Biocatalysis@TUDelft

Applied and Environmental Microbiology, Volume 92, Issue 3, March 2026.

A chemoenzymatic process employing pyranose 2-oxidase generates in situ hydrogen peroxide for sustainable synthesize of iodinated phenolics from palm oil mill effluent waste.

Sustainable valorization of biomass-derived aromatics is essential for green chemical manufacturing. Palm oil mill effluent (POME), a major agro-industrial wastewater, contains phenolic acids such as p-hydroxybenzoic acid (p-HBA) that inhibit anaerobic digestion but represent valuable chemical feedstocks. Here, we demonstrate a scalable chemoenzymatic platform for selective iodination of p-HBA, vanillic acid (VA), and ferulic acid (FA) using pyranose 2-oxidase (P2O) to generate hydrogen peroxide in situ under mild aqueous conditions. Buffer screening revealed that tris(hydroxymethyl)aminomethane hydrochloride, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, and sodium phosphate best promoted monoodination of p-HBA, VA, and FA, respectively. Compared to direct H₂O₂ addition, P2O-mediated oxidant supply improved selectivity for 3-iodo-p-HBA (3-IHBA) (15% vs. 21%), suppressed di-iodination, and prevented oxidative coupling of FA. Liter-scale reactions retained small-scale efficiency, achieving >99% conversion and 93% yield for VA, and 89% conversion with 88% yield for FA. Moreover, direct conversion of POME-derived p-HBA yielded 3-IHBA (19%), confirming compatibility with real wastewater matrices. This work establishes P2O-driven iodination as a mild, selective, and scalable route for synthesizing iodinated aromatics from waste-derived phenolics, providing a green alternative to conventional halogenation and advancing sustainable biorefinery development.

Nature Communications, Published online: 02 February 2026; doi:10.1038/s41467-026-68907-1

The reliance on natural plasmid replication mechanisms limits plasmid tunability, compatibility, and modularity. Here the authors refactor the natural pMB1 origin and create plasmids with customizable copy numbers with synthetic RNA regulators to implement independent copy control.

Enzymes catalyze diverse chemical transformations and offer a sustainable approach to both breaking and making chemical bonds. However, finding an enzyme capable of performing a specific chemical reaction remains a challenge. We developed a new framework, Enzyme-toolkit (Enzyme-tk), that integrates 23 open-source tools to enable the discovery of enzymes that have activity toward a specific target reaction. Additionally, we introduce two new methods to facilitate enzyme discovery: (1) Func-e, an ML tool that searches large databases for enzymes that potentially catalyze a specific chemical transformation and (2) Oligopoolio, a gene assembly approach that reduces the cost of accessing protein sequences and thus the barrier to their experimental validation. We applied Enzyme-tk to find enzymes for chemical degradation of two man-made pollutants, di-(2-ethylhexyl) phthalate (DEHP) and triphenyl phosphate (TPP). We demonstrate that new, previously unannotated enzymes with favorable characteristics, such as high thermostability, can be identified using Enzyme-tk for reactions that are dissimilar to the training set.

Science Advances, Volume 12, Issue 5, January 2026.

A one-pot enzymatic platform enables the biosynthesis and site-specific incorporation of chalcogen-containing tyrosine analogues (O, S, Se) into proteins in E. coli. Engineered tyrosine phenol lyase (TPL) variants and orthogonal synthetases are combined to expand the genetic code with redox-active residues, paving the way for designer proteins with tunable electronic and catalytic properties.

ABSTRACT

Expanding the genetic code with unnatural amino acids (UAAs) offers powerful opportunities to engineer proteins with novel redox and catalytic functions, but is often limited by the need for multistep UAA synthesis and inefficient cellular uptake. Here, we report an integrated biosynthetic–genetic incorporation strategy for chalcogen-containing proteins from the respective phenols. Structure-guided engineering of tyrosine phenol lyase (TPL) enabled the enzymatic production of 3-methoxy-, 3-methylthio-, and 3-methylseleno-L-tyrosine (MeSeY) directly in living cells. Using evolved orthogonal aminoacyl-tRNA synthetases, these analogues were site-specifically incorporated into green fluorescent protein (GFP), as confirmed by fluorescence assays, spectroscopy, and mass spectrometry. We further established a one-pot in vivo system that unifies analogue biosynthesis with translation, reducing precursor requirements and cellular toxicity. This work introduces selenium as a genetically encoded handle for protein engineering and establishes a scalable strategy that couples biocatalysis with genetic code expansion to access redox-active designer proteins. Importantly, installation of MeSeY at the GFP chromophore residue Tyr66 provides redox-responsive fluorescence. In a circularly permuted GFP (cpGFP) scaffold, improved chromophore accessibility enables reversible redox switching under H₂O₂/thiol cycling.

The 100 kDa hexokinase (HK) enzyme family represents an attractive model to investigate the molecular origins of allosteric regulation in multidomain enzymes. Extant HK homologs are subject to various allosteric phenomena, including activation and inhibition by both homotropic and heterotropic ligands. Here, we report the results of a phylogenetic investigation of this enzyme family using the recently developed Topiary ancestral sequence reconstruction pipeline. The results agree with prior studies that used a smaller number of sequences from individual HK domains and suggest that modern HK3 isozymes diverged first from a 100 kDa ancestor, followed by gene duplication and divergence of the HK2 isozymes. A subsequent gene duplication event led to divergence of HK1 and the hexokinase domain containing protein 1 (HKDC1). To probe the ability of Topiary to yield functional, allosterically regulated ancestral enzymes, we resurrected and biochemically characterized two HKs from early vertebrate evolution, Anc1 and Anc2. Both enzymes were functionally similar to extant HK1, and possessed a low activity, regulatory N-terminal domain that governs allosteric regulation of the C-terminal active site by two heterotropic effectors, glucose 6-phosphate and inorganic phosphate. Neither ancestor was subject to homotropic regulation by substrate glucose, a characteristic observed in several extant HK3 family members. Our phylogenetic analysis provides a foundation for investigating the evolution of allostery in this enzyme family. It also demonstrates the need to sequence and biochemically characterize additional full-length HKs, especially those from jawless vertebrates, to enable more robust inferences of ancestral regulatory traits.

Sucrose synthase (SuSy) has been suggested as a key enabling enzyme for uridine diphosphate glucose (UDP-Glc) regeneration in glycosyltransferase-catalyzed biotransformations. However, its stability and efficiency in industrially relevant conditions have not been characterized or engineered, limiting its industrial readiness. Here, we combined enzyme discovery and characterization with comprehensive semi-rational enzyme engineering strategies, to optimize SuSys catalytic activity, thermostability, solvent tolerance, and soluble expression. The engineered variants were significantly more stable than wild-type, with up to 13.6 {degrees}C increase in melting temperature, over two orders of magnitude improvement in half-lives at elevated temperatures, and approximately three orders of magnitude increase in total turnover number. Additionally, the optimized variants retained up to 75% relative activity at 60 {degrees}C in the presence of 25% (v/v) DMSO, which the wild-type shows near complete loss of activity. Structural and molecular dynamics analyses reveal how mutations modulate conformational dynamics and hydrophobic packing, favoring catalytically competent conformations. Using methyl anthranilate glycosylation as a representative biotransformation, we demonstrate that the engineered SuSy variants consistently outperform both wild-type SuSy and stoichiometric UDP-Glc systems, enabling efficient UDP-Glc regeneration at reduced enzyme and sugar donor loadings. Finally, techno-economic and environmental assessments further indicate that implementation of engineered SuSy reduces reaction cost by approximately 6- and 2-fold relative to UDP-Glc and wild-type systems, respectively, while achieving average reductions of 3- and 2-fold in environmental end-point impacts. These results established SuSy engineering as a critical enabler for sustainable glycosylation reactions.

A novel noncanonical amino acid, DiZAAsu, possessing photo-cross-linking, biotinylation, and alkaline-cleavable functionalities, is genetically encoded by an engineered pyrrolyl-tRNA synthetase mutant. The alkyl ester moiety expands the design space of cleavable photo-cross-linkable amino acids.

Genetically encoded photo-cross-linkable amino acids (PAAs) are powerful tools for analyzing direct protein–protein interactions (PPIs) in mammalian cells. Cleavable PAAs are particularly useful, enabling covalent capture and subsequent release of interacting partners, which facilitates the characterization of interaction interfaces using mass spectrometry. However, the limited options for cleavable linker structures have restricted the design of PAAs. In this study, we genetically encoded a novel trifunctional PAA, DiZAAsu, which contains three distinct chemical groups: diazirine, alkyne, and alkaline-cleavable alkyl ester moieties. An archaeal pyrrolysyl-tRNA synthetase was engineered to incorporate DiZAAsu efficiently into proteins in mammalian cells. We demonstrated the in-cell photoreactive function of diazirine by cross-linking the DiZAAsu-introduced GRB2 protein to its binding partner, SHC. Using the alkyne group for biotinylation, we established a tandem affinity purification strategy that enabled efficient enrichment of the cross–linked complex, thereby reducing nonspecific protein contamination. The alkaline-based cleavage of the ester group in DiZAAsu was also demonstrated, confirming its potential for the dissociation of covalently linked complexes. This system thus expands the design space of multifunctional PAAs and adds alkaline-based dissociation to the limited repertoire of available cleavage strategies.

Communications Chemistry, Published online: 29 January 2026; doi:10.1038/s42004-026-01915-w

Ribosomally synthesized and post-translationally modified peptides (RiPPs) require precise proteolytic cleavage to generate bioactive natural products, yet the diversity of serine proteases involved remains underexplored. Here, the authors identify and characterize the serine protease WprP2 from Streptomyces venezuelae NPDC049867, revealing its unique cleavage activity on precursor peptides WprA2 involved in the biosynthesis of cyclophane RiPP natural products.

Nature Catalysis, Published online: 29 January 2026; doi:10.1038/s41929-026-01497-9

Author Correction: A polyketide-based biosynthetic platform for diols, amino alcohols and hydroxy acids

An acyltransferase from Chromobacterium sphagni (CsATase) was identified that catalyzes the regioselective formylation of resorcinol substrates. The formylation of substituted resorcinol derivatives yielded mono-formylated products with up to 99% conversion and up to 74% isolated yield. The structure of CsATase was elucidated by X-ray crystallography, providing insight into its active site.

ABSTRACT

Although aromatic formylation reactions are highly valuable from a synthetic perspective, a biocatalytic version has not yet been reported. Here, the cofactor-independent multimeric three-component acyltransferase from Chromobacterium sphagni (CsATase) was identified to enable the nonnatural promiscuous regioselective C-formylation of polyphenolic substrates, especially resorcinol derivatives, and thus extending the reaction scope of acyltransferases. Formylation of 4- and 5-substituted resorcinol derivatives gave access to regioselectively mono-formylated products with up to 99% conversion and up to 74% isolated yield. Formylation of phloroglucinol led to the di-formylated product with 99% conversion, outperforming chemical methods. Structural analysis of CsATase by X-ray crystallography provided insights into its active site.

Science, Volume 391, Issue 6784, January 2026.

Nature, Published online: 30 January 2026; doi:10.1038/d41586-026-00275-8

Engineered Escherichia coli could open the door to more sustainable routes to new drugs and other chemicals.