Biocatalysis@TUDelft

Communications Chemistry, Published online: 12 December 2025; doi:10.1038/s42004-025-01783-w

The sustainable and scalable synthesis of chiral amines remains a significant challenge in industrial chemistry, particularly due to the limitations of current biocatalytic processes in achieving high productivity and selectivity at scale. This study reports a dynamic kinetic resolution strategy that integrates flash thermal racemization with enzymatic resolution in a continuous flow system, achieving unprecedented productivity, excellent enantioselectivity, and industry-relevant green chemistry metrics at scales up to 100 grams.

The power of catalytic promiscuity: The enantioselective conjugate hydrocyanation of enals remains a long-standing challenge for biocatalysis. Here, we report the redesign of 2-deoxy-D-ribose-5-phosphate aldolase for the asymmetric conjugate hydrocyanation of aromatic enals, expanding the reaction scope of iminium-based enzyme catalysis to include an additional new-to-nature reaction.

ABSTRACT

The enantioselective conjugate hydrocyanation of α,β-unsaturated aldehydes remains a long-standing challenge in synthetic chemistry. Here, we report the redesign of 2-deoxy-D-ribose-5-phosphate aldolase (DERA) into an efficient biocatalyst capable of promoting the asymmetric conjugate addition of hydrogen cyanide (generated in situ from trimethylsilyl cyanide) to aromatic enals via an iminium activation pathway. The evolved variant DERA-CN enables the efficient formation of various C4-nitriles with high conversions (up to 99%) and good enantioselectivity (up to 98% e.e.). Control experiments revealed a stepwise process involving enzyme-catalyzed conjugate hydrocyanation followed by spontaneous 1,2-addition of cyanide. Substrates with various electron-donating and electron-withdrawing groups are tolerated, providing access to various enantioenriched nitriles. This work expands the scope of DERA-promoted iminium catalysis and provides a rare enzymatic platform for asymmetric conjugate hydrocyanation under mild aqueous conditions.

The strobilurin iterative PKS assembles a remarkable EZE triene by varying its stereoselectivity during synthesis. Stereoselective 2R-methylation of the triketide intermediate by the C-terminal C-methyltransferase domain induces the KR and DH domains to invert their native stereoselectivities and produce a Z-configured intermediate.

Abstract

Type I Iterative polyketide synthases (PKS) use a limited set of catalytic extension and β-processing domains to create complex polyketides. A remarkable case is that of the strobilurin PKS where a single dehydratase (DH) domain creates an EZE triene over three dehydration cycles. Here we dissect the strobilurin PKS and assay catalytic domains individually and in combination with stereo-defined synthetic substrates in vitro, to reveal the complex and varying selectivities of methylation, ketoreduction and dehydration that lead to this remarkable result. At the diketide stage all stereoselectivities are consistent with those known for other related systems, giving an E product. But at the triketide stage, 2R-methylation is selectively achieved, that is followed by rapid keto-reduction to give an unusual 3-L-alcohol. In-turn, this is eliminated to give the unusual Z-alkene. These selectivities are flexible and change in response to the structure of the substrate at every stage. This uncovers the complete and intricate regio- and stereo-selectivities of a highly reducing iterative Type I PKS for the first time, and highlights the important differences to the well-studied cis-AT modular PKS β-processing enzymes that appear to have inflexible selectivities.

Enzyme catalysis: The di-nickel enzyme GdmH catalyzes guanidinium hydrolysis through a mechanism involving nucleophilic attack, proton transfer, and C-N bond cleavage. The positive charge of guanidinium is essential for initiating the reaction. In contrast, GdmH demonstrates significantly reduced efficiency in catalyzing urea hydrolysis.

Abstract

The nitrogen-rich compound guanidine is widely distributed in nature, but its utilization is hindered by strong resonance stabilization. GdmH, a binuclear nickel enzyme from Synechocystis sp. PCC 6803, is capable of directly converting guanidinium cation into urea and ammonium. In this study, we employed density functional calculations to investigate the reaction mechanism of GdmH using a chemical model derived from the enzyme's X-ray crystal structure. The calculations revealed that the GdmH-catalyzed guanidinium hydrolysis proceeds through a nucleophilic attack by the di-nickel bridging hydroxide on the guanidinium carbon forming a tetrahedral intermediate, two proton transfer steps from the hydroxyl to an amino facilitated by Asp203, C─N bond cleavage yielding urea and ammonia, and regeneration of the bridging hydroxide accompanied by ammonium release. The rate-limiting step is the first proton transfer from hydroxyl to Asp203, with an energy barrier of 11.8 kcal mol⁻¹. Comparative analyses demonstrated that neutral guanidine cannot be hydrolyzed by GdmH due to the absence of a positive charge, which is essential for effective catalysis. Further investigations showed that GdmH is inefficient in catalyzing urea hydrolysis. These findings enhance our understanding of the catalytic specificity of GdmH and the role of nickel cofactors in biological enzymatic processes.

Use of a H₂-driven flavin recycling system based on the O₂-tolerant [NiFe] hydrogenase 1 from E. coli supports efficient halogenation catalyzed by the flavin-dependent halogenase, PyrH. Operation under atmospheric H₂ with controlled delivery of safe, low-level O₂ enables intensification of biocatalytic halogenation.

Abstract

We report a simplified, H₂-driven method for operating biocatalytic halogenation by a flavin-dependent halogenase, PyrH, which enables intensification of biocatalytic conversion of L-tryptophan to its 5-halo product. Flavin-dependent halogenases are gaining traction in biotechnology as their substrate scope is expanded by enzyme discovery and engineering, but their application remains impeded by a particularly complex electron transfer chain and the fact that full conversion is generally only achieved at sub-millimolar substrate concentrations. Here, we apply nickel-iron hydrogenase and ambient pressure H₂ in place of the NAD(P)⁺, glucose, glucose dehydrogenase and reductase which are normally used to supply reduced FAD to halogenases. Together with controlled delivery of O₂, which is needed for generating a hypohalous acid intermediate, we achieve full conversion of 5.5 mM tryptophan, with a PyrH total turnover number of 275, and PyrH turnover frequency of 0.76 min⁻¹ over a 6 h reaction, comparable with rates sustained only for short reaction times using the conventional glucose-driven system. This should help to facilitate application of flavin-dependent halogenases in fine chemical synthesis.

A class III phosphoribosyl pyrophosphate synthetase (PfPRS) from the hyper thermophilic archaeon Pyrolobus fumarii 1A was identified and characterized. PfPRS exhibited exceptional thermal and pH stability, retaining high activity even at 100 °C. Its robust catalytic features, along with exploratory co-immobilization for nucleotide regeneration, highlight its promise for biocatalytic applications.

Phosphoribosyl pyrophosphate (PRPP) functions as a central metabolic intermediate, supplying ribose-5-phosphate moieties for the biosynthesis of nucleotides, certain amino acids, and a range of essential cofactors. In this study, a thermostable phosphoribosyl pyrophosphate synthetase (PfPRS) was identified from the hyper thermophilic archaeon Pyrolobus fumarii 1A, a hyper thermophilic archaeon that grows optimally at 90–113 °C. The prs gene was heterologously expressed in Escherichia coli, and the recombinant enzyme was purified and characterized. Peak catalytic activity of PfPRS was observed at approximately pH 7.5 and 55 °C and retained over 85% of its activity after 2 h of incubation across pH 4.0–10.5. PfPRS exhibited high thermal stability. The enzyme exhibited half-lives of 12 h at 90 °C, 5 h at 95 °C, and 3 h at 100 °C. Among the nucleotides tested as diphosphate donors, PfPRS showed a strong preference for ATP, whereas ADP served as an effective inhibitor. Kinetic analysis revealed K _m values of 35 µM for R5P and 46 µM for ATP, with turnover rates (k _cat) of 71 s⁻¹ and 56 s⁻¹. PfPRS was co-immobilized with polyphosphate kinase 2 (DrPPK2) from Deinococcus radiodurans using a cross-linked enzyme aggregate (CLEA) system to enable ATP regeneration and to explore the feasibility of using PfPRS for PRPP biosynthesis.

Applied and Environmental Microbiology, Volume 92, Issue 1, January 2026.

We show that Limosilactobacillus fermentum NKN51 isolated from fermented Himalayan yak milk converts cholesterol to coprostanol through a cholesterol 5β reductase (5βChR). The structure and mutation analysis of the enzyme confirms the residues involved in binding to NADPH and cholesterol. Phylogenetic analysis revealed that 5βChR classified as a new class of microbial short-chain dehydrogenases. Diabetic cohort metagenomic study highlights 5βChR abundance in healthy participants and its importance for human physiology.

Cholesterol serves as a fundamental molecule in various structural and biochemical pathways; however, high cholesterol levels are linked to cardiovascular diseases. Some selected strains of Lactobacilli are known for modulating cholesterol levels. However, the molecular mechanism underlying cholesterol transformation by lactobacilli has remained elusive. This study describes the discovery and function of a microbial 3β-OH-Δ^5-6-cholesterol-5β-reductase (5βChR) from Limosilactobacillus fermentum NKN51, which directly converts cholesterol to coprostanol, thereby unraveling this longstanding mystery. Protein engineering of the reductase enzyme identified the cholesterol and NADPH interacting amino acid residues, detailing the catalytic mechanism of 5βChR. Phylogenetic analyses highlight the prevalence of 5βChRs in gut commensal lactobacilli, which share a common evolutionary origin with plant 5β reductases. Meta-analysis of microbiomes from healthy individuals underscores the importance of 5βChR homologs, while a cohort study demonstrates an inverse association between 5βChR abundance and diabetes. The discovery of the 5βChR enzyme and its molecular mechanism in cholesterol metabolism paves the way for a better understanding of the gut-associated microbiome and the design of practical applications to ameliorate dyslipidemia.

Better enzymes are needed to develop sustainable methods to recycle plastics with C-X heterobonds such as polyurethane (PUR) and nylon, for which no industrial-scale solutions exist. Current methods rely largely on sequence mining based on a small number of known enzymes. Here we expand the pool of PURases and nylonases by bioprospecting legacy plastic waste with fluorophore plastic mimics combined with FACS. We identify 29 plastic-degrading bacteria, from which 12 enzymes are identified by mass spectrometry and homology searches. Compared to existing enzymes, these enzymes are superior in thermostability and the ability to hydrolyse different high-molecular weight PUR oligomers and nylon textiles. To our knowledge, this is the first reported example of enzymes capable of hydrolysing longer chains of PUR and nylon. This study significantly increases the number of known PURases and nylonases and provides starting points for optimization campaigns through protein engineering and for in silico discovery.

The conversion of CO₂ into value-added chemicals using sunlight is a major goal in sustainable chemistry. Here, we report the design and chemogenetic optimization of a Re(I)-based artificial metalloenzyme (ArM) for the photocatalytic reduction of CO₂ to CO in aqueous solution. By incorporating a biotinylated Re(I)-phenanthroline catalyst into a tetrameric streptavidin scaffold, we achieved a significant enhancement in catalytic turnover compared to free catalyst. Mutational screening at viable positions Ser112 and Lys121 revealed a range of catalytic activities, highlighting the tunability of the system. Through a combination of molecular dynamics simulations and experimental characterization, we demonstrate that the catalytic turnover number correlates strongly with the accessibility of CO₂ to the catalyst's active site, a factor not considered in free solution catalyst design, and further computation reveals the variable activation of a bound CO2 among mutants. This work establishes a clear design principle for enhancing Re(I)-based photocatalysis by leveraging the second coordination sphere of a protein scaffold to confer aqueous stability and control substrate access.

The biosynthesis of Ahp-bicyclodepsipeptides featuring an N51-C15 bridge linking citrulline and tyrosine residues is reported. A single CYP450 enzyme, Dlm16, has been biochemically characterized to perform this intriguing macrocyclization through sequential aromatic hydroxylation and C─N coupling. The enzymatic C─H arene amidation mediated by Dlm16 involves a primary amide as the nitrogen donor and an aromatic ring as the acceptor.

Abstract

We report the biosynthesis of FR901277 (1) and delmomycin A2 (2), two 3-amino-6-hydroxypiperidone (Ahp)-containing bicyclodepsipeptides featuring an N-C bridge linking the citrulline and tyrosine residues. This intriguing side-chain macrocyclization is catalyzed by Dlm16, a cytochrome P450 monooxygenase (CYP450), through a sequential process initiated by ortho-hydroxylation of the tyrosine ring, followed by intramolecular C─N coupling between the resulting catechol moiety and the terminal NH₂ of the ureido group. Structure-function analyses and site-directed mutagenesis confirmed the catalytic importance of identified key residues, enabling the proposal of plausible macrocyclization mechanisms. Functional characterization of eight additional Dlm16 homologs further revealed a CYP450 subfamily capable of catalyzing C─N bond formation, underscoring the prevalence of this unusual macrocyclization in cyclodepsipeptide biosynthesis. Our work highlights nature's strategies for macrocycle construction and provides another example of CYP450-catalyzed C─N coupling via direct C─H functionalization.

Identification and protein engineering on an imine reductase (IRED) for both (R)- and (S)-selective synthesis of nonbiaryl amine atropisomers via dynamic kinetic resolution (DKR) have been achieved. Enantiocomplementary ADHs were also identified to catalyze the reductive DKR processes. The axially chiral styrenes were produced in high atroposelectivity and yields with broad substrate scope.

Abstract

Enzymatic synthesis of atropisomers has recently attracted considerable research attention, with most studies focusing on axially chiral biaryls. We report a less explored atroposelective dynamic kinetic resolution (DKR) of nonbiaryl styrenes catalyzed by imine reductases (IREDs) and alcohol dehydrogenases (ADHs). The IR189 wild type enzyme was identified to be highly active and selective; furthermore, the inversion of atroposelectivity was achieved with protein engineering. Additionally, two ADHs with enantio-complementary selectivity for the reductive DKR were identified and applied in the synthesis of axially chiral styrenes. Both IREDs and ADHs exhibited broad substrate scope, affording up to 99:1 e.r. and 99% yields for up to 29 examples. Scaled-up reactions and derivatization of optically pure products demonstrated the synthetic utility of these axially chiral styrenes. Molecular recognition mechanisms were elucidated by molecular dynamics (MD) simulations. The current strategy expands the scope of enzymatic DKR of atropisomeric compounds and significantly advances the field of biocatalytic synthesis of axially chiral compounds.

Designing novel proteins that share no homology with natural sequences, but which nonetheless provide life sustaining functions, is an important goal for synthetic biology. Towards this goal, we previously reported Syn-F4, the first de novo enzyme capable of catalyzing a life-sustaining reaction, both in vitro and in vivo. Syn-F4 catalyzes hydrolysis of the siderophore, ferric enterobactin, thereby releasing iron and enabling growth in iron-limited media of an otherwise inviable {Delta}fes strain of Escherichia coli. Although Syn-F4 provides a direct enzymatic replacement of the natural ferric enterobactin esterase encoded by Fes, it has a dramatically different structure and enzymatic mechanism than the natural Fes enzyme. The novel Syn-F4 enzyme forms a 4-helix bundle, comprising a homodimer of two -helical hairpins. Here we describe the engineering of a covalent peptide linkage into the homodimer to generate a single chain 4-helix bundle. As expected, the resulting linked protein (Syn-F4-Link) also rescued {Delta}fes cells in iron-limited media. Moreover, X-ray crystallography revealed a 3D structure similar to the parental homodimer. Surprisingly, however, the linked protein was not enzymatically active. Instead, Syn-F4-Link rescues {Delta}fes cells by upregulating biosynthesis of the enterobactin siderophore thereby enabling assimilation of sufficient iron to sustain cell growth. These findings demonstrate that two very similar de novo proteins can sustain cell growth using dramatically different biological mechanisms.

Among the >300 skeletally distinct products derived from farnesyl pyrophosphate (FPP) reported to date, meroterpenoids derived from the humulene core are of great interest due to their potent biological activities. Whereas the most available (E,E,E)-humulene core and derivatives thereof have been well-studied, comparatively little is known of the chemistry and biological properties of the related (E,E,Z)-core and natural products derived from this humulene isomer. Herein, we detail the first synthesis of (E,E,Z)-(10S)-hydroxyhumulene and elaborate this macrocycle through a chemoenzymatic route to (±)-deoxyeupenifeldin and neosetophomone B, and further accomplish the chemical synthesis of pughiinin A. The (E,E,Z)-(10S)-hydroxyhumulene was accessed via a selective oxidative double-bond isomerization from the (E,E,E)-humulene core. Additionally, when studying this macrocycle in subsequent hetero-Diels Alder (hDA) cycloadditions, it was observed that the (E,E,Z)-core imparted more biased diastereoselectivity when compared to previous studies of the corresponding (E,E,E)-core. Our findings demonstrate scalable access to (E,E,Z)-(10S)-hydroxyhumulene and the ability to build meroterpene natural products from this humulene isomer.

The generation of completely new enzyme functionalities in non-catalytic protein scaffolds through sequence alterations alone, i.e., without resorting to the recruitment of catalytic metals or cofactors, remains an unsolved fundamental problem in protein engineering. In a biotechnology context, our insufficient understanding of the mechanisms of de novo enzyme generation hampers efforts to design novel enzymes for non-natural, anthropogenic reactions. In an evolutionary context, it makes it difficult to understand how the huge diversity of enzyme activities required for life emerged at a primordial stage. Yet, amino acids and peptides may function as organocatalysts.10-13 Therefore, proteins may already harbor a diversity of low-level enzyme functionalities even in the absence of previous design or selection. In support of this notion, we show that two TIM-barrel glycosidases and the redox enzyme thioredoxin consistently catalyze Kemp elimination, ester hydrolysis, peptide bond cleavage and phosphoester hydrolysis. These results expose a biomolecular mechanism that bypasses the de novo generation bottleneck in enzyme evolution, suggest solutions to the puzzle of the emergence of primordial enzymes and reveal a source of de novo-like activities that may provide starting points for the engineering of novel enzymes.

The catalytic activity of β-lactamases presents the basis for the most important mechanism of antibiotic resistance, the hydrolysis of β-lactam drugs. Given the clinical relevance of these enzymes, there is a pressing need to find general concepts that allow predicting quantitatively how enzymatic efficiency is tuned along the evolution towards new resistances. Using a molecular dynamics-(MD)-based approach and direct comparison to experimental observables, we provide insight into the evolutionary trajectory of TEM1 to TEM52 β-lactamases, i.e. from narrow spectrum resistance against penicillin G (PenG) to broad-spectrum resistance. Combing molecular docking and MD simulations with the fixed-charge GAFF and multipolar, polarizable AMOEBA force field, we identify three distinct PenG sidechain conformations in the Michaelis complex. These conformations modulate active site electric fields that are relevant to electrostatic catalysis and emerge as active-site heterogeneity increases during evolution. Importantly, the AMOEBA-derived electric fields and computational infrared spectra reproduce quantitatively prior spectroscopic vibrational Stark effect shifts. Therefore, relating our results to catalytic rate constants corroborate that the transition to broad-spectrum resistance is governed by two competing factors: (i) active-site electric fields enhance catalytic efficiency via electrostatic catalysis, while (ii) ligand conformational flexibility promotes noncatalytic, off-pathway intermediates that reduce turnover.