Biocatalysis@TUDelft

Nature Chemical Biology, Published online: 21 July 2025; doi:10.1038/s41589-025-02001-3

Publisher Correction: Nonheme Fe 1,3-nitrogen migratases for asymmetric noncanonical amino acid synthesis

Nature, Published online: 16 July 2025; doi:10.1038/s41586-025-09291-6

A redox reaction network, comprising concurrent oxidation and reduction pathways, is described that can drive autonomous unidirectional motion about a C–C bond in a structurally simple synthetic molecular motor based on an achiral biphenyl.

Nature, Published online: 23 July 2025; doi:10.1038/s41586-025-09185-7

The salicylic acid (SA) biosynthesis pathway—involving ligation of benzoyl coenzyme A and benzyl alcohol to give benzyl benzoate; hydroxylation of benzyl benzoate to give benzyl salicylate; and cleavage of benzyl salicylate to give SA—is highly conserved in plants.

Nature, Published online: 23 July 2025; doi:10.1038/s41586-025-09280-9

Diverse plant species synthesize salicylic acid from phenylalanine through a pathway that includes a conserved triple-enzyme module that converts benzoyl-CoA to salicylic acid.

Nature, Published online: 23 July 2025; doi:10.1038/s41586-025-09175-9

Characterization of four SALICYLIC ACID-DEFICIENT GENEs in rice identifies an ancestral pathway for biosynthesis of salicylic acid from phenylalanine and provides genetic targets for engineering disease resistance in crop plants.

Nature, Published online: 24 July 2025; doi:10.1038/d41586-025-01627-6

It took time and rejections to understand what granting agencies look for. This is how I picked up application-writing skills.

Nature Catalysis, Published online: 24 July 2025; doi:10.1038/s41929-025-01391-w

Biocompatible Lossen rearrangement

Nature Catalysis, Published online: 24 July 2025; doi:10.1038/s41929-025-01353-2

Changing the catalytic metal centre of a non-haem iron dioxygenase to copper results in an enzyme capable of Lewis acid catalysis of new-to-nature enantioselective Conia-ene reactions.

Enantioenriched α-/β-/γ-thiocarboxylic acids featuring C–S chirality are valuable targets in organic synthesis. This review outlines recent progress in their asymmetric synthesis, including chiral pool approaches, metal- /organo-catalysis, and emerging biocatalytic strategies.

Enantiomerically pure α-/β-/γ-thiocarboxylic acids bearing a stereocentre at the C–S bond represent a class of compounds of notable significance in organic and medicinal chemistry. The chirality at the C–S bond presents both challenges and opportunities in synthetic chemistry. Traditionally, the enantioselective synthesis of these α-/β-/γ-thiocarboxylic acids has relied on chiral pool strategies using naturally derived precursors such as amino acids and hydroxycarboxylic acids. In recent years, the expansion of organo- and metal-catalysed methodologies, employing catalysts such as cinchona alkaloids, copper, and rhodium complexes, has led to strategies with improved efficiency and stereocontrol. Additionally, advances in biocatalysis have enabled more sustainable routes toward s these valuable compounds, using enzymes such as hydrolases and nitrilases. Given the growing academic and industrial interest in these compounds, this review provides an overview of the methods developed and the progresses made over the past two decades in the asymmetric synthesis of thiocarboxylic acids bearing a stereocentre at C–S bond.

We report synthetic routes that yield prodrugs consisting of two carbapenems connected by benzyl carbonate linkers. A mass spectrometry assay was elaborated to monitor, in a single kinetic experiment, the acylation of a bacterial target (a peptidoglycan transpeptidase) by these prodrugs, the resulting cleavage of the linker, and the release of their warhead moiety in an active form.

Abstract

β-lactam antibiotics remain the best treatment to fight bacterial infections. Among various antibiotics used today, β-lactams belonging to the carbapenem class are last resort drugs active against many multiresistant Gram-negative bacteria. Carbapenem antibiotics are prone to hydrolysis by certain β-lactamases, leading to the opening of the β-lactam ring and drug inactivation. This mechanism can be repurposed for targeted molecular fragmentation. Here, we report the synthesis of model prodrugs consisting of two carbapenems connected by original self-immolative linkers. A mass spectrometry assay was developed to simultaneously monitor acylation of a β-lactam target by the prodrugs and by their sacrificial and warhead moieties. The linker connecting the two carbapenems was optimized with respect to prodrug stability and effective release of the activated warhead upon carbapenem ring opening.

Reaction sequence for the synthesis of methylated products through enzymatic decarboxylation of bio-based hydroxycinnamic acids and subsequent direct methylation of the resulting styrene intermediates without any further isolation and purification steps. This approach enables the clean and facile production of methylated functional styrene compounds with unique properties capable of cationic (photo)polymerization.

Abstract

Enzymatic decarboxylation of phenolic acids enables the production of biobased phenolic styrenes under mild reaction conditions. However, the free para-phenolic group can lead to undesirable side products during polymerization, giving protection of the free phenolic OH group critical importance for the application in adhesives. Here we present a one-pot two-step cascade reaction in which phenolic acid decarboxylase from Bacillus subtilis (BsPAD) catalyzes the decarboxylation of coumaric acid, caffeic acid, ferulic acid, and sinapic acid, followed by O-methylation of the intermediate phenolic styrenes by resveratrol O-methyltransferase from Vitis vinifera (VvROMT). The reaction sequence avoids the isolation and purification of the reactive intermediate hydroxystyrenes. Characterization of variants with amino acid substitution in the active-site cavity led to the identification of variants without activity toward the four phenolic acids. This avoids undesired O-methylation of the starting material, which is not desirable since the decarboxylase does not convert the formed p-methoxyphenolic acids. Furthermore, variation of amino acids in the active site led to the identification of variants with improved activity for all four phenolic styrenes. The results constitute an important step toward the synthesis of biomass-derived methoxystyrene derivatives whose photopolymerization yields polymers with outstanding adhesion properties.

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

Designing artificial metal sites in proteins is challenging due to the need to place the metal site in precise positions and to tailor the coordination environment of the metal. In article 10.1002/cbic.202500208, Matteo Tegoni and co-workers use a modular approach using the SpyTag/SpyCatcher technology to provide the Spy construct with a Cu²⁺/ATCUN site. The reconstituted copper protein showed enhanced ROS catalytic activity. This strategy enables versatile metalloprotein design by shifting complexity from a protein to a peptide.

In light of its superior metabolic stability, the thiophosphate motif is widely regarded as a valuable substitute for its native phosphate counterpart. This property has led to the incorporation of thiophosphates and phosphorothioates in various therapeutic modalities, such as antisense oligonucleotides and cyclic dinucleotide analogs. Biochemically, thiophosphates can be installed on proteins by combining protein kinases and ATPγS, but the method has never been explored on additional substrate classes and is impractical to scale up because it requires superstoichiometric amounts of the expensive ATPγS. We report the invention of an ATPγS recycling strategy, which circumvents these limitations through the use of a specially designed creatine derivative to turn over the kinase reaction byproduct. The developed protocol is compatible with many kinases, enabling the design of multi-enzyme cascades to synthesize a diverse range of thiophosphate-containing small and macromolecules.

The reduction of carboxylic acids to alcohols is a key chemical transformation. We present a whole-cell biocatalyst employing carboxylic acid reductases and H2 for cofactor regeneration. H2 as a reductant ensures high atom economy, enabling a more sustainable route to alcohols. Notably, we showcase that the reaction proceeds under oxygen-limited conditions, demonstrating potential for safer, non-explosive bioprocesses.

Phenolic acid decarboxylases (PADs) have significant potential for converting bio-based hydroxycinnamic acids (e.g. ferulic acid, p-coumaric acid, caffeic acid, and sinapic acid) into valuable hydroxystyrene monomers. These monomers are in high demand in various industries, including additives in polymer production, cosmetics, and flavoring. PADs offer an efficient and scalable method for producing these compounds under mild reaction conditions. Establishing a viable industrial process requires a thermostable enzyme that shows robustness under operational conditions. Therefore, in this study, we assessed five thermostable ancestors towards their activity and stability for conversion of ferulic acid and sinapic acid at different temperatures. A combinatorial active site library was prepared for the most thermostable ancestor. Among the diverse hydroxystyrene monomers, especially 4-vinyl syringol (4-VS) the decarboxylation product from sinapic acid, exhibits interesting properties. Its polymers demonstrate similar thermal stability characteristics and higher glass transition temperatures compared to those based on vinyl guaiacol, the decarboxylation product from ferulic acid. For this potential, we expanded the substrate scope of the selected PAD ancestor to include sinapic acid through directed mutagenesis. A trade-off between ferulic/caffeic acid and sinapic acid was observed and further investigated via molecular dynamics simulations. The most thermally stable ancestor was identified with a half-life time of 3.65 days, analyzed at 50 °C. We found the Ile29Ser-Leu80Ser-Ile93Ala triple mutation (SSA) to effectively expand the substrate scope with an almost 7-fold increase in activity for sinapic acid, with a half-life time of 1.12 days at 50 °C, being approximately 1,610-fold higher than the PAD from Bacillus subtilis.

[FeFe]-hydrogenases are known for their bias towards proton reduction and the formation of dihydrogen with exceptional reaction rates, which opens a pathway to green hydrogen formation. To achieve the high turnover numbers found in the natural environments, researchers have to mimic the biological electron donors by using artificial electron delivery systems. In this sense, the use of an externally applied electrochemical potential is of particular interest, for which redox mediators often have to be included to effectively shuttle electrons between the electrode(s) and the enzyme(s). The class of viologens, with methylviologen (MV) as the most prominent candidate, has shown to perform efficient electron transfer. However, the reduced forms of MV are restricted in terms of stability and accessibility of higher reduced, low-potential species. Herein, we report the electrochemical and spectroscopic characterization of a modified viologen-based derivative, which is capable of storing two electrons at a low potential of −0.75 V vs. SHE. In combination with the [FeFe]-hydrogenase CpI, an activity of up to 10’480 μmolH2 mgCpI −1 min−1 (TOF = 11’179 s−1) could be achieved, 10 times higher than the parent methylviologen system under the same conditions.

Exogenous transition metal catalysts can be combined with natural enzymes to perform designed, non-natural tandem reactions within living mammalian cells. The resulting abiotic metabolic network can process exogenous substrates to yield fluorescent or bioactive products, only in those cells that harness both catalytic systems.

This study reveals a highly regioselective and cofactor-independent enzymatic approach to terpene and terpenoid hydration using a carotenoid 1,2-hydratase from Rubrivivax gelatinosus. Beyond detailing reaction optimization strategies, an extensive substrate scope, and scale-up potential, it explores mutagenesis and structural modeling to identify key catalytic residues and improve enzymatic activity.

Abstract

Terminally hydrated terpenes are highly sought-after compounds in the flavor and fragrance industries. However, their selective synthesis remains a considerable challenge in catalysis. Regioselective hydration of non-activated C─C double bonds is typically hindered by poor selectivity and low atom efficiency in conventional methods. In this study, we harness the underexplored potential of the acyclic carotenoid 1,2-hydratase from Rubrivivax gelatinosus IL144, employing it as a whole-cell biocatalyst for cofactor-independent terminal hydration of a diverse range of terpenes. This enzyme demonstrates exceptional activity across more than 20 C₁₂─C₂₀ terpenes and shows notable tolerance to various functional groups, establishing it as a valuable tool for sustainable organic synthesis. We emphasize the critical influence of expression system choice in maximizing enzymatic performance, enabling high-yield transformations on the gram scale. Through a combination of homology modeling, consensus analysis, and targeted mutagenesis, essential residues involved in catalytic activity were identified. Notably, enhanced catalytic efficiency was only achievable through the epistatic effect of three specific mutations. These findings highlight the biocatalytic potential of acyclic carotenoid hydratase, offering a green and efficient route to the production of valuable tertiary alcohols.

Enzyme engineering has produced numerous methods to optimize enzymes for biotechnological processes; however, less is known about how natural evolution creates new functionalities. We investigate the evolutionary emergence of enantioselectivity in plant borneol dehydrogenases (BDHs), which feature hydrophobic active-sites and are enantioselective towards dibornane-type monoterpenols. Ancestral sequence reconstruction provided a trajectory from the oldest unselective BDH ancestor N30 (E=12) toward the youngest selective ancestor N32, involving 19 mutations: 18 mutations are peripheral, one (I111L) occurs in the active-site. The mutation L111I in the hydrophobic pocket increased the selectivity of N30, while the back-mutation I111L decreased the selectivity of N32. Additional peripheral mutations (V136L/G169A/V183I) were required for high selectivity. Crystal structures suggested that protein dynamics, rather than structural changes shape these catalytic properties. Molecular simulations with funnel-metadynamics revealed a correlation between the active-sites solvent-accessible surface area (SASA) and selectivity. This potential evolutionary pathway shapes enantioselectivity, and guides future enzyme engineering campaigns.