Biocatalysis@TUDelft

This review evaluates engineered (semi)autonomous cell systems for the biosynthesis and incorporation of noncanonical amino acids (ncAAs) into proteins. While semi-autonomous cells convert supplied precursors into ncAAs autonomous cells integrate biosynthetic pathways that produce these building blocks intracellularly. Such integrated approaches significantly reduce process costs, can increase protein yields, and overcome challenges such as the limited membrane permeability of ncAAs.

Autonomous cells are engineered biological systems capable of biosynthesising and directly incorporating noncanonical amino acids (ncAAs) into proteins. These systems have the potential to extend the applicability of the genetic code to enable large-scale fermentative production of proteins carrying ncAAs. This work evaluates approaches for the generation of autonomous and semi-autonomous cells. Semi-autonomous cells rely on the external addition of a precursor, which is enzymatically converted in vivo to an ncAA that is directly incorporated. In contrast, autonomous cells have a metabolic system that produces and directly incorporates an ncAA in vivo. Through a critical evaluation of the state of the art, the reader is provided with an opinion on the future development of the field.

Proteiniphilum saccharofermentans, a Gram-negative facultative anaerobe, produces an N-acetyl transferase (5THNAT) that acetylates 5-thiohistidine using acetyl-CoA. This reaction competes with OvoC-mediated Nπ-methylation of the same substrate. The production ratio of ovothiol to Nα-acetyl 5-thiohistidine is likely regulated by the relative availability of SAM and acetyl-CoA.

Ovothiol A is a 5-thiohistidine derivative biosynthesized by a broad range of prokaryotic and eukaryotic organisms. Its redox-active mercaptoimidazole side chain is believed to protect cells from oxidative stress. The three enzymes that produce ovothiol A from histidine, cysteine, and S-adenosyl methionine have been identified and characterized. In contrast, no enzymes are known that produce other 5-thiohistidine derivatives. Here, a small family of acetyl-coenzyme A-dependent transferases is described that produce N-acetyl-5-thiohistidine. The discovery of these enzymes from Proteiniphilum saccharofermentans and related Bacteroidota provides evidence that the 5-thiohistidine class may be structurally and functionally more diverse than previously thought.

A putative thrazarine biosynthetic gene cluster is discovered in the genome sequence of Streptomyces coerulescens MH802-fF5. In vivo and in vitro analyses of ThzN, a hydrazine synthetase with cupin and methionyl-tRNA synthetase-like domains encoded in the cluster, showed that it synthesizes a key intermediate of thrazarine by catalyzing condensation between l-threonine and N ⁶-hydroxylysine, followed by intramolecular rearrangement.

Hydrazine synthetases (HSs), consisting of cupin and methionyl-tRNA synthetase (MetRS)-like domains, catalyze hydrazine formation in the biosynthesis of various nitrogennitrogen (NN) bond-containing secondary metabolites. The structural diversity of the NN bond-containing secondary metabolites synthesized using this system is attributed to the diversity of amino acids (e.g., l-Glu, d-Glu, l-Ala, l-Tyr, l-Ser, and Gly) that are recognized by the MetRS domain. However, there are still many HS genes in the genome database whose substrates are unknown. This study identifies a putative biosynthetic gene cluster (BGC) for thrazarine, a diazo group-containing secondary metabolite with antitumor activity, by whole-genome sequencing of the thrazarine producer Streptomyces coerulescens MH802-fF5. In vivo and in vitro analyses showed that ThzN, an HS encoded by this BGC, synthesizes N-((5-carboxy-5-(amino)pentyl)amino)threonine from l-Thr and N ⁶-hydroxylysine. This is the first example of l-Thr-utilizing HS. Sequence alignment analysis and structure prediction using Boltz-1 indicated that the space near Gly417 is important for the accommodation of the threonine side chain. The comparison of thrazarine BGC with azaserine BGC indicated that the biosynthetic mechanism of the diazo group of thrazarine is different from that of azaserine. This study expands the diversity of HSs and provides new insights into the biosynthesis of diazo groups.

We report a one-pot, three-step biocatalytic cascade to synthesize trihydroxy-piperidine and -azepane iminosugars directly from monosaccharides, employing transaminase (ATA) and galactose oxidase (GOase) enzymes.

Abstract

Biocatalytic cascades offer products of multistep synthesis without the use of protecting groups or isolation of intermediates. This strategy is particularly amenable to the synthesis of iminosugars, where traditional routes typically suffer from lengthy and inefficient steps. We have developed a one-pot, three-step biocatalytic cascade to synthesize trihydroxypiperidine and azepane iminosugars directly from monosaccharides, employing transaminase and galactose oxidase enzymes. An in situ LC/MS method for monitoring the key reaction intermediates that have never been observed was also developed.

The vanadium-dependent haloperoxidase from Curvularia inaequalis (CiVCPO) enables the selective introduction of halo-atoms into the products via Hunsdiecker-type reaction and halogenation reaction. A broad range of diversely halogenated compounds was prepared under environmentally friendly conditions.

Abstract

Compounds containing halogen atoms exhibit notable bioactivities and have found widespread applications across various fields. In this study, we report a method for synthesizing such compounds with diverse halogen atoms using vanadium-dependent haloperoxidase from Curvularia inaequalis (CiVCPO). Under the optimized reaction conditions, CiVCPO efficiently converts a broad range of halogen-containing carboxylic acids, phenols, and anilines into the corresponding diversely halogenated products. These reactions achieved up to 99% conversion and 99% chemoselectivity under mild conditions, with a turnover number of 47750 and a turnover frequency of 27.5 s⁻¹ for CiVCPO. This study provides an efficient and environmentally friendly strategy for synthesizing structurally diverse halo-compounds.

Nature, Published online: 25 June 2025; doi:10.1038/s41586-025-09110-y

In locusts, the aggregation pheromone 4-vinylanisole is derived from dietary phenylalanine, and its production is dependent on two 4-vinylphenol methyltransferases that are potential targets for locust pest control.

Nature, Published online: 26 June 2025; doi:10.1038/d41586-025-01986-0

Study highlights potential for sustainable synthesis of paracetamol.

Nature Chemistry, Published online: 23 June 2025; doi:10.1038/s41557-025-01845-5

Biocompatible chemistry merges chemo-catalytic reactions with cellular metabolism for sustainable small-molecule synthesis. Now a biocompatible Lossen rearrangement has been demonstrated to control bacterial cell growth and chemistry and applied to the remediation and upcycling of polyethylene terephthalate plastic waste in whole-cell reactions and fermentations to produce valuable industrial chemicals, including the drug paracetamol.

An engineered CO dehydrogenase (CODH) that remains active in air through a multilayered gas tunnel is illustrated on the cover. The architecture selectively blocks O₂ while allowing CO/CO₂ to access the FeS cluster, thereby maintaining redox balance and preventing oxidative destabilization. The visual contrast between inactive and active forms highlights the tunnel's protective role. This work offers a tunnel-guided strategy for engineering air-stable metalloenzymes with practical potential, as described by Suk Min Kim, Hyung Ho Lee, Yong Hwan Kim et al. in their Research Article (e202508565).

H³CP contributes to sustainable synthesis by integrating regioselective enzymatic halogenation, Pd-catalyzed Heck coupling, and enzymatic hydrolysis. TPGS-705-M micelles enable the Pd-catalyzed step, allowing seamless compatibility with enzymatic reactions in one vessel under aqueous conditions. Using this platform, diverse simple aromatic substrates are readily transformed into valuable acrylic acid building blocks.

Abstract

The combination of bio- and chemo-catalytic processes has tremendous potential for the sustainable production of important synthetic building blocks. Such approaches capitalize on the often-high selectivity of enzymes and the broad arsenal of synthetic transformations to access highly functionalized molecules from simple precursors. However, the strict requirement of enzymes for an aqueous reaction environment, often combined with their lack of stability under harsh reaction conditions, makes the development of efficient chemo-enzymatic cascade reactions challenging. Within this work, we developed a chemo-enzymatic platform that combines regioselective enzymatic H alogenation, chemo-catalytic H eck coupling, and an enzyme-catalyzed H ydrolysis reaction (H³ Catalytic Platform = H³CP) in a single reaction vessel. H³CP uses water as a reaction solvent to facilitate the enzymatic transformations, with the cross-coupling reaction taking place in dynamic and protective super-molecular reaction vessels composed of the commercially available TPGS-705-M. Our work utilizes various halogenating enzymes (FDHs and VHPOs), selectively functionalizing diverse substrates, thus enabling subsequent metal-catalyzed cross-coupling followed by enzymatic hydrolysis. H³CP gives access to a broad range of valuable acrylic acid building blocks with diverse functionalization at the aromatic portion.

Planar chirality represents a distinct and underexplored form of molecular asymmetry that arises from restricted three-dimensional conformations. It holds significant potential for expanding chemical space and enabling the development of novel chiral reagents, catalysts, and functional materials. Among the most prominent scaffolds exhibiting planar chirality is the rigid and conformationally stable [2.2]paracyclophane (PCP), known for its exceptional photophysical and electronic properties. In this work, we present a simple, cost-effective, and scalable biocatalytic desymmetrization strategy for the efficient synthesis of two key disubstituted PCP intermediates, the pseudo-geminal and pseudo-para isomers, directly addressing the current lack of catalytic methods for inducing planar chirality on this scaffold. This practical approach enables access to structurally complex PCP frameworks and underscores the largely overlooked potential of biocatalysis as a powerful, sustainable route to planar chiral molecules.

Nitrogen-nitrogen (N-N) bond-forming enzymes are rare but play vital roles in both primary and secondary metabolism. Guided by a nitric oxide synthase (NOS)-based genome mining strategy, we report the discovery and characterization of a new heme-dependent enzyme system that catalyzes intermolecular N-N bond formation. Using both in vivo and in vitro reconstitution approaches, we demonstrated that a protein complex, comprising a heme enzyme and a 2[4Fe-4S] ferredoxin partner, mediates the coupling of the -amine group of L-aspartate with inorganic nitrogen oxide species, such as nitrite or nitric oxide, to generate hydrazinosuccinic acid, a key biosynthetic precursor in several natural product pathways. Structural modeling and site-directed mutagenesis suggest a plausible catalytic mechanism involving the formation of a reactive nitrogen intermediate, potentially a heme-bound nitrene species. These findings reveal a new family of N-N bond-forming biocatalysts that leverage inorganic nitrogen sources, offering valuable tools for genome mining and the synthetic biology.

Magnetic Resonance Imaging (MRI) is a cornerstone of modern clinical diagnostics, often enhanced by contrast agents. Traditionally, these agents are chemically synthesized, which can involve complex, costly, and environmentally unfriendly processes. Here, we report a novel biocatalytic approach for the efficient, safe, and eco-friendly synthesis of 5-methyl-5,6-dihydrothymidine (5-MDHT), a potent Chemical Exchange Saturation Transfer (CEST) MRI probe for imaging in vivo expression of the Herpes Simplex Virus Type-1 Thymidine Kinase (HSV1-TK) reporter gene. We demonstrate that 5-MDHT can be biosynthesized via one- or two-step enzymatic reactions using human purine nucleoside phosphorylase (hPNPase) and the SgvMVAV SAM-dependent methyltransferase. hPNPase catalyzed the base-exchange reaction with catalytic efficiencies (kcat/KM) between 138-316 s-1 M-1, while SgvMVAV methylation of 5,6-dihydrothymidine yielded 5-MDHT with a catalytic efficiency of 26 s-1 M-1. Molecular dynamics simulations supported the enzymatic binding and selectivity observed experimentally. The resulting 5-MDHT was validated using CEST-MRI, showing a distinct exchangeable imino proton signal at 5.3 ppm. These findings highlight the chemo- and regioselectivity of the biocatalysts and establish biocatalysis as a viable platform for producing clinically relevant MRI contrast agents.

The cofactor-free arylmalonate decarboxylase (AMDase) is a valuable biocatalyst for synthesizing α-aryl and α-alkenylcarboxylic acids with excellent stereoselectivity. We engineered a new hydrophobic pocket in (S)-selective AMDase variants, creating AMDase ICPLLG with enhanced activity. For the investigation of the mechanism, we synthesized isotope-labelled, pseudochiral 2-methyl-2-vinyl malonate via an auxiliary-based asymmetric route using a chiral imidazolidinone to enable stereoselective bis-alkylation of malonates. Our results reveal striking substrate-dependent stereochemical behavior: AMDase ICPLLG decarboxylates prochiral aromatic malonates with retention of configuration at the α-carbon, but decarboxylates the corresponding alkenyl malonate with inversion of configuration. Kinetic isotope effect measurements and QM/MM metadynamics calculations suggest that alkenyl malonates adopt an alternative binding mode and undergo decarboxylation via a borderline concerted mechanism instead of a stepwise mechanism. This new pathway changes the stereochemical preference. We exploited this strategy to decarboxylate sterically hindered alkenyl malonates – substrates not converted by wild-type AMDase – with high stereoselectivity. The engineered hydrophobic pocket in (S)-selective AMDase variants expands the substrate scope for synthesizing enantiomerically pure α-aryl and α-alkenyl butanoic acids. This work demonstrates a new approach – a mechanistic change – to engineer the substrate range and stereoselectivity of enzymes.

The elaboration of amine substrates through C–C bond forming reactions is important in the synthesis of bioactive small mole-cules. Pyridoxal-5'-phosphate (PLP)-dependent enzymes have emerged as valuable biocatalysts for this class of reactions, due to their high stereoselectivity and ability to forge new C–C bonds on unprotected α-amino acid substrates. However, the use of abiological primary amines as pronucleophiles with enzymes such as threonine aldolase has been unexplored, moderating the utility of a biocatalytic approach in the synthesis of diverse 1,2-amino alcohols. In this report, we disclose the discovery and engineering of a PLP-dependent aldolase which accepts (2-azaaryl)methanamines in an aldol-type transformation. The 1,2-amino alcohol products generated, which contain representative heteroaromatic pharmacophores, are delivered with control over both the diastereoselectivity and enantioselectivity in the C–C bond forming event. Protein engineering provided variants with improved binding affinity for the abiological substrate and decreased affinity for the native α-amino acid, overcoming inhibition of the abiotic reaction by components of lysate, a major challenge in reaction discovery with PLP-dependent enzymes such as threonine aldolases. This work represents the first known example of C–C bond formation on non-amino acid sub-strates with threonine aldolase and provides a platform for further development of complexity building biocatalytic reactions with abiotic amine substrates.

Polyethylene terephthalate (PET) hydrolases offer a promising enzymatic route to plastic waste degradation under mild conditions. Among these, the engineered FAST-PETase variant exhibits superior catalytic efficiency and thermostability compared to the wild-type IsPETase, yet the molecular origins of these enhancements remain debated. In this work, we employ empirical valence bond (EVB) simulations in conjunction with semi-macroscopic PDLD/S-LRA calculations to investigate the rate-determining acylation step in PET dimer hydrolysis catalyzed by both wild-type and FAST-PETase. Our results successfully reproduce the experimentally observed trend in catalytic rate enhancement between the two systems. While prior interpretations attributing the improved activity to a strengthened hydrogen-bond network involving Asp106 and His237, we demonstrate that the distal N233K mutation in FAST-PETase induces long-range electrostatic changes that enhance catalytic efficiency by modulating the active site dipolar environment. More importantly, we show that the elevated performance of FAST-PETase at higher temperatures is not due to reduced flexibility in the mutant region but arises from enhanced thermal stability, which allows the enzyme to operate effectively at elevated temperatures and thus accelerate reaction rates. These findings underscore the central role of electrostatics and stability in enzyme engineering and suggest that data-driven methods, such as maximum entropy models, may enable the rational identification of further stability-enhancing mutations for improved PET depolymerization.

Visible light photocatalysis that exploits the reactivity of molecules at their excited state has induced a paradigm shift in organic synthesis by enabling unique chemical transformations, but controlling their enantioselectivity has proven difficult. A promising strategy involves linking a synthetic transition metal photocatalyst within the chiral architecture of a biomolecule to create a highly selective artificial photoenzyme. However, such a biohybrid system that combines the merits of biocatalysis and metallo-photocatalysis to promote abiological reactions fueled by visible light with high enantioselectivity is still unknown. Here, we report on an artificial metallo-photoDNAzyme resulting from covalently anchoring a blue light absorbing iridium-based photocatalyst within a double-stranded DNA helix that exhibits efficient triplet-triplet energy transfer and high levels of enantioselectivity in [2+2] intramolecular cycloadditions.

Nature Catalysis, Published online: 25 June 2025; doi:10.1038/s41929-025-01371-0

Locking in lipases

This study reveals that MarE, a heme-dependent aromatic oxygenase, favors dioxygenation of β-methyl-l-tryptophan in the absence of ascorbate and demonstrates how structural and redox tuning shifts its reactivity toward selective monooxygenation, yielding a 2-oxindole scaffold. These findings offer new insights into enzyme-controlled indole oxidation in tryptophan-derived natural products.

Abstract

MarE, a heme-dependent aromatic oxygenase with a histidyl axial ligation, catalyzes the monooxygenation of β-methyl-l-tryptophan to form a 2-oxindole scaffold central to maremycin biosynthesis. Although structurally similar to tryptophan 2,3-dioxygenase (TDO), which initiates l-tryptophan catabolism via dioxygenation, MarE exhibits distinct reactivity modulated by ascorbate. While ascorbate has no effect on TDO, it promotes selective monooxygenation by MarE. In its absence, MarE favors dioxygenation and formation of pyrroloindoline products, revealing latent catalytic versatility. Active-site loop sequences differ between the two enzymes, SLGGR in MarE versus GTGGS in TDO, prompting loop-swapping experiments to probe structure-function relationships. Substituting GTGGS in TDO to MarE-like sequences (GTGGA or SLGGS) shifted reactivity toward monooxygenation and formation of C3-hydroxylated, non-oxindole products that underwent further cyclization into tricyclic structures. Conversely, replacing SLGGR in MarE with GTGGS resulted in enhanced C2,C3-dioxygenation nearly 4-fold. These results underscore the active-site loop as a key determinant of oxidation outcome, alongside the modulatory role of ascorbate. By revealing the true catalytic identity of MarE and delineating the roles of small-molecule effectors and loop architecture, this study advances mechanistic understanding and predictive capabilities within the oxygenase superfamily.

EDDS lyase was subjected to iterative cycles of site-saturation mutagenesis and screened for increased activity for the addition of 2-((methylamino)methyl)aniline to fumarate. The final mutant, EDDS lyase CEA, accepts various N-methyl-1-phenylmethanamines derivatives, yielding N,N-disubstituted L-aspartic acids, as well as several ortho-substituted anilines, producing N-arylated L-aspartic acids.

Abstract

Optically pure N-functionalized α-amino acids are valuable chiral building blocks for pharmaceuticals, nutraceuticals, and agrochemicals. Ethylenediamine-N,N-disuccinic acid lyase from Chelativorans sp. BNC1 catalyzes the addition of a wide range of aliphatic and aromatic primary amines to fumarate, producing the corresponding enantioenriched N-substituted L-aspartic acids. In this work, the enzyme was subjected to iterative cycles of site-saturation mutagenesis and screened for increased activity for the addition of 2-((methylamino)methyl)aniline to fumarate. The final variant displayed an activity of three orders of magnitude higher compared to the wild-type enzyme. Unexpectedly, the enzyme catalyzed the hydroamination of fumarate with the aliphatic secondary amine of the starting substrate, rather than with the aromatic primary amine, leading to the formation of a tertiary amine. Exploring the substrate scope showed that the enzyme accepts various substituted N-methyl-1-phenylmethanamines for the hydroamination of fumarate, yielding N,N-disubstituted L-aspartic acids in high optical purity (up to >99% ee). Furthermore, we showed that the enzyme accepts several ortho-substituted anilines that were previously not accepted by the wild-type enzyme, yielding the corresponding N-arylated L-aspartic acids in high enantiomeric excess (>99% ee). This serendipitous finding enables a new strategy for the biocatalytic synthesis of tertiary amines, unlocked within the C-N lyase toolbox.

Chiral amines are ubiquitous in pharmaceuticals and agrochemicals, making their efficient and selective synthesis a significant synthetic challenge. Threonine aldolases synthesize chi- ral amines via stereoselective C–C bond formation, however, they are restricted to small amino acids as pro-nucleophiles, limiting their utility in chemical synthesis. Here, we report an engineered threonine aldolase capable of 𝛼-functionalizing benzylamines. The evolved enzyme has excellent catalytic efficiency and accepts a broad range of (heterocyclic)benzyl amines and structurally diverse aldehydes to yield single-enantiomers of 1,2-amino alcohols in high-yield and diastereoselectivity. Mechanistic and crystallo- graphic studies provide a rationale for how these mutations enable this previously unknown function. Moreover, beneficial mutations can be transferred to a related pyridoxal-dependent protein, high- lighting the generality of these insights.

Nature Catalysis, Published online: 24 June 2025; doi:10.1038/s41929-025-01347-0

Remote C–H bond formation via photoenzymatic hydrogen atom transfer has enabled the precise installation of remote stereocentres but is still in its infancy. Here, the authors report the photoenzymatic stereoablative enantioconvergence of γ-chiral oximes using repurposed flavin-dependent ene-reductases.

A synthetic cascade with two consecutive biocatalytic steps consisting of a short-chain alcohol dehydrogenase and an ene reductase in a biphasic system provides (R)-citronellal with >90% conversion and >99% enantiomeric excess from inexpensive geraniol.

The development of synthetic routes to produce enantiopure (R)-citronellal as a key intermediate for the synthesis of (–)-menthol and other valuable terpenoids is highly relevant in the pharmaceutical, flavor, and fragrance industries. Herein, we showcase a cascade with two consecutive biocatalytic steps performed separately using the inherent selectivity of a short-chain alcohol dehydrogenase (SDR) and an ene reductase (ERED) from the Old Yellow Enzyme (OYE) family. The first reaction involves the AaSDR1-catalyzed oxidation of relatively inexpensive geraniol in a biphasic system, providing geranial as an intermediate. The organic phase containing geranial is then extracted and transferred to the second step, where the ERED variant OYE2_Y83V catalyzes the asymmetric reduction of geranial to produce (R)-citronellal, achieving >90% conversion and >99% enantiomeric excess. The use of n-heptane in a two-liquid phase system not only facilitates substrate and product solubilization but also minimizes geranial isomerization. This biocatalytic cascade therefore enables the synthesis of enantiopure (R)-citronellal.