Biocatalysis@TUDelft

A diazo-forming enzyme (stFur5) facilitates deamination in the biosynthesis of furaquinocins and other polyketide-derived meroterpenoids.

Meroterpenoids are known for their distinct structure and hybrid biosynthetic origin. The biosynthetic gene clusters of several well-characterized meroterpenoids contain three genes whose functions have remained elusive. Recent studies on nonmeroterpenoid pathways suggest that these genes may be involved in nitrite-dependent NN bond formation. In this study, it is shown that one of these genes, stfur5, is essential for the biosynthesis of the representative meroterpenoid furaquinocin M. By leveraging a cell-free protein synthesis platform, it is found that stFur5 catalyzes the transformation of 8-amino-flaviolin (8-AF) into diazo-flaviolin, which subsequently undergoes nonenzymatic deamination to yield the downstream intermediate flaviolin. The findings suggest that stFur5, together with the nitrite-generating enzymes stFur15 and stFur16, facilitates the deamination of 8-AF via diazotization in furaquinocin biosynthesis. We further identified the nitrite-binding pocket within stFur5 and proposed a catalytic mechanism in which nitrite is activated through adenylation. The findings enrich the understanding of the role of diazo-forming enzymes in natural product biosynthesis.

2-oxoglutarate (2OG) analogs are rationally designed to elucidate structure–activity relationships (SARs) in 2OG-dependent nonheme iron (NHFe) enzymes. These analogs are tested against prolyl hydroxylase domain 2 (PHD2). Activity assays reveal that certain analogs compete with 2OG for the PHD2 active site. Computational and mutagenesis studies reveal crucial binding residues, demonstrating the utility of these analogs to derive SARs.

2-oxoglutarate (2OG)-dependent nonheme iron (NHFe) enzymes constitute a family of enzymes that use 2OG and oxygen to hydroxylate unactivated C(sp ³)–H bonds. These enzymes are biologically important and therapeutically relevant due to their role in key cellular processes. However, selective targeting remains challenging due to high structural conservation in their active sites. Herein, two classes of 2OG analogs are rationally designed and used as tools to investigate the active site of a 2OG-dependent NHFe enzyme, prolyl hydroxylase domain 2 (PHD2). Using an activity assay in conjunction with steady-state kinetics, a new class of aryl-conjugated 2OG analogs is identified that exhibits 12-fold varied inhibition and competes with 2OG for the PHD2 active site. Immunoblot studies suggest that these analogs are biologically active and can target PHD2 intracellularly. Furthermore, computational modeling studies reveal that the analogs bind to the active site in a “flipped” conformation relative to 2OG, and functional group placement is responsible for their different inhibition capabilities. Mutagenesis studies further validate this unique binding mode and suggest several interactions that are crucial for inhibition. Overall, these studies provide a toolkit of 2OG analogs to establish structure–activity relationships and identify interactions that can be useful for PHD2 inhibitor design.

This perspective outlines the required steps to translate laboratory biocatalytic reactions into laboratory processes, with the required performance metrics prior to scaling for pilot testing. A range of tools are presented with the aim of using enzyme and process engineering synergistically.

Abstract

The extraordinary developments in protein and enzyme engineering today are giving new opportunities for biocatalysis, including the synthesis of pharmaceuticals with new modalities, as well as new synthetic cascades and even the conversion of waste materials. Nevertheless, a significant proportion of these enzyme-catalyzed reactions remain in the laboratory as a curiosity rather than being implemented at a larger scale. One clear reason for this is the need for design and scale-up guidelines, as widely used in more conventional chemical processing. In this perspective, some of the tools and technologies available to enable such implementation for enzyme-catalyzed processes are outlined.

Bifidobacterium longum subsp. infantis converts tryptophan into indole-3-lactate, indole-3-acetate and indole-3-carboxaldehyde. Production is substrate-dependent, with higher tryptophan levels enhancing yields. This study reveals metabolic modulation in nutrient-rich and resting states, emphasising B. infantis' role in producing beneficial indole derivatives for gut health.

ABSTRACT

The metabolic processes of Bifidobacterium longum subsp. infantis, an early coloniser of the human gut, are essential for gut health, mainly due to the production of indole derivatives from tryptophan. This study investigates the capacity of B. infantis ATCC 15697 to biosynthesise indole-3-lactate (ILA), indole-3-acetate (IAA), and indole-3-carboxaldehyde (I3CA) and the regulatory effects of substrate availability on these pathways. The tryptophan catabolic profile of B. infantis ATCC 15697 under a non-growing but metabolically active state was investigated. Through HPLC-PDA and LC–MS analyses, we confirmed for the first time the production of IAA and I3CA by B. infantis ATCC 15697. The results revealed a dose-dependent relationship between tryptophan availability and the production of indole derivatives, highlighting the nutrient-driven effect of these metabolic pathways. By integrating genomic analysis with metabolic profiles, we proposed potential pathways underlying the biosynthesis of IAA and I3CA from tryptophan. These findings enhance our understanding of the role of B. infantis ATCC 15697 in human health, with ILA, IAA, and I3CA contributing to immune modulation and gut health. We also provide a platform for using B. infantis ATCC 15697 as a biocatalyst for the biosynthesis of beneficial indole derivatives through whole-cell bioconversion, which was further demonstrated in B. infantis ATCC 25962 and ATCC 15702. Future in vivo studies will help clarify the impact of these metabolites on the gut environment and inform dietary and probiotic strategies for enhancing indole derivatives production.

In this work, we aimed to enhance the hydroxylase activity of PhzO by heterologous expression of the flavin reductase Fre in Pseudomonas chlororaphis. This strategy not only improved PhzO's catalytic efficiency but also unexpectedly led to the discovery of a novel dihydroxylated derivative, 3,4-dihydroxy-phenazine-1-carboxylic acid. In vitro assays further verified that PhzO exhibits FAD-dependent catalytic promiscuity, simultaneously generating 2-OH-PCA and 3,4-OH-PCA. Furthermore, in vitro and in vivo assays demonstrated that substrate concentration affected the distribution of products.

ABSTRACT

Phenazines are bioactive secondary metabolites with antifungal, anticancer, and insecticidal properties, while hydroxylated derivatives often exhibit enhanced bioactivity. 2-hydroxyphenazine (2-OH-PHZ), which is synthesised by the flavin-dependent monooxygenase PhzO from phenazine-1-carboxylic acid (PCA), shows better bioactivity against the pathogenic fungus Gaeumannomyces graminis vars. tritici. However, the low catalytic efficiency (10%–20% conversion) of PhzO limited 2-OH-PHZ production. To boost PhzO activity, engineering flavin reductase (Fre)-mediated FADH₂ regeneration was applied to Pseudomonas chlororaphis LX24AE. Remarkably, this approach improved catalytic efficiency from 25% to 40% and increased the production of a novel dihydroxylated derivative. Then, it was first characterised by UPLC-MS and NMR, and identified as 3,4-dihydroxyphenazine-1-carboxylic acid (3,4-OH-PCA). Next, the Fre-PhzO module through heterologous co-expression in P. putida KT2440 demonstrated a 4.5-fold enhancement in hydroxylation efficiency relative to the PhzO mono-component system, which also confirmed that PhzO and flavin reductase are essential for 3,4-OH-PCA biosynthesis. Moreover, in vitro assays further verified that PhzO exhibits FAD-dependent catalytic promiscuity, simultaneously generating 2-OH-PCA and 3,4-OH-PCA. Furthermore, in vitro and in vivo assays demonstrated that substrate concentration affected the distribution of products. Finally, cytotoxicity evaluation of the isolated 3,4-OH-PCA was performed, and it showed substantial inhibition against oesophageal cancer TE-1 cells and human cervical cancer HeLa cells with an IC₅₀ value of 8.55 μM and 17.69 μM, respectively. This work redefines PhzO as a promiscuous biocatalyst capable of dual hydroxylation, offering a modular platform for engineering bioactive phenazine derivatives.

Science Advances, Volume 11, Issue 25, June 2025.

Thermophilic anaerobic organisms, particularly species that can naturally degrade lignocellulosic biomass, show great promise for next generation bioprocessing. This has led to the development of nascent genetic systems to metabolically engineer these non-model organisms. However, a major challenge remains a lack of reliable reporter systems compatible with the combination of thermophilic and anaerobic growth conditions. Additionally, native glycoside hydrolases in these organisms limit the usefulness of traditional glycosidic enzyme reporters (e.g. LacZ) because of the native background activity present on para-nitrophenyl glucoside substrates. Here we describe the development of a straightforward and robust enzymatic reporter system that overcomes these challenges in Anaerocellum (f. Caldicellulosiruptor) bescii, an anaerobic, extremely thermophilic (Topt [~]78 {degrees}C), lignocellulolytic bacterium. Our method is based on heterologous expression of hyperthermophilic archaeal galactosidases: an -galactosidase from Pyroccous furiosus (Pfgal), and a {beta}-galactosidase from Caldivirga maquilingensis (Cm{beta}gal). We show that these reporters produce strong, orthogonal signals on colorimetric substrates at high temperatures ([≥]90{degrees}C) that eliminate background activity from endogenous galactosidases. We then demonstrate the capability of Cm{beta}gal, the stronger of the two reporters, to distinguish differences in levels of expression between A. bescii promoter sequences, which we verify through qRT-PCR. With its high signal to noise ratio and ease of use, this reporter system offers a reliable method for assessing protein expression in anaerobic thermophilic organisms, opening doors to improved genetic tools and metabolic engineering applications for industrial biotechnology.

Cytochrome bd is a terminal oxidase expressed under low oxygen conditions and central for the survival of many pathogens. Here we characterise the first qOR-2 type bd oxidase, the cyt bd-II from Mycobacterium smegmatis, by combining biochemical studies with cryo-electron microscopy (cryo-EM), and multiscale simulations. By over-expressing the appCB operon in its native host, we produce a highly active bd-II (kcat=30 e-s-1) that together with a high-resolution (2.8 [A]) cryo-EM structure and multiscale simulations reveal unique proton pathways and oxygen channels responsible for its function. We propose that O2-scavenging activates a pH-dependent molecular switch, involving coordination changes of heme d and surrounding bulky residues that regulate substrate access into the active site. Taken together, our findings provide detailed mechanistic insight of qOR-2 type bd oxidases, and a basis for understanding the evolution of the superfamily.

Nature Chemistry, Published online: 23 June 2025; doi:10.1038/s41557-025-01862-4

Chemists first synthesized acylsulfenic acids in the 1990s, but natural products containing this labile moiety have so far not been isolated. The identification of a new natural product, sulfenicin, and the characterization of its biosynthesis now suggest this functional group is widespread among bacteria, hinting at an undiscovered subclass of natural products.

Nature Chemistry, Published online: 23 June 2025; doi:10.1038/s41557-025-01863-3

Synthetic and biological chemistry are traditionally seen as separate fields. Now, a biocompatible chemical reaction enables an engineered microbe to convert plastic waste into valuable compounds under mild, cell-friendly conditions.

The integration between visible-light photocatalysis and biocatalysis has the potential to greatly improve modern organic synthesis by combining the production of valuable synthetic intermediates under mild photochemical conditions with the high degree of stereoselectivity reached by enzymes. The review collects the recent efforts in the photoenzymatic asymmetric synthesis of alcohols and amines.

The stereocontrolled formation of CO and CN bonds is important for the preparation of agrochemicals, pharmaceuticals, fragrances, and flavors. In this field, enzymes have become a valid alternative to transition metal complexes and organocatalysts due to their high degree of stereoselectivity and desirable sustainability features. In this scenario, the increasing interest toward sustainable and efficient chemical processes has also led to the combination of complementary catalytic systems. The merge of biocatalysis and photocatalysis is among the most recent approaches. While photocatalysis allows the production of valuable synthetic intermediates and reactive species under mild conditions, biocatalysis exploits highly specific enzymes to catalyze reactions with high (stereo)-selectivity and minimal byproduct formation. This review summarizes the progress in photoenzymatic catalysis (also photobiocatalysis), emphasizing their complementary mechanisms in producing chiral alcohols and amines, and highlights how such integration not only enhances the sustainability of catalytic systems but also expands the scope of reactions accessible under milder and ultimately more sustainable conditions.

The catalytic M-clusters of nitrogenase enzymes exhibit characteristic electrostatic patterns around the sites involved in N₂ fixation. Specifically, a strong local electric field pointing away from the Fe2 site is consistently identified, as well as a minor field pointing toward the Fe6 sites. Furthermore, a significant long-range field along the Fe2–Fe6 axis is computed. These patterns may significantly impact the chemical reactivity of these clusters.

Abstract

Nitrogen fixation is a fundamental, and yet challenging, chemical transformation due to the intrinsic inertness of dinitrogen. Whereas industrial ammonia synthesis relies on the energy-intensive Haber–Bosch process, nitrogenase enzymes achieve this transformation under ambient conditions—yet at the expense of a remarkably high ATP demand. Understanding their mode of operation could inspire the development of more efficient synthetic catalysts. In this study, we scrutinize the electrostatic environment surrounding nitrogenase's active site, the so-called M-cluster. Strikingly, we observe that all types of M-clusters exhibit similar trends, with distinct patterns around the individual metal sites that have been proposed as potential N₂-coordination sites. Specifically, a strong local electric field pointing away from the Fe2 site is identified, as well as a minor field pointing toward the Fe6 sites. Furthermore, a significant oriented long-range field along the Fe2–Fe6 axis is computed across the entire family of nitrogenases. In the final part of the manuscript, we discuss how the observed electrostatic patterns may impact chemical reactivity, and how they can be connected to previously made mechanistic hypotheses. Overall, this study provides further evidence for the ubiquitousness of local electric fields in enzyme catalysis, even when substrates that seemingly have only limited electrostatic susceptibility are involved.

Backbone Thz moieties prevail in bioactive peptidic natural products and play important roles in their biological functions. Here, we report an in vitro selection platform for Thz-containing macrocyclic peptides, established through a posttranslational chemoenzymatic transformation. This study establishes a robust system to expedite ligand development in pseudo-natural peptide discovery.

Abstract

Backbone thiazole (Thz) moieties prevail in bioactive peptidic natural products and play important roles in their biological functions. However, the de novo discovery of artificial Thz-containing peptide ligands remains challenging. Here, we report an mRNA display-based selection platform for Thz-containing macrocyclic peptides (^ThzteMP), established through a dedicated posttranslational chemoenzymatic transformation. This method exploits the unique reactivity of ribosomally incorporated thioamides, enabling enzyme-free spontaneous heterocyclization to form thiazoline (Thn), which is further oxidized using the substrate-tolerant azoline dehydrogenase (GodE) to yield a Thz moiety. By integrating this chemoenzymatic process with chloroacetyl-mediated thioether macrocyclization and mRNA display, we have successfully discovered Thz-containing macrocyclic peptide ligands with high binding affinities against p21-activated kinase 4 (PAK4). This study establishes a robust system to expedite ligand discovery of pseudo-natural peptides and to investigate the functional benefit of their backbone Thzs.

Enzymatic synthesis of the iodo-thiobenzoate of calicheamicin γ₁ ^I was reconstituted in vitro from malonyl-CoA, involving the orsellinate biosynthesis by CalO5 and subsequent modifications by CalO6, CalO3, CalO2, and CalO1. A TEV site was introduced into CalO5, enabling the successful detection of the intermediate or product attached to the ACP domain through intact-protein mass spectrometric analysis.

Abstract

Flavin-dependent halogenases (FDHs) play important roles in natural product biosynthesis, particularly chlorinases and brominases. Apart from ubiquitous mammalian iodination in thyroxine biosynthesis, iodinated natural products are extremely rare, and enzymes responsible for iodination are even less well described. A notable exception is calicheamicin γ₁ ^I, a potent antitumor compound containing an aromatic iodide, for which a halogenase has been proposed to mediate iodine incorporation. Despite predictions regarding the enzymes involved in iodinated aryl ring biosynthesis, experimental evidence remains limited due to challenges in substrate identification and reaction monitoring. In this study, we successfully reconstituted the enzymatic activities required for iodination and all embellishments of the highly substituted benzene ring in calicheamicin γ₁ ^I. Using intact-protein mass spectrometry combined with protease cleavage, we demonstrated formation of the orsellinate thioester followed by sequential C-2 O-methylation, C-5 iodination, C-3 oxidation, and C-3 O-methylation. This research characterizes the first flavin-dependent iodinase that acts on a carrier protein-dependent substrate, identifies the natural substrates of a cytochrome P450 oxygenase and two O-methyltransferases, and provides valuable insights for biocatalysis. Additionally, these findings could facilitate the engineering of other polyketide biosynthetic pathways and contribute to optimizing fermentation conditions to generate new calicheamicin derivatives.

Enzymes are powerful catalysts. Unfortunately, computational enzyme design remains challenging. In their Research Article, H. Adrian Bunzel and co-workers develop AI.zymes (e202507031), a modular platform integrating state-of-the-art computational tools through evolutionary design. AI.zymes boost enzymes by optimizing biocatalytic features such as transition-state binding, protein stability, and electrostatic catalysis. Its modular architecture will facilitate the integration of emerging design algorithms and enable addressing diverse design challenges.

Developing unnatural functions and optimizing catalytic properties of enzymes are challenging tasks that require extensive engineering and screening of biocatalysts. In this study, we report the first example of enantiocomplementary intramolecular crossed aldehyde-ketone benzoin cyclizations catalyzed by benzaldehyde lyase. Benefiting from the utilization of structure prediction and molecular simulations, we efficiently designed and optimized enzyme mutants capable of achieving this novel catalytic function with high yields (up to 97%) and excellent enantioselectivity (up to 99:1 e.r).

In the search for reliable biomarker in other planetary environments such as Mars, we need molecules that are stable over geological timescales while preserving indicative information about their potential biological origin. Thiophene-bearing quinones fulfill these requirements, and thiophenes, which are their basic moieties, have been found on Mars. However, thiophenes can be produced abiotically and have been found in meteorites. Furthermore, the Martian environment may alter their molecular structure over time. To evaluate whether there could be a distinction between biotically and abiotically produced thiohenes considering the harsh environmental conditions on Mars, we cultivated the extremophilic archaeon Acidianus manzaensis on ESA01-E Mars analog material. We then exposed the cell-mineral material to one month of desiccation and Mars-like conditions in a Mars simulation chamber to analyze changes in the composition of thiophene-bearing quinones using mass spectrometry-based metabolomics, and evaluated their potential for cell recovery after exposure to extreme conditions. Recultivations after one month of desiccation and two weeks of exposure to Mars-like conditions was successful, proving the durability of the organism and its potential for cell recovery. Analysis of their thiophene-bearing quinone composition showed a heterogeneous distribution of possible oxidation states used as an adaptation to environmental stressors. While additional analysis of the headgroup moieties might clarify the traces of biologically produced thiophene remnants, this study showed the durability of Acidianus manzaensis to recover after exposure to extreme conditions, paving the way for further investigation.

Fish protein hydrolysates hold great promise as nutraceuticals, yet their application as food ingredients or nutraceuticals is currently limited by their fish-like odor. This odor is mainly due to the presence of trimethylamine (TMA), a volatile biogenic amine resulting from the breakdown of naturally occurring trimethylamine-N-oxide (TMAO) in marine fish. The bacterial trimethylamine monooxygenase mFMO can oxidize TMA into TMAO using molecular oxygen and the cofactor nicotinamide adenine dinucleotide phosphate (NADPH). We have established an enzyme cascade which takes advantage of glucose dehydrogenase to recycle NADPH from NADP+, significantly decreasing the cost of the reaction and paving the way for using the enzyme system in fish protein hydrolysates targeted for human consumption. We demonstrate that the dual enzyme system works in an industrially relevant substrate. Salmon protein hydrolysate treated with an mFMO/glucose dehydrogenase cocktail showed a 75% reduction in TMA. A trained sensory panel perceived an improved odor across several parameters, including a reduction in the characteristic TMA smell.

Enzymes play a pivotal role in "green chemistry" as tools for biocatalysis. Oxidoreductase enzymes are especially useful for carrying out key electron transfer (redox) steps towards a wide range of chemical transformations (e.g., asymmetric hydrogenation, oxygenation, hydroxylation, epoxidation, or Baeyer-Villiger oxidation) that might not otherwise be available to chemists through conventional (nonbiological) synthetic approaches. The ability to screen oxidoreductase activity is important in identifying useful biocatalysts from nature, and also towards engineering novel ones through directed evolution. Many valuable redox enzymes are dependent upon NAD(P)H as an electron donating co-substrate (or conversely, upon NAD(P)+ as an electron acceptor), and the common method to detect their activity is to monitor the change in absorbance at 340 nm as NAD(P)H is converted to NAD(P)+ (or vice versa). The limited sensitivity of this method presents a challenge in detecting very low levels of oxidoreductase activity, and this can prove very difficult to begin engineering enzymes as improved biocatalysts when the rates of natural enzymes may be slow for a desired redox reaction. Herein, we report a fluorescence-based, enzyme cascade-coupled system that we have developed to detect oxidoreductase activity with orders of magnitude more sensitivity than conventional absorbance-based assays. While recycling NAD(P)H from NAD(P)+, the coupled enzyme cascade triggers cleavage of a fluorogenically labeled probe, releasing a strong fluorescent signal. This allows detection of very low levels of a specific oxidoreductase activity that we may wish to magnify by directed evolution using our assay in high-throughput screening.

PsiK from Psilocybe cubensis phosphorylates a diverse set of substituted phenols and benzenediols beyond its native substrate. High-throughput screening of active site mutant libraries using DESI-MS enabled protein engineering of PsiK, further expanding its substrate scope. The scalability of PsiK-catalyzed reactions was demonstrated, further highlighting its biocatalytic utility for selective phosphorylation.

Abstract

Phosphorylation plays important roles in biology by modulating the structure, reactivity, and biological function of a broad range of molecules. Biocatalytic phosphorylation has attracted attention from synthetic chemists due to its selectivity and mild reaction conditions using ATP as a phosphate donor. Given the potential synthetic utility of kinases with activity on small molecule substrates, we explored the activity of PsiK, the enzyme responsible for selective 4-O-phosphorylation of 4-hydroxytryptamine or psilocin in psylocybin biosynthesis by Psilocybe cubensis. We find that PsiK has good activity on a range of substituted phenols and benzenediols beyond its native substrate, enabling preparative phosphorylation of different substrates, and substantially expands the substrate scope of biocatalytic phosphorylation. We also show that active site mutations can further expand substrate scope and improve site-selectivity. This engineering effort was greatly expedited using DESI-MS screening, which enabled analysis of 2688 reactions in only 40 min. Finally, gram-scale phosphorylation of a representative substrate was achieved with a turnover number over 10 000. Together, these results highlight the biocatalytic utility of PsiK and derivatives thereof for selective phosphorylation of phenols and benzenediols under mild conditions.

When cellular iron is low, bacterioferritin (Bfr) releases its stored iron through a reductive process dependent on the [2Fe–2S] ferredoxin Bfd. Why accessing stored iron should depend on an iron-requiring protein when iron is scarce is unclear. Here, we show that the Bfd [2Fe–2S] cluster is sensitive to oxidative stress, suggesting that it may act as a “biological fuse”, shutting down iron release under conditions where free iron is toxic.

Abstract

Iron is in an essential micronutrient in living systems. However, it is also potentially toxic and so the concentration of chelatable iron within cells is tightly regulated to prevent catalytic formation of harmful reactive oxygen species (ROS). Ferritins play a key role in iron homeostasis by storing excess iron as an insoluble ferric mineral within the protein. When bacterial cells become iron deficient, this store may be accessed by reduction/solubilisation of the iron. Bacterioferritins utilise heme, bound at an inter-subunit site, to support electron transfer to the stored mineral. Electrons for heme reduction are shuttled from NADPH via Bfd, a [2Fe–2S] cluster-containing ferredoxin. This raises the paradox that the synthesis of an iron-dependent protein co-factor is required under conditions of iron-deficiency so that stored iron can be utilised. Here, we show that exposure of Bfd to ROS suppresses the capacity of the protein to stimulate iron release from bacterioferritin. We propose that reliance of iron release on Bfd evolved to ensure that chelatable iron levels do not increase under oxidative stress conditions. Thus, the Bfd iron–sulfur cluster functions as a “biological fuse” in providing a fail-safe that immediately halts iron release once ROS accumulate to damaging concentrations.