Biocatalysis@TUDelft

Biocatalysis has emerged as an effective method for synthesizing chiral compounds due to its mild reaction conditions and excellent selectivity. However, enzyme stereoselectivity is often limited by natural substrate specificity, which can be addressed by protein engineering techniques. Machine learning-assisted protein engineering can identify patterns from biocatalytic reaction data and provide reliable predictions of stereoselectivity. This approach is expected to disrupt the traditional trial-and-error research paradigm in biocatalysis and lead to a more efficient and sustainable asymmetric synthesis process.

Nature, Published online: 08 July 2025; doi:10.1038/d41586-025-02054-3

A computational workflow designs proteins with catalytic efficiencies comparable to those of some natural enzymes — a landmark result for the field.

Flavins are organocatalyst that can be employed as photocatalyst, but also as cofactor regeneration systems when coupled with alcohol dehydrogenases (ADHs) for alcohol oxidation reactions. In previous work, we developed a confined (organo)enzymatic system where ADH catalyzes NAD⁺-dependent regioselective diol oxidation, while FMN enables ground-state NAD⁺ regeneration using molecular oxygen as the electron acceptor. Despite co-immobilization on the same support, the flavin induces premature ADH inactivation during discontinuous use of the heterogeneous hybrid catalyst. Through experimental and computational methods, we herein investigate how FMN interacts with ADHs triggering their inactivation. We found that the isoalloxazine ring and the ribityl moiety of flavins disrupt ADH’s quaternary structure via nonspecific binding at subunit interfaces, creating an oxidative microenvironment that oxidizes surface-exposed cysteine residues. By immobilizing and compartmentalizing flavins and ADHs through a cationic polymer layer, we enhanced the operational stability of this hybrid heterogenous catalyst, maintaining its maximum oxidative performance for three consecutive reaction cycles, outperforming flavin-dependent oxidation systems previously reported. Collectively, this work shows that microscopic catalyst compartmentalization via rational immobilization overcomes flavin–dehydrogenase incompatibilities in cascade reactions, enabling a robust integration of chemo- and biocatalysts for concurrent reaction routes.

Nature Catalysis, Published online: 10 July 2025; doi:10.1038/s41929-025-01364-z

Chemical synthesis of chiral β-hydroxy-α-amino acids usually requires multiple steps. Now a biomimetic enantioselective aldol reaction of glycinate and aldehydes catalysed by a chiral pyridoxal has been achieved, providing efficient access to a large number of chiral β-hydroxy-α-amino esters.

N-alkylated heteroarenes are key structural motifs in bioactive compounds, but their regioselective synthesis via coupling of readily available azoles with haloalkanes remains very challenging. Here, we present a mild biocatalytic approach that proceeds on gram-scale, is highly chemo- and regioselective, offering rapid access to valuable N-alkylated building blocks and enabling demanding late-stage alkylations.

Abstract

The alkylation with electrophilic haloalkanes is a key methodology in chemical synthesis to build desired molecules. Although alkylation of compounds bearing a single nucleophilic site is routine, the selective alkylation of polyfunctional molecules with multiple competing nucleophilic positions of comparable reactivity is often very challenging. In this work, we report a generalizable solution for selective alkylation chemistry that combines the selectivity of enzyme catalysis with the reactivity of off-the-shelf alkylation reagents. We employ engineered transferases in a modular cyclic cascade and use functionalized N-heteroarenes as challenging proof-of-concept substrates. This catalytic alkylation approach is mild, highly chemo- and regioselective, proceeds on gram-scale, provides rapid access to important N-alkylated heterocyclic building blocks and enables challenging late-stage alkylations. This study demonstrates a generalizable strategy to streamline synthetic routes to many pharmaceutically important compounds by selective biocatalytic alkylation of polyfunctional molecules and ambident nucleophiles.

A metalloenzyme-catalyzed asymmetric transfer hydrogenation platform has been developed for the stereoselective synthesis of chiral amines. In contrast to natural NAD(P)H-dependent C═N bond reductases, this strategy employs carbonic anhydrase or P450 as a catalyst in combination with a silane-reducing agent, offering a fully orthogonal alternative to conventional NAD(P)H-dependent cellular processes.

Abstract

Chiral amines are prevalent in natural products, pharmaceuticals, and organic catalysts. Their increasing demand has driven the advancement of synthetic methods. In this study, we developed a metalloenzyme-catalyzed asymmetric transfer hydrogenation method for the synthesis of chiral amines. Given the challenges of traditional chemical synthesis, which relies on precious metals and complex synthetic ligands, our approach utilizes base metals derived from natural metalloenzymes for transfer hydrogenation and employs protein scaffolds to achieve stereochemical control. Furthermore, in contrast to natural NAD(P)H-dependent C═N bond reductases, this strategy utilizes silanes as reducing agents and is entirely orthogonal to conventional NAD(P)H-dependent cellular functions. This reactivity highlights the potential to develop new-to-nature enzymatic functions capable of addressing challenges in both organic synthesis and biosynthesis.

Enzymatic catalysis promises the achievement of functional group reactivity selectivity with the conjunctional expansive acquirement of reactivity diversity, through the conformal mutagenesis tuning of catalytic cavity. However, essentially all of the functional group reactivity diversification systems demonstrated thus far are centered on the inter-substrate-shift regime, with the enzymatic catalysis adapted to alternative substrate and associated target functional group in totality. Herein, we report an enzymatic stringency-relaxation strategy for effecting reactivity diversification into the intra-substrate orthogonal functional group reactivity selectivity regime. The stringency-relaxation strategy operates in either the amino acid relaxation or functional group relaxation format, by a working sequence of initial stringency amino acid catalytic access to the catalytically most demanding functional group and subsequent relaxation amino acid catalytic access to the catalytically less demanding functional group. In particular herein, through this strategy, α,β-unsaturated carbonyls have been reduced with ketoreductase, in a selective manner, at either the carbonyl group site or the alkenyl group site. A broad substrate scope has been established for the alkenyl reduction of α-cyano-α,β-unsaturated esters, showcasing enzymatic stringency-relaxation as a prospective platform for programming reactivity diversification and reaction development.

 A self-sufficient heterogeneous biocatalyst (ssHB) composed of two NADH-dependent dehydrogenases co-immobilized with NADH on porous supports enables the redox disproportionation of HMF, yielding both 2,5-bis(hydroxymethyl)furan (BHMF) and 5-hydroxymethyl-2-furancarboxylic acid (HMFCA)

Abstract

5-Hydroxymethyl furfural (HMF) is a key molecule in biorefineries, but its conversion into valuable products faces technological challenges. Biocatalysis offers a sustainable solution, yet high cofactor costs and poor enzyme recyclability hinder industrial implementation. In this work, we develop a self-sufficient heterogeneous biocatalyst (ssHB) composed of two NADH-dependent dehydrogenases co-immobilized with NADH on porous supports. This system enables redox disproportionation of HMF, yielding both 2,5-bis(hydroxymethyl)furan (BHMF) and 5-hydroxymethyl-2-furancarboxylic acid (HMFCA). The reaction relies on NADH/NAD⁺ hydride transport, transferring hydrogen between HMF molecules. Volumetric productivities of 0.75–1.5 g L⁻¹ h⁻¹ and an 80% overall yield (40% per product) were achieved using boronic acid–functionalized supports. The system maintained the same yield over two cycles without additional cofactor, demonstrating efficient cofactor recycling with total turnover numbers (TTN_NADH) of 100–160, around 5 times higher than using exogenous cofactor. This work highlights the potential of self-sufficient heterogeneous biocatalysts to enhance cofactor efficiency and reduce process costs, paving the way for more sustainable biocatalytic valorization of HMF.

We identified and characterised AMM-1, a novel metallo-β-lactamase from Alteromonas mangrovi in the Yangtze River Estuary. AMM-1 clusters with clinically relevant MBLs. Structural modelling reveals a conserved di-zinc active site essential for β-lactam hydrolysis; AMM-1 shows distinct active-site flexibility compared with representative MBLs, potentially affecting substrate recognition and resistance evolution.

ABSTRACT

Carbapenem resistance driven by metallo-β-lactamases (MBLs) poses a formidable global challenge as these enzymes can degrade a wide range of β-lactam antibiotics, including last-line carbapenems. Despite extensive documentation of MBL-producing pathogens, their evolutionary origins and ecological reservoirs are still poorly understood. Here, we report the discovery and in-depth characterisation of AMM-1, a previously unrecognised B1.2 MBL identified within a metagenome-assembled genome of Alteromonas mangrovi obtained from the Yangtze River Estuary. Comparative sequence analyses and phylogenetics reveal that AMM-1 clusters closely with clinically significant MBLs, underscoring its potential impact to human health. Structural modelling confirms the presence of a conserved di-zinc binding site critical for β-lactam hydrolysis, while heterologous expression in Escherichia coli (E. coli) demonstrates a marked increase in resistance against multiple β-lactam classes, including carbapenems. Phylogenetic depth analysis and ancestral reconstruction delineate AMM-1's distinct evolutionary path, placing it deeper than IMP-1 and SPM-1 but shallower than NDM-1. Flexibility simulations reveal unique active-site loop dynamics (L3 and L10), with reduced mobility in key regions that shape substrate binding stability and spectrum. Notably, AMM-1 is stably located on the host chromosome without flanking mobile genetic elements, suggesting that it may have persisted as a vertically inherited trait rather than a recently acquired component of a mobile resistome. These findings highlight the capacity of environmental microbes to serve as long-standing, cryptic reservoirs of potent resistance determinants, emphasising the need for integrated environmental surveillance and preemptive stewardship strategies. By unveiling the molecular and functional properties of AMM-1, this work provides critical insights into how resistance elements can reside, evolve and potentially mobilise within natural habitats, ultimately informing efforts to predict and mitigate the future emergence of carbapenem-resistant bacterial pathogens.

Applied and Environmental Microbiology, Volume 91, Issue 8, August 2025.

Nature Synthesis, Published online: 08 July 2025; doi:10.1038/s44160-025-00835-2

One-carbon compounds, including carbon dioxide and methane, represent a sustainable resource for chemical conversions. This Review highlights recent advances in the biochemical upgrading of one-carbon substrates to value-added products using a combination of cellular, cell-free and abiotic catalysis strategies.

We describe single-stranded DNA-assisted double-stranded DNA nicking by DNAzymes (DANDA), in which DNAzymes are used to sequence-specifically nick or cleave superhelical plasmids, with help from assisting single-stranded DNAs. The DANDA system can be used for PCR-free site-directed mutagenesis on plasmids to create mutations on difficult targets such as repetitive sequences.

Abstract

Site-directed mutagenesis (SDM) is a key driver for many biochemical investigations and biotechnological applications. Despite decades of development, it is still difficult to perform SDM on some sequences, including highly repetitive ones, because polymerase chain reaction (PCR) is often used as the most effective method for SDM, but results in complicated products with these sequences. To overcome this limitation, we report herein a PCR-free SDM method that uses DNA-cleaving DNAzymes. Since these DNAzymes lack the capability of cleaving double-stranded DNA, we employed single-stranded DNAs as assisting DNAs to open superhelical plasmids, allowing the DNA-cleaving DNAzymes to cleave the plasmid. Such a system, named single-stranded DNA-assisted double-stranded DNA nicking by DNAzymes (DANDA), expanded the substrate scope of DNAzymes to double-stranded, superhelical plasmids. We showed that DANDA is highly customizable and target-specific, which allows successful generation of mutations on a plasmid containing PCR-incompatible repetitive sequences. By using solely unmodified DNA oligos that are more cost-effective and easier to prepare than other systems that employ either PNA or restriction enzymes, this DANDA system further expands the applications of DNA-cleaving DNAzymes in synthetic biology for different biochemical and biotechnological applications.

A new genetically encoded amino acid is chemically converted into homocysteine (Hcy) under mild conditions to probe and to activate protein active sites. Surprisingly, Hcy can replace cysteine or serine residues in several enzymes. Its potential for a widespread use in proteins is further underlined by rendering Escherichia coli dependent on Hcy, providing the first link between cell survival and a noncanonical amino acid directly involved in biocatalysis.

Abstract

Noncanonical amino acids (ncAAs) incorporated into proteins by stop codon suppression are powerful tools to probe and expand protein structure and function. Although homocysteine (Hcy) is a ubiquitous, naturally occurring amino acid, it was excluded from the universal genetic code. Hcy is very interesting, yet mostly unexplored, for probing protein active sites because of its subtle structural and electronic differences from cysteine and serine, which are widespread catalytic residues in enzymes. We report the genetic encoding of a new protected Hcy precursor, HcyX, that can be conveniently deprotected by chemical reductants or bioorthogonal reagents. We find varying and sometimes remarkable levels of activity for different purified enzymes with Hcy at catalytic positions. By exploiting partial intracellular deprotection to Hcy, we show that two proteins rendered Hcy-dependent, an intein and thymidylate synthase, can rescue growth of Escherichia coli by catalyzing a reaction essential for cell survival. To the best of our knowledge, these are the first examples in which cell growth is linked to a genetically incorporated ncAA directly involved in catalysis. We further demonstrate that Hcy-based disulfide bonds are chemically more stable than cysteine disulfides. Together, these findings open new paths for the experimental evolution of the genetic code.

Enzymatic detoxification of organophosphate (OP) insecticides can confer resistance in some insects, yet the precise molecular basis of this trait, and how it has evolved, remains poorly understood. In certain dipteran species, a G[->]D mutation in the oxyanion hole of -carboxylesterases (CBEs) enhances OP hydrolysis, yet this adaptation is not widespread despite the presence of orthologous CBEs in other insect species that are also exposed to OPs. The extent, and molecular basis, of evolutionary contingency and epistasis in this catalytic OP resistance has not been explored, and how further mutations might optimize OP detoxification in the future is not clear. Here, we systematically compare OP hydrolysis and analyse structures of CBE orthologs across several dipteran species, revealing that the success of the G137D mutation is sequence context-dependent. We employed laboratory-directed evolution to enhance OP turnover over 1000-fold vs. the wild-type enzyme and tested these variants in transgenic Drosophila melanogaster, demonstrating that improved catalytic rates do not directly translate to increased resistance. By highlighting the trade-off between organophosphate affinity and turnover, this work further clarifies the complex evolutionary trajectories determining why a particular resistance mechanism may evolve in some species but not others. SignificanceThis study reveals the intricate evolutionary path to insecticide resistance in insects, highlighting why a potent resistance mutation is effective in some species but not others. We show that the mutations success is contingent on the enzymes pre-existing structural features, highlighting the strong intramolecular epistasis. Using laboratory evolution, we enhanced the enzymes detoxification activity over 1000-fold, yet discovered this did not translate to increased resistance in transgenic flies. This surprising result demonstrates that effective real-world resistance requires a delicate balance between an enzymes ability to bind an insecticide (affinity) and its speed at breaking it down (turnover), providing crucial insights into the constraints governing molecular adaptation.

UbiA-type terpene synthases, traditionally annotated as prenyltransferases, have been shown to catalyze terpene cyclization in recent years, expanding their catalytic repertoire beyond primary metabolism. Here, we report on the genome-guided discovery and functional characterization of bacterial UbiA diterpene synthases (diTSs). Using a geranylgeranyl diphosphate (GGPP)-overproducing E. coli system, we screened 32 candidate enzymes and identified five that generate structurally diverse diterpenes, two of which represent the first examples of cyathane synthases from bacteria. Site-directed mutagenesis uncovered active- site residues that influence product formation, directing cyclization towards mono- or tricyclic products. This study expands the known catalytic repertoire of UbiA enzymes and highlights their untapped potential in bacterial terpenoid biosynthesis. Our findings suggest that bacteria may produce diverse and bioactive diterpenoids using UbiA TSs for the first committed biosynthetic step, warranting further exploration of UbiA TSs for natural product discovery.

To simulate enzyme reactions, multiscale quantum mechanics/molecular mechanics (QM/MM) approaches are well established and popular. However, accurately and efficiently estimating enzyme activity is a challenge, because in general, precise methods are too computationally expensive. Here, we demonstrate that enzyme catalysis can be captured by coupling efficient machine-learned potentials (MLPs) for a reaction to the wider enzyme environment using electrostatic machine-learning embedding (EMLE). Our EMLE model is first applied to the natural Diels-Alderase AbyU, showing that it correctly differentiates the catalytic action on different enzyme-substrate conformations. Then, we show that training a reaction-specific EMLE model allows us to accurately capture the enzyme catalytic effects of the conversion of chorismate to prephenate, a reaction with a highly polarizable and charged transition state. In both cases, in contrast to mechanical embedding approaches, EMLE embedding allows accurate and efficient predictions of enzyme activity, agreeing with high-level QM/MM reference calculations. This approach facilitates the use of gas phase-trained MLPs in MLP/molecular mechanics (ML/MM) simulations and should thus be highly beneficial for computational activity screening of enzyme biocatalysts.

By mining an alcohol dehydrogenase that can convert NADH to NAD+, we developed a strategy that dynamically adjusts the cellular NADH/NAD+ ratio to balance cell growth and antibiotic biosynthesis. This work establishes a NAD-based dynamic engineering strategy and provides an effective platform to boost secondary metabolite titers in actinomycetes.

The halide motif is ubiquitous in organic synthesis, facilitating fine-tuning of molecular properties and functionalization using substitution and cross-coupling reactions. Combination of different flavin-dependent tryptophan halogenases enables the installation of novel halogenation patterns on l-tryptophan, which can be orthogonally addressed in Suzuki–Miyaura coupling reactions, yielding a plethora of halogenated biaryl and triaryl building blocks for the synthesis offine chemicals.

Flavin-dependent halogenases provide an environmentally friendly, highly regioselective toolkit for the halogenation of various aryl compounds. While previous works characterize the substrate scope of many halogenases individually, exploration of their combinatorial use has thus far been at best superficial. In this study, combinations of the Trp-halogenases PyrH, Thal, RebH, and AetF are used to synthesize a range of novel di- and trihalogenated tryptophan derivatives. All tested enzymes accept some halotryptophan derivatives well, while others are mostly rejected. This behavior is rationalized using crystallographic data in conjunction with molecular docking. Upscaling using halogenase combiCLEAs provides facile access to nine novel di- and even trihalogenated tryptophan derivatives. The more reactive bromine and iodine substituents prove orthogonal to chlorine substituted positions during Suzuki–Miyaura cross-coupling, readily giving access to chlorinated biaryl scaffolds. Finally, the strategic selection of two different boronic acids enables the selective synthesis of indole-containing triaryl compounds.

Alpinetin ((2S)-7-Hydroxy-5-methoxyflavan-4-one) is a natural flavonoid found in various medicinal herbs and is frequently used in Chinese patent medicines. It exhibits a wide range of bioactivities, including anti-inflammatory, cardiovascular protective, lung protective, antiviral, hepatoprotective, and antitumor effects. Alpinetin features a 5-methyl group on the A ring, a rare characteristic among methylated flavonoids. The limited abundance of Alpinetin in plant biomass, its laborious extraction and purification from this biomass, and the toxicity and lack of regio- and chemoselectivity in organic synthesis render its biosynthesis in engineered microbes an attractive alternative. In this study, we aimed to achieve the de novo biosynthesis of alpinetin in Escherichia coli. In a series of optimization steps, we varied the selection of pathway enzymes, plasmid configurations, medium composition, and fermentation strategies. Lastly, we applied a directed evolution campaign to the 5-O-methyltransferase to successfully enable the de novo biosynthesis of alpinetin in E. coli for the first time. Moreover, we demonstrated the ability of O-methyltransferase to methylate a broad range of flavonoid substrates, leading to the production of valuable O-methylated flavonoids. Our study represents the first example of alpinetin biosynthesis in a heterologous host and paves the way to produce other valuable O-methylated flavonoids enzymatically.

The multifunctional cytochrome P450 DoxA, central to anthracycline biosynthesis, demonstrates unprecedented catalytic versatility by performing both 13- and 14-hydroxylation of daunorubicin precursors to form doxorubicin (DXR). Structural elucidation (2.8 Å resolution) reveals a classic P450 fold with an open, flexible substrate-binding pocket and six substrate recognition sites, enabling steric accommodation of bulky intermediates. Remarkably, DoxA employs three distinct catalytic strategies: (1) conventional redox protein-dependent electron transfer, (2) direct NADPH-mediated H₂O₂ generation driving peroxygenase-like activity, and (3) exogenous H₂O₂ utilization, with hydroxylation efficiencies scaling with peroxide concentration. Tunnel engineering targeting H₂O₂ access pathways yielded mutants (e.g., R303G) with 4-fold enhanced activity at low H₂O₂ (5 mM), mitigating oxidative damage. This work establishes DoxA as a paradigm of P450 mechanistic adaptability, combining structural plasticity with multi-pathway catalysis, while its engineered variants offer biotechnological potential for streamlined DXR production.

Enterobacter cloacae is a Gram-negative nosocomial human pathogen that inhabits diverse ecological niches. Its genome encodes a conserved set of putative chitin-active enzymes, including a peculiar lytic polysaccharide monooxygenase (LPMO), termed EcLPMO, which we functionally characterized in this study. EcLPMO is a tetra-modular protein consisting of an auxiliary activity family 10 (AA10) catalytic domain, two central domains of unknown function (DUF-A and DUF-B), and a C-terminal carbohydrate-binding module (CBM73). Functional assays using full-length EcLPMO and its truncated variants demonstrated that the AA10 domain oxidatively cleaves chitin at the C1 position. The CBM73 module enhances chitin binding and promotes synergy with endogenous chitinases. Notably, EcLPMO displayed a particularly strong synergistic effect with the unimodular chitinase EcChiA, leading to up to 14-fold and 60-fold increases in GlcNAc release from - and {beta}-chitin, respectively. Deletion of both DUFs reduced EcLPMO activity. While DUF-A alone and the association of DUF-A and DUF-B showed limited chitin binding, DUF-B alone exhibited no binding, suggesting a distinct role. Unexpectedly, using state-of-the-art structural modelling (AlphaFold3), we observed that the DUF-B domain contains two highly conserved histidines that coordinate the AA10-bound copper, forming a previously unreported inter-domain tetra-histidine copper coordination center. These findings highlight the structural and functional complexity of EcLPMO and suggest that its accessory domains, particularly DUF-B, may contribute to enzyme stability and substrate interaction. We speculate that DUF-B may protect the LPMO active site from oxidative damage, a feature that could prove crucial in its ecological and pathogenic contexts.

Several bacterial pathogens secrete multi-domain enzymes known as lytic polysaccharide monooxygenases (LPMOs) that are important for virulence. One example is the Vibrio cholerae virulence factor GbpA (VcGbpA), in which an N-terminal LPMO domain is followed by two domains of unknown function called GbpA2 and GbpA3, and a C-terminal chitin-binding domain, called CBM73. In-depth functional characterization of full-length and truncated variants of VcGbpA and a homologue from V. campbellii (previously V. harveyi; VhGbpA) showed that the catalytic LPMO domains of these proteins exhibit properties similar to natural single-domain LPMOs with established roles in chitin degradation. Interestingly, binding to chitin and efficient degradation of this substrate were affected by the presence of the GbpA2 and GbpA3 domains. Combined with structural predictions and analyses of sequence conservation, our data suggests that GbpA3 has evolved to interact with the reduced copper site to prevent off-pathway reactions in the absence of substrate. Substrate binding by CBM73 weakens this interaction, enabling the activation of the LPMO only when substrate is present. These observations shed new light into the functionality of these multi-domain LPMOs and uncover a novel mechanism for regulating LPMO activity.