Biocatalysis@TUDelft

Science, Volume 389, Issue 6767, September 2025.

Enzyme fusion constructs comprised of formate dehydrogenase and azoreductase can produce hydrogen peroxide especially in the presence of FMN. These enzyme fusions are utilized for in situ hydrogen peroxide production and are coupled to an unspecific peroxygenase for a slower peroxide delivery. Results show that the rate of hydrogen peroxide delivery and concentration can affect C─H oxyfunctionalization reactions, primarily affecting enantioselectivity.

Abstract

Unspecific peroxygenases (UPO) have been widely studied for different reactions as they are often considered as the dream biocatalyst. UPOs only require H₂O₂ to perform C─H oxyfunctionalization reactions. However, excessive supply of peroxides can also lead to enzyme inactivation. Therefore, strategies to slowly supply the peroxide are being investigated. Here, we report a bifunctional biocatalyst that is comprised of a formate dehydrogenase and an azoreductase with a peptide linker in-between. It was shown that the fusion constructs can be used as an in situ H₂O₂ generator to fuel the unspecific peroxygenase from Collariella viriscens (CviUPO). Fusion protein can also inherently produce H₂O₂ but addition of free FMN led to 10-fold production of peroxides. Moreover, it was found that by coupling the fusion protein with CviUPO, supply level and delivery of H₂O₂ can affect C─H oxyfunctionalization reaction and even enantioselectivity. Ethylbenzene and thioanisole were used as model substrates to demonstrate the importance of H₂O₂ delivery. CviUPO was not active with ethylbenzene even with just 1 mM H₂O₂ but coupled reactions with the FDH-AzoRo fusion showed improved activities with preferences for the R-enantiomer. This study demonstrates how the rate and the delivery of peroxides are crucial to enantioselective reactions.

Cobalamin-dependent aryl methyl ether O-demethylases have high potential for biocatalytic applications, including lignin valorization and synthetic chemistry. In this review, we provide a detailed overview of such O-demethylase systems identified to date from various microorganisms, including their mechanism, substrate scope and selectivity, and further discuss their potential for biocatalytic applications.

Abstract

Cobalamin-dependent aryl methyl ether O-demethylase is a multi-component enzyme system that converts O-methylated aromatic compounds into demethylated phenolics. The central enzyme of the system is a cobalamin-dependent protein that interacts with methyltransferase enzymes for transferring the methyl group between O-methyl groups of aryl methyl ethers and various methyl acceptors. Besides their role in energy metabolism of certain anaerobic bacteria, O-demethylases possess high potential for biocatalysis, including lignin valorization and use in organic synthesis for reversible (de)methylation reactions. An increasing number of cobalamin-dependent O-demethylase enzyme systems from various bacteria, including gut microorganisms, with different substrate scopes and regioselectivity profiles have been identified in the recent decade. Moreover, biocatalytic studies have been carried out on O-demethylase systems demonstrating their potential in synthetic applications. In this review, we provide a comprehensive overview of the cobalamin-dependent aryl methyl ether O-demethylase systems identified to date in various microorganisms. We present the mechanism, biological function, substrate scope and selectivity of the studied systems and discuss their potential for biocatalytic applications.

This review focuses on the latest research progress of mechanoenzymatic reactions and their green metrics in line with the Principles of Green Chemistry, and also discusses the challenges and prospects.

Abstract

Mechanoenzymology is a green chemistry technology that has emerged in recent years, which can efficiently promote enzymatic hydrolysis reactions through mechanical force under low-solvent conditions. Mechanoenzymatic reactions has the green metrics of reducing solvent usage, minimizing waste generation, potentially improving reaction efficiency, and mild reaction conditions, which conform to the Principles of Green Chemistry. In this review, the focus is on the latest research progress of mechanoenzymatic reactions and the green aspects based on the Principles of Green Chemistry. The challenges and prospects of mechanoenzymology are discussed to further promote its development and application.

Nature Chemistry, Published online: 30 September 2025; doi:10.1038/s41557-025-01949-y

Azetidine is a pharmacophore present in drug-related molecules. Here the authors unveil a two-metalloenzyme cascade leading to the azetidine-containing polyoximic acid, in which PolE functions as an Fe2+/pterin-dependent l-isoleucine desaturase, while PolF is a haem-oxygenase-like diiron oxidase, orchestrating the sequential desaturation and cyclization. These findings expand our knowledge of metalloenzymes.

Alcohol dehydrogenases (ADHs) are best known for reducing ketones to chiral alcohols, but their oxidative potential is rarely exploited. Here, we show that selected ADHs catalyze newly discovered promiscuous transformations: the oxidative coupling of primary alcohols with amines or thiols, enabling the direct and efficient synthesis of a broad range of amides and thioesters under mild reaction conditions.

Abstract

Amide and thioester moieties are prevalent in pharmaceuticals, natural products, and functional materials, but their chemical synthesis suffers from poor atom economy and ungreen conditions, while biocatalytic methods require ATP-dependent enzymes, activated intermediates, or show limited scope and activity. Here, we report the oxidative coupling of alcohols with ammonia or amines catalyzed by alcohol dehydrogenases (ADHs) via hemiaminal intermediates to form primary and secondary amides at pH 9.5–10.5. Pf-ADH preferably converted linear aliphatic or arylaliphatic alcohols (up to 90% conversion), while Pp-ADH and Aa-ADH preferably converted branched or aromatic alcohols (up to 99% conversion). Preparative-scale synthesis of an N-methyl amide gave >99% conversion and 87% isolated yield. The method was extended to thioacid and thioester formation via hemithioacetal intermediates using hydrogen sulfide or thiols at pH 7. Pf-ADH favored linear aliphatic alcohols (up to 93% conversion), Pp-ADH branched alcohols (up to 82% conversion), and Aa-ADH aromatic alcohols (up to 98% conversion). A KPi/MTBE biphasic system enabled the reaction with poorly soluble long-chain thiols. Structure-guided engineering of Aa-ADH led to the Y151A and L186A variants with expanded activity toward longer-chain amines or thiols. This work highlights how enzyme promiscuity with protein engineering can enable new-to-nature synthetic pathways for the production of valuable compounds.

Proceedings of the National Academy of Sciences, Volume 122, Issue 39, September 2025.
SignificanceCannabinoids are the primary active compounds inCannabis sativaand hold significant medical potential. However, their high lipophilicity limits bioavailability, an issue which can be resolved by promiscuous glycosylation. Here, we reveal ...

Controlling enzyme activity with molecular precision remains a fundamental challenge. Here, we present a DNA-based strategy for the dynamic and programmable regulation of enzyme function in response to arbitrary, user-defined chemical cues. We report the development of single-molecule DNA tweezers (SMDTs)—structures that can be programmed to bind and inhibit enzymes, then release upon sensing specific signals, restoring activity.

Abstract

Engineering allosteric control sites into enzymes typically requires extensive protein modification. Here, we introduce single-molecule DNA tweezers (SMDTs), which enable programmable, allosteric-like regulation of enzyme activity in response to user-defined chemical cues, without altering the enzyme itself. SMDTs consist of two aptamers connected by a tunable, stimuli-responsive DNA linker. By binding non-covalently to two distinct sites on an enzyme, the SMDT adopts a “pinched” conformation, reminiscent of mechanical tweezers, that inhibits enzymatic activity. Upon exposure to specific molecular triggers, the SMDT undergoes a conformational change that releases the inhibitory aptamer, restoring function. The degree of inhibition and reactivation efficiency can be finely tuned by adjusting the DNA linker's length, sequence, flexibility, and geometry. Operating at nanomolar concentrations, the system exhibits high specificity, capable of discriminating between closely related inputs, including single-base mismatches in nucleic acids. Importantly, SMDTs can be programmed to respond not only to molecular abundance but also to molecular activity. We show the versatility of this platform by regulating enzymes using diverse triggers, including nucleic acids, transcription factors (TATA-binding protein [TBP], cellular myelocytomatosis [c-Myc]), signaling proteins (platelet-derived growth factor [PDGF]), small molecules (kanamycin), and metal ions (Mn2+). These results establish a generalizable framework for designing responsive protein binders that translate molecular recognition into functional outcomes.

Through a two-step genome mining strategy, a conserved and widely distributed gene cluster family was discovered from Fusarium sp., which was demonstrated for synthesising a group of new and rare terpenoid-isonitrile amino acid hybrid natural products (namely, chlamonitriles A–F) by heterologous expression and enzymatic biochemical characterisation. These compounds present a group of unusual structural features, including a highly oxidised guaiane-type sesquiterpene skeleton connected to a Cα–Cβ dehydrogenation isonitrile isoleucine moiety via an ester bond.

Abstract

Terpenoid nonproteinogenic amino acid hybrid natural products usually have complex chemical structures and important biological functions. Herein, we discovered a group of conserved and widely distributed clusters of sesquiterpenoid-isonitrile isoleucine hybrid compounds from Fusarium through a two-step genome mining strategy. An investigation of the function of the isc cluster from Fusarium chlamydosporum revealed a variety of unusual enzymatic transformations, which importantly include 1) a single-module nonribosomal peptide synthase (NRPS) IscF featuring an unusual C-terminal transferase domain that catalyses the esterification reaction on C2─OH of a highly oxidised guaiane-type 5/7-bicyclic sesquiterpene precursor 4 with T domain-bound l-isonitrile isoleucine 11a to yield terpenoid-isonitrile isoleucine hybrid natural product 9a (chlamonitrile A) and 2) cytochrome P450 (CYP450) IscD, which unexpectedly catalyses the Cα–Cβ dehydrogenation of the isonitrile isoleucine moiety of 9a, resulting in the final products 13a/13b (chlamonitriles E/F). In addition, phytotoxic evaluation experiments revealed that 9a and its structural analogues are new phytotoxins of Fusarium. Our work provides the first example of the discovery of terpenoid-isonitrile amino acid hybrid compounds from fungi, expands our knowledge of the new functions of NRPS domains and fungal CYP450s, and uncovers the possible biological functions of these compounds in Fusarium.

De novo designed proteins offer a malleable platform for the development of stereoselective transformations not found in biochemistry. Here, we report the de novo design and directed evolution of helical bundle protein catalysts for enantioselective germylation through Ge-H insertion, a transformation not previously achieved by enzymatic catalysis. Comparative computational analysis revealed that, relative to Si-H insertion, the Ge-H insertion reaction proceeds through an earlier and more flexible transition state, introducing distinct challenges for stereocontrol. Using a fully de novo designed truncated four-helix bundle scaffold as the starting point, directed evolution afforded a quadruple mutant that catalyzes Ge-H insertion with high efficiency, enantioselectivity, and broad substrate scope. Molecular dynamics simulations indicated that beneficial mutations introduced from directed evolution enhanced active-site preorganization and modulated local back-bone flexibility, contributing to improved transition-state complementarity with fine-tuned binding pocket size and more stable cofactor positioning regulated by hydrogen bonding interactions. These findings showcase the excellent potential for de novo proteins to achieve stereoselective transformations previously unknown to biocatalysts and underscore the importance of active-site remodeling of de novo protein scaffolds via directed evolution in achieving selective catalysis involving flexible transition states. Table of Contents artwork O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/679279v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@162187forg.highwire.dtl.DTLVardef@13665a4org.highwire.dtl.DTLVardef@4afff1org.highwire.dtl.DTLVardef@1e92f92_HPS_FORMAT_FIGEXP M_FIG C_FIG

Polyethylene terephthalate (PET) is the fourth most commonly used plastic worldwide. Like all plastics, post-consumer PET is poorly managed and accumulates in the environment, posing significant ecological threats. After 70 years of accumulation, microorganisms capable of degrading and assimilating PET have been isolated, demonstrating that PET can be broken down and converted into valuable cellular biomass or metabolic products. These natural isolates, however, are poorly characterized and challenging to genetically manipulate, which limits their further optimization and applicability. Here, we engineer a well-established synthetic biology chassis for the biodegradation and assimilation of PET. We modified the bacterium Pseudomonas putida KT2440 to heterologously express an active PET-hydrolytic enzyme extracellularly and to metabolize PET biodegradation products. The resulting strain, named PETBuster, was capable of growing on PET as the sole carbon source on solid and liquid media. We achieved 91% PET degradation after 21 days of culture, with a doubling time of 3.6 days, under mesophilic conditions. In this way, we demonstrate that PET fermentation is feasible, opening the door to the production of valuable chemicals from waste.

Ketosynthase domains govern chain transfer and substrate selectivity in modular polyketide synthases (PKS), yet their functional tunability in native contexts remains poorly understood. We performed phylogenetically guided mutagenesis of the KS5 domain from the Streptomyces cinnamonensis monensin PKS and evaluated 72 variants in vivo across wild type and reductive loop null backgrounds. This revealed discrete active site motifs that control productivity, redox state specificity, and extender unit selection, functions traditionally ascribed to other PKS domains. AlphaFold3 structural mapping linked these motifs to substrate tunnel and catalytic core features, providing a mechanistic basis for the observed phenotypes. Our findings demonstrate that KS domains can be rationally re tuned to overcome productivity bottlenecks and alter specificity in intact PKSs, offering a route to improved yields and expanded chemical diversity in engineered polyketides.

Terpenes are a structurally and functionally highly diverse class of natural products found across all living organisms. Pseudomonas species possess significant potential for the biosynthetic assembly of farnesyl pyrophosphate (FPP)-derived terpenes with unusual carbon skeletons. However, their structural and biosynthetic diversity is largely unexplored. Here, we report the discovery of grimophan, an unprecedented C17 terpene featuring a deltacyclane skeleton. The compound was accessed by heterologous expression of the pgr biosynthetic gene cluster from Pseudomonas grimontii DSM 17515 in E. coli. The roles of the enzymes involved in grimophan biosynthesis were elucidated through in vitro reconstitution of the entire biosynthetic pathway. In-depth functional studies on the involved SAM-dependent methyltransferases led to the discovery of novel methyltransferase-like enzymes that do not perform functional group transfer, but rather significantly enhance the production titer of non-canonical terpenes. These enzymes thus constitute valuable tools for increasing biotechnological production levels of terpenes. The function of the cognate terpene synthase PgrE was evaluated by targeted point mutations within the conserved Asp-rich motif D92DMPLG97 to map effects of active-site residues on product formation. These investigations facilitated redirecting product selectivity, leading to alternative products. Overall, our work sheds light on the general biosynthetic logic leading to non-canonical C17 terpenes, provides a basis for their targeted discovery by genome mining and heterologous expression, and for engineering C17 terpene structural frameworks.

Nature Chemistry, Published online: 25 September 2025; doi:10.1038/s41557-025-01937-2

Visible-light-driven pyridoxal radical biocatalysis offers a promising approach for developing stereoselective intermolecular radical reactions that have no known precedent in biology or chemistry. Now, building on the engineering of pyridoxal-dependent carboligases, a multienzyme photobiocatalytic cascade enables the stereoselective synthesis of polysubstituted unnatural prolines, including 2,5-anti-stereoisomers that remain challenging to access by other methods.

A flow electrolyzer employing formate dehydrogenase on a porous TiO₂-carbon felt cathode is developed for paired electrolysis of CO₂ and waste (plastic and biomass) to produce formate. The electrolyzer operates with an initial cell faradaic efficiency toward formate of almost 200% at a low cell voltage of −1.5 V, which also enables bias-free operation with a commercial solar cell. The electrogenerated formate is used directly for photocatalytic carbon chain extension of styrene to phenylpropanoic acid.

Abstract

Paired electrolysis enables the simultaneous coupling of CO₂ reduction with anodic waste upcycling to form valuable products. However, achieving selective, efficient, and stable product formation and coupling to downstream valorization remains a challenge. In this study, W-containing formate dehydrogenase from Nitratidesulfovibrio vulgaris Hildenborough is immobilized onto a cathode made from carbon felt coated with porous TiO₂ and paired with a commercial Ni foam anode to assemble a semiartificial flow electrolyzer for the simultaneous conversion of CO₂ and waste (plastic and biomass) to the single product formate. The enzymatic flow electrolyzer achieved an initial cell faradaic efficiency toward formate of almost 200%, a maximum CO₂ conversion yield of 18% and can operate at a low full-cell voltage of −1.5 V for 122 h, which allows for bias-free operation with a silicon photovoltaic cell. The aqueous formate produced in the enzymatic electrolyzer was subsequently utilized downstream as a C₁ building block in the photocatalytic hydrocarboxylation of alkenes, providing a path for the domino valorization of CO₂ and waste toward bulk and fine chemical synthesis.

Direct carbonyl desaturation to prepare a,b-unsaturated carbonyl compounds is an important area of research in organic synthesis due to the significance of these molecules in medicinal chemistry and chemical biology. Despite numerous methods developed for this transformation, approaches that enable precise control over the site-selectivity of the reaction on substrates containing multiple potential reactive sites remain extremely rare, limiting their applications in late-stage functionalization of complex molecules. We report herein the engineering of ‘ene’-reductases (EREDs) for the direct carbonyl desaturation of diverse cyclic ketones to their corresponding enones with unprecedented divergent and exquisite site-selectivity. This study leverages the distinctive ability of EREDs to differentiate the stereochemical environments of hydrogens at the carbonyl b-positions. The synthetic utility of this biocatalytic platform is further demonstrated through the successful late-stage dehydrogenation on terpenoids with complementary site- selectivity to existing methods. Additionally, the method could efficiently prepare chiral enones with a b-all carbon quaternary stereogenic center via novel biocatalytic desaturative kinetic resolution. Mechanistic studies elucidate key enzyme-substrate interactions responsible for the unrivaled enzyme-controlled site-divergence.

Nature, Published online: 24 September 2025; doi:10.1038/d41586-025-03071-y

Genome editors are molecular machines that can rewrite the genetic code in cells, but sometimes they produce errors in the form of unintended sequence insertions or deletions, collectively known as indel errors. Genome editors have now been engineered that make up to 60-fold fewer indel errors than previous ones did.

Nature Catalysis, Published online: 24 September 2025; doi:10.1038/s41929-025-01419-1

Substrate promiscuity fuels biosynthesis success

Nature Catalysis, Published online: 24 September 2025; doi:10.1038/s41929-025-01412-8

Hexopyranose cleavage is a crucial step in carbon metabolism. Here the authors report the discovery and characterization of metalloenzyme Art22, which is involved in the sugar moiety modification of aurantinin B, an antibacterial agent from Bacillus.

Nature Catalysis, Published online: 24 September 2025; doi:10.1038/s41929-025-01413-7

A TIM-barrel metalloenzyme — Art22 — involved in the sugar-moiety modification of the antibiotic aurantinin B (ART B) has been discovered. This enzyme activates 4-keto ART B to ART B through rapid isomerization. Additionally, Art22 slowly converts ART B into inactive products through oxidative cleavage of the 3-keto hexopyranose.

Nature Catalysis, Published online: 24 September 2025; doi:10.1038/s41929-025-01400-y

The 1913 study ‘Die Kinetik der Invertinwirkung’, by Michaelis and Menten, marked a pivotal advancement in enzymology by illustrating the application of mechanistic models and quantitative kinetics to biocatalysis. The foundational framework described back then continues to have a strong impact on enzymology, with profound influences that range from undergraduate education to structure–function studies and the format and content of contemporary kinetic databases.

Nature Catalysis, Published online: 24 September 2025; doi:10.1038/s41929-025-01415-5

The 2025 RepArtZymes conference featured the latest developments in the design and development of artificial and repurposed enzymes for synthetic and biotechnological applications. These contributions illustrate the impact of this rapidly expanding research area towards addressing key challenges in organic synthesis, medicinal chemistry, polymer chemistry, energy conversion, and environmental remediation.