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The natural heterogeneity of guaiacyl (G) and syringyl (S) compounds resulting from lignin processing hampers their direct use as plant‐based chemicals and materials. Herein, we explore six short polyphenol oxidases (PPOs) from lignocellulose‐degrading ascomycetes for their capacity to react with G‐type and S‐type phenolic compounds. All six PPOs catalyze the ortho‐hydroxylation of G‐type compounds (guaiacol, vanillic acid, and ferulic acid), forming the corresponding methoxy‐ortho‐diphenols. Remarkably, a subset of these PPOs is also active towards S‐compounds (syringol, syringic acid, and sinapic acid) resulting in identical methoxy‐ortho‐diphenols. Assays with 18O2 demonstrate that these PPOs in particular catalyze ortho‐hydroxylation and ortho‐demethoxylation of S‐compounds and generate methanol as a co‐product. Oxidative (ortho−) demethoxylation of S‐compounds is a novel reaction for PPOs, which we propose occurs by a distinct reaction mechanism as compared to aryl‐O‐demethylases. We further show that addition of a reducing agent can steer the PPO reaction to form methoxy‐ortho‐diphenols from both G‐ and S‐type substrates rather than reactive quinones that lead to unfavorable polymerization. Application of PPOs opens for new routes to reduce the heterogeneity and methoxylation degree of mixtures of G and S lignin‐derived compounds. [ABSTRACT FROM AUTHOR]

Plant biomass conversion by saprotrophic fungi plays a pivotal role in terrestrial carbon (C) cycling. The general consensus is that fungi metabolize carbohydrates, while lignin is only degraded and mineralized to CO2. Recent research, however, demonstrated fungal conversion of 13C-monoaromatic compounds into proteinogenic amino acids. To unambiguously prove that polymeric lignin is not merely degraded, but also metabolized, carefully isolated 13C-labeled lignin served as substrate for Agaricus bisporus, the world's most consumed mushroom. The fungus formed a dense mycelial network, secreted lignin-active enzymes, depolymerized, and removed lignin. With a lignin carbon use efficiency of 0.14 (g/g) and fungal biomass enrichment in 13C, we demonstrate that A. bisporus assimilated and further metabolized lignin when offered as C-source. Amino acids were high in 13C-enrichment, while fungal-derived carbohydrates, fatty acids, and ergosterol showed traces of 13C. These results hint at lignin conversion via aromatic ring-cleaved intermediates to central metabolites, underlining lignin's metabolic value for fungi. [ABSTRACT FROM AUTHOR]

Dimethylallyl tryptophan synthases (DMATSs) are aromatic prenyltransferases that catalyze the transfer of a prenyl moiety from a donor to an aromatic acceptor during the biosynthesis of microbial secondary metabolites. Due to their broad substrate scope, DMATSs are anticipated as biotechnological tools for producing bioactive prenylated aromatic compounds. Our study explored the substrate scope and product profile of a recombinant RePT, a novel DMATS from the thermophilic fungus Rasamsonia emersonii. Among a variety of aromatic substrates, RePT showed the highest substrate conversion for L-tryptophan and L-tyrosine (> 90%), yielding two mono-prenylated products in both cases. Nine phenolics from diverse phenolic subclasses were notably converted (> 10%), of which the stilbenes oxyresveratrol, piceatannol, pinostilbene, and resveratrol were the best acceptors (37-55% conversion). The position of prenylation was determined using NMR spectroscopy or annotated using MS 2 fragmentation patterns, demonstrating that RePT mainly catalyzed mono-O-prenylation on the hydroxylated aromatic substrates. On L-tryptophan, a non-hydroxylated substrate, it preferentially catalyzed C7 prenylation with reverse N1 prenylation as a secondary reaction. Moreover, RePT also possessed substrate-dependent organic solvent tolerance in the presence of 20% (v/v) methanol or DMSO, where a significant conversion (> 90%) was maintained. Our study demonstrates the potential of RePT as a biocatalyst for the production of bioactive prenylated aromatic amino acids, stilbenes, and various phenolic compounds. KEY POINTS: • RePT catalyzes prenylation of diverse aromatic substrates. • RePT enables O-prenylation of phenolics, especially stilbenes. • The novel RePT remains active in 20% methanol or DMSO., (© 2024. The Author(s).)

Plant biomass conversion by saprotrophic fungi plays a pivotal role in terrestrial carbon (C) cycling. The general consensus is that fungi metabolize carbohydrates, while lignin is only degraded and mineralized to CO 2 . Recent research, however, demonstrated fungal conversion of 13 C-monoaromatic compounds into proteinogenic amino acids. To unambiguously prove that polymeric lignin is not merely degraded, but also metabolized, carefully isolated 13 C-labeled lignin served as substrate for Agaricus bisporus , the world's most consumed mushroom. The fungus formed a dense mycelial network, secreted lignin-active enzymes, depolymerized, and removed lignin. With a lignin carbon use efficiency of 0.14 (g/g) and fungal biomass enrichment in 13 C, we demonstrate that A. bisporus assimilated and further metabolized lignin when offered as C-source. Amino acids were high in 13 C-enrichment, while fungal-derived carbohydrates, fatty acids, and ergosterol showed traces of 13 C. These results hint at lignin conversion via aromatic ring-cleaved intermediates to central metabolites, underlining lignin's metabolic value for fungi.

Arabinoxylo-oligosaccharides (AXOS) are non-digestible dietary fibers that potentially confer a health benefit by stimulating beneficial bacteria in the gut. Still, a detailed overview of the diversity of gut bacteria and their specificity to utilize structurally different AXOS has not been provided to date and was aimed for in this study. Moreover, we assessed the genetic information of summarized bacteria, and we extracted genes expected to encode for enzymes that are involved in AXOS hydrolysis (based on the CAZy database). The taxa involved in AXOS fermentation in the gut display a large variety of AXOS-active enzymes in their genome and consequently utilize AXOS to a highly different extent. Clostridia and Bacteroidales are generalists that consume many structurally diverse AXOS, whereas Bifidobacterium are specialists that specifically consume AXOS with a low degree of polymerization. Further complexity is evident from the fact that the exact bacterial species, and in some cases even the bacterial strains (e.g. in Bifidobacterium longum ) that are stimulated, highly depend on the specific AXOS molecular structure. Furthermore, certain species in Bifidobacterium and Lactobacillaceae are active as cross-feeders and consume monosaccharides and unbranched short xylo-oligosaccharides released from AXOS. Our review highlights the possibility that (enzymatic) fine-tuning of specific AXOS structures leads to improved precision in targeting growth of specific beneficial bacterial species and strains in the gut.