained from the nematode worms tended to yield a total yellow pigment aurovertin production exceeding the inhibitory concentration shown in nematicidal bioassays. Aurovertin D was abundant in the pigment metabolites of P. chlamydosporia strains. Aurovertin D showed strong toxicity toward the root-knot nematode M. incognita and exerted profound and detrimental effects on the viability of Caenorhabditis elegans even at a subinhibitory concentration. Evaluation of the nematode mutation in the β subunit of F1-ATPase, together with the application of RNA interference in screening each subunit of F1FO-ATPase in the nematode worms, demonstrated that the β subunit of F1-ATPase might not be the specific target for aurovertins in nematodes. The resistance of C. elegans daf-2(e1370) and the hypersensitivity of C. elegans daf-16(mu86) to aurovertin D indicated that DAF-16/FOXO transcription factor in nematodes was triggered in response to the aurovertin attack. These findings advance our understanding of the roles of aurovertin production in the interactions between nematodes and the pathogen fungus P. chlamydosporia.

Prior chemical analysis of obligate thermophilic fungus Talaromyces thermophilus led to the discovery of thermolides A–F, six previously undescribed members of the lactam-bearing macrolactone class. A combination of chemical screening, genome analyses, and genetic manipulation led to the identification of the thermolide biosynthetic genes from sister thermophilic fungi T. thermophilus and Thermomyces lanuginosus and a new thermolide. The biosynthetic locus for the thermolides’ mixed polyketide/amino acid structure encodes a hybrid polyketide synthase–nonribosomal peptide synthetase (PKS–NRPS). Our results reveal the first fungal hybrid iterative PKS–NRPS genes involved in the biosynthesis of bacterial-like hybrid macrolactones instead of typical fungal tetramic acids-containing metabolites. The finding provides an insight into the convergent biosynthetic end products that bridge the gap between the modular and iterative PKS–NRPS hybrids.