Biocatalysis@TUDelft

Nature Biotechnology, Published online: 07 May 2025; doi:10.1038/s41587-025-02655-3

A combination of engineering approaches is used to improve the function of IscB for a variety of editing activities in vivo.

Science, Volume 388, Issue 6747, Page 665-670, May 2025.

Aldolases and ThDP-dependent enzymes have played important roles in the C─C bond formation of formaldehyde, a versatile C1 molecule. In this review, we summarize the recent progresses in the enzymatic C─C bond formation reactions of formaldehyde and multi-enzyme cascade reactions, that have enabled the synthesis of complex multifunctional compounds from formaldehyde via hydroxymethylation.

Abstract

Given the importance of multifunctional compounds and the versatile reactivity of one-carbon (C1) molecule formaldehyde, the synthesis of value-added multifunctional compounds from formaldehyde and other simple molecules via C─C bond formation has been the focus of intensive investigations. In view of the uncontrollable reactivity of formaldehyde, the employment of formaldehyde in the C─C bond formation is considered as one of the most challenging tasks in organic synthesis. Aldolases and thiamine pyrophosphate (ThDP)-dependent enzymes have been regarded as the most potential biocatalytic tools for carbon–carbon (C─C) bond formation. In the present review, aldolases and ThDP-dependent enzymes are shown to be the simplest and powerful biocatalytic platforms for controlling the reactivity of formaldehyde to effectively produce multifunctional compounds. The recent advances in the construction of multienzymatic cascade reactions for the synthesis of complex multifunctional compounds from formaldehyde via C─C bond formation have also been presented.

This research examines polyethylene terephthalate (PET)/cotton textiles biodegradation, using different stressors as enzymatic incubation. It reveals that aggressive chemical treatments (alkali conditions) cause nonspecific degradation, affecting both PET and cotton fibers. UV–ozone pretreatment synergizes with PETase activity, improving terephthalic acid yield, while natural sunlight shows minimal effects, emphasizing the effectiveness of stronger pretreatment methods for enhancing PET recycling processes.

The rapid growth of the fashion industry has led to increasing textile waste, exacerbating environmental pollution and climate change. To support sustainability and circular economy goals, this study investigates the enzymatic degradation of cotton/polyethylene terephthalate (PET) mixed textiles using PETase, comparing wild-type and mutant (MUT S238F/W159H) variants. To improve enzyme accessibility, three pretreatment strategies are evaluated: alkali treatment, UV–ozone (UVO) exposure, and natural sunlight weathering. The effects are assessed by measuring textile weight loss, surface morphology (scanning electron microscopy), Fourier transform infrared spectroscopy, and yields of terephthalic acid (TPA) and mono-(2-hydroxyethyl) terephthalic acid. Alkali treatment produces the highest weight loss, while UVO pretreatment moderately degrades textiles and significantly enhances enzymatic TPA production. In contrast, prolonged sunlight exposure has negligible effects. ¹H NMR analysis of supernatants confirms the formation of oxidized PET products following UVO exposure, indicating surface chemical modifications that increase enzymatic susceptibility. The results reveal differential effects on PET and cotton fibers, highlighting UVO as a promising, selective pretreatment for mixed textile waste. This study demonstrates the potential of combining photochemical oxidation and enzymatic processes for targeted PET degradation, contributing to more efficient textile recycling strategies.

Biocatalysis has become a sustainable and cost-competitive alternative to established chemical synthesis, enabling the enzyme-based production of not only commodity chemicals but (non-natural) amino acids, (rare) sugars, as well as synthetic nucleotides. These building blocks give access to highly complex molecules with versatile industrial and therapeutic applications.

Abstract

Supported by rapid technological advancements, biocatalytic applications have matured into sustainable, scalable, and cost-competitive alternatives to established chemical catalysis. This review presents the most recent examples of enzyme-based solutions for the manufacturing of molecules with extended carbon–carbon frameworks and multiple stereogenic centers at commercial scale, including peptide building blocks, (rare) sugars, synthetic (oligo)nucleotides, and terpenoids, such as (–)-Ambrox^®. Novel enzyme classes are highlighted along with their potential applications—the synthesis of DNA/RNA, the depolymerization of synthetic plastics, or fully enzymatic protection/deprotection schemes—pointing toward the diversification and broader industrial utilization of biocatalysis-based processes.

Nature Synthesis, Published online: 14 April 2025; doi:10.1038/s44160-025-00788-6

A repurposed non-haem, iron-based dioxygenase enables the stereoconvergent reduction of alkenes with excellent selectivity. Mechanistic studies support an iron hydride pathway and reveal the molecular mechanism of stereoconvergence.

We explore KslB, a Pictet–Spenglerase (PSase) capable of accepting ketones, in preparing 1,1′-disubstituted-ß-carbolines. Biocatalytic optimizations show its broad promiscuity, excellent stereoselectivity, stability (TTN > 4 38 000), and application in an enzyme-cascade with tryptophan synthase. X-ray characterization demonstrate conserved features among bacterial PSases like McbB and KslB and offered insights into KslB's ability to accept ketones.

Abstract

In light of the ubiquity of 1,1′-disubstituted tetrahydro-ß-carboline (THBC) motif in alkaloid natural products, developing asymmetric methodology for its preparation is highly valuable. Despite the immense progress toward achieving stereoselective Pictet–Spengler reaction with aldehydes, the analogous reaction with ketones is still underdeveloped. Exploiting KslB, a Pictet–Spenglerase from the biosynthesis of kitasetaline, we develop a general, diastereoselective, and protecting-group free method for the construction of densely functionalized THBCs with α-quaternary center by coupling tryptophan derivatives and α-keto acids. We determine the stereochemistry of kitasetalic acid, KslB's physiological product and a key biosynthetic intermediate toward kitasetaline, and established that KslB's selectivity is opposite to what is achieved chemically. Our investigations of KslB show its high activity (total turnover number >438000), substrate promiscuity, and tolerance for high substrate concentrations (0.1 M). Additionally, a TrpB-KslB cascade enables the construction of complex tricyclic products from simple indoles in one-pot. X-ray structural characterization of KslB sheds light on potential active site interactions to account for its stereoselectivity and ability to accept ketone substrates.

A new-to-nature biocatalytic radical alkylation of 2-acyl imidazoles is achieved through the cross-integration of a Lewis-acid (LA)-type artificial metalloenzyme (ArM) and a photobiocatalysis strategy. Directed evolution leads to enantiodivergent synthesis with different mutants. Detailed mechanistic studies illustrate a difference in reactivity and enantiomeric preference between the illuminated and dark conditions.

Abstract

Artificial metalloenzymes (ArMs) and photoenzymatic catalysis represent two cutting-edge approaches to creating new enzyme reactivity. However, the potential of merging these two strategies remains underdeveloped for enantiocontrolled biotransformations. Herein, we develop a synergistic metalloenzymatic and photoredox catalysis platform to enable enantiodivergent radical alkylation of 2-acyl imidazoles. Specifically, cupin proteins are redesigned to function as copper(II)-based Lewis-acid-enzymes (LAses), which, in synergy with tripyridinyl-ruthenium-based photoredox catalysis, precisely control the generation, reactivity, and selectivity of abiological radicals, thereby unlocking non-natural enzyme reactivity. Powered by protein engineering, repurposed photo-LAses facilitate the green and efficient synthesis of diverse enantioenriched α-chiral ketones in high enantioselectivity (both enantiomers accessible, up to 97% yield and 98.5:1.5 enantiomeric ratio [er]). Detailed mechanistic studies suggest a radical addition to the metalloenzymatic enolate pathway and explain the switched selectivity from dark to photoconditions.

Human cis-prenyltransferase (hcis-PT) synthesizes long-chain isoprenoids essential for N-linked protein glycosylation. This heteromeric complex comprises the catalytic subunit DHDDS and the regulatory Nogo-B receptor (NgBR). Although NgBR dramatically enhances DHDDS activity, the molecular basis for this allosteric regulation remains unclear. Here, we combined crystallography, hydrogen-deuterium exchange mass spectrometry (HDX-MS), molecular dynamics simulations, and network analysis to uncover the structural dynamics and communication pathways within hcis-PT. By solving the apo structure of hcis-PT, we reveal only a localized flexibility at the active site and the NgBR C-terminus. However, HDX-MS demonstrated widespread substrate-induced stabilization, particularly at the NgBR {beta}D-{beta}E loop, highlighting it as an allosteric hub. Functional mutagenesis scanning identified NgBRS249 as critical for enzymatic activity, independent of structural perturbations. Network analysis of MD simulations pinpointed this residue as a central node in inter-subunit communication, with perturbations disrupting downstream allosteric pathways, altering enzymatic activity. Our findings reveal a dynamic regulatory network centered at the inter-subunit interface, wherein specific NgBR residues modulate DHDDS activity through allosteric signaling. This work elucidates a conserved mechanism of subunit coordination in long-chain cis-prenyltransferases and suggests novel avenues for therapeutic targeting of hcis-PT-related disorders.

Protein language models (pLMs) enable generative design of novel protein sequences but remain fundamentally misaligned with protein engineering goals, as they lack explicit understanding of function and often fail to improve properties beyond those found in nature. We introduce Reinforcement Learning from eXperimental Feedback (RLXF), a general framework that aligns protein language models with experimentally measured functional objectives, drawing inspiration from the methods used to align large language models like ChatGPT. Applied across five diverse protein families, RLXF improves generation of high-functioning variants beyond pre-trained baselines. We demonstrate this with CreiLOV, an oxygen-independent fluorescent protein, where RLXF-aligned models generate sequences with significantly enhanced fluorescence, including the most fluorescent CreiLOV variants reported to date. Our results indicate that RLXF-aligned models effectively integrate the evolutionary knowledge encoded in pre-trained pLMs with experimental observations, improving the success rate of generated sequences and enabling the discovery of synergistic mutation combinations that are difficult to identify through zero-shot or evolutionary approaches. RLXF provides a scalable and accessible approach to steer generative models toward desired biochemical properties, enabling function-driven protein design beyond the limits of natural evolution.

Metalloenzyme superfamilies achieve diverse functions within a shared structural framework, and similar functional variety may be achievable in designed proteins. We have previously reported a computational approach that enables the de novo design of symmetric protein assemblies around metal centers with pre-defined coordination geometries. Here, we demonstrate that an artificial protein trimer termed Tet4, whose structure was designed around an idealized tetrahedral His3/H2O-ZnII coordination motif, enables the high-affinity binding of several other divalent first-row transition metal ions in the same geometry as for ZnII. We then follow the proposed evolutionary path of a natural metalloenzyme family by engineering a pseudosymmetric, single chain variant of Tet4, scTet425. scTet425 allows us to introduce asymmetric point mutations that influence the catalytic properties of the metal center. We also demonstrate that we can further tune the enzymatic activity of Tet4 by designing a substrate pocket that improves Zn-Tet4’s affinity for a hydrolysis substrate, 4-methylumbelliferyl acetate.

We introduce a solvent-free transesterification of rose geranium oil using wool-immobilised Pseudomonas fluorescens lipase (WPFL) as a greener and more sustainable alternative to conventional ester synthesis. Targeting the United Nations’ 12th Sustainable Development Goal and aligning with green chemistry principles, the system eliminates solvents, designs an inherently safer process, and utilizes nature's own catalysts: enzymes. WPFL demonstrates catalytic efficiency comparable to Novozym 435, a widely used commercial lipase, while offering a significant cost advantage. Characterisation through ESEM and CLSM imaging confirms that wool provides a uniform enzyme distribution, enhancing both catalytic efficiency and structural stability. The system achieves full conversion of geraniol in five hours and 88% conversion of citronellol, effectively doubling productivity compared to solvent-based systems. The final ester yields are independent of rose geranium oil concentration, underscoring the robustness of the system for processing real flower waste oils which is an essential feature for industrial applications. Sensory analysis showed the enzymatic modification enhanced the fragrance profile by intensifying floral and fruity notes while removing undesirable herbal undertones. Finally, we present a novel parallel Ping pong bi-bi mechanism incorporating specificity constants (kcat/KM) that describes the parallel reactions of citronellol and geraniol, providing a robust framework for comparing enzyme efficiency and selectivity in terpene ester production from diverse floral waste streams. This work establishes WPFL as a cost-effective, scalable, and sustainable alternative to traditional lipases, with transformative potential for industrial terpene ester synthesis.

Polyketides are a diverse class of natural products with a wide range of therapeutic uses, serving as antibiotics, anticancer agents, and immunosuppressants. A major subset of these molecules is synthesized by Type I Assembly-Line Polyketide Synthases (T1ALPKSs), large enzyme complexes that catalyze their stepwise construction. Understanding the selectivity and scope of T1ALPKS domains could greatly expand the diversity of products generated by these synthases and their components. This study explores the reactivity and selectivity of the terminal domains of T1ALPKSs, known as thioesterases (TEs). A panel of 25 wild-type and 10 mutant (Ser->Cys) thioesterases was reacted with semi-synthetic substrates. The selectivity with which these TEs form 12- and/or 14-membered macrolactones and a pair of diastereomeric 14-membered macrolactones was quantified. The results indicate that the selectivity with which wild-type TEs react with unnatural substrates correlates with their parent T1ALPKSs, enabling the assignment of TEs into groups. Additionally, our findings reveal that mutant (Ser->Cys) TEs often exhibit expanded scopes compared to their wild-type counterparts. We demonstrated the synthetic applications of TEs with kinetic resolutions and preparative-scale reactions. This study provides a framework for organizing TEs and predicting their reactivity and selectivity, facilitating future research on structure-activity relationships and supporting directed evolution studies.

The ribosomal incorporation of backbone-modified amino acid analogs into peptides and proteins enables the programmed synthesis of sequence-defined biopolymers with tunable properties. However, the successful use of backbone-modified monomers as substrates by the ribosome requires coordination across multiple parts of the translation machinery, including aminoacyl-tRNA synthetases, translation factors, and finally the ribosome itself. β-hydroxyacids are particularly interesting monomers because they have potential to support the programmed biosynthesis of both polyesters (plastics) and depsipeptides (therapeutics). Previous work has reported that both enantiomers of β2-hydroxy-Nε-Boc-Lysine (β2-OH-BocK) are in vitro substrates for the orthogonal M. alvi PylRS/tRNA pair, but only one enantiomer is introduced into protein in vivo and with substantially lower yield than expected. We sought to determine whether there is a structural basis for the diminished yield as well as the preferential incorporation of one β2-OH-BocK enantiomer over the other. Here we report high-resolution cryo-EM structures of the Escherichia coli (E. coli) ribosome complexed with either (R)- or (S)-β2-OH-BocK. These structures reveal that both enantiomers are well positioned to undergo bond formation within the ribosome active site and are likely equally reactive. In vitro translation experiments confirm that orthogonal tRNAs acylated with (R)- or (S)-β2-OH-BocK are ribosome substrates, implying that the preferential incorporation of one enantiomer over the other in vivo results from deficiencies in other translation steps, such as tRNA acylation efficiency in cells or delivery to the ribosome by elongation factor Tu (EF-Tu). Taken together, this work demonstrates the plasticity of the E. coli ribosome and its tolerance for diverse substrates.

Lactonases, a class of metalloenzymes that exhibit catalytic promiscuity, have been extensively studied from a biological perspective, yet their application as biocatalysts remains underexplored. In this study, we disclose the biocatalytic activity of lactonase enzymes in the hydrolysis and deracemization of chiral C3-substituted-γ-thiolactones and the asymmetric synthesis of γ-thio-α-substituted-carboxylic acids. The thiolactonase activity of lactonases from different protein superfamilies was investigated. The biocatalyst GcL, from the metallo-β-lactamase-like lactonase family, catalysed the enzymatic kinetic resolution (EKR) of homocysteine (Hcy) thiolactones with excellent enantioselectivity (E-value up to 136), yielding enantioenriched Hcy thiolactones and γ-thio-α-amino-carboxylic acids with high ees. Additionally, the biocatalyst N9 Y71G, a rationally engineered variant of the reconstructed ancestral paraoxonase enzyme N9, catalysed the dynamic kinetic resolution (DKR) of C3-thio-γ-thiolactones, yielding γ-thio-α-thio-carboxylic acids in enantioselective manner with high ees (up to >99%) and yields (up to >99%). Insights on the mechanism and the stereoselectivity of the lactonase biocatalysts were gained through computational and site-directed mutagenesis studies.

FrazP2, a cytochrome P450, catalyzes cyclohexane ring formation in forazoline A, a marine-derived antifungal from Actinomadura sp. WMMB-499. Structural and mechanistic studies have uncovered a radical-mediated cyclization, unlocking opportunities to engineer P450s for the generation of novel antifungal forazoline analogues.

Abstract

Forazoline A, produced by the marine actinomycete Actinomadura sp. WMMB-499, is a unique PK/NRP hybrid macrolactone with promising antifungal in vivo efficacy through a previously unreported mechanism. Although a PKS/NRPS gene cluster was identified as a candidate for forazoline production, the precise biosynthetic pathway and the functions of the tailoring enzymes remain unclear. In this work, the functions of three cytochrome P450 mono-oxygenases (FrazP1P2P3) were characterized. Notably, FrazP2 was found to mediate cyclohexane ring formation from an 1,3,6-triene precursor during forazoline A biosynthesis, as confirmed by genetic and biochemical analysis. To gain structural and mechanistic insight into the activity of FrazP2, the crystal structure of a FrazP2-substrate complex has been solved at 2.3 Å resolution. The molecular dynamics simulations and DFT calculations revealed an unprecedented enzyme-catalyzed oxidative cyclization reaction by FrazP2. These findings expand our understanding of the catalytic diversity of cytochrome P450s, contributing to the diversification of natural products and enabling the creation of unnatural derivatives with increased antifungal potency.

Nature Chemistry, Published online: 07 May 2025; doi:10.1038/s41557-025-01809-9

Qinglong Shi and Juntao Ye discuss the history, structure, and reactivity of flavin mononucleotide in natural and non-natural reactions.

Nature Chemistry, Published online: 06 May 2025; doi:10.1038/s41557-025-01820-0

Recent studies have shown that energy transfer photoenzymes can be engineered to promote stereocontrolled [2 + 2] cycloadditions; however, existing systems rely on ultraviolet light and display limited photochemical efficiencies. A generation of thioxanthone-containing photoenzymes now harnesses visible light to drive challenging photochemical conversions with high efficiencies and selectivities.

The pikromycin polyketide synthase (PKS) catalyzes formation of 12-membered macrolactone 10-deoxymethynolide, and 14-membered macrolactone narbonolide. Herein, we show the efficient diversification of novel 14-membered macrolactones from a series of unnatural pentaketides using the PikAIII/PikAIV PKS in vitro system. New macrocycles were further elaborated by the addition of D-desosamine and late-stage C-H hydroxylation. Molecular dynamics (MD) simulations and density functional theory (DFT) calculations were conducted to probe the reactivity and selectivity of this terminal catalytic step on the assembled unnatural macrolides. This approach demonstrates the flexibility and applicability of sequential biocatalytic steps for chemoenzymatic creation of complex antibiotic scaffolds.

Azaspiro[2.y]alkanes are increasingly valuable scaffolds in pharmaceutical drug discovery; however, an asymmetric catalytic method for their synthesis remains unknown. Here, we present a stereodivergent carbene transferase platform for the cyclo-propanation of unsaturated exocyclic N-heterocycles to provide structurally-diverse and pharmaceutically-relevant azaspi-ro[2.y]alkanes in high yield (21–>99% yield) and excellent diastereo- and enantioselectivity (dr from 52.5:47.5 to >99.5:0.5; er from 51:49 to >99.5:0.5). These engineered protoglobin-based enzymes operate on gram scale, with no organic co-solvent, at substrate concentrations up to 150 mM (25 g/L) using lyophilized E. coli lysate as the catalyst. This platform represents a practical, scalable, and stereodivergent biocatalytic synthesis of azaspiro[2.y]alkanes using low-cost engineered protoglobins and their native iron-heme cofactors.