Biocatalysis@TUDelft

Novel thermophilic PPases from Thermoleophilia bacterium and Thermoprotei archaeon exhibit broad temperature adaptability, with flexible active sites that enhance catalytic efficiency under moderate temperatures, thus presenting promising prospects for applications in scientific research and biomanufacturing.

Inorganic pyrophosphatases (PPases) are ubiquitously present in living organisms, where they prevent the pathological accumulation of inorganic pyrophosphate (PPi) in cells by catalyzing its efficient hydrolysis. This study reports the cloning, expression, and biochemical characterization of two novel thermostable soluble PPases derived from Thermoleophilia bacterium (PPase_Tba) and Thermoprotei archaeon (PPase_Tar). These enzymes demonstrate robust catalytic activity toward PPi, along with exceptional thermostability and pH tolerance, exhibiting optimal activity at 85°C and pH 8.5. Notably, they retain 60–70% of their maximal activity even at 25°C, indicating a broad temperature adaptability that is uncommon among known thermostable enzymes. The efficient PPi-hydrolyzing capability under both moderate and high temperatures effectively alleviates thermodynamic inhibition in biosynthetic systems. These properties highlight the significant potential of PPase_Tba and PPase_Tar in industrial biocatalysis, molecular diagnostics, and RNA-based biotechnology, presenting broad prospects for applications in biomedical research and biomanufacturing.

Structure-guided scanning of an engineered IscB RNA-guided nuclease (IscB*) pinpointed V367Y as an activity hotspot. The V367Y variant yields IscB*-Act with stronger nuclease editing and also boosts an IscB* adenine base editor to increase A-to-G conversion in mammalian cells.

Compact RNA-guided nucleases such as IscB represent an attractive foundation for next-generation genome editors, yet their application in mammalian cells has been constrained by suboptimal activity. Here, instead of re-engineering enzymes, we establish a mutant-initiated, structure-guided optimization strategy to generate second-generation high-activity IscB editors. Using AlphaFold3 to model the engineered IscB*-ωRNA–DNA complex, we reveal remodeling of the nucleic-acid-binding interface induced by activity-enhancing substitutions. Guided by this predicted structure, we perform a focused mutational scan and identify V367 as an activity hotspot. Saturation mutagenesis at this position yields a single substitution, V367Y (IscB*-Act), which increases mean editing efficiency by 34% and achieves up to 2.1-fold improvement across endogenous targets in mammalian cells. Importantly, the V367Y substitution is transferable to an IscB-based adenine base editor, elevating A-to-G conversion by 68% on average and up to 4.46-fold at individual loci without altering the intrinsic editing window. Targeted off-target profiling at loci suggests that V367Y does not substantially increase off-target indels or A-to-G conversion. Together, our work demonstrates a practical framework for second-generation refinement of compact genome editors, bridging deep-learning-enabled structural prediction with interpretable protein engineering, and expands the functional potential of miniature IscB systems for both nuclease and base editing applications.

This study presents a novel strategy inspired by the spatial compartmentalization of cellular metabolic pathways to address the challenge of coenzyme regeneration in enzyme–photocatalyst hybrid systems. Glucose dehydrogenase was encapsulated within silica nanoparticles, exploiting the short half-life and limited diffusion distance of hydroxyl radicals to protect the enzymes against photocatalytic oxidation stress. The encapsulated enzyme maintained catalytic activity and achieved continuous NAD⁺/NADH regeneration cycles.

Coenzyme regeneration is essential for oxidoreductase-based biocatalysis, yet integrating enzymatic and photocatalytic systems remains challenging due to ROS-mediated enzyme inactivation. Here, we developed a metabolic-inspired compartmentalization strategy that enables sustainable NAD⁺/NADH cycling by mimicking the spatial organization of cellular redox metabolism. Glucose dehydrogenase (GDH) was encapsulated within silica nanoparticles, exploiting the intrinsic instability of hydroxyl radicals and their limited diffusion distance to shield the enzyme from TiO₂-generated ROS while preserving substrate and cofactor accessibility. The encapsulated GDH maintained catalytic competence and achieved five consecutive coenzyme regeneration cycles, whereas the free enzyme lost activity after a single photocatalytic exposure. Beyond cofactor recycling, NADH generated by this system functioned as an endogenous antioxidant, protecting cells from photocatalytic oxidative stress. This work demonstrates that principles from cellular biochemistry, including compartmentalization and redox homeostasis, can be translated into artificial systems for sustainable biocatalysis and cell protection.

Characterization of the first marine-derived fungal monofunctional class I diterpene synthase, combined with phylogenetic analysis and heterologous coexpression, leads to production of a 5-8-5 tricyclic diterpene. Its absolute configuration is first established. Structural modeling and site-directed mutagenesis reveal that residue F65 serves as a pivotal bidirectional functional switch between tricyclic and bicyclic systems, and a distinct dehydrogenation mechanism was proposed.

The first marine-derived fungal monofunctional class I diterpene synthase RousA was characterized by a combination of bioinformatics, phylogenetic analysis, and heterologous coexpression. RousA resides on a remote branch of phylogenetic tree, indicating it is an uncharacterized terpene cyclase. Heterologous coexpression of RousA and RousB in engineered yeast led to the production of a 5-8-5 tricyclic diterpene scaffold 1. Its absolute configuration was first established by ECD calculation. Structural modeling and site-directed mutagenesis demonstrated that several amino acid residues in nonconserved motif have a strong influence on 5-8-5 accumulation and F65 residue serves as pivotal “bidirectional functional switch” between tricyclic and bicyclic systems. A cyclization process of tricyclic product 1 exhibits a deprotonation mechanism distinct from those of known enzymes. The antimicrobial and plant immune activities were also evaluated.

A mevalonate kinase (MvaK) variant with 10-fold enhanced isoprenol phosphorylation activity was developed through directed evolution and rational design, enabling efficient lycopene biosynthesis (83.0 mg/L) via the two-step isoprenol phosphorylation pathway.

Efficient supply of terpenoid precursors remains a key challenge in metabolic engineering. The two-step isoprenol phosphorylation pathway, which converts isoprenol to isopentenyl diphosphate and dimethylallyl diphosphate via two kinase reactions, offers a streamlined alternative to native biosynthetic routes, yet its efficiency is critically limited by the first phosphorylation step (Kinase-1). Conventional Kinase-1 enzymes exhibit poor catalytic efficiency toward isoprenol, with kcat/Km values approximately four orders of magnitude below those of typical metabolic enzymes. Here, we establish mevalonate kinase (MvaK) as a novel Kinase-1 alternative for the two-step pathway in Escherichia coli. Through bioinformatic screening and lycopene-based functional evaluation, MvaK from Kitasatospora griseola was identified as the most promising candidate among five tested kinases. Two rounds of directed evolution yielded beneficial mutations that improved lycopene production approximately threefold. Molecular docking revealed that poor isoprenol accommodation stems from electrostatic mismatch at residues E33 and H34. Structure-guided rational design on the evolved variant identified E33A/H34L as the best-performing variant among those tested, achieving a lycopene titer of 83.0 ± 1.1 mg/L, representing a 10-fold improvement over wild-type. This work demonstrates that combining bioinformatic mining, directed evolution, and rational design effectively improves enzyme activity toward non-native substrates in terpenoid biosynthesis.

We coencapsulated glucose oxidase (GOX) and horseradish peroxidase (HRP) within silica nanoconfinement using an enzyme-friendly synthesis method. The coencapsulated system demonstrated faster cascade kinetics than spatially separated configurations, providing direct evidence for beneficial proximity effects on intermediate transfer. This platform enables quantitative studies on enzyme behavior under nanoscale confinement.

Enzyme cascade reactions, where the product of one enzyme serves as the substrate for another, are fundamental to cellular metabolism and essential for constructing efficient multi-enzyme systems. Spatial confinement of sequential enzymes can enhance cascade efficiency through proximity effects and substrate channeling, mimicking the organization of natural metabolic pathways. Here, we coencapsulated glucose oxidase (GOX) and horseradish peroxidase (HRP) within silica nanocapsules (SiNCs) using an enzyme-friendly synthesis method. The encapsulated enzymes retained high catalytic activity, and Michaelis-Menten kinetics analysis revealed decreased Km for GOX, indicating enhanced substrate affinity under confinement. The coencapsulated system demonstrated faster cascade kinetics compared to spatially separated enzyme configurations, providing direct evidence for the beneficial effects of enzyme proximity on intermediate transfer. Enhanced thermal stability of confined enzymes was also observed, presumably due to preserved hydration shells. This platform enables quantitative studies on enzyme behavior under nanoscale confinement, offering insights into the biochemical principles governing multi-enzyme cascade systems in both natural and artificial environments.

Plastic waste from diverse sources can undergo direct biocatalytic depolymerization and upcycling to generate short-chain monomers, oligomers, and platform chemicals, including terephthalic acid, ethylene glycol, 6-aminohexanoic acid, phenylacetic acid, and phthalates. In parallel, downstream biological valorization of these intermediates can yield value-added products such as polyhydroxyalkanoates, β-ketoadipic acid, and catechol through enzyme- and whole-cell-based biocatalysis. Emerging technologies, including AI-enabled catalyst design and hybrid chemo-bio platforms, are expected to further advance plastic circularity.

ABSTRACT

The rapid growth in global plastic production has resulted in unprecedented accumulation of plastic waste, posing severe environmental, ecological, and human-health challenges. Conventional end-of-life management strategies, including landfilling, incineration, and mechanical recycling, remain insufficient to establish a truly circular plastics economy because of material downcycling, high energy demand, and greenhouse-gas emissions. In this context, biological deconstruction, direct upcycling, and downstream valorization of intermediates derived from plastic-waste streams have emerged as compelling catalytic strategies that exploit the selectivity and efficiency of enzymes and microbial systems to depolymerize waste plastics and transform the resulting intermediates into value-added chemicals, fuels, and functional materials under mild conditions. This review critically examines recent advances in the biocatalytic degradation, upcycling, and downstream valorization of intermediates from major synthetic polymers, including polyethylene terephthalate, polyurethanes, polyamides, polyethylene, polypropylene, polystyrene, and poly(vinyl chloride). Emphasis is placed on enzyme discovery and engineering, structure–activity relationships, metabolic pathway design, and integrated chemo-bio catalytic platforms that enable selective depolymerization and downstream valorization. We further analyze current limitations related to polymer recalcitrance, feedstock heterogeneity, catalyst stability, process scalability, and techno-economic feasibility. Finally, future opportunities are outlined, highlighting the roles of machine-learning-guided enzyme design, synthetic biology, hybrid catalytic systems, and decentralized recycling infrastructures in advancing sustainable plastic circularity. Collectively, this review positions biocatalytic deconstruction, direct upcycling, and downstream biological valorization as key catalytic pillars for next-generation circular plastics technologies.

Nature Synthesis, Published online: 27 May 2026; doi:10.1038/s44160-026-01094-5

Stereoselective cross-coupling using photoredox and metalloenzyme catalysis

Nature Communications, Published online: 23 May 2026; doi:10.1038/s41467-026-73618-8

Genetic code expansion can be limited by the need to supply non-standard amino acids. The authors design bacteria that perform integrated steps of converting supplemented aldehydes to L-phenylalanine derivatives and incorporating them within proteins.

Nature Chemical Biology, Published online: 22 May 2026; doi:10.1038/s41589-026-02210-4

The enzymatic depolymerization of synthetic polyamides remains a major challenge. Here Bell et al. review the current biocatalytic nylon recycling landscape, highlighting the biochemical, structural and material science bottlenecks that limit polyamide deconstruction.

We report a scalable biocatalytic cascade that converts CO₂ and acetaldehyde to pyruvate via a newly characterized pyruvate decarboxylase, followed by stereoselective reduction to lactic acid. The system enables fully switchable enantioselectivity using either L- or D-lactate dehydrogenases.

Biocatalytic cascades offer a promising route for CO₂-fixation into valuable chemicals, addressing the urgent need for efficient, sustainable technologies to reduce CO₂ emissions. This paper describes an enzymatic route converting gaseous CO₂ and acetaldehyde into enantiopure lactic acid, widely used in diverse industries. A newly characterized pyruvate decarboxylase from Neoasia chiangmaiensis (NcPDC) enabled acetaldehyde carboxylation to pyruvate. To suppress the competing carboligation to acetoin, acetaldehyde was reversibly trapped with Tris. Pyruvate was reduced to lactate by lactate dehydrogenase, coupled with glucose dehydrogenase for NADH regeneration via D-glucose oxidation to D-gluconic acid. Up to 65% lactate yield was achieved. Repeated acetaldehyde dosing resulted in a 27 mM titer, representing a >100-fold improvement over previous reports. At 0.5 L scale, using a gas mixture mimicking industrial-grade CO₂, we obtained 21 mM D-(–)-lactic acid, 42% yield and >98% e.e., demonstrating scalability and robustness. Finally, replacing the D-(–)-selective lactate dehydrogenase with an L-(+)-selective variant at small scale enabled production of L-(+)-lactic acid at 41% yield and >93% e.e, allowing switchable access to either enantiomer. A volumetric productivity of 1.1 × 10⁻² g L⁻¹ h⁻¹ ranks among the most efficient minimal enzymatic routes developed to date for CO₂-to-lactate conversion.

We report a non-genetic strategy for bacterial cell surface enzyme immobilisation using cholesterol-based artificial lipids containing Ni²⁺-NTA binding groups that can integrate into biological membranes. The engineered cells can capture His-tagged enzymes directly from cell lysates while preserving intracellular and surface activity, allowing for recyclable enzyme catalysis and single-cell cascade reactions.

ABSTRACT

The cell membrane is a prime target for the introduction of novel cellular functionalities, as it is a complex system with many routes for surface modification. Several chemical coating and genetic engineering methods have thus been developed for this purpose. Here, a distinct way to enable enzyme-binding onto the surface of bacterial cells is explored using biomimetic lipids that integrate within the cell membrane. E. coli cells were equipped with a cholesterol-based artificial lipid containing a nitrilotriacetic acid (NTA) group which, when loaded with Ni²⁺ ions, selectively binds His-tagged enzymes through affinity interactions. This interaction is stable and selective for tagged proteins including green fluorescent protein, enabling their direct one-step purification and immobilisation from cell lysates. Furthermore, the process is biocompatible and preserves both intracellular and cell-surface enzymatic activity. This strategy further enables binding of benzaldehyde lyase or amine transaminase enzymes to the surface of bacterial cells for recyclable single-step enzymatic reactions. Importantly, it allowed the creation of a single-cell system for the two-step cascade reaction from benzyl alcohol to (R)-benzoin using both intracellular and surface-immobilised enzymes. This provides a solid proof of concept for the streamlined development of cascade reaction systems in a single cell through non-genetic cell surface enzyme immobilisation.

Electroenzymatic CO₂ fixation enables energy-efficient, highly selective synthesis of complex molecules. Unlocking its full potential requires fundamental understanding of electrode-coupled reductases and carboxylases. This review critically discusses available enzymes, product scope, and key thermodynamic and kinetic considerations, highlighting current opportunities and limitations in electroenzymatic CO₂ fixation.

ABSTRACT

Replacing fossil resources with renewable alternatives for chemical production requires highly integrated, energy-efficient, and selective CO₂ utilization processes. Electroenzymatic CO₂ fixation offers a promising pathway, combining high energy efficiency with unparalleled product selectivity, making it a viable approach for synthesizing complex molecules. This review outlines the fundamental principles of bioelectrocatalytic CO₂ conversion using reductases and carboxylases, providing an overview of the available enzymes, their product range, and key thermodynamic and kinetic considerations. By highlighting both the potential and limitations of electrochemical CO₂ fixation, this review aims to inform future progress in the discovery, engineering, and application of CO₂-converting enzymes, with the ultimate goal of realizing the full potential of electroenzymatic CO₂ fixation for the sustainable synthesis of fine and specialty chemicals.

Sinefungin is a potent nucleoside antimetabolite of S-adenosylmethionine (SAM), yet its biosynthesis has remained unclear for decades. Here we detail the identification and characterization of the complete sinefungin biosynthetic gene cluster (BGC) from Streptomyces incarnatus NRRL 8089. In vitro and in vivo analyses demonstrate that the defining carbon-carbon (C-C) bond is formed not by the long-hypothesized PLP-dependent process, but by a vitamin B12-dependent radical SAM enzyme. Using isotope-labeled cofactors and substrates, we provide evidence that the adenosyl group of sinefungin atypically originates from adenosylcobalamin via a homolytic SH2 substitution, establishing a rare instance where adenosylcobalamin is enzymatically consumed during the reaction. Furthermore, the pathway utilizes a cryptic phosphorylation-dephosphorylation strategy to control intermediate processing and substrate recognition. We also characterize two peptide aminoacyl-tRNA ligases (PEARLs) that append alanines onto the nucleoside scaffold using tRNA-activated amino acids. The PEARLs act directly on small molecules rather than macromolecular substrates, with one PEARL capable of iterative elongation. Finally, we leverage these enzymes in a reduced multi-enzyme cascade to biosynthesize sinefungin. Together, these findings redefine radical-mediated C-C bond formation and pearlin enzyme versatility, unlocking biocatalytic possibilities to produce amino acid-nucleoside conjugates. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/726688v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1e2508borg.highwire.dtl.DTLVardef@115dbc5org.highwire.dtl.DTLVardef@f7ec3org.highwire.dtl.DTLVardef@14b73d6_HPS_FORMAT_FIGEXP M_FIG C_FIG

Glycyl radical enzymes (GREs) catalyze challenging chemical reactions using a post-translationally installed glycyl radical cofactor. One such enzyme, indoleacetate decarboxylase (IAD), performs the radical-based decarboxylation of indole-3-acetate (I3A) to form the malodorant molecule skatole. In addition to being an odor nuisance, skatole is a human and livestock lung toxin, a suspected carcinogen, and a mosquito attractant, all of which impact human health, agriculture, food production, and wastewater treatment. Here, we use cryogenic electron microscopy to solve a 2.45-[A] resolution structure of indoleacetate decarboxylase from the gut bacterium Olsenella uli. We observe IAD in a homotetrameric form with the substrate I3A bound in all four protomers. The positioning of the I3A in the active site is unexpected and is more consistent with a Kolbe-type decarboxylation mechanism, i.e. a decarboxylation initiated by a 1-electron oxidation of the carboxylate moiety rather than being initiated by hydrogen atom transfer (HAT). Previously, a high deuterium content in skatole from IAD assays in D2O was used to support a HAT mechanism over a Kolbe-type mechanism. However, we show here that deuterium content does not necessarily inform on mechanism as IAD can catalyze the exchange of skatoles 3'-methyl hydrogens post-turnover. Structural comparisons show that both IAD and HPAD display structural features that are not found in other characterized GREs, suggesting that they represent a distinct GRE-subclass. Collectively, these insights will inform IAD inhibitor design aimed at decreasing skatole production. Significance StatementGlycyl radical enzymes (GREs) are a superfamily of enzymes that catalyze challenging chemical reactions in anaerobic environments. One such enzyme, indoleacetate decarboxylase (IAD) performs a C-C bond cleavage and decarboxylation reaction on the tryptophan metabolite indole-3-acetate (I3A). Decarboxylation of I3A by IAD forms the malodorant molecule skatole which negatively impacts human health, livestock health, odor emissions, the environment, and food production. Here, we present the first structure of IAD with I3A bound, revealing the substrate binding mode and active site architecture. This work provides new insight into the catalytic mechanism of the enzyme and illuminates new avenues for control of the odor nuisance skatole.

{beta}-galactosidases (BGs) are essential enzymes widely used in the food industry, particularly in the production of lactose-free products. Among them, the BG from Aspergillus oryzae is of industrial relevance due to its activity at acidic pH and moderate thermal tolerance. However, enhancing its catalytic performance remains a key challenge. Traditional enzyme engineering methods are time-consuming and resource-intensive, limiting their scalability. Recent advances in Artificial Intelligence (AI), particularly those based on Natural Language Processing, offer a promising alternative by enabling efficient exploration of protein sequence space and prediction of beneficial mutations. In this study, we introduce an ensemble-based, zero-shot Protein Language Model pipeline that reconciles predictions from six independent models (ESM2 and the five ESM1v variants) combined with a diversity-aware candidate selection strategy. Applied to the BG from A. oryzae, this approach identified beneficial mutations leading to novel enzyme variants with up to a four-fold increase in catalytic efficiency on oNPGal, a two-fold increase on lactose, and, independently, a T338I variant with markedly enhanced thermostability ({approx}80% residual activity after 24 h at 60 {degrees}C), all without requiring supervised fine-tuning on experimental fitness data. Our results demonstrate that consensus across an ensemble of PLMs can efficiently enrich beneficial substitutions in industrially relevant enzymes and substantially reduce the number of wet-lab candidates that need to be screened. Table of Contents graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/726739v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@815717org.highwire.dtl.DTLVardef@17cb392org.highwire.dtl.DTLVardef@1f16110org.highwire.dtl.DTLVardef@1b71d2_HPS_FORMAT_FIGEXP M_FIG C_FIG

Nature Synthesis, Published online: 22 May 2026; doi:10.1038/s44160-026-01087-4

The combination of biocatalysts with electro- and photocatalysis provides a compelling route towards sustainable chemical and energy production driven by renewable electricity or light. We reflect on the challenges and opportunities associated with combining these technologies.

Nature Synthesis, Published online: 22 May 2026; doi:10.1038/s44160-026-01080-x

Sijia Dong, Assistant Professor at Northeastern University, talks to Nature Synthesis about the computational studies and design of photoenzymatic catalysts.