Biocatalysis@TUDelft

We developed biofoundry workflows for efficient enzyme engineering, integrating a simple and accessible computational tool with automation to reduce time and resources. Using this approach, isoprene synthase was engineered for enhanced catalytic efficiency and thermostability, and also enhanced methane-to-isoprene conversion when applied to methanotrophs, highlighting its potential for methane-based biomanufacturing.

TheA is a moderate thermostable flavin-dependent monooxygenase which accepts, besides l-ornithine a number of related molecules for the regio-selective N-hydroxylation. The combination with a NADP⁺-reducing formate dehydrogenase variant (FDH M4) and a H₂O₂ degrading catalase allows in vitro biocatalytic applications.

The N-hydroxylating monooxygenase (NMO) TheA from Thermocrispum agreste catalyzes the N-hydroxylation step of l-ornithine, which is the first step in the thermochelin siderophore biosynthesis. Characterization of this enzyme revealed a significant thermostability up to 50 °C and activity with the non-native substrate d-ornithine with kinetic parameters (K _m = 4.06 ± 0.31 mM, k _cat = 0.057 ± 0.001 s⁻¹, and k _cat/K _m = 0.007 s⁻¹mM⁻¹) and a coupling rate of 81%. The enzyme is applied in a one-pot reaction with a formate dehydrogenase variant for NADPH regeneration and catalase for H₂O₂ detoxification. Optimization of the reaction conditions resulted in activity with various non-native substrates such as d-ornithine, l-lysine, S-(2-aminoethyl)-l-cysteine, and l-arginine. Products are confirmed through LC-MS/MS, and mutagenesis experiments gave insight on the potentially underlying mechanisms. This work identifies a thermotolerant NMO that is suitable for application and as a starting point for enzyme engineering.

Nature Synthesis, Published online: 10 September 2025; doi:10.1038/s44160-025-00882-9

Molecular-dynamics-simulation-guided evolution of flavoenzymes produces efficient catalysts for non-C2-symmetric biaryl synthesis with excellent atroposelectivity, offering promise for natural product synthesis and pharmaceutical applications.

Synthetic methods that are applicable to a broad range of substrates are sought after, owing to their utility in industrial settings. This minireview describes considerations associated with how chemists define and identify general methods, especially with the emergence of modern analytical, high-throughput, and data science tools in chemistry, and gives the reader an overview of workflows that have been used to expedite this pursuit.

Abstract

The term “generality” has recently been popularized in synthetic chemistry, owing largely to the increasing use of high-throughput technology for producing vast quantities of data and the emergence of data science tools to plan and interpret these experiments. Despite this, the term has not been clearly defined, and there is no standardized approach toward developing a method with a diverse (general) scope. This minireview will examine different emerging strategies toward achieving generality using selected examples and aims to give the reader an overview of modern workflows that have been used to expedite this pursuit.

Inspired by nature, we report a visible light-driven membrane-bound compartment composed of photoisomerizable phenylazothiazole lipids and phospholipids, which enables reversible and graded regulation of enzyme activity. This biomimetic strategy operates without enzyme modification and mutagenesis, offering a versatile and biocompatible platform applicable to a broad range of enzymes.

Abstract

Photo-responsive systems provide a powerful tool to reversibly regulate enzyme activity. However, inhibitor-based strategies, though widely used, are often restricted to specific enzymes. Noninhibitor strategies, such as enzyme surface modification or genetic mutation, often compromise structural integrity or residual activity. Inspired by the gating mechanisms of biological membranes, we reported a visible light-driven membrane-bound compartment system constructed from phenylazothiazole gated lipids and phospholipids. In this design, phenylazothiazole lipids undergo reversible isomerization between trans and cis configurations under alternating purple and green light, generating continuous nanomechanical motions that transiently enhance membrane permeability. This dynamic gating behavior enables substrate diffusion across the membrane under light exposure and allows the activity of encapsulated enzymes to be switched on and off in a noninvasive, temporally defined manner. This system requires no chemical modification or mutagenesis, thus preserving the native structure and activity of encapsulated enzymes. Beyond binary regulation, precise modulation of the irradiation pattern permits graded control over enzyme activity, offering an advanced level of functional tunability. Using carbonic anhydrase, catalase, and glucose oxidase as models, we demonstrate that enzyme activity can be reversibly and quantitatively regulated via programmable light inputs. This strategy offers a broadly applicable and biocompatible platform for spatiotemporal enzyme regulation.

Biocatalysis is a branch of catalysis that has allowed the development of a diverse range of sophisticated synthetic methodologies that target geometrically demanding structures such as pharmaceuticals, natural products, and their analogues. The routes are often more efficient due to enzymes innately high degree of chemo-, regio- and stereoselectivity, while making the overall process more sustainable when compared to their chemical equivalent, as enzymes are naturally biodegradable and operate under physiological conditions. Herein, we demonstrate the power of this catalytic approach via the development of a chemoenzymatic one-pot process that allowed access to the fungal metabolites tenuipesones A and B, along with their enantiomeric counterparts, in good yields (60-72 %) and excellent enantioselectivity (>99 % ee). The stereochemical outcome of the products was controlled through careful selection of the biocatalysts, enabling control of the configuration of the key C7 chiral center. This novel chemoenzymatic cascade process allowed access to all four possible stereoisomers, with their absolute stereochemistry being evaluated to confirm the true configuration of the natural isolates.

We report a one-pot biocatalytic cascade for the conversion of allylic alcohols into enantioenriched secondary amines through a sequence involving oxidation followed by conjugate reduction–reductive amination (CR–RA). This transformation, catalyzed by cholesterol oxidase (ShCO) and an ene-imine reductase (EneIRED), proceeds under mild conditions and accommodates a broad substrate scope. In several cases, the cascade outperforms the stepwise method and exhibits substrate-dependent selectivity, highlighting the advantages of tandem enzymatic systems for efficient and streamlined chiral amine synthesis.

Nature, Published online: 03 September 2025; doi:10.1038/d41586-025-02722-4

Reductase enzymes catalyse the conversion of the greenhouse gas nitrous oxide (N2O) to environmentally benign dinitrogen gas. The discovery of a microbial N2O reductase reveals a previously unknown N2O sink and creates opportunities for innovative biotechnologies to counter the effects of N2O emissions.

Nucleotide sugar short-chain dehydrogenases/reductases (NS-SDRs) are involved in pseudomurein and capsular polysaccharide formation in methanogenic Archaea. Methanothermobacter thermautotrophicus possesses several NS-SDRs labelled Mth375, Mth380, Mth373, Mth631 and Mth1789. Utilising X-ray crystallography, molecular modelling and phylogenetics we shed light on the NS-SDRs of M. thermautotrophicus and their potential enzyme activities.

Epimerases and dehydratases are widely studied members of the extended short-chain dehydrogenase/reductase (SDR) enzyme superfamily and are important in nucleotide sugar conversion and diversification, for example, the interconversion of uridine diphosphate (UDP)-linked glucose and galactose. Methanothermobacter thermautotrophicus contains a cluster of genes, the annotations of which indicate involvement in glycan biosynthesis such as that of cell walls or capsular polysaccharides. In particular, genes encoding UDP-glucose 4-epimerase related protein (Mth375), UDP-glucose 4-epimerase homologue (Mth380) and dTDP-glucose 4,6-dehydratase related protein (Mth373) may be involved in the biosynthesis of an unusual aminosugar in pseudomurein. In this paper, we present the structures of Mth375, an archaeal sugar epimerase/dehydratase protein (WbmF) determined to a resolution of 2.0 Å. The structure contains an N-terminal Rossmann-fold domain with bound nicotinamide adenine dinucleotide hydride (NADH) and a C-terminal catalytic domain with bound UDP. We also present the structure for Mth373 co-crystallised with uridine-5′-diphosphate-xylopyranose to a resolution of 1.96 Å as a NAD⁺-dependent oxidative decarboxylase (UDP-xylose synthase; EC4.1.1.35). Molecular modelling has also allowed for the identification of Mth380 as a UDP-N-acetylglucosamine 4-epimerase (WbpP; EC5.1.3.7), Mth631 as a UDP-glucose 4-epimerase (GalE; EC5.1.3.2) and Mth1789 as a classical dTDP-d-glucose 4,6-dehydratase (EC4.2.1.46). The UDP–sugar specificity of each archaeal nucleotide sugar short-chain dehydrogenase/reductase (NS-SDR) was elucidated via sequence, molecular modelling and structural analyses. Overall, these structures potentially shed light on the formation of the glycan portion of pseudomurein and capsular polysaccharide in Archaea.

Type III polyketide synthases (T3PKSs) produce diverse compounds of ecological and clinical importance. Here, the activity of 37 fungal T3PKSs was profiled, revealing unexpected products and generating several pharmaceutical precursors. The machine learning model trained on the activity data accurately extrapolates the predictions to other enzymes and substrates, facilitating the development of biocatalytic routes towards (un)natural polyketides.

Abstract

Type III polyketide synthases (T3PKSs) are enzymes that produce diverse compounds of ecological and clinical importance. While well-studied in plants, only a handful of T3PKSs from fungi have been characterised to date. Here, we developed a comprehensive workflow for kingdom-wide characterisation of T3PKSs. Using publicly available genomes, we mined more than 1000 putative enzymes and analysed their active site architecture and genomic neighbourhood. From there, we selected 37 representative PKS candidates for cell-free expression and prototyping with a diverse set of Coenzyme A activated substrates, revealing unique patterns in substrate and cyclisation specificity, as well as the preferred number of malonyl-Coenzyme A extensions. Using the 341 enzyme-substrate pairs generated in this study, we trained a machine learning model to predict T3PKS substrate specificity and experimentally validated it with an extended panel of non-natural substrates. In addition, we applied the model to identify two more promiscuous T3PKSs from fungi. We anticipate that the ML model will be useful for in silico screening of T3PKSs, while the insight into the product scope of these enzymes offers interesting starting points for further exploration.