Journal of Natural Products

Natural products are a rich source of bioactive molecules and undiscovered chemical scaffolds with significant potential for novel drug discovery. Among these, fungi are particularly promising, offering diverse metabolites and undiscovered structural motifs. Large, well-curated collections of crude extracts, or "libraries", are central to fungal natural product discovery, serving as starting material for bioassay-guided isolation of new compounds. However, the systematic influence of fungal selection strategies, culturing methods, and environmental factors on chemical diversity remains underexplored. In this study, we analyzed several large fungal libraries to assess how geographic origin, and phylogenetic classification shape fungal chemical profiles. We also evaluated whether culturing conditions that more closely mimic natural environments can enhance metabolite diversity. Our findings offer practical guidelines for optimizing fungal natural product library design, improving drug development efficiency and access to novel chemotypes for future drug discovery. Summary Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=60 SRC="FIGDIR/small/709592v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@70a0e0org.highwire.dtl.DTLVardef@51f84eorg.highwire.dtl.DTLVardef@184dd90org.highwire.dtl.DTLVardef@1ee2813_HPS_FORMAT_FIGEXP M_FIG C_FIG

Actinoplanes teichomyceticus is a well-established producer of bioactive secondary metabolites, including the glycopeptide antibiotic teicoplanin. Although its antibiotic biosynthetic capacity has been extensively investigated, its siderophore diversity and any additional biological functions of these iron-chelating metabolites remain comparatively underexplored. We identified a reproducibly bioactive, teicoplanin-independent fraction that inhibited Bacillus spizizenii. Molecular networking applied to this fraction identified hydroxamate ferrioxamine and desferrioxamine-type siderophores as the dominant metabolites, including acylated analogs detected as Al3+- and Fe3+-chelated species. Robust siderophore secretion was confirmed by the CAS assay. Notably, siderophore-enriched fractions exhibited selective antibacterial activity against Gram-positive bacteria, with minimum inhibitory concentrations of approximately 16 {micro}g/mL against B. spizizenii and partial inhibition of Staphylococcus aureus, while no activity was observed against Escherichia coli. Synthetic C7 and C9 acyl-desferrioxamine analogs showed enhanced antibacterial activity upon Al3 chelation, indicating a metal-dependent bioactivity. These findings reveal an unexpected antibacterial role for ferrioxamine-type siderophores produced by A. teichomyceticus, extending their function beyond iron acquisition, possibly through a "Trojan horse" (or "Trojan metal") mechanism.

Mushroom production generates large amounts of by-products, particularly stipes, which can represent up to half of the fruiting body biomass. Due to their similar composition to mushroom caps, these residues represent a promising substrate for the development of value-added foods. In this study, oyster mushroom stipes were used as a substrate for solid-state fermentation (SSF) with a Neurospora crassa strain isolated in Albacete to produce a novel meat analogue inspired by the oncom. Fermentation generated a cohesive matrix bound by hyphae that adopted the shape of the mold and exhibited a meat-like color, although with a softer texture. Nutritional analysis revealed a product with relatively low protein content but a complete amino acid profile, enriched in dietary fiber and containing unsaturated fatty acids. These results demonstrate that SSF with N. crassa provides a strategy to upcycle oyster mushroom by-products into fiber-rich meat analogues with potential applications in sustainable food systems.

Mixed-linkage {beta}-glucans (MLGs) are emerging as promising biopolymers with significant biotechnological potential due to their unique structural and rheological properties. In rhizobia, MLG biosynthesis is controlled by the second messenger cyclic di-GMP (c-di-GMP) and mediated by the bicistronic operon bgsBA. However, the full composition of the biosynthetic machinery and strategies for enhanced production remain incompletely understood. In this study, we demonstrate that the outer membrane protein TolC is essential for MLG production in Sinorhizobium meliloti. Genetic disruption of tolC abolished MLG synthesis, while its complementation restored production. We propose that TolC forms a tripartite complex with BgsA and BgsB, enabling efficient polymer synthesis and export. Furthermore, co-overexpression of tolC, bgsBA, and a constitutively active diguanylate cyclase (pleD*) yielded a 10-fold increase of MLG over a control plasmid without tolC, reaching up to [~]10 g/L under bioreactor conditions. Additionally, this genetic module enabled de novo MLG production in otherwise non-producer rhizobial hosts (e.g. Mesorhizobium japonicum), allowing bacterial chassis exchanges and highlighting its portability and potential for synthetic biology applications. Overall, our findings identify TolC as a key component of the MLG biosynthetic machinery and provide a robust platform for the scalable production of this valuable biopolymer. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/721817v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@1da8e1org.highwire.dtl.DTLVardef@13a7b06org.highwire.dtl.DTLVardef@62d6eeorg.highwire.dtl.DTLVardef@10cc02d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Polyketide biosynthesis is typically initiated by loading a starter unit onto an acyl carrier protein (ACP). In type I modular polyketide synthases (PKSs), responsible for the assembly of diverse bioactive metabolites in bacteria, this ACP is usually incorporated into a chain initiation module, alongside a starter unit loading domain. In several cases, the starter unit undergoes structural modification prior to the initiation of chain assembly. Gladiolin, an antibiotic with promising activity against bacterial and fungal pathogens, is assembled by a trans-acyltransferase (AT) PKS in Burkholderia gladioli. It appears to incorporate a succinyl starter unit, but the gladiolin PKS lacks a conventional loading module, making it unclear how this happens. The gladiolin biosynthetic gene cluster encodes an AT of unassigned function (GbnB), an ACP (GbnA), and an asparagine synthetase homolog (GbnC) with similarity to enzymes that amidate the malonyl-ACP starter unit in glutarimide antibiotic biosynthesis. Here, we elucidate a cryptic starter unit amidation mechanism in gladiolin biosynthesis involving these three proteins. GbnB loads a succinyl unit onto the phosphopantetheinyl arm of GbnA, which is subsequently amidated by GbnC using glutamine as the nitrogen donor. After the fully assembled polyketide chain is released, GbnM hydrolyzes the amide, yielding mature gladiolin. Phylogenetic analyses, coupled with gene cluster reannotation revealed analogous enzymatic machinery likely responsible for cryptic succinamyl and malonamyl starter unit incorporation into etnangien and sorangicin A, respectively. Retro-biosynthetic analyses suggest succinamyl and malonamyl starter units may be involved in the assembly of other metabolites, such as the sorangiolides and azumamides.

The prevalence of naturally occurring C13-acetoxy taxanes, together with the presence of a native C13-acetyltransferase in yew trees, suggests that the natural biosynthetic pathway for paclitaxel may involve a cryptic C13-O-deacetylation step. However, whether a putative taxane C13-O-deacetylase (T13dA) acts in the pathway of paclitaxel biosynthesis remains elusive. Here we functionally characterized two novel taxane C13-O-deacetylases (T13dA1 and T13dA2) from Taxus x media cell cultures, providing experimental evidence for the molecular and biochemical plausibility of C13-O-deacetylation in paclitaxel biosynthesis in Taxus species. Also, we identified a previously uncharacterized bifunctional taxane C7{beta}-O-, C9-O-deacetylase, designated T79dA, which demonstrates the functional promiscuity by enabling stepwise deacetylation at taxane C7{beta} and C9 positions in a single enzymatic reaction. Furthermore, T7dA1, a novel taxane C7{beta}-O-deacetylase with higher activity than the reported T7dA was discovered and characterized here. Moreover, we reconstituted two new pathways (an 18-gene and a 19-gene pathway) enabled by the integration of a C13-O-acetylation-deacetylation module for the de novo biosynthesis of baccatin III in Nicotiana benthamiana leaves. These pathways with the previously established 17-gene baccatin III pathway, further allow paclitaxel biosynthesis to be a network. Our reconstituted 19-gene pathway achieves a baccatin III yield of up to 23 g g-1 dried weight (DW) in N. benthamiana leaves, which is comparable to the yield reported for the 17-gene pathway. This work facilitates a better understanding, elucidation and reconstruction of metabolic network of paclitaxel biosynthetic pathway, and provides new enzymes and strategies for artificial pathway reconstruction and efficiently bio-chemical production of paclitaxel.

Enzymatic degradation of synthetic polymers has attracted broad interest because it offers environmental and manufacturing advantages compared to traditional mechanical and chemical breakdown approaches. Enzymes are highly specific and reaction conditions are generally aqueous and require low pressure and temperature, resulting in lower energy consumption and lower chemical waste production. Here we report the biochemical and structural characterization of three newly discovered enzymes capable of Nylon hydrolysis: Nyl10, Nyl12 and Nyl50. Using solution characterization techniques, we found that the enzymes adopt a single oligomeric state consistent with a tetramer over a wide range of concentrations. X-ray crystallographic structures of all three enzymes support the association into tetramers. Comparison of ligand-bound X-ray crystal structures of Nyl10 and Nyl12 with the previously determined structure of Nyl50 identified key structural determinants involved in ligand binding. Noticeably, a flexible loop found in several polyamide degrading enzymes is observed to flip towards (closed conformation) and away (open conformation) from the active site upon ligand binding. Analysis of adduct and surrogate substrate-bound enzyme complex structures provide a model for substrate binding directionality. Finally, activity assays showed that both Nyl10 and Nyl12 can hydrolyze ester bonds, and that Nyl12 has the highest activity toward PA66, identifying it as the best candidate for protein engineering for efficient nylon hydrolysis.

Antibiotic resistance in Mycobacterium tuberculosis is a pressing global health challenge demanding new therapeutic strategies. The bacterial respiratory chain comprises promising antibacterial targets, with dual inhibition of the terminal oxidases cytochrome bcc:aa3 and cytochrome bd (cyt bd) showing bactericidal activity. While bcc:aa3 inhibitors such as Q203 have advanced clinically, cyt bd remains underexplored due to difficulties in assigning activity of the purified enzyme and structurally resolving the quinol substrate binding site. Here, we report a rapid in vitro screening platform for cyt bd inhibitors by engineering a minimal respiratory system that couples the activity of cyt bd to that of a type 2 NADH dehydrogenase. This coupled assay enables spectroscopic monitoring of NADH oxidation as a proxy for cyt bd activity, allowing rapid screening of over 10,000 compounds. Screening identified WSL017, a fragment with low micromolar potency against both M. tuberculosis and E. coli cyt bd. Kinetic and structural analyses revealed competitive inhibition at the quinol-binding site, providing the first structural insights into cyt bd inhibition by a non-quinone scaffold. WSL017 displayed growth inhibition of M. tuberculosis H37ra, corroborating oxidase inhibition as a promising therapeutic strategy. This work establishes a pipeline for cyt bd inhibitor discovery and highlights new opportunities for structure-guided drug development against cytochrome bd oxidases.

Rhamnogalacturonan-II (RG-II) is considered the most complex glycan in nature. It forms part of an intricate network of complex glycans in the plant cell wall where it plays a critical role in plant growth, development and defence. It has been identified as an important nutrient source for the human gut microbiota (HGM), a key modulator of human health and disease status. Increasing evidence also suggests that RG-II can modulate plant-microbe interactions. Given its importance and potential, detailed studies of RG-IIs structure-function relationships and metabolism are required to underpin future crop-improvement strategies and to harness its benefits for plant and human health. Progress in this field is however hampered by RG-IIs structural complexity and limited access to enabling tools, in particular chemically defined RG-II-derived oligosaccharide (CDRO) substructures. Achieving targeted, efficient, and scalable production of CDROs remains a significant challenge and is indeed one of the major reasons why RG-II and glycomic research in general, significantly lag behind genomic and proteomic research. Here, we have genetically engineered as well as screened a diverse set of genetic strains, including transposon (Tn) mutants of the prominent model human gut microbe Bacteroides thetaiotaomicron (B. theta) and its gut and plant-associated relatives for new CDRO-generating and/or RG-II-utilising strains. Several CDROs, some of which had never been produced before by any other means (including chemical synthesis), where generated and characterised by a combination of high-resolution mass spectrometry (MS), enzymatic profiling and 2D-NMR. In addition to expanding the CDRO toolbox, we identified key genetic strains that will serve as a base or platform for the production of an unprecedented amount of CDROs covering the complexity and diversity of chemical modifications in RG-II. CDROs were later exploited to gain new insights into the microbial metabolism of RG-II in the human gut, revealing key aspects of its chemical structure that drive or limit its metabolism in B. theta. Notably, we generated new evidence in support of an alternative operational paradigm for polysaccharide utilisation systems that are widespread in the Bacteroidota phylum. We confirmed the presence of pathways for the metabolism of RG-II and/or RG-II core sugars D-apiose (D-Apif), and 3-deoxy-D-manno-2-octulosonic acid (D-Kdo) in aerobic plant-associated microbes including fungi and Flavobacterium spp., highlighting their potential to be exploited as cost-effective alternatives to B. theta for the generation of CDROs.

Forest tree seeds are mass produced for afforestation and forest restoration programs, but are mostly underutilized beyond propagation. Here, we aimed to evaluate the antioxidant, anti-inflammatory, anticancer, and tyrosinase-inhibitory activities of seed extracts of seven economically important forest tree species in the Republic of Korea to explore their potential as multifunctional natural bioresources. The seed extracts of Alnus japonica, Chamaecyparis obtusa, Cornus kousa, Phellodendron amurense, Pinus densiflora, Prunus sargentii, and Quercus glauca were comparatively assessed using multiple in vitro assays. The results revealed clear species-dependent functional profiles rather than uniform bioactivities across species. Quercus glauca exhibited strong antioxidant activity and significant anti-inflammatory and tyrosinase-inhibitory activities, suggesting multifunctional potential, while C. obtuse presented considerable anticancer activity against several cancer cell lines. Alnus japonica exhibited the highest tyrosinase-inhibitory activity, followed by Q. glauca and C. obtuse; A. japonica extract also showed a strong antioxidant capacity. Overall, the results demonstrated that forest tree seed extracts possess diverse and complementary bioactivities, supporting their potential as underexplored multifunctional natural materials. By focusing on seed resources generated within existing afforestation systems, we highlight a sustainable approach to valorize forest-derived by-products without additional pressure on natural ecosystems. Nevertheless, as bioactivities were evaluated using crude extracts, further studies are required to identify and elucidate the active compounds and their mechanisms of action.

Streptomyces are prolific producers of bioactive compounds and increasingly recognized as members of insect microbiomes, yet the microbiome of cadaveric fly larvae remain an overlooked system for discovering metabolically versatile Streptomyces species. Here, we conduct targeted bacterial isolations from the microbiome of fly larvae collected from pig cadavers, generating 42 Streptomyces isolates of interest, and systematically evaluated their metabolic potential through genomic analysis, antimicrobial screening, biosynthetic gene cluster assessment, untargeted LC-MS/MS metabolomics, and compound purification. The Streptomyces isolates spanned nine species, including underrepresented lineages for which we added genomic representatives. Streptomyces from carrion fly larvae exhibited broad-spectrum antimicrobial activity and substantial BGC diversity, supported by metabolomic detection of antimycins, surugamides, and macrotetrolides. From a deep phylogenetic lineage, we purified JBIR-68 and Simamycin and demonstrated their potent anthelmintic activity against Brugia malayi microfilariae. GNPS molecular networking revealed three additional JBIR-68 analogs, establishing the first taxonomically resolved Streptomyces lineage capable of producing these rare metabolites. Our findings position cadaveric fly larvae as a rich ecological reservoir for discovering Streptomyces with the potential to produce chemically diverse natural products with biomedical applications.

Biological metal chelators are of great interest for investigation due to their capacity to retain or mobilize metals from the environment. While some biological and bioinspired chelators find use in medical applications, others are promising platforms for the mining or recycling of technologically important metal ions. In particular, the siderophores, which are primarily iron chelators, have been studied. Four siderophores of relevance are schizokinen and its derivatives, which have been isolated from bacterial and algae cultures, in addition to soil. These siderophores have shown metal chelating activity with different metals such as iron, copper, and aluminum. In the time of metabolomics, it is required to unambiguously determine the identity of the produced siderophores as quickly as possible. Thus, Liquid Chromatography coupled to High Resolution Mass Spectrometry and mass-tandem fragmentation (LC-HRMS-MS) provides a quick and applicable alternative for identification of schizokinen and its derivatives. Here, we report an analytical method for the identification and potential quantification of the schizokinen siderophore series. We developed a working method through LC-HRMS-MS, which provides the unequivocal identification of the four schizokinen derivatives, which has not been reported to date. Additionally, we constructed the molecular network for the four molecules to enable their identification using the Global Natural Products Social Molecular Networking (GNPS) platform. Most importantly, this contribution can help speed up the characterization of schizokinen producers and facilitate the dereplication process of siderophores.

Streptomyces bacteria produce a variety of secondary metabolites that hold clinical and agricultural value, yet their biosynthetic potential remains unrealized as many biosynthetic gene clusters are not expressed under standard laboratory conditions. Expression of these clusters is tightly regulated, often by cluster situated transcription factors. The TetR family are regulators whose activity is modulated by small molecule elicitors. Although many TetRs have been characterized, elicitors have only been identified for a small fraction of them. This lack of data presents a limitation in our ability to exploit elicitor-regulator pairs for activation of silent clusters and underscores the need for predictive and testable strategies for elicitor identification. In this work, we test the use of sequence similarity networks (SSNs) as a predictor of elicitor identity using the well characterized TetR protein, JadR2, that has a known elicitor, chloramphenicol. We utilized SSNs to identify JadR2 homologs that may also be elicited by chloramphenicol. We developed a heterologous Escherichia coli reporter system in which regulator activity was monitored using an EGFP readout of DNA binding activity. Using this system, we screened JadR2 and four homologs for responsiveness to chloramphenicol. We found that 3 homologs were elicited by chloramphenicol, all of which were formerly uncharacterized. These results demonstrate that TetR-family proteins can share elicitor responsiveness and that SSNs can be used to prioritize regulators for functional screening. This work establishes a genomics-informed and bioinformatics-guided framework for linking elicitors to their regulator, expanding the toolkit for natural product discovery by unlocking regulatory information across Streptomyces.

In light of the "silent" AMR pandemic, new avenues to combat pathogenic bacteria are needed. In this work, we screened a large molecule library (n=35 684 unique compounds) with the aim of identifying molecules being able to bind and block translation of the prfA-thermosensor transcript in the bacterial pathogen Listeria monocytogenes. Using a thiazole-orange displacement approach, 468 ([~]1.3% of all molecules) showed the ability to reduce fluorescence. After dose response testing, 32 compounds remained promising and eight of them showed sufficient purity and availability to be further validated. Interestingly, four compounds, being structurally very similar, showed specificity for prfA at a varying degree. All four compounds carried 3 aromatic rings with one connecting amine between two of the rings and an amide linking an aliphatic amine side chain. The most selective compounds, M5, showed a Kd of [~]0.8 {micro}M for the prfA RNA at 35{degrees}C. However, none of the eight most efficient compounds were able to inhibit prfA translation in vitro, suggesting that the molecules are able to bind but not affect the stability of the overall structure. Through this work, we have been able to identify a set of molecules, able to bind the prfA thermosensor RNA selectively, but without affecting translation. These molecules could constitute an important scaffold for further drug development.

Endophytic fungi residing within medicinal plants are emerging as prolific sources of structurally diverse bioactive secondary metabolites with applications in drug discovery. Azadirachta indica (Neem) and Melia azedarach (Melia), members of the Meliaceae family, are renowned for their rich phytochemical composition; however, the contribution of their endophytic fungi communities to this chemical diversity remains largely unexplored. Herein, endophytic fungi were isolated from leaves and bark of Neem and Melia collected in Kenya and cultured under distinct physical conditions, solid (plates) and liquid (broth) media to assess how culture environment influences compound production. Compounds were extracted and analyzed using gas chromatography-mass spectrometry (GCMS) to profile the chemical diversity associated with each endophytic fungi, physical culturing state and host plant. GCMS analysis revealed that while the host plant identity influences the presence of specific compounds, the dominant determinant of chemical diversity was intrinsic biosynthetic capacity of the endophytic fungi themselves. Several compounds were unique to endophytic fungi cultures, highlighting their role as independent sources of bioactive compounds. Culture conditions moderately influence metabolite profiles, demonstrating the importance of optimizing growth environments in experimental design and natural product bioprospecting. From the Neem samples, we found 53 compounds uniquely present in the broth samples (consisting of Neem powder and endophytic fungi), 22 found exclusively with the endophytic fungi from the Neem, and 31 compounds shared between the broth and the endophytic fungi samples. In Melia samples, 109 compounds were uniquely present in broth samples from Melia plant (consisting of Melia powder and endophytic fungi), 22 compounds were found exclusively with the endophytic fungi from the Melia, and 55 were shared between the broth and the endophytic fungi samples. Our comparative analysis assessed the Neem and Melia endophytic fungi exclusive samples and reported 12 shared compounds. 10 compounds were unique to Neem and 10 unique to Melia; however, their identities varied between the two categories. While GCMS enabled the identification of volatile and semi-volatile metabolites, future studies employing complementary metabolomic approaches, such as liquid chromatography-mass spectrometry (LCMS), ultra-high-performance liquid chromatography MS/MS (UHPLC MS/MS), or nuclear magnetic resonance (NMR) spectroscopy, would expand coverage to non-volatile, polar, and high molecular weight compounds, providing a more comprehensive understanding of endophyte-derived chemical diversity. These findings provide insights into the interplay between medicinal plants and their endophytes and establish a foundation for leveraging endophytic fungi from Neem and Melia as scalable sources of structurally complex natural products for pharmaceutical and biotechnological applications while minimizing ecological impact.

Licorice (Glycyrrhiza) is a medicinal plant widely used in approximately 70% of traditional Japanese Kampo formulations and is known to produce a wide array of specialized metabolites with diverse pharmacological properties. Although hundreds of metabolites have been reported, the overall chemical diversity of Glycyrrhiza remains poorly characterized. Here, using mass spectrometry data obtained from fully 13C-labeled leaves and roots of Glycyrrhiza uralensis and Glycyrrhiza glabra, we determined the carbon number, followed by molecular formula and substructure prediction in combination with MS/MS similarity-based molecular networking. After excluding redundant ions, including isotopic peaks, adducts, and in-source fragments, we extracted 3,060 unique metabolite features with assigned carbon numbers. Among these, substructure information was assigned to 1,015 features (33%) across the four plant tissues, revealing the tissue-specific metabolome profiles. Furthermore, we discovered five previously unreported alkaloids, homopipecolic acid-conjugated flavonoids, in the roots of G. uralensis and G. glabra, and Glycine max, another member of the Fabaceae family. Two of these structures were validated using nuclear magnetic resonance spectroscopy. We further proposed a biosynthetic route involving a spontaneous reaction between 1-piperideine and malonyl glycoside substrates and confirmed the formation of the conjugated product using authentic standards.

The ability of SAM-dependent enzymes to accept S-adenosyl-D-methionine [D-SAM, (SS,RC)-SAM] instead of the native cofactor S-adenosyl-L-methionine [L-SAM, (SS,SC)-SAM] remains largely unexplored. Challenging the stereochemical preference of SAM-dependent enzymes, we investigated the ability of different enzyme classes to accept D-SAM. Contrary to common assumptions, the tested N- and O-methyl transferases (MTs), as well as one of the examined C-MTs accepted D-SAM. Docking studies suggest that acceptance of D-SAM by C-MTs may be influenced by the angle between the transferable methyl group of SAM and the nucleophilic carbon of the substrate, along with enzyme and substrate flexibility. In addition to conventional MTs, the radical SAM glutamine C-MT QCMT showed low but detectable methylation activity with D-SAM. Furthermore, the azetidine-2-carboxylic acid synthase AzeJ not only uses D-SAM but also incorporates the stereocentre of D-methionine into the cyclic amino acid product. The pyridoxal 5'-phosphate (PLP)-dependent enzyme 1-aminocyclopropyl-1-carboxylic acid synthase (ACCS) also showed detectable turnover with D-SAM. These findings broaden the understanding of enzyme stereoselectivity, provide an overview of D-SAM-utilising enzymes, and identify first enzyme systems that may serve as starting points for engineering efforts aimed at shifting cofactor preference towards D-SAM.

The hydrolytic breakdown of cellobiose into glucose, catalysed by {beta}-glucosidases, is the last and rate-limiting step in cellulose saccharification for producing fermentable glucose in the bioethanol industry. This limitation arises because {beta}-glucosidase activity is inhibited by factors such as temperature, pH, and glucose accumulation in reactors. Enzyme inactivation leads to the buildup of cello-oligosaccharides, which, in turn, inhibit upstream cellulases. Therefore, glucose-tolerant {beta}-glucosidases are preferred for the formulation of industrial cellulase cocktails. In this study, we have recombinantly expressed, purified, and biochemically characterised a {beta}-glucosidase from the cellulolytic fungus Fusarium odoratissimum (FoBgl-WT). FoBgl-WT exhibits optimal cellobiose hydrolysis over a broad pH range (4.5-7.5), an important and industrially desirable property for its application in bioreactors. However, the glucose tolerance of FoBgl-WT was [~]0.56 M. Structure-based analyses were carried out to map the residues lining the active site of FoBgl, and their roles in stabilising the product glucose (or even the substrate, cellobiose) were elucidated through a series of site-specific mutations, followed by biochemical characterisation of the resulting FoBgl mutants. Among all the mutants generated, FoBgl-K256I-Y325F exhibits >2.5-fold greater glucose tolerance ([~]1.4 M) than FoBgl-WT. Further, we have observed that the FoBgl-K256W and FoBgl-K256I mutants exhibit improved kinetic properties, such as catalytic efficiencies. The structure-based rational engineering efforts improve glucose tolerance and the kinetic properties of FoBgl mutants, making it a useful and promising candidate enzyme for industrial cellulase cocktails.

Cationic polymers, which mimic the structure of antimicrobial peptides (AMPs), are increasingly recognized as promising antimicrobial materials. Here, we report the synthesis and evaluation of a new class of cationic lipid-terminated oligomers (CLOs), comprised of 2C18-hydrophobic lipid tails, and short oligomeric cationic chains synthesised via Cu(0)-mediated reversible-deactivation radical polymerization (RDRP). Two 2-vinyl-4,4-dimethyl-5-oxazolone (VDM) oligomers with degrees of polymerization (DP) of 20 or 50 were synthesized using the lipid functional initiator (R)-3-((2-bromo-2-methylpropanoyl) oxy)propane-1,2-diyl dioctadecanoate (2C18-Br). Post-polymerization modification of the pendant oxazolone moieties was carried out using reactive amines, including N-Boc-ethylenediamine (BEDA) and N,N-dimethylethylenediamine (DMEN). Subsequent deprotection of the BEDA groups and quaternization of DMEN groups enabled the synthesis of six functional CLOs exhibiting distinct cationic functionalities. Antimicrobial assays against a panel of WHO bacterial and fungal priority pathogens (methicillin-resistant Staphylococcus aureus [MRSA], Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Candida albicans, and Cryptococcus neoformans) revealed that these CLOs exhibited potent and selective structure-dependent antibacterial activity, particularly against MRSA, with minimum inhibitory concentrations (MICs) in the clinically relevant range, below 4 {micro}g mL-1, comparable to antibiotics vancomycin and colistin. Among these, BEDA-functionalized CLOs demonstrated the strongest antimicrobial profile, which was significantly increased by increasing DP, as evidenced by a reduction in MIC values from 64 {micro}g mL-1 (for DP20) to [≤] 4 {micro}g mL-1 (for DP50) against A. baumannii. Biocompatibility assays against red blood cells and HEK293 cells indicated negligible toxicity, with haemolytic (HC50) and cytotoxic (CC50) values exceeding 512 {micro}g mL-1 across all CLOs. All CLOs displayed minimal activity against C. albicans (MIC [≥] 512 {micro}g mL-1). In contrast, activity against C. neoformans was influenced by both cationic functionality and DP, with DMEN-based CLOs exhibited superior antifungal activity at higher DP relative to their BEDA-based counterparts. Most CLOs displayed high selectivity (SI) toward MRSA (SI >128), while 2C18-O(BEDA)50 exhibited the broadest spectrum, showing potent antimicrobial activity and high selectivity against E. coli (MIC [≤] 4 {micro}g mL-1, SI [≥] 128), A. baumannii (MIC [≤] 4 {micro}g mL-1, SI [≥] 128), and MRSA (MIC [≤] 4 {micro}g mL-1, SI [≥] 128), along with moderate activity against P. aeruginosa (MIC = 32 {micro}g mL-1, SI > 16). Taken together, these findings elucidate the combined influence of end-group lipidation, cationic functionality, and polymer length in modulating antimicrobial activity, thereby establishing 2C18-terminated CLOs as a rationally tunable and biocompatible platform for antimicrobial material development.

Glycyrrhiza uralensis is a widely used medicinal plant present in more than 70% of Kampo formulations in Japan owing to its diverse pharmacological activities, including immunomodulatory, antitumor, and antioxidant effects. Isoliquiritigenin (ILG), a major chalcone constituent of G. uralensis, exhibits anti-inflammatory activity; however, its molecular mechanism remains unclear. Here, we employed an activity-based protein profiling approach to identify the molecular targets of ILG. Given that the ,{beta}-unsaturated carbonyl moiety of ILG can covalently react with reactive cysteine residues via nucleophilic addition, we used an iodoacetamide-based probe to globally profile cysteine-reactive proteomes. The comparative analysis between ILG- and vehicle-treated RAW 264.7 macrophages identified cysteine 65 (Cys65) of lipocalin-type prostaglandin D2 synthase (L-PGDS) as a potential covalent target. ILG treatment did not alter L-PGDS expression levels, indicating that reduced probe labeling reflects direct covalent competition rather than changes in expression. Consistently, ILG significantly suppressed prostaglandin D2 (PGD2) production, comparable to the selective L-PGDS inhibitor AT-56. Although both ILG and AT-56 reduced interleukin-6 expression, ILG exerted a stronger inhibitory effect. Our results demonstrate that covalent inhibition of L-PGDS and subsequent suppression of PGD2 production represent a key mechanism underlying the anti-inflammatory activity of ILG.