Biocatalysis@TUDelft

This five-step enzymatic cascade for indigo synthesis is derived from the natural biosynthetic route starting from anthranilate. A two-phase system addresses enzyme incompatibility, and process engineering through stepwise parameter optimization for each phase results in a titer of 183 mg L⁻¹ indigo. This work establishes a proof-of-concept enzymatic platform that expands the toolbox for designing bio-based indigo synthesis.

Herein, an enzymatic in vitro synthesis route to indigo, the dye that gives denim its renowned color, is described. The proposed five-step enzymatic pathway is derived from the natural biosynthesis route starts from anthranilate. In the context of the bio-based economy, this method provides an alternative to the current method of indigo synthesis, which relies on petroleum-based building blocks. First, the enzymes are assessed by stepwise addition to detect intermediate compounds, to ensure cascade function. A two-phase system is designed in response to differences in melting temperatures of the enzymes. A titer of 183 mg L⁻¹ indigo is reached through stepwise parameter optimization for each phase, followed by process engineering to enhance the indigo titer. Continuous supplementation of the cosubstrate phosphoribosyl pyrophosphate is necessary to maintain indole-3-glycerol phosphate (IGP) formation in the first phase. During the second phase, complete uptake of IGP by ZmBX1 remains challenging, and formation of significant byproducts is observed. Despite attempts to resolve these issues, the underlying mechanism remains unclear. Although the yield must be significantly improved for an economically viable process, an enzymatic cascade based on bio-based materials remains a potential alternative for sustainable denim dye synthesis by circumventing host toxicity observed in whole-cell approaches.

Nature, Published online: 03 December 2025; doi:10.1038/s41586-025-09747-9

A hybrid machine learning and atomistic modelling strategy enables one-shot design of efficient enzymes to catalyse diverse biological and non-biological chemical transformations.

Nature, Published online: 03 December 2025; doi:10.1038/s41586-025-09746-w

A generative artificial intelligence-powered method enables de novo design of highly active enzymes based on information about the geometry of residues in the active site, without requiring protein backbone or sequence information.

A multifunctional CYP450 enzyme from Antrodia camphorata, AcCYP1, not only catalyzes the C21 and C15 oxidation of lanosterol, but also mediates a C14–C15 methyl migration to produce a rare triterpene skeleton. This finding reveals a novel enzymatic approach to expand the diversity of triterpene skeleton and facilitates the de novo heterologous synthesis of high-value lanostane triterpenoids.

Abstract

Lanostane triterpenoids, key therapeutic components of medicinal mushrooms, such as rare Antrodia camphorata, face heterologous biosynthesis barriers due to the lack of enzymes for essential C21/C15 oxidation and skeletal rearrangement, limiting access to these rare therapeutics. In this study, we deciphered these missing steps through the characterization of a single CYP450 enzyme, AcCYP1. Notably, AcCYP1 not only catalyzes the indispensable C21 and C15 oxidations, but also represents the first CYP450 enzyme identified to directly rearrange a triterpenoid backbone. This rearrangement generates the uncommon lanostane skeleton characterized by a Δ¹⁴⁽¹⁵⁾ double bond and a C15 methyl group by disrupting canonical hydroxyl rebound and triggering cation-initiated rearrangement. Mechanistically, the catalytic performance of AcCYP1 is regulated by proximal active-pocket geometry and distal hydrophobicity. Mutating the key residue N520 markedly enhanced enzymatic activity, enabling controllable yeast-based production of lanostane triterpenoids with expanded structural diversity for more efficient than conventional artificial cultivation. Collectively, this work uncovers a non-canonical route for triterpenoid structural diversification beyond oxidosqualene cyclases, establishes a systematic strategy for deciphering biosynthetic pathways, and provides scalable, sustainable access to rare natural products.

Comprehensive analyses of two enzyme classes from the amidohydrolase superfamily (AHS) revealed that catalysis proceeds either via 1,4 or 1,6 nucleophilic conjugate addition and that this property defines substrate specificity. Moreover, although all substrates and products are entirely achiral, this mechanistic difference results in an inverted enantioselectivity for fleeting chiral intermediates in the two analyzed AHS classes.

Abstract

The sequencing of numerous genomes has led to the identification of open reading frames for millions of enzymes, many of which use unknown substrates. Hence, the identification of both primary and promiscuous activities remains a major challenge for enzyme research. Here, we identified the mechanistic basis of substrate specificity for members of the amidohydrolase superfamily (AHS). Comprehensive analyses of two AHS classes revealed that catalysis proceeds either via 1,4 or 1,6 nucleophilic conjugate addition mediated by a glutamine that is located at two different positions within the active site thereby shaping substrate scope in these enzymes. These different enzymatic properties result in an inverted enantioselectivity for fleeting chiral intermediates, which are transient chiral species on the reaction pathway from an achiral substrate to an achiral product. Moreover, we demonstrated that catalysis focuses on conserved core structures rather than on all moieties of a given substrate, which increases the degree of promiscuity and evolvability in these enzymes. Using site-directed mutagenesis, we showed that an enzyme specialized in a specific nucleophilic conjugate addition can both readily adapt to secondary substrates and accommodate novel substrates by few amino acid exchanges. Hence, our study reveals mechanistic principles that underly substrate specificity, promiscuity, and enantioselectivity.

Clopidogrel is a widely used antiplatelet prodrug to treat acute coronary syndromes. However, its clinical efficacy is hampered by ineffective bioactivation to produce the pharmacologically active metabolite (AM), leading to variability in antiplatelet response among different ethnic groups. To overcome the shortcomings of clopidogrel, DT-678 was developed by conjugating the AM to 3-nitropyridine-2-thiol via a mixed disulfide bond. It has been challenging to produce the conjugate in high yield by chemical synthesis. Here, we report the first de novo biosynthesis of DT-678 using engineered CYP102A1 variants. We applied structure-based computational design using UniDesign to generate three variants (UD4, UD5, and UD6) that enhanced the catalytic activity and selectivity toward DT-678 synthesis. Among them, UD6 demonstrated the highest total turnover number and DT-678-specific productivity under optimized conditions. Mechanistic analysis revealed that rapid enzyme inactivation, driven by reactive oxygen species (ROS) such as superoxide and hydrogen peroxide, limited overall yield. Remarkably, we found that ascorbic acid significantly protected CYP102A1 variants from inactivation and hence increased production yield. This work establishes a scalable enzymatic strategy for DT-678 biosynthesis and highlights the importance of combining protein engineering with redox control to overcome limitations in CYP-catalyzed reactions.

The past decade has witnessed groundbreaking developments in metalloenzyme-catalyzed free radical transformations which were previously unknown or uncommon in native metalloenzymology. Guided by mechanistic understandings from organic, organometallic and biochemistry, an array of radical reactions has been developed using various metalloprotein catalysts based on first-row transition metal cofactors including Fe, Co and Cu. The structural and functional diversity and the readily tunable active site environment of metalloproteins offer an excellent opportunity to solve the challenging chemo-, regio- and stereoselectivity problems in radical-mediated transformations facing synthetic chemists. In this Review, we summarize metalloprotein-catalyzed radical reactions based on the reactive intermediates involved, including carbon-centered radicals, nitrogen-centered radicals, oxygen-centered radicals, and metal carbenoids and nitrenoids with radical character. We further survey the reaction mechanism, enzyme engineering strategies, and substrate scope of these metalloprotein-catalyzed radical transformations, providing an overview of the current status of metalloenzymology unknown or uncommon in native biochemistry.

Via in silico docking of pyrichalasin H on G- and F-actin, the C21-OAc moiety is identified as a modifiable site tolerated for actin binding. Fluorescent [11]cytochalasan probes from pyrichalasin H and epoxycytochalasin C are obtained by selective C21-OAc hydrolysis and acylation. The conjugates retain actin binding and bioactivity, establishing C21-OAc as a versatile handle for functionalization.

The actin cytoskeleton plays a central role in cellular organization and dynamics, yet the tools available for targeting actin function remain limited. Cytochalasans represent a large class of actin-targeting natural products that are readily cell permeable with varying cytotoxicity and actin-targeting capabilities in mammalian cells. Their pharmacologic exploitation not only requires an understanding of their mode of action but also exact knowledge of how they can be derivatized without affecting their bioactivity. Herein, the design, synthesis, and evaluation of five fluorescently labeled [11]cytochalasan derivatives generated from pyrichalasin H (PyriH) and 19,20-epoxycytochalasin C (EpoxyCytoC) are reported. Guided by molecular docking, the C21-OAc moiety is identified as a promising site for tag attachment without disrupting actin binding. Semisynthetic modifications enable the conjugation of different dyes to PyriH and EpoxyCytoC scaffolds. Moreover, the effect of linkers separating cytochalasans and dyes is analyzed. Biological evaluation supplemented by in vitro assays to additionally interrogates their activities on the assembly of pure actin filaments revealing distinct activity profiles. Together, this study compares permeability, cytotoxicity, and actin binding of five novel [11]cytochalasan probes bearing substitutions of the C21-OAc moiety. These findings establish the C21-OAc position as a versatile functionalization site and provide a framework for developing next generation cytochalasan-based actin-targeting probes.

A versatile microdevice loaded with an immobilized amine transaminase can be operated as either a packed-bed or fluidized microreactor depending on the flow direction. This setup is very useful to characterize the performance of immobilized enzymes under different reactor configurations.

Microfluidics is a very attractive discipline for implementing more versatile high-throughput screening methods to improve enzymes. However, state-of-the-art microfluidics set-ups are mainly devoted to screening enzymes in solution, while screening of enzyme immobilization protocols using microfluidics is scarce. In this work, a microreactor device is designed, fabricated and applied to test different heterogeneous biocatalysts with transaminase activity. This microsystem set-up can be operated in two different modes: packed-bed (PBR) and fluidized (FBR) microreactors without exchanging the sample. Using the same sample to run the two modes, the fidelity of the comparison is increased between the two fluid dynamic regimes. Moreover, by testing different carriers under different modes, a Histagged transaminase is found from Pseudomonas fluorescens immobilized on agarose porous microbeads functionalized with cobalt-chelates and operated as FBR maximizes the STY, and minimizes the equilibration times. This device exemplifies the potential of microreactors for prototyping more efficient heterogeneous biocatalysts.

Plastics such as polyamides (PAs) possess unique physicochemical properties that make them indispensable in modern society. However, their energy-intensive production and challenging end-of-life management highlight the urgent need for efficient recycling or remanufacturing solutions. Enzymatic depolymerization offers a promising route toward circular recycling, yet remains constrained by limited enzyme characterization, lack of validation under industrially relevant conditions, and overall performance. Here we optimized the reaction conditions for three recently discovered nylon-degrading enzymes. One of them, Nyl12, achieved product titers with PA6 and PA66 that exceed previously reported values, without enzyme engineering or substrate pretreatment. We further demonstrated the scalability of the process and its application to complex PA-based materials used in microelectronic components. Analysis of substrate features, including surface area and particle size, revealed key parameters governing enzymatic activity and provided a framework for future pretreatment and process optimization efforts. In combination, these efforts provide a new benchmark for enzymatic nylon recycling.

Polyphenylene sulfide (PPS) is a sulfur-containing polymer widely used in high-performance materials. Its stability makes PPS challenging to recycle. Herein, we report a computational study of the catalysis of the oxidation of dimeric PPS (DPS) to its sulfoxide derivative (DPSO) by DszC, a flavin-dependent monooxygenase from the 4S pathway. Molecular dynamics (MD) simulations reveal that DPS preferentially localizes near the flavin C4a-hydroperoxide intermediate within DszC’s hydrophobic cavity. Analysis of hydrogen-bond networks involving the C4a-hydroperoxide hydrogen and oxygen atoms highlighted five distinct starting structures for hybrid QM/MM Nudged Elastic Band calculations involving intramolecular stabilization with the D-ribitol tail, intermolecular contacts with His391 and Tyr96, and two water-mediated networks. Free energy barrier analysis demonstrated that enhanced stabilization of the proximal oxygen of the flavin C4a-hydroperoxide lowers the proximal O-O bond cleavage barrier. Indeed, the lowest barrier is observed when both His391 and Tyr96 residues are involved in the hydrogen bond network with the reactant. Moreover, a water molecule also stabilizes the transition state via hydrogen bonding with the distal oxygen, yielding a comparable barrier. These findings suggest that enzymatic DPS oxidation by DszC protein is feasible, opening the way of new biodegradation pathway of PPS polymers.

The design of artificial photoenzymes by incorporating synthetic chromophores into proteins represents a promising strategy to achieve new-to-nature biocatalytic transformations with high levels of stereocontrol. Selecting an appropriate protein scaffold is a crucial step in this approach, which so far has been limited to naturally occurring proteins. Here, we tested the suitability of computationally designed scaffolds for this purpose. We chose a de novo helical bundle protein that has a central cavity for small molecule binding but no inherent catalytic activity. To generate a starting point for photoenzyme engineering, we installed a thioxanthone-based triplet sensitizer via cysteine bioconjugation. Guided by computational modeling and molecular dynamics (MD) simulations, three rounds of directed evolution toward the [2+2] photocycloaddition of a 3-alkenyloxy-substituted quinolone resulted in highly efficient enzyme variants with opposite enantioselectivity. Upon visible-light irradiation, both product enantiomers were accessible with excellent yield and >90:10 enantiomeric ratio. Furthermore, we obtained high-resolution crystal structures of the evolved designer enzymes. When exposing crystals of substrate-bound protein to blue light, we observed product formation in crystallo and could rationalize the enantioselectivity. Our work highlights the potential of de novo designed protein scaffolds to efficiently generate and evolve stereoselective artificial photoenzymes.

Peptide lipidation can enhance stability and permeability, yet biocatalysts for late-stage, site- and mode-selective modification remain limited. We established a streamlined activity-profiling system that rapidly assesses peptide-prenylating activities using an artificial substrate set. Screening 19 recombinant, putative cyanobactin prenyltransferases (PTases) uncovered 14 active enzymes with diverse catalytic properties, including five unprecedented modes of prenylation, bifunctionality toward Trp/Tyr, and expanded donor utilization such as farnesylation. Co-crystal structures of seven PTases reveal how subtle active-site changes enable dual substrate recognition and accommodation of bulkier donors, highlighting the evolutionary diversification of catalytic functions. The enzymes tolerate non-native substrates, enabling late-stage, site- and mode-selective lipidation to construct pseudo-natural peptides. Together, these findings drastically expand the enzymatic and mechanistic landscape of cyanobactin PTases, substantially enlarging their functional repertoire and revealing the structural principles that govern the diversification of peptide prenylation chemistry.

Nature Chemical Biology, Published online: 01 December 2025; doi:10.1038/s41589-025-02078-w

Radical FeII/α-ketoglutarate-dependent halogenases are promising biocatalysts for C(sp3)–H functionalization, which can replace a substrate H atom with a bound anion. In this study, we have shown that the amino acid halogenase HalA uses dynamic active site coordination to control the reaction cycle, promoting both C–H activation and subsequent anion transfer.

Non-canonical glycopeptides of the proteoglycan linkage region are accessible by the enzymes B3GalT6 and GlcAT-1 confirming a recently discovered rescue mode in glycosaminoglycan (GAG) biosynthesis. The crystal structure of B3GalT6 revealed a covalent dimer linked by a disulfide.

Abstract

We selected the N,O-glycosylated proteoglycan bikunin as a model to establish a chemoenzymatic approach to defined proteoglycans using native chemical ligation. Overexpression of the human linkage region glycosyltransferases B4GalT7, B3GalT6 and B3GlcAT-1 as N-terminal SUMO-fusions gave high yields of soluble and active enzymes in E. col i. When starting with xylosylated bikunin peptides the transferases performed well in enzymatic cascade reactions and provided the desired linkage region tetrasaccharide glycopeptides. B3GalT6 and B3GlcAT-1 led to side products with N,O-glycosylated bikunin peptides revealing unexpected promiscuity of both enzymes towards complex type N-glycans. Additionally, B3GalT6 was found to synthesize short poly-β3 Gal structures. B3GlcAT-1 can slowly convert the biosynthetic intermediate Gal-Xyl to the non-canonical trisaccharide GlcA-Gal-Xyl. This reaction independently confirmed the recently detected biosynthetic bypass to GAGs in the case of dysfunctional B3GalT6 (spondylodysplastic Ehlers-Danlos-syndrome). The three linkage region glycosyltransferases B4GalT7, B3GalT6 and B3GlcAT-1 were dimeric in solution and the crystal structure of B3GalT6 was solved showing a covalent dimer linked by a disulfide in the center of the large dimerization domain. This motif appears to be conserved in higher organisms and reinforces the concept of dimeric glycosyltransferases lining the Golgi.