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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 MS2 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. Graphical abstract: (Figure presented.)

The utility of 3D-small intestinal organoid (enteroid) models for evaluating effects of e.g. food (related) compounds is limited due to the apical epithelium facing the interior. To overcome this limitation, we developed a novel 3D-apical-out enteroid model for mice, which allows apical exposure. Using this model, we evaluated the effects on the enteroids’ intestinal epithelium (including cytotoxicity, cell viability, and biotransformation) after exposure to glabridin, a prenylated secondary metabolite with antimicrobial properties from licorice roots (Glycyrrhiza glabra). Apical-out enteroids were five times less sensitive to glabridin exposure compared to conventional apical-in enteroids, with obtained cytotoxicities of 1.5 mM and 0.31 mM, respectively. Apical-out enteroids showed a luminal/apical layer of fucose rich mucus, which may contribute to the protection against potential cytotoxicity of glabridin. Furthermore, in apical-in enteroids IC50 values for cytotoxicity were determined for licochalcone A, glycycoumarin, and glabridin, the species-specific prenylated phenolics from the commonly used G. inflata, G. uralensis, and G. glabra, respectively. Both enteroid models differed in their functional phase II biotransformation capacity, where glabridin was transformed to glucuronide- and sulfate-conjugates. Lastly, our results indicate that the prenylated phenolics do not show cytotoxicity in mouse enteroids at previously reported minimum inhibitory concentrations (MICs) against a diverse set of Gram positive bacteria. Altogether, we show that apical-out enteroids provide a better mimic of the gastrointestinal tract compared to conventional enteroids and are consequently a superior model to study effects of food (related) compounds. This work revealed that prenylated phenolics with promising antibacterial activity show no harmful effects in the GI-tract at their MICs and therefore may offer a new perspective to control unwanted microbial growth.

This Comment critically addresses the article by Gao et al. ( Gao, K. , et al. J. Agric. Food Chem. 2015, 63, 1067−1075 ), providing the structural elucidation of three phenolamide dimers (neolignanamides) from the fruits of Lycium barbarum. A more recent article published by Chen et al. ( Chen, H. , et al. J. Agric. Food Chem. 2023, 71, 11080−11093 ) incorporates these structures into further research on the bioactivity of these compounds. Although the analytical techniques used by Gao et al. are adequate, in our opinion, the nuclear magnetic resonance (NMR) spectroscopic data have not been interpreted correctly, resulting in incorrect structures for three neolignanamides from the fruits of L. barbarum. In this Comment, an alternative interpretation of the NMR spectroscopic data and the corresponding structures are proposed. The proposed structures feature linkage types that are much more common for neolignanamides than the linkage types in the originally reported structures of these compounds.