ACS Catalysis

Glycoside hydrolases (GHs) play central roles in carbohydrate metabolism and are widely exploited for industrial and biomedical applications. However, they are often not optimal for applications due to their constrained function and strict stereochemical specificity, necessitating the discovery and optimization of distinct enzymes for each glycosidic configuration. Members of glycoside hydrolase family 1 (GH1) are archetypal retaining {beta}-glycosidases, while -specific activity is rare within this family. Here, I demonstrate that a retaining GH1 enzyme can be engineered to hydrolyze both {beta}- and -configured substrates without altering its canonical catalytic residues. Using a well-characterized {beta}-glycosidase and computational protein design strategies targeting second-shell residues surrounding the active site, a bifunctional {beta}-/-glycosidase containing 45 mutations was generated. The engineered variant acquired the ability to hydrolyze the -configured substrate 4-nitrophenyl--D-glucopyranoside while retaining activity toward the originals {beta}-substrates, with reduced catalytic efficiency and thermostability. Structural modeling and docking analyses reveal that the engineered enzyme preserves the original fold and accommodates substrates within the catalytic pocket in a similar manner to the wild type. These findings provide direct evidence that stereochemical constraint in retaining GH is more flexible than previously appreciated and can be modulated through targeted engineering.

MilM from the mildiomycin biosynthetic pathway is a PLP-dependent enzyme, previously annotated as an aminotransferase, but has recently been demonstrated as L-arginine oxidase cum C-hydroxylase. Here, we report detailed biochemical, biophysical, structural modeling, and molecular dynamics simulation-based investigations of MilM from Streptoverticillium rimofaciens B-98891 to elucidate the mechanisms of substrate binding, catalysis, and the role of the active site residues involved in these processes. Our experimental findings confirmed that MilM functions as a stable homodimer, requiring the PLP cofactor and molecular oxygen to transform the L-arginine substrate into 5-guanidino-4-hydroxy-2-oxovaleric acid and 5-guanidino-2-oxovaleric acid through the intermediacy of a possible superoxide radical anion species, while generating H2O2 and NH3 as reaction by-products. Our labeling studies also established that the hydroxyl group in the product is obtained from the solvent, water. The structure-based three-dimensional modeling and simulation of MilM coupled with site-directed mutagenesis further confirmed that, in addition to the catalytic residues Lys232 and His31, active site residues from both the protomers are crucial for stabilization of the PLP cofactor (Ser92, Phe116, Asn164, Asp195, Lys240 from chain A and Tyr89 from chain B) and the substrate (Thr14, Glu17, Asn118, and Arg364 from chain A and Thr259, Ser260 from chain B). Moreover, the molecular dynamics simulation uncovered a dimer-mediated alternating lid mechanism in which large-scale, concerted motions of the dimer interface helices reciprocally expose and occlude the two active sites. This see-saw-like dynamics controls the substrate entry and product release through transiently formed tunnels, while preserving a catalytically protective environment, a critical phenomenon previously left unnoticed in similar PLP-dependent oxidases/hydroxylases. Overall, these findings provide new insights into the substrate/cofactor stabilization and the catalytic mechanism of MilM, a recent member of an emerging family of remarkable PLP-dependent oxidases and help us decode a key puzzle in the mildiomycin biosynthetic pathway.

Phosphoketolases can be used to convert non-phosphorylated sugars to the high energy compound acetyl phosphate and the versatile metabolic precursor acetyl-CoA. The performance of these pathways is limited by low catalytic activity of natural phosphoketolases towards these sugars. Here, we report the rational engineering of the phosphoketolase from Bifidobacterium adolescentis (Bad.F6Pkt) to enhance its activity and affinity towards glycoaldehyde (GA) and D-erythrulose (ERU) through re-organisation of the protein electric field to reproduce the role of terminal phosphate groups in cognate substrates. Guided by predicted induced side-chain pKa shifts, visualisation of electrostatic potential difference maps alongside molecular modelling and sequence variation analyses, we identified mutations that could promote in situ ring opening of the pre-dominant cyclic GA dimer form in solution. This approach to the electrostatic inverse design problem yielded the GA-specific double mutant H142N:E153D, exhibiting a ten-fold improved affinity and slightly enhanced catalytic efficiency (KM = 4.4 mM, kcat/ KM = 26.3 s-1 M-1) compared to the previously reported H142N variant (KM = 42.3 mM, kcat/ KM = 20.6 s-1 M-1). We additionally constructed a H256Y:H260Y:H548Y variant comprising long-range electrostatic mutations with a 3.8-fold increased catalytic efficiency (kcat/ KM = 49.6 s-1 M-1) on the acylic four-carbon ERU ketose compared to the wild-type enzyme. The engineered enzymes were evaluated in cell-free enzyme cascades for ATP regeneration via acetyl phosphate formation. The H142N variant enabled efficient ATP regeneration from GA and ethylene glycol, whereas the H142N:E153D mutant exhibited reduced stability under synthesis conditions. Furthermore, coupling of a highly GA-specific D-threose aldolase and a D-threose isomerase with the PKT triple mutant enabled rapid conversion of GA into C4 sugar intermediates and significantly improved ATP regeneration from GA.

Enzyme-directed evolution increasingly relies on computational tools to prioritize mutations, yet their practical value is difficult to assess because kinetic data are often aggregated across heterogeneous assay conditions, inflating apparent generalization. Here we introduce EnzyArena, a curated benchmark that groups kinetic parameters (kcat, Km, kcat/Km) into condition-matched experimental subsets to enable realistic evaluation. Using this resource, we benchmark 10 representative models from two arising strategy families--zero-shot fitness prediction and supervised kinetic-parameter prediction--across BRENDA- and SABIO-RK-derived subsets and 25 independent mutagenesis datasets. Kinetic-parameter predictors perform strongly on database-derived subsets but lose their advantage on independent datasets, whereas zero-shot predictors show more consistent generalization. A simple consensus of multiple zero-shot models further improves the precision of identifying beneficial mutants. We prospectively validated these findings in a wet-lab campaign (150 mutants) comparing random mutants, UniKP-prioritized mutants and ESM-1v-prioritized mutants (representing supervised kinetic-parameter prediction and zero-shot fitness prediction, respectively), where ESM-1v achieved the highest utility and UniKP underperformed the random baseline. Together, this study establishes realistic baselines for computational mutant prioritization and highlights consensus zero-shot strategies as a practical starting point for enzyme engineering.

The enzymatic depolymerization of polyethylene terephthalate (PET) presents a sustainable route for plastic circularity, but its industrial viability is disadvantaged by the need for thermostable enzymes that remain active under mild, energy-efficient conditions. While the Polyester Hydrolase Leipzig 7 (PHL7) rapidly degrades amorphous PET near its melting point, its poor protein expression, inactivation issues at temperatures above 60{degrees}C and slow depolymerization activity below 60{degrees}C limit its practical application. Here, we employ inverse folding models ProteinMPNN and LigandMPNN, informed by structural and evolutionary information, to redesign the sequence of PHL7, aiming to improve protein expression, thermal stability and activity. From 36 designed variants, we identified two (termed D5 and D11) with significantly enhanced PET depolymerization rates at lower temperatures, where enzymatic performance is typically limited. Remarkably, design D5 at 50{degrees}C achieved the same product yield as PHL7 at 70{degrees}C in 24 h PET microparticle degradation assays, with a shifted product profile favoring mono-(2-hydroxyethyl) terephthalate (MHET) over terephthalic acid (TPA). Molecular dynamics simulations revealed that the active redesigns exhibit enhanced local flexibility in key active site regions at 50{degrees}C, providing a mechanistic understanding of their low-temperature catalysis. This work demonstrates that computational sequence redesign can optimize biocatalysts for lower production costs and milder operational conditions. Furthermore, the D5 variant enables a potential route to resynthesize virgin PET via MHET polycondensation, offering an efficient circular economy pathway.

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.

Sucrose synthase (SuSy) has been suggested as a key enabling enzyme for uridine diphosphate glucose (UDP-Glc) regeneration in glycosyltransferase-catalyzed biotransformations. However, its stability and efficiency in industrially relevant conditions have not been characterized or engineered, limiting its industrial readiness. Here, we combined enzyme discovery and characterization with comprehensive semi-rational enzyme engineering strategies, to optimize SuSys catalytic activity, thermostability, solvent tolerance, and soluble expression. The engineered variants were significantly more stable than wild-type, with up to 13.6 {degrees}C increase in melting temperature, over two orders of magnitude improvement in half-lives at elevated temperatures, and approximately three orders of magnitude increase in total turnover number. Additionally, the optimized variants retained up to 75% relative activity at 60 {degrees}C in the presence of 25% (v/v) DMSO, which the wild-type shows near complete loss of activity. Structural and molecular dynamics analyses reveal how mutations modulate conformational dynamics and hydrophobic packing, favoring catalytically competent conformations. Using methyl anthranilate glycosylation as a representative biotransformation, we demonstrate that the engineered SuSy variants consistently outperform both wild-type SuSy and stoichiometric UDP-Glc systems, enabling efficient UDP-Glc regeneration at reduced enzyme and sugar donor loadings. Finally, techno-economic and environmental assessments further indicate that implementation of engineered SuSy reduces reaction cost by approximately 6- and 2-fold relative to UDP-Glc and wild-type systems, respectively, while achieving average reductions of 3- and 2-fold in environmental end-point impacts. These results established SuSy engineering as a critical enabler for sustainable glycosylation reactions.

Assembly-line enzymes carry out multi-step synthesis of diverse metabolites by using a handful of catalytic motifs in which minor structural differences control substrate specificity and reaction order. Here we examine differences in substrate binding to FabB and FabF, the two {beta}-ketoacyl-ACP synthases (KSs) responsible for fatty acid elongation in Escherichia coli, by exploring a peculiar mutational effect. In FabB, a blocking mutation in the acyl binding pocket yields a shifted, but broad product profile, while in FabF, the same mutation disrupts the binding of acyl chains longer than eight carbons (C8). X-ray crystal structures of the FabB mutant provide an explanation: a second, previously unobserved binding pocket allows medium-to-long acyl chains ([≥] C8) to bind with an alternate conformation. Molecular simulations suggest that this pocket is more stable in FabB than in FabF, where mutations reduce the catalytic competency of longer chains instead of shifting them to the alternate pocket. Our findings indicate that homologous KSs differ not only in their primary binding sites but also in the availability of alternative binding modes that can buffer against mutational effects and enable functional diversification.

Enzymes used on industrial scale are routinely engineered for best performance. However, exhaustive mutagenesis campaigns using the twenty canonical proteinogenic amino acids rapidly reach an evolutionary ceiling, where gains in activity compromise other critical properties such as thermal endurance. Although non-canonical amino acids (ncAA) expand the chemical space, most are costly for use on an industrial scale and significantly perturb structure. Here, we demonstrate that the evolutionary ceiling of highly optimized polyethylene terephthalate (PET) hydrolases (PETases) can be broken with azatryptophans that (i) differ minimally from their canonical tryptophan, (ii) are genetically encoded, and (iii) are produced in high yield by enzymatic biosynthesis from inexpensive precursors. The first genetic encoding systems are described for 4-azatryptophan, 5-azatryptophan, and 6-azatryptophan, achieving single, site-selective isosteric CH [->] N substitutions that enhancing the catalytic activity while preserving thermal stability. The fluorescence of 6AW provides a uniquely sensitive reporter of side-chain solvent exposure, which is critical for PETase activity and shown to vary between five different PETases. Furthermore, Azatryptophan-bearing enzymes are inexpensive to produce. To benchmark PETase activity, a rapid fluorescence-based kinetic assay, PETra, is introduced, which delivers consistency and reproducibility by using a soluble substrate yet correlates strongly with the hydrolysis of solid PET.

The surface of gram-positive bacteria is a highly regulated environment with specific attachment of proteins required for viability. Sortase enzymes are cysteine transpeptidases that recognize and ligate substrates to the peptidoglycan layer in these microorganisms, which can be highly pathogenic (e.g., Staphylococcus aureus, Streptococcus pyogenes, etc.). As such, sortases represent a potentially novel target for antibiotic development. In addition, the catalytic activity of sortase enzymes is utilized in sortase-mediated ligation (SML) engineering approaches for a variety of uses. In SML experiments, engineered variants of Staphylococcus aureus sortase A (saSrtA) are the most widely used enzymes. One of the mutated amino acids in the previously engineered pentamutant (or saSrtA5M) enzyme is P94. Structural analyses of experimental saSrtA structures revealed that P94 interacts directly with Y187 when saSrtA is in its inactive conformation. While saSrtA5M, developed via directed evolution, contains a P94R mutation, we wanted to interrogate this position further and ask if other single P94 mutations may reveal a greater effect on activity and/or substrate specificity. We created 18 P94X mutations (excluding P94C), and tested relative activity using a fluorescence resonance energy transfer (FRET) assay for 4 substrate sequences: LPATG, LPETG, LPKTG, and LPSTG. We identified several P94 variants that outperformed the single mutant P94R for all peptides tested, including P94A, P94D, P94E, P94G, P94H, P94N, P94Q, P94S, and P94T. We further observed that the reactivity of substrates with variations in the central position of the pentapeptide recognition motif (LPXTG) can be sensitive to the identity of the P94X residue. We tested P94A and P94D saSrtA5M variants and found that, depending on LPXTG sequence, these variants could outperform saSrtA5M in activity > 3-fold. Finally, we compared saSrtA5M and P94D saSrtA5M in a model sortase-mediated ligation reaction using a LPKTG substrate and saw [~]2-fold greater product formation. Taken together, we characterized an important position that modulates substrate access and activity in saSrtA. Furthermore, we argue that future studies which combine rational design and high throughput approaches, e.g., directed evolution, may result in sortase variants with increased SML potential.

De novo metabolic pathways open possibilities for sustainable biotransformations in microbes. However, the in vivo-implementation of such new-to-nature pathways is highly challenging and heavily relies on adaptive laboratory evolution (ALE) of the hosts native metabolic network. Here, we assess how much this need for host-centric ALE can be overcome and/or complemented through the informed design of the newly introduced pathway. Exemplifying for a synthetic CO2-fixation module via methyl-succinate, we established methylsuccinate-dependent growth of Escherichia coli over six months by ALE of E. colis native metabolism. In parallel, we developed a machine-learning guided workflow (MEVIS) for the automated engineering of the synthetic pathway, resulting in methylsuccinate-dependent growth within three weeks. Critically, performing MEVIS in the background of the ALE-evolved strain is necessary to further approach wild-type like growth, demonstrating how ALE in combination with machine-learning-guided lab automation holds great potential to accelerate and improve design-build-test-learn cycles in contemporary metabolic engineering.

Carbohydrate-binding modules (CBMs) play crucial roles in carbohydrate-active enzymes by promoting substrate recognition and proximity, particularly for insoluble polysaccharides. Here, we report the discovery and characterization of a novel {beta}-trefoil structured CBM associated with a GH87 -1,3-glucanase from Flavobacterium johnsoniae, which accordingly was designated FjCBMXXXGH87. The full-length enzyme efficiently hydrolyzed -1,3-glucan (mutan) and -1,3/-1,6-glucan (alternan), whereas the catalytic domain alone displayed reduced activity, indicating that FjCBMXXXGH87 enhances substrate interaction. Pull-down assays confirmed that FjCBMXXXGH87 binds -1,3-linked glucans, and structural investigation together with site-directed mutagenesis identified two distinct binding sites essential for protein-ligand interactions. Phylogenetic analysis showed that CBMXXX homologs are present together with enzymes from families GH87, GH13, GH16, and GH99, and potentially may comprise up to three binding sites. Together, these findings establish FjCBMXXXGH87 as the founding member of a new CBM family, which may have broad functional versatility in polysaccharide recognition. This discovery expands the repertoire of {beta}-trefoil CBMs and provides new insights into carbohydrate recognition strategies relevant to -glucan degradation.

Polyurethanes (PURs) represent a significant challenge in plastic waste management due to their chemical resilience and limited recycling options. In this study, we report the identification and characterization of six novel bacterial urethanases, expanding the enzymatic repertoire for targeted PUR depolymerization. These enzymes demonstrated carbamate-cleaving activity optimally under alkaline conditions, maintaining stability across a pH range of 7 to 10 and varying thermal and solvent tolerances. Among the candidate enzymes, u17, u10, and u15 collectively exhibited high activity, catalytic efficiency, and thermostability, establishing a strong foundation for further optimization. Building on these results, u15 emerged as particularly notable for its catalytic efficiency on the carbamate model substrate di-urethane ethylene methylenedianiline, DUE-MDA, with a kcat/KM of 51.8 {+/-} 0.1 (s-1mM-1). and this motivated its selection for detailed structural analysis. High-resolution crystallography of u15 revealed key active-site architecture, including the conserved amidase signature catalytic triad and flexible loop regions that influence substrate binding and specificity. Molecular docking and molecular dynamics simulations further elucidated substrate binding determinants of u15 during urethane bond hydrolysis. Docking of DUE-MDA revealed two distinct substrate orientations (Pose A and Pose B) differing in the positioning of the carbamate group relative to Ser177. Pose A was more stable and catalytically competent, maintaining the substrate within the oxyanion hole and sustaining optimal geometry for nucleophilic attack by Ser177. Comparable behavior was observed for the partially hydrolyzed intermediate mono-urethane ethylene methylenedianiline, MUE-MDA, indicating a conserved binding mode across substrates. Collectively, these findings highlight amidase signature urethanases as valuable scaffolds for advancing sustainable and scalable biocatalytic recycling of polyurethanes. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/705263v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@127bf23org.highwire.dtl.DTLVardef@75c29corg.highwire.dtl.DTLVardef@13bbf30org.highwire.dtl.DTLVardef@18504a4_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

Lignin is a promising alternative to petroleum as a feedstock for the chemical industry. Emergent strategies for lignin valorization involve tandem processes in which biomass is chemo-catalytically fractionated followed by biotransformation of the depolymerized lignin by microbial cell factories. A rate-limiting step in this biotransformation is O-demethylation of the lignin-derived monomers. The reductive catalytic fractionation of hardwood biomass generates high yields of two classes of monomers: 4-alkylguaiacols and 4-alkylsyringols. To better understand the biotransformation of these monomers, we studied AgcA, a cytochrome P450, and AgcB, the cognate reductase, that together catalyze the O-demethylation of 4-alkylguaiacols. A 1.82 [A] resolution crystal structure of AgcAEP4 from Rhodococcus rhodochrous EP4 in complex with 4-ethylguaiacol identified residues Leu78, Ala293 and Phe166 as potential specificity determinants. Substitution of Ala293 and Leu78 decreased the specificity of AgcAEP4 for alkylguaiacols. Substitution of Phe166 yielded a variant that bound 4-propylsyringol but did not transform it. In contrast, the corresponding variant in the Rhodococcus aromaticivorans RHA1 homolog, AgcARHA1 Y166A, catalyzed the O-demethylated of both methoxy groups of 4-propylsyringol with a kcat/Km of 8500 M-1 s-1 for the first O-demethylation, nearly 7-fold higher than WT AgcARHA1. A strain of RHA1 harboring the variant did not grow on 4-propylsyringol but consumed it at approximately the same rate as 4-propylguaiacol and transformed some of it to pentanoyl-CoA, consistent with metabolism via the meta-cleavage pathway that catabolizes 4-alkylguaiacols. These studies improve our understanding of a critical lignin-degrading enzyme system and facilitate its efficient implementation into biocatalysts. SignificanceLignin is a highly abundant source of aromatic carbon and a promising alternative to petroleum to generate materials. Fulfilling this promise depends on technological advances in areas such as catalytic fractionation and biocatalysis. Catalytic fractionation of hardwood biomass generates mixtures of aromatics enriched in 4-propylguaiacol and 4-propylsyringol. Here, we biochemically and structurally characterized a cytochrome P450 that initiates 4-propylguaiacol catabolism. Informed by the structure, we engineered the enzyme to have dual activity on both 4-propylguaiacol and 4-propylsyringol, and implemented this enzyme into a bacterial biocatalyst. Metabolomic analysis of this strain provided insights into the catabolism of both aromatics. Overall, these findings greatly facilitate the engineering of P450s and bacteria to biocatalytically upgrade lignin.

Polyketide biosynthesis relies on the conformational adaptability of type II polyketide synthases and accessory enzymes, which direct chain folding and regiospecific cyclization. The aromatase/cyclase TcmN from Streptomyces glaucescensis catalyzes the first two ring closures of tetracenomycin C. Still, the molecular basis by which conformational dynamics regulate substrate binding and product release remains unresolved. Understanding how conformational transitions control ligand recognition and prevent aggregation is crucial for deciphering the molecular bases of polyketide biosynthesis and for guiding engineering strategies to synthesize novel natural products. Here, we investigated how ligand interactions modulate the conformational equilibrium of TcmN and the mechanistic consequences for catalysis. Using NMR spectroscopy (STD, CSP, relaxation dispersion), calorimetry, molecular docking, and microsecond-scale molecular dynamics simulations, we mapped the conformational ensembles of apo TcmN and its complexes with naringenin (a substrate/product analogue) and intermediate 12 (INT12). Apo TcmN samples both open and closed conformations. Naringenin preferentially stabilizes the closed state, a conformation thought to protect hydrophobic residues from solvent exposure. In contrast, INT12 shifts the equilibrium toward the open state, characterized by an expanded cavity that permits substrate entry, product release, and accommodation of extended intermediates. Hydrogen-bond analysis highlighted conserved catalytic residues (R82, E34, Q110, T133) as key anchors for productive poses. These results establish that TcmN functions through a ligand-gated breathing mechanism, in which successive intermediates selectively tune the cavity volume and shape, balancing catalytic efficiency with protection against aggregation. Conformational adaptability emerges as a central determinant of aromatase/cyclase function, providing molecular insights relevant for polyketide biosynthetic engineering. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/708631v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@5646aorg.highwire.dtl.DTLVardef@39016org.highwire.dtl.DTLVardef@1e8c285org.highwire.dtl.DTLVardef@3aba20_HPS_FORMAT_FIGEXP M_FIG C_FIG

Fluorinases have high potential for industrial biofluorination but any applications have been precluded by low catalytic efficiency and resistance to active site engineering. In this work, we employed PRIZM, a computational workflow utilizing an existing low-N dataset and zero-shot models for in silico prediction of activity-enhancing mutations at distal sites. The combination of these predictions with expert opinion led to the identification of 21 fluorinase mutants with enhanced relative activities, while 3 variants showed increased melting temperatures. A mutation in the hexameric interface, K237R, resulted in the largest stability gain, a more than 3.2-fold improvement in catalytic efficiency at 57{degrees}C, and an 8-fold increase in relative activity at 62{degrees}C. These results highlight the potential of distal fluorinase engineering for improving properties required to realize its industrial applications.

Xanthan gum is a widely used industrial polysaccharide employed as a thickening and stabilizing agent in food, pharmaceutical, and technological applications. Its biosynthesis involves membrane-associated glycosyltransferases that assemble the repeating unit at the cytoplasmic side of the inner membrane. Among them, GumH and GumI catalyze consecutive reactions using the same donor substrate, guanosine 5-diphospho-alpha-D-mannose, but with opposite stereoselectivity. Despite their biochemical characterization, structural insights into their catalytic mechanisms and membrane interactions remain limited, hindering a detailed understanding of their function and future engineering efforts. In this work, we combined artificial intelligence-based structure prediction with atomistic molecular dynamics simulations to investigate the structural organization and substrate-binding modes of GumH (family GT4) and GumI (family GT94). The predicted apo structures exhibit a conserved GT-B fold but differ in interdomain flexibility and membrane-anchoring strategies. GumH displays a more structured interdomain linker and a defined clamp-like region in the acceptor-binding domain, consistent with stable membrane interaction, whereas GumI shows a more flexible linker and an open groove architecture. Modeling of the donor-bound complexes reveals distinct substrate-binding modes. In GumH, it adopts a geometry consistent with its retaining stereochemical outcome, positioning the sugar close to the conserved catalytic residue. In contrast, GumI exhibits a different donor orientation, lacking a clearly positioned catalytic base near the reactive center, suggesting a substrate-assisted catalytic mechanism. Although the predicted ternary complexes show limited stability in our simulations, they provide chemically reasonable conformations and offer structural insights into substrate recognition, membrane association, and stereochemical control in these two glycosyltransferase families. Significance statementXanthan gum is an industrially important polysaccharide widely used in food and other technological products. Although several enzymes in its biosynthetic pathway have been studied, structural information remains limited. Using AI-based structure predictions and molecular simulations, we revealed how these enzymes sit in the membrane and bind sugar substrates. These structural insights clarify xanthan biosynthesis and could help improve or engineer its production.

Carbon monoxide dehydrogenases (CODHs) are metalloenzymes central to microbial CO metabolism and CO2 fixation. We report the heterologous production and characterisation of Clostridium pasteurianum BC1 CODH-III (CpBC1CODH-III), from the phylogenetic clade E, co-expressed with its maturation machinery CooCTJ. CpBC1CODH-III shows moderate CO oxidation (150 U/mg) and CO2 reduction (0.568 U/mg) activities. Electron paramagnetic resonance (EPR) spectroscopy under varying redox conditions identified a rhombic signal at g {approx} 2.0, characteristic of reduced B-clusters, and a C-clusters at different stages (g {approx} 1.75, g {approx} 1.72), indicative of a bound CO2. Investigation of maturation effects showed that co-expression of CooCTJ stabilised CpBC1CODH-III production, but did not enhance maximum activity, which was primarily influenced by nickel availability. Comparative operon analysis with the well-studied clade F Rhodospirillum rubrum CODH (RrCODH) revealed high structural similarity in CODH and CooC, but significant divergence in CooJ, with conserved metal-binding regions identified via AlphaFold3 modelling and dot plot analysis. CpBC1CODH-III represents a unique example of a clade E CODH within a clade F genomic context, demonstrating intrinsic robustness in maturation and activity

Monascus spp., a food fermentation microorganism, produced valuable secondary metabolites including Monascus pigments (MPs) which served as natural food colorants. However, rational metabolic engineering to enhance MPs production remained limited by the lack of regulatory targets that govern metabolic branching. Mitogen-activated protein kinase cascades, particularly the STE20-STE11-STE7 core module, regulated fungal growth and metabolism, but their roles in MPs biosynthesis remain unexplored. In this study, we functionally characterized MpSte11A, the first STE11 homolog identified in Monascus spp., through bioinformatic analysis and genetic manipulation. Most importantly, deletion of mpSte11A triggered a profound metabolic shift, which resulted in a 22-fold increase in MPs production. Integrated transcriptomic and metabolomic analysis revealed that MpSte11A functioned as a metabolic gatekeeper where its deletion redirected carbon flux from primary to MPs biosynthesis by controlling the TCA cycle. These findings not only elucidated the signaling role of the MAPK cascade in Monascus spp. specialized metabolism but also provided a robust strategy for re-engineering carbon partitioning to maximize the output of high-value secondary metabolites in filamentous fungal cell factories. ImportanceFilamentous fungi are versatile cell factories for the production of diverse high-value secondary metabolites, but the rational enhancement of these compounds is often limited by a lack of universal regulatory targets. In this study, we employed the food-fermentation fungus Monascus spp. as a model and identified MpSte11A as a master "metabolic gatekeeper" that governed the trade-off between fungal growth and secondary metabolism. By disrupting this single signaling node, we achieved a remarkable 22-fold increase in compound production. This significant enhancement resulted from a systematic redirection of carbon flux from primary growth (the TCA cycle) to secondary biosynthesis. This work provided a precise molecular blueprint for the reprogramming of fungal metabolism. It also demonstrated that the tuning of core MAPK modules is a powerful and broadly applicable strategy for the engineering of robust fungal cell factories producing a wide array of bioproducts. Graphical Abstract (For Table of Contents Only) O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/703344v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1b062a5org.highwire.dtl.DTLVardef@11c4cd6org.highwire.dtl.DTLVardef@f8a34eorg.highwire.dtl.DTLVardef@1a97f69_HPS_FORMAT_FIGEXP M_FIG C_FIG