Biochemical and genetic basis of orsellinic acid biosynthesis and prenylation in a stereaceous basidiomycete.

The prenylphenols are a class of natural products that have been frequently isolated from basidiomycetes, e.g., from the genus Stereum (false turkey tail fungi) and other Russulales as well as from ascomycetes. Biosynthetically, these compounds are considered hybrids, as the orsellinic acid moiety is a polyketide and the prenyl side chain originates from the terpene metabolism, although no literature on the genetic and biochemical background of the biosynthesis is available. In a stereaceous basidiomycete, referred to as BY1, a new prenylphenol, now termed cloquetin, was identified and its structure elucidated by mass spectrometry and nuclear magnetic resonance spectroscopy. Genes for two non-reducing polyketide synthases (PKS1 and PKS2) were identified in the BY1 genome, and heterologously expressed in Aspergillus niger. Product formation identified both PKSs as orsellinic acid synthases. A putative prenyltransferase gene (BYPB) found in the BY1 genome was expressed in Escherichia coli. In vitro characterization showed that BYPB activity depends on bivalent cations and that it uses orsellinic acid as acceptor substrate for the transfer of a prenyl group. The two orsellinic acid synthases support the emerging notion that fungi secure individual metabolic steps or entire pathways by redundant enzymes.

Unexpected Metabolic Versatility in a Combined Fungal Fomannoxin/Vibralactone Biosynthesis.

The secondary metabolome of an undescribed stereaceous basidiomycete (BY1) was investigated for bioactive compounds. Along with a known fomannoxin derivative and two known vibralactones, we here describe three new compounds of these natural product families, whose structures were elucidated using 1D and 2D NMR spectroscopy and high-resolution mass spectrometry. The new compound vibralactone S (4) shows a 3,6-substituted oxepin-2(7H)-one ring system, which is unprecedented for the vibralactone/fomannoxin class of compounds. Stable isotope labeling established a biosynthetic route that is dissimilar to the two published cascades of oxepinone formation. Another new compound, the antifungal methyl seco-fomannoxinate (6), features a 2-methylprop-1-enyl ether moiety, which is only rarely observed with natural products. The structure of 6 was confirmed by total synthesis. (13)C-labeling experiments revealed that the unusual 2-methylprop-1-enyl ether residue derives from an isoprene unit. The diversity of BY1's combined fomannoxin/vibralactone metabolism is remarkable in that these compound families, although biosynthetically related, usually occur in different organisms.

Involutin is an Fe3+ reductant secreted by the ectomycorrhizal fungus Paxillus involutus during Fenton-based decomposition of organic matter.

Ectomycorrhizal fungi play a key role in mobilizing nutrients embedded in recalcitrant organic matter complexes, thereby increasing nutrient accessibility to the host plant. Recent studies have shown that during the assimilation of nutrients, the ectomycorrhizal fungus Paxillus involutus decomposes organic matter using an oxidative mechanism involving Fenton chemistry (Fe(2+) + H2O2 + H(+) → Fe(3+) + ˙OH + H2O), similar to that of brown rot wood-decaying fungi. In such fungi, secreted metabolites are one of the components that drive one-electron reductions of Fe(3+) and O2, generating Fenton chemistry reagents. Here we investigated whether such a mechanism is also implemented by P. involutus during organic matter decomposition. Activity-guided purification was performed to isolate the Fe(3+)-reducing principle secreted by P. involutus during growth on a maize compost extract. The Fe(3+)-reducing activity correlated with the presence of one compound. Mass spectrometry and nuclear magnetic resonance (NMR) identified this compound as the diarylcyclopentenone involutin. A major part of the involutin produced by P. involutus during organic matter decomposition was secreted into the medium, and the metabolite was not detected when the fungus was grown on a mineral nutrient medium. We also demonstrated that in the presence of H2O2, involutin has the capacity to drive an in vitro Fenton reaction via Fe(3+) reduction. Our results show that the mechanism for the reduction of Fe(3+) and the generation of hydroxyl radicals via Fenton chemistry by ectomycorrhizal fungi during organic matter decomposition is similar to that employed by the evolutionarily related brown rot saprotrophs during wood decay.

Injury-induced biosynthesis of methyl-branched polyene pigments in a white-rotting basidiomycete.

A stereaceous basidiomycete was investigated with regard to its capacity to produce yellow pigments after physical injury of the mycelium. Two pigments were isolated from mycelial extracts, and their structures were elucidated by ESIMS and one- and two-dimensional NMR methods. The structures were identified as the previously undescribed polyenes (3Z,5E,7E,9E,11E,13Z,15E,17E)-18-methyl-19-oxoicosa-3,5,7,9,11,13,15,17-octaenoic acid (1) and (3E,5Z,7E,9E,11E,13E,15Z,17E,19E)-20-methyl-21-oxodocosa-3,5,7,9,11,13,15,17,19-nonaenoic acid (2). Stable-isotope feeding with [1-(13)C]acetate and l-[methyl-(13)C]methionine demonstrated a polyketide backbone and that the introduction of the sole methyl branch is most likely S-adenosyl-l-methionine-dependent. Dose-dependent inhibition of Drosophila melanogaster larval development was observed with both polyenes in concentrations between 12.5 and 100 µM. GI50 values for 1 and 2 against HUVEC (K-562 cells) were 71.6 and 17.4 µM (15.4 and 1.1 µM), respectively, whereas CC50 values for HeLa cells were virtually identical (44.1 and 45.1 µM).

A gene cluster responsible for biosynthesis of phomenoic acid in the plant pathogenic fungus, Leptosphaeria maculans.

Phomenoic acid, a long chain aliphatic carboxylic acid is a major metabolite produced by Leptosphaeria maculans, a fungal pathogen of Brassica napus (canola). This fungus has 15 predicted polyketide synthases (PKS) and seven of them have the appropriate domains for the biosynthesis of phomenoic acid. The most highly expressed PKS gene after 7 days growth in 10% V8 juice, PKS2, was silenced and the resultant mutant produced very low levels of phomenoic acid, indicating that this PKS is involved in phomenoic acid biosynthesis. This gene is part of a co-regulated cluster of genes. Reduced expression of an adjacent gene encoding the transcriptional regulator C6TF, led to reduced expression of genes for PKS2, P450, a cytochrome P450 monoxygenase, YogA, an alcohol dehydrogenase/quinone reductase, RTA1, a lipid transport exporter superfamily member and MFS, a Major Facilitator Superfamily transporter, as well as a marked reduction in phomenoic acid production. Phomenoic acid is toxic towards another canola pathogen Leptosphaeria biglobosa 'canadensis', but not towards L. maculans and only moderately toxic towards the wheat pathogen Stagonospora nodorum. This molecule is detected in infected stems and stubble of B. napus, but biosynthesis of it does not appear to be essential for pathogenicity of L. maculans. Phomenoic acid may play a role in allowing L. maculans to outcompete other fungi in its environmental niche.