Biocatalysis@TUDelft

A flexible active-site loop that is involved in shaping the substrate and product cleft as well as the active-site pocket was identified for the MDR-related ene/yne-reductase CaeEnR1 from Cyclocybe aegerita. Double mutations of this loop showed an up to 20-fold increase in conversion rates toward cinnamaldehyde-like substrates and a 2-fold increase in conversion toward an aliphatic and aromatic alkyne.

Abstract

Medium-chain dehydrogenase/reductase-superfamily related (MDR) ene-reductases are highly neglected biocatalysts that have not yet been systematically studied in terms of rational protein engineering. Therefore, we crystallized the MDR-related CaeEnR1 in its apo- and binary-complex and searched for secondary structures that might influence enzyme activity toward specific substrate classes. By comparing the apo- and binary-complexes, a flexible active-site loop was identified that is involved in shaping the substrate and product cleft as well as the active-site pocket. Furthermore, we could show that an active-site loop undergoes drastic re-arrangement after cofactor/substrate binding, revealing an open–closed mechanism. Based on these results, we subjected this active-site loop to an alanine-scan. The identified hits were then designed toward substituted cinnamaldehyde-like substrates and an aliphatic/aromatic alkyne. From all tested amino acids P64, Y69, and S70 were identified as the most influential amino acid residues. Particularly the double mutations P64F/Y69A and Y69F/S70A showed an up to 20-fold increase in conversion rates toward cinnamaldehyde-like substrates. In contrast, a 2-fold increase in conversion toward the aliphatic and aromatic alkyne was achieved with the S70F variant. With this study we state the foundation of protein engineering of the neglected MDR-related ERs which opens up a new pathway for tailored biocatalysts.

In a study of the biosynthesis of the polyether ionophore lysocellin, the P450 enzyme LyoI was found to catalyze a rare oxidation of a hydro-2,2′-bifuran moiety to its hemiketal form. The oxidation was identified as the key for unlocking the bioactivity of lysocellin. The sequence of LyoI lacks a key acidic residue, essential for its oxidative capabilities, which was utilized to identify a range of non-canonical P450 enzymes.

Abstract

Polyether ionophores are potent antimicrobials, albeit also cytotoxic against mammalian cells. We have identified several polyether ionophores containing a common hydro-2,2′-bifuran-2-ol (hemiketal) moiety, which cannot be derived from the canonical biosynthetic steps observed for the compound class, suggesting an unusual oxidative transformation. To identify the responsible enzyme, we applied CRISPR–BEST to knock out genes in the lysocellin-producing strain S. longwoodensis. This allowed us to propose the first annotation of the lysocellin biosynthetic gene cluster and identify the responsible P450 enzyme, LyoI, through reconstitution of the function in vivo. LyoI knockout provided access to the non-oxidized precursor (pre-lysocellin) which allowed both in vitro validation of the unusual direct hydro-2,2′-bifuran to hydro-2,2′-bifuran-2-ol oxidation and investigation of the impact on biological activity. Interestingly, absence of the LyoI-mediated oxidation greatly reduced the biological potency of the compound. Closer investigation of the sequence revealed that LyoI lacks a key conserved acidic residue, which proved essential for the unusual oxidative function of the enzyme. Through a sequence similarity network of LyoI, we were able to identify a wide range of non-canonical P450 enzymes, highlighting the possibilities of a biosynthesis-focused approach to discovering novel enzymes.

This review outlines major advances in the design, execution, analysis, and data management phases of high-throughput experimentation (HTE). The limitations and potential opportunities of applying modern HTE to organic synthesis are highlighted.

Abstract

High-throughput experimentation (HTE), the miniaturization and parallelization of reactions, is a valuable tool for accelerating diverse compound library generation, optimizing reaction conditions, and enabling data collection for machine learning (ML) applications. When applied to organic synthesis and methodology, HTE still poses various challenges due to the diverse workflows and reagents required, motivating advancements in reaction design, execution, analysis, and data management. To address these limitations, cutting-edge technologies, automation, and artificial intelligence (AI) have been implemented to standardize protocols, enhance reproducibility, and improve efficiency. Additionally, strategies to reduce bias and promote serendipitous discoveries have further strengthened HTE's impact. This review highlights recent advances at every stage of the HTE workflow, including the development of customized workflows, diverse analysis, and improved data management practices for greater accessibility and shareability. Furthermore, we examine the current state of the field, outstanding challenges, and future directions toward transforming HTE into a fully integrated, flexible, and democratized platform that drives innovation in organic synthesis.

Progress in enzymology is enabled by the development of high-throughput platforms for kinetic measurements. Presented herein is a method for one-shot ultra-high-throughput quantification of enzymatic kinetics using mRNA display and next-generation sequencing. The technique was leveraged to quantify kcat/KM values for 2.86 × 105 peptide substrates of a promiscuous dehydroamino acid reductase.

The single-domain auxiliary activity family 12 (AA12) pyrroloquinoline quinone-dependent oxidoreductases and free AA8 modules are prevalent in cellulolytic fungi, however, their function in polysaccharide biodegradation is still confused. Here, we characterized three single-domain AA12 oxidoreductases and one free AA8 module from Thermothelomyces thermophilus and Thermothielavioides terrestris. All three single-domain AA12 oxidoreductases are restrict dehydrogenases with trace oxidase activity. All three single-domain AA12 enzymes could directly transfer electrons to lytic polysaccharide monooxygenase (LPMO) and drive NcLPMO9C activity. Furthermore, inter-protein electron transfer between single-domain AA12 enzymes and the AA8 module was observed. The AA12 enzyme-driven NcLPMO9C efficiency could be significantly enhanced by the addition of free AA8 module TthAA8B, probably attributing to the acceleration of electron transfer from AA12 enzymes to NcLPMO9C and the attenuation of H2O2 accumulation mediated by TthAA8B. Our findings highlight the potential role of single-domain AA12 enzyme and free AA8 modules in the biodegradation system of LPMOs.

Intrinsically disordered proteins (IDPs) challenge the traditional structure-function paradigm by lacking a stable three-dimensional structure 1. While their roles as dynamic effectors, scaffolds, and molecular switches are well-established, it has been widely accepted that enzymatic activity requires a stably folded catalytic center 2. Here, we challenge this dogma by demonstrating that a 284-amino acid intrinsically disordered domain of the cytoskeleton-associated protein 2 (CKAP2) is sufficient to catalyze both microtubule polymerization and depolymerization. CKAP2 promotes tubulin incorporation without high-affinity tubulin binding, suggesting a transition-state-based catalytic mechanism distinct from known microtubule polymerases. These findings establish, for the first time, that an intrinsically disordered domain can function as a bona fide enzyme, expanding our understanding of the functional repertoire of disordered proteins and their roles in cellular processes.

Nature Chemical Biology, Published online: 08 July 2025; doi:10.1038/s41589-025-01953-w

Nonheme Fe enzymes with open coordination sites hold the potential for advancing new-to-nature reactions. Here a plant-derived nonheme Fe enzyme, 1-aminocyclopropane-1-carboxylic acid oxidase, is evolved and repurposed to catalyze 1,3-nitrogen migration reactions, enabling the enantioselective synthesis of noncanonical amino acids.

Nature Chemical Biology, Published online: 22 August 2025; doi:10.1038/s41589-025-02004-0

Engineering non-natural functions into enzymes has opened unexpected avenues for chemical synthesis. Whereas past efforts in repurposing natural enzymes have predominantly focused on heme- and flavin-dependent enzymes, latest work further highlights the advantages and potential of non-heme iron enzymes for organic synthesis.

Amino acid steric engineering as an enzyme engineering strategy is generally indispensable for the creation of an ideally configured catalytic cavity with maximized catalytic effect. The conventional approach in the typical form of hypo-steric engineering relies on the intentional decrease of steric size of an amino acid as a control handle for the purposeful increase of catalytic cavity size and accordingly, elevation of catalytic adaptability. Herein, we have developed a hyper-steric engineering method, with the intentional increase of steric size of an amino acid exploited as a superior alternative handle for the targeted increase of catalytic cavity size and correspondingly, elevation in catalytic activity and/or enantioselectivity. In particular, a carboxylesterase has been engineered for the achievement of highly efficient acyl transfer to alcohols. With the R386V,R390V variant (VIII) of a metagenomically identified family VIII carboxylesterase, EstFF1, a high 105:1 alcohol over VIII molar ratio and a low 2:1 vinyl acetate over alcohol molar ratio are sufficient for effecting highly productive acyl transfer to a broad scope of primary alcohols over hydrolysis. The hyper-steric engineering of G359 to L359 (IX) on the R386V variant (II) enables acyl transfer to a broad scope of both aromatic/alkyl and alkyl/alkyl secondary alcohols, at the quantitative or near-quantitative S-enantioselectivity level. The high catalytic activity and/or enantioselectivity demonstrated herein promise hyper-steric engineering as a powerful conceptual framework for expanding the toolbox of enzymatic catalysis.

Lytic polysaccharide monooxygenase (LPMO) is an enzyme that has enormous potential for industrial applications. It has a synergistic effect with the cellulase enzyme complex. The LPMO enzymes typically adopt a compact {beta}-sandwich fold that consists of a 7 to 9 {beta}-strand with a flat active site containing copper in its active site. There are a few loops in LPMO, among them the L2 loop is reported to take a key role in shaping the active site and substrate binding. Now, in this work, we want to investigate the role of the L2 loop in enzymatic activity and synergistic effect. In achieving our goal, we have replaced the L2 loop of our concerning AfLPMO16 with other L2 loops from different LPMOs: HiLPMO9B (PDB: 5NNS), McLPMO9 (PDB: 7NTL) and CsLPMO9 (PDB: 7EXK). Interestingly, L2 loop replacement from CsLPMO9 (PDB: 7EXK) showed enhanced activity and synergism compared to others. The secondary structural analysis by circular dichroism also suggested that it changed the structure significantly. Moreover, this is the first report of complete L2 loop engineering in LPMO.

ChlH, a flavin-dependent tryptophan halogenase, was reconstituted in vitro and capable of modifying a wide array of diverse peptidic substrates. This suggests a potential use in the biocatalytic production of chlorinated peptides.

Abstract

Amino acids undergo numerous enzymatic modifications. However, the broad applicability of amino acid-modifying enzymes for synthetic purposes is limited by narrow substrate scope and often unknown regulatory or accessory factor requirements. Here, we characterize ChlH, a flavin-dependent halogenase (FDH) from the chlorolassin biosynthetic gene cluster. Unlike characterized peptide-modifying FDHs, which are limited to either specifically modified peptides or the termini of linear peptides, ChlH halogenates internal Trp residues of linear peptides, as well as N- and C-terminal Trp. Scanning mutagenesis of the substrate peptide ChlA revealed Trp was tolerated by ChlH at nearly every position. Molecular dynamics simulations corroborated the importance of a C-terminal motif in ChlA and provided insight into the lack of Trp14 chlorination in native chlorolassin. Furthermore, halogenation of disparate ribosomally synthesized and post-translationally modified peptide (RiPP) precursor peptides, pharmacologically relevant peptides, and special examples of internal Trp within proteins was achieved using wild-type ChlH. A rapid cell-free biosynthetic assay provided insight into ChlH's preferences. In contrast to characterized FDHs, ChlH halogenates diverse peptide sequences, and we predict this promiscuity may find utility in the modification of additional peptide and protein substrates of biotechnological value.

Multicompartmental protocell models with complex architectures are constructed through a versatile structural transformation from membrane-free coacervates to robust hybrid microreactors, which enables spatiotemporal regulation of individual biocatalysis or divergent cascades with high reaction efficiency.

Abstract

Advancing the design and construction of artificial protocells with organized complexity, diverse functionality and practical applicability is urgently demanded in vitro synthetic biology and bioengineering but remains a grand challenge. Here, we present a versatile Pickering emulsion-based encapsulation approach to transform membraneless coacervate compartments into robust multicompartmental hybrid microreactors, which concurrently assimilate the expected attributes of hierarchically compartmentalized structure, molecularly crowded environment, selectively permeable ability and mechanically reinforced stability. Single or multiple biological and non-biological catalytic species can be spatially sequestered in specific domains of the hybrid microreactor, enabling spatiotemporal regulation of individual biocatalysis or divergent cascades with high reaction efficiency. As proof of concept, we not only demonstrate the markedly improved catalytic activity (1.9–9.2 folds enhancement), strengthened thermostability (up to 100 °C) and impressive long-term durability (1600 h) of the obtained microreactors in lipase-driven kinetic resolution of alcohol medicine intermediates, but also showcase their superior capability in processing chemo-enzymatic cascade of ketone hydrogenation-kinetic resolution and multi-enzymatic cascade of oxidation reactions. Macromolecular crowding and confinement effects arising from structural features of the hybrid microreactors are identified as the dominant factors for the promotion of catalytic functions.

Engineered luciferases have transformed biological imaging and sensing, yet optimizing NanoLuc luciferase (NLuc) remains challenging due to the inherent stability-activity trade-off and its limited sequence homology with characterized proteins. We report a hybrid approach that synergistically integrates computational deep learning with structure-guided rational design to develop enhanced NLuc variants that improve thermostability and thereby activity at elevated temperatures. By systematically analyzing libraries of engineered variants, we established that modifications to termini and loops distal from the catalytic center, combined with preservation of allosterically coupled networks, effectively enhance thermal resilience while maintaining enzymatic function. Our optimized variants - notably B.07 and B.09 - exhibit substantial thermostability enhancements (increases of 4.2 degrees Celsius and 5.2 degrees Celsius at 50 % solubility), leading to increased activity at elevated temperatures (320 % and 370 % of wild-type at 55 degrees Celsius). These variants maintain NLuc's pH tolerance and retain improved activity with the alternative substrate coelenterazine. Molecular dynamics simulations and protein folding studies elucidate how these mutations favorably modulate conformational landscapes without perturbing substrate binding architecture. Beyond providing superior tools for bioluminescence applications, our integrated methodology establishes a broadly applicable framework for engineering enzymes where traditional homology-based approaches fail, and stability-activity constraints present formidable barriers to improvement.

Plants have evolved to produce diverse molecules that inhibit protein translation. A lead example is homoharringtonine (HHT), both a key tool for ribosomal profiling and an FDA-approved treatment for chronic myeloid leukemia. HHT is commercially produced through semi-synthesis by esterifying the alkaloid core cephalotaxine (CET) extracted from endangered Cephalotaxus species. Despite its medicinal significance, a biosynthetic pathway to CET and HHT has not been described. Here, we use paired untargeted metabolomics (stable-isotope labeled precursor feeding) and transcriptomics to elucidate a near-complete biosynthesis to CET without prior knowledge of intermediates and biosynthetic genes. We show that while CET alkaloid core is actively biosynthesized only in growing root tips, both CET and HHT accumulate throughout the plant. We discovered and characterized seven CET pathway intermediates and six novel biosynthetic enzymes that, together, can be used to produce cephalotaxinone, the likely direct precursor of CET. Included are non-canonical cytochrome P450s, an atypical short-chain dehydrogenase, and a 2-oxogluatrate-dependent dioxygenase that together result in carbon excision and the formation of the characteristic pentacyclic backbone of HHT alkaloids. Our data support a model where cephalotaxinone is the last pathway intermediate produced specifically in the root tips, and its distribution throughout the plant is likely the starting point for subsequent elaboration to HHT. This study not only establishes a metabolic route to the core scaffold of HHT-enabling future sustainable, large-scale production of this valuable drug-but also suggests how Cephalotaxus species employ a whole plant coordination to regulate the biosynthesis of eukaryotic ribosomal toxins.

S-Adenosyl-L-methionine (SAM) is the second most used enzyme cofactor and vital for numerous cellular reactions such as methylation or polyamine synthesis. While most stereocentres of the biologically active (SS,SC)-SAM are fixed, epimerisation at the methyl sulfonium centre is driven by heat, yielding biologically inactive (RS,SC)-SAM. This SAM diastereomer disturbs SAM-dependent pathways, posing a metabolic threat especially to thermophilic organisms. In vitro analysis shows that SAM hydrolases cleave the biologically inactive (RS,SC)-SAM, thereby constituting to a metabolic salvage pathway. For further analysis of the biological relevance, we characterised two archaeal SAM hydrolases from the thermophilic Sulfolobus acidocaldarius and the halophilic Haloferax volcanii, confirming their selectivity towards (RS,SC)-SAM in vitro. Genetic manipulation in the native hosts supports a significant role of the SAM hydrolases in decreasing the share of intracellular (RS,SC)-SAM to sustain cellular functions in thermophilic organisms.

Enzymes offer unparalleled selectivity and sustainability for chemical synthesis, yet their widespread industrial application is often hindered by the slow and uncertain process of discovering and optimizing suitable biocatalysts. While directed evolution remains the gold standard for enzyme optimization, its success hinges on the availability of a starting enzyme with measurable activity, a persistent bottleneck for many desired functions. Designing libraries likely to contain such functional starting points remains a major challenge. In this work, we use the GenSLM protein language model (PLM) along with a series of filters to generate novel sequences of the {beta}-subunit of tryptophan synthase (TrpB) that express in Escherichia coli, are stable, and are catalytically active in the absence of a TrpA partner. Many generated TrpBs also demonstrated significant substrate promiscuity, accepting non-canonical substrates typically inaccessible to natural TrpBs. Remarkably, several outperformed both natural and laboratory-optimized TrpBs on native and non-canonical substrates. Comparative analysis of the most active and promiscuous generated TrpB and its closest natural homolog confirmed that this enhanced functional versatility does not stem from the natural enzyme, highlighting the creative potential of generative models. Our results demonstrate that the model can generate enzymes which not only preserve natural structure and function but also acquire non-natural properties, establishing PLMs as powerful tools for biocatalyst discovery and engineering, with the potential in some cases to bypass further optimization.