Laura S. Mazzaferro

Postharvest losses in aromatic crops often result from the discard of biomass exposed to environmental conditions that affect visual quality but not necessarily its biochemical value. In oregano ( Origanum vulgare L.) production systems, plant material left in the field after harvest is frequently exposed to nocturnal dew and subsequently rejected. This study evaluated the effect of dew exposure on essential oil yield, chemical composition, physicochemical characteristics, and biological activity. Dew exposure reduced essential oil yield by approximately 40%, indicating an effect on extraction efficiency. However, no major changes were observed in the chemical composition or physicochemical parameters of the oils, with thymol remaining the dominant compound. In addition, antioxidant capacity, polyphenol oxidase inhibition, and antifungal activity against Aspergillus parasiticus , Fusarium verticillioides , and Eurotium amstelodami were preserved. These results demonstrate that biomass currently discarded based on visual criteria retains its chemical identity and functional properties. From a biomass valorization perspective, dew-exposed oregano represents a viable raw material to produce value-added essential oils, contributing to reduced postharvest losses and improved resource efficiency in aromatic crop systems. Statement of Novelty The novelty of this study lies in demonstrating that oregano biomass discarded after nocturnal dew exposure retains its chemical identity and biological functionality, despite a significant reduction in essential oil yield. While postharvest losses in aromatic crops are typically assessed based on visual quality, this work provides experimental evidence that such criteria may not reflect the true value of the biomass. By integrating compositional, physicochemical, and bioactivity analyses, the study shows that dew-exposed material remains a viable source of functional essential oils. This shifts the perspective from quality loss to resource recovery, supporting biomass valorisation strategies and offering a practical approach to reduce postharvest waste in oregano production systems.

The fungal diglycosidase α-rhamnosyl-β-glucosidase I (αRβG I) from Acremonium sp. DSM 24697 catalyzes the glycosylation of various OH-acceptors using the citrus flavanone hesperidin. We successfully applied a one-pot biocatalysis process to synthesize 4-methylumbellipheryl rutinoside (4-MUR) and glyceryl rutinoside using a citrus peel residue as sugar donor. This residue, which contained 3.5 % [w/w] hesperidin, is the remaining of citrus processing after producing orange juice, essential oil, and peel-juice. The low-cost compound glycerol was utilized in the synthesis of glyceryl rutinoside. We implemented a simple method for the obtention of glyceryl rutinoside with 99 % yield, and its purification involving activated charcoal, which also facilitated the recovery of the by-product hesperetin through liquid-liquid extraction. This process presents a promising alternative for biorefinery operations, highlighting the valuable role of αRβG I in valorizing glycerol and agricultural by-products. Keypoints: • αRβG I catalyzed the synthesis of rutinosides using a suspension of OPW as sugar donor. • The glycosylation of aliphatic polyalcohols by the αRβG I resulted in products bearing a single rutinose moiety. • αRβG I catalyzed the synthesis of glyceryl rutinoside with high glycosylation/hydrolysis selectivity (99 % yield). Graphical

In the biosynthesis of menaquinone in bacteria, the thiamine diphosphate‐dependent enzyme MenD catalyzes the decarboxylative carboligation of α‐ketoglutarate and isochorismate to (1R,2S,5S,6S)‐2‐succinyl‐5‐enolpyruvyl‐6‐hydroxycyclohex‐3‐ene‐1‐carboxylate (SEPHCHC). The regioisomer of SEPHCHC, namely (1R,5S,6S)‐2‐succinyl‐5‐enolpyruvyl‐6‐hydroxycyclohex‐2‐ene‐1‐carboxylate (iso‐SEPHCHC), has been considered as a possible product, however, its existence has been doubtful due to a spontaneous elimination of pyruvate from SEPHCHC to 2‐succinyl‐6‐hydroxy‐2,4‐cyclohexadiene‐1‐carboxylate (SHCHC). In this work, the regioisomer iso‐SEPHCHC was distinguished from SEPHCHC by liquid chromatography‐tandem mass spectrometry. Iso‐SEPHCHC was purified and identified by NMR spectroscopy. Just as SEPHCHC remained hidden as a MenD product for more than two decades, its regioisomer iso‐SEPHCHC has remained until now.

Diglycosidases are endo-β-glucosidases that hydrolyze the heterosidic linkage of diglycoconjugates, thereby releasing in a single reaction the disacch

Diglycosidases hydrolyze the heterosidic linkage of diglycoconjugates, releasing the disaccharide and the aglycone. Usually, these enzymes do not hydrolyze or present only low activities towards monoglycosylated compounds. The flavonoid degrading fungus Acremonium sp. DSM 24697 produced two diglycosidases, which were termed 6-O-α-rhamnosyl-β-glucosidase I and II (αRβG I and II) because of their function of releasing the disaccharide rutinose (6-O-α-L-rhamnosyl-β-D-glucose) from the diglycoconjugates hesperidin or rutin. In this work, the genome of Acremonium sp. DSM 24697 was sequenced and assembled with a size of ~ 27 Mb. The genes encoding αRβG I and II were expressed in Pichia pastoris KM71 and the protein products were purified with apparent molecular masses of 42 and 82 kDa, respectively. A phylogenetic analysis showed that αRβG I grouped in glycoside hydrolase family 5, subfamily 23 (GH5), together with other fungal diglycosidases whose substrate specificities had been reported to be different from αRβG I. On the other hand, αRβG II grouped in glycoside hydrolase family 3 (GH3) and thus is the first GH3 member that hydrolyzes the heterosidic linkage of rutinosylated compounds. The substrate scopes of the enzymes were different: αRβG I showed exclusive specificity toward 7-O-β-rutinosyl flavonoids, whereas αRβG II hydrolyzed both 7-O-β-rutinosyl- and 3-O-β-rutinosyl- flavonoids. None of the enzymes displayed activity toward 7-O-β-neohesperidosyl- flavonoids. The recombinant enzymes also exhibited transglycosylation activities, transferring rutinose from hesperidin or rutin onto various alcoholic acceptors. The different substrate scopes of αRβG I and II may be part of an optimized strategy of the original microorganism to utilize different carbon sources.

Chorismate and isochorismate constitute branch‐point intermediates in the biosynthesis of many aromatic metabolites in microorganisms and plants. To obtain unnatural compounds, we modified the route to menaquinone in Escherichia coli. We propose a model for the binding of isochorismate to the active site of MenD ((1R,2S, 5S,6S)‐2‐succinyl‐5‐enolpyruvyl‐6‐hydroxycyclohex‐3‐ene‐1‐carboxylate (SEPHCHC) synthase) that explains the outcome of the native reaction with α‐ketoglutarate. We have rationally designed variants of MenD for the conversion of several isochorismate analogues. The double‐variant Asn117Arg–Leu478Thr preferentially converts (5S,6S)‐5,6‐dihydroxycyclohexa‐1,3‐diene‐1‐carboxylate (2,3‐trans‐CHD), the hydrolysis product of isochorismate, with a >70‐fold higher ratio than that for the wild type. The single‐variant Arg107Ile uses (5S,6S)‐6‐amino‐5‐hydroxycyclohexa‐1,3‐diene‐1‐carboxylate (2,3‐trans‐CHA) as substrate with >6‐fold conversion compared to wild‐type MenD. The novel compounds have been made accessible in vivo (up to 5.3 g L⁻¹). Unexpectedly, as the identified residues such as Arg107 are highly conserved (>94 %), some of the designed variations can be found in wild‐type SEPHCHC synthases from other bacteria (Arg107Lys, 0.3 %). This raises the question for the possible natural occurrence of as yet unexplored branches of the shikimate pathway.

The structure of the carbohydrate moiety of a natural phenolic glycoside can have a significant effect on the molecular interactions and physicochemical and pharmacokinetic properties of the entire compound, which may include anti‐inflammatory and anticancer activities. The enzyme 6‐O‐α‐rhamnosyl‐β‐glucosidase (EC 3.2.1.168) has the capacity to transfer the rutinosyl moiety (6‐O‐α‐l‐rhamnopyranosyl‐β‐d‐glucopyranose) from 7‐O‐rutinosylated flavonoids to hydroxylated organic compounds. This transglycosylation reaction was optimized using hydroquinone (HQ) and hesperidin as rutinose acceptor and donor, respectively. Since HQ undergoes oxidation in a neutral to alkaline aqueous environment, the transglycosylation process was carried out at pH values ≤6.0. The structure of 4‐hydroxyphenyl‐β‐rutinoside was confirmed by NMR, that is, a single glycosylated product with a free hydroxyl group was formed. The highest yield of 4‐hydroxyphenyl‐β‐rutinoside (38%, regarding hesperidin) was achieved in a 2‐h process at pH 5.0 and 30 °C, with 36 mM OH‐acceptor and 5% (v/v) cosolvent. Under the same conditions, the enzyme synthesized glycoconjugates of various phenolic compounds (phloroglucinol, resorcinol, pyrogallol, catechol), with yields between 12% and 28% and an apparent direct linear relationship between the yield and the pK_a value of the aglycon. This work is a contribution to the development of convenient and sustainable processes for the glycosylation of small phenolic compounds.