Biocatalysis@TUDelft

By comparing the biosynthetic gene clusters for natural products with 2,4-diamino-3-hydroxybenzoic acid (2,4,3-DAHBA) moiety and heterologous expression in Streptomyces albus, we identified nine genes required for the biosynthesis of 2,4,3-DAHBA, including nitrite biosynthesis genes. Further analysis showed that three genes (nybN, nybO, and nybC) are responsible for an unprecedented aromatic amination using nitrite to synthesize 2,4,3-DAHBA.

Aromatic rings bearing amino groups provide natural products with structural diversity and potent biological activities. Although aromatic amination is a useful reaction in organic synthesis, knowledge of biological aromatic amination remains limited. In this study, we identified an unprecedented nitrite-dependent aromatic amination in nybomycin biosynthesis. By comparing biosynthetic gene clusters whose products have a diamino phenol scaffold, we hypothesized that nine genes, including two nitrite biosynthetic genes, are responsible for the biosynthesis of this scaffold. Using heterologous expression in Streptomyces albus, we identified the minimum number of enzymes required for 2,4-diamino-3-hydroxybenzoic acid (2,4,3-DAHBA) biosynthesis. Further analysis revealed that three enzymes (NybN, NybO, and NybC) were responsible for converting 3-hydroxyanthranilic acid (3-HAA) into 2,4,3-DAHBA using nitrite. In vitro assays revealed that NybO, an ATP-dependent ligase, catalyzes the diazotization of 3-HAA to form 2-diazo-3-hydroxybenzoic acid (2,3-DHBA) and that NybC, an NADPH-dependent oxidoreductase, catalyzes the reduction of 2,3-DHBA to form 2-hydrazino-3-hydroxybenzoic acid. Taken together with other experimental results, we propose two possible biosynthetic pathways for 2,4,3-DAHBA synthesis from 3-HAA. This study provides important insights into nitrite-mediated aromatic amination, expanding the availability of nitrite for natural product biosynthesis.

Resurrecting ancestral enzymes through ancestral sequence reconstruction (ASR) can unlock newcatalytic activities, offering an evolutionary-guided strategy to overcome functional specialization barriers in biocatalyst development. Furthermore, although a large body of work on imine reductases has been reported, imine reductases capable of catalyzing three types of reactions have still not been reported (sequential three-step enzymatic reactions enabled by a single enzyme). In addition, multifunctional biocatalysts streamline traditional biocascade reaction development by reducing thenumber of enzymes required and simplifying the enzyme engineering process.

Traditional biocatalytic cascades typically require discrete enzymes for each synthetic step. Here, we report unprecedented trifunctional imine reductases (IRED) that conduct three sequential transformations—alkene reduction, intramolecular reductive amination, and imine reduction—all within a single catalytic cycle. This elegant single-enzyme catalytic system directly transforms linear substrates into enantiomerically pure 2-aryl pyrrolidines via a concerted cascade without intermediate isolation. Combining density functional theory (DFT) calculations and mechanistic studies, we elucidate how the IRED achieves step-selective catalysis. Our findings establish a proof-of-concept for simplifying complex biocatalytic cascades using multifunctional enzymes, offering a powerful strategy to streamline synthetic pathways.

Nature Synthesis, Published online: 29 January 2026; doi:10.1038/s44160-025-00989-z

Macrocyclization typically proceeds via thioesterase mediation in type I polyketide synthases. Now, using genome mining and crystallographic analysis, an alternative mechanism for stereoselective macrocyclization in the akaeolide biosynthetic pathway is reported. The mechanism is proposed to proceed via an iminium-catalysed tandem Michael addition and Knoevenagel condensation, using nuclear transport factor 2-like enzymes.

Database mining using principal component analysis (PCA)-based clustering enabled discovery of bacterial enzymes capable of catalyzing stereodivergent cyclopropanation of styrene. Statistical analyses of sequence data further revealed characteristic structural properties of these enzymes, highlighting the potential of the bioinformatics tools for exploring enzyme candidates for abiotic reactions beyond the scope of natural biological function.

ABSTRACT

Protein engineering is a practical approach to providing enzymes with an “abiotic” catalytic activity. However, it remains difficult to explore the full diversity of natural sequence space through the engineering of a single specific protein. As an alternative to these protein engineering approaches, we here demonstrate a database mining approach using a principal component analysis (PCA)-based clustering method to facilitate the identification of promising enzyme candidates. As a proof of concept, we applied this method to the cyclopropanation of styrene, and the sequence space of bacterial globins in the database was extensively investigated. By screening 275 globins from 171 different organisms, we successfully discovered enzymes capable of catalyzing stereodivergent carbene transfer reactions. Furthermore, statistical analyses of sequence data allowed us to detect characteristic structural properties of these globins, which determine the unique stereoselectivity of cyclopropanation. While these bioinformatics tools have primarily been applied to predict enzymes’ natural biological functions, this study demonstrates their applicability to exploring enzyme candidates for abiotic reactions unrelated to their native biological activity. Given the increasing interest in biocatalytic applications beyond natural reactivity, this PCA-based mining approach provides a promising direction for expanding the functional diversity of biocatalysts.

Proceedings of the National Academy of Sciences, Volume 123, Issue 4, January 2026.
SignificanceCbl-dependent radical SAM methylases (RSMs) use two SAM molecules to methylate unactivated carbons. One SAM is used to methylate cob(I)alamin, generating methylcobalamin (MeCbl). A second SAM molecule is used to generate a 5′-deoxyadenosyl ...

Cyclopeptide alkaloids are an expanding class of plant peptide natural products defined by a macrocyclic ether-crosslink via a tyrosine-derived phenol. Classical cyclopeptide alkaloids are characterized by strained 13- to 15-membered cyclophanes and terminal modifications such as N-methylation and C-terminal styrylamine moieties. While synthetic access to many classical cyclopeptide alkaloids has been established, no biosynthetic route has been reported. Here, we elucidate the biosynthetic pathway of a 14-membered cyclopeptide alkaloid, lotusine A, from Chinese date tree (Ziziphus jujuba) which features peptide cyclization on a ribosomal precursor peptide by a split burpitide cyclase, non-heme-iron and 2-oxoglutarate-dependent oxidative decarboxylation affording the C-terminal hydroxystyrylamine, and SAM-dependent N-terminal -N,N-dimethylation. We apply discovered Z. jujuba enzymes in combination with a clubmoss cyclopeptide alkaloid cyclase for biosynthesis and diversification of analgesic adouetine X and anxiolytic sanjoinine A by combining in planta and in vitro reactions. Our work expands the biocatalytic repertoire of non-heme-iron- and 2-oxoglutarate-dependent enzymology to oxidative peptide decarboxylation and primes scaled metabolic engineering and chemoenzymatic synthesis of 14-membered cyclopeptide alkaloids with terminal posttranslational modifications.

Harnessing transient, unstabilized alkyl radical intermediates for the enantioselective construction of valueadded chemical entities remains a fundamental challenge in biocatalysis. Through the repurposing and directed evolution of pyridoxal phosphate (PLP)-dependent tryptophan synthases, we advanced an open-shell enzyme platform capable of intercepting transient alkyl radicals for the efficient and enantioselective synthesis of aliphatic non-canonical amino acids. Engineering an orthogonal pair of radical PLP enzymes allowed unstabilized alkyl radicals, generated from diverse aliphatic organoboronates, to undergo dehydroxylative C(sp3)-C(sp3) coupling with a common L-serine donor, affording either L- or D-amino acids with excellent enantiopurity in an enzyme-controlled fashion. Mechanistic and computational investigations employing radical clock substrates and unusual radical-mediated rearrangement processes revealed that the radical intermediates generated in this system exhibit unexpectedly long lifetimes, highlighting the power of this dual enzyme-photocatalyst platform to engage unactivated alkyl radicals. Collectively, these findings delineate a potentially general strategy for generating and utilizing unstabilized alkyl radicals and underscore the synthetic potential of radical pyridoxal biocatalysts for stereodivergent amino acid construction.

Sulfur oxidation of β-vinyl sulfides into β-vinyl sulfones activates the C═C bond for ene-reductase ENE-101 catalysis, enabling highly enantioselective formation of C(sp³)–S(VI) stereocentres (up to >99% ee). Sulfur oxidation enhances alkene electrophilicity and optimizes substrate–enzyme interactions, providing a general strategy to expand the catalytic scope of ene-reductase enzymes in the stereoselective C–S bond construction.

ABSTRACT

The enantioselective construction of C(sp³)─S stereocentres remains a major challenge in catalysis due to the distinct electronic and steric features of sulfur, compared to oxygen or nitrogen atoms, which complicate both stereocontrol and configurational stability at the C─S bond. Here, we report that the ene-reductase biocatalyst ENE-101 catalyses the highly enantioselective reduction of β-vinyl sulfones, enabling the direct formation of C(sp³)–S(VI) stereocentres in excellent yields and enantiomeric excesses (up to >99% ee). Whereas the corresponding β-vinyl sulfides are unreactive towards ENE-101, the S(II) to S(VI) oxidation activates the C═C bond toward enzymatic reduction. Computational studies reveal that the sulfone moiety enhances alkene electrophilicity and promotes favourable substrate orientation and binding within the ENE-101 active site. The biocatalyst exhibits broad substrate scope, tolerating diverse β-vinyl sulfones. This work establishes sulfur(VI) activation as an effective strategy to expand the reactivity landscape of ene-reductase biocatalysts for the asymmetric C─S bond formation.

A set of β-ketosulfides has been selectively reduced to optically active β-hydroxysulfides employing KREDs as biocatalysts in the presence of Deep Eutectic Solvents. By appropriately selecting both the biocatalyst and the reaction medium, both alcohol enantiomers can be obtained in high yields and optical purities. The use of the DES ChCl:Gly (1:2) at 30% v/v generally provided superior results in terms of activity and/or selectivity.

ABSTRACT

The β-hydroxysulfide motif is present in both natural and synthetic compounds with notable bioactivities. Herein, the synthesis of a set of optically active (R)- and (S)-β-hydroxysulfides starting from β-ketosulfides using ketoreductases (KREDs) in nonconventional media aqueous buffer/deep eutectic solvents (DESs) has been developed. Several Type III DESs have been tested as cosolvents, being observed for most of the biotransformations higher conversions and enantiomeric excesses in the presence of some glycerol- or ethylenglycol-based DESs. Bioreductions can be performed up to 70% v/v of DESs with good conversions, demonstrating the high performance of these nonconventional media in biocatalyzed reductive processes. Additionally, it was observed that the lipase CalB partially retained activity in DES-containing media for the hydrolysis of β-alkylsulfide enol esters, thus being possible to develop a one-pot, two-enzyme cascade that combined CaalB-catalyzed hydrolysis of a β-alkylsulfide enol ester with the subsequent KRED reduction of the β-ketosulfide obtained to the (R)-β-hydroxysulfide in both sequential and concurrent ways, affording high stereoselectivity.

Monoalkylated Cu(II) oxacyclen complexes show a dual DNA effect: shorter alkyl chains enable ROS-mediated DNA cleavage, whereas longer chains cause strong DNA condensation/aggregation. Thus, chain length controls activity. Using circular dichroism, UV/visible and fluorescence spectroscopy, atomic force microscopy, dynamic light scattering, and molecular dynamics, we demonstrate that amphiphilic Cu(II) complexes are promising DNA modulators.

Cu(II) complexes with monoalkylated oxacyclen ligands (C₁₂, C₁₆, and C₁₈) have been investigated regarding their interaction with DNA by different methods: circular dichroism, UV/VIS (ultraviolet-visible) and fluorescence spectroscopy as well as by gel electrophoresis. The results demonstrate that the complexes can cleave DNA through both hydrolytic and oxidative mechanisms, with hydroxyl radicals and hydrogen peroxide identified as the reactive oxygen species involved. The targeted incorporation of alkyl chains significantly enhances the DNA-binding affinity of the Cu(II) complexes, and the length of the alkyl substituents plays an important role, as they can interact with the major groove of the DNA. Alkylation is the determining structural factor responsible for the enhanced DNA interaction, since such an interaction is not observed with unsubstituted complexes. Moreover, the length of the alkyl chains significantly influences this behavior, as longer substituents induce a concentration-dependent DNA aggregation, a phenomenon absent in the nonalkylated analog. This aggregation and condensation behavior is examined using atomic force microscopy and dynamic light scattering. Moreover, DNA/small molecule interactions are also investigated using molecular dynamics simulations.

Steroid hormones are key signaling molecules regulating growth, metabolism, reproduction, and stress adaptation and are widely used as essential pharmaceuticals. Traditional production from sterol feedstocks through multistep chemical or microbial transformations is limited by inefficiency and scalability. Recent advances in synthetic biotechnology enable de novo biosynthesis of steroid hormones from simple carbon sources in yeasts and fungi. This review highlights metabolic rewiring to increase flux, cytochrome P450 enzyme engineering for side-chain cleavage, and hydroxylation to overcome rate-limiting bottlenecks of steroid hormone biosynthesis. We also discuss strategies to redesign steroid-transport pathways to alleviate intracellular accumulation and improve membrane export. Looking ahead, we envision integrating metabolic, enzyme, and transport engineering to build a scalable, data-driven ‘intelligent’ platform for sustainable steroid hormone biomanufacturing.

Nature Communications, Published online: 23 January 2026; doi:10.1038/s41467-026-68816-3

Securinega alkaloids, comprising a distinctive tetracyclic scaffold, have long been studied, but their biosynthesis has remained largely unknown. Here, the authors employ chemical insights with single-cell transcriptomics to reveal key biosynthetic steps of securinega alkaloids in Flueggea suffruticosa.

Nature Catalysis, Published online: 23 January 2026; doi:10.1038/s41929-025-01470-y

Light-driven enzymatic catalysis has enabled important abiological transformations in vitro. Now a cellular ene-reductase photoenzyme is integrated with a de novo-designed olefin biosynthetic pathway for photoinduced hydroalkylation, hydroamination and hydrosulfonylation reactions within cells.

Asymmetric hydrogen-atom transfer (HAT) is challenging due to early, weakly organized transition states that lead to small energy differences and competing racemic pathways. This mini-review is intended to provide a mechanistically unified framework for asymmetric HAT by classifying strategies into five regimes according to where enantioselection is set: donation-controlled termination, radical-centered control, abstraction-controlled HAT, cooperative bimetallic catalysis, and enzyme-mediated HAT.

Abstract

Hydrogen-atom transfer (HAT) lies at the heart of radical chemistry, yet asymmetric HAT has been difficult because the high reactivity of radicals often forces H-transfer to proceed through early, weakly organized transition states, yielding small ΔΔG^‡ and allowing rapid racemic background pathways to compete. Recent advances across small-molecule, metalloradical, cooperative, peptide, and enzymatic catalysis show that high enantioselectivity is attainable when the catalyst is engineered to exert stereocontrol precisely at the H-transfer step that sets configuration. In this minireview, we organize asymmetric HAT into five regimes—donation-controlled termination, radical-centered control, abstraction-controlled HAT, cooperative bimetallic catalysis, and enzyme-mediated HAT—each specified by where chiral information is introduced during H-transfer. Through representative cases, we illustrate how catalysts achieve enantioselection by defining radical geometry, guiding H-delivery, enforcing selective hydrogen abstraction, or confining donor–acceptor pairs within organized chiral environments. This mechanistic framework provides a unified lens spanning synthetic and biocatalytic systems, clarifies the distinct stereochemical logics in each regime, and highlights emerging opportunities for expanding asymmetric radical chemistry through precisely orchestrated H-atom transfer.

YUCCA enzymes are well known to catalyze the main step of auxin biosynthesis in plants. Here, a hitherto undescribed dual function was discovered, revealing that some YUCCAs also act in chlorophyll degradation. In vitro feedback regulation furthermore suggests a link between chlorophyll degradation and hormone homeostasis and a physiological role of accumulating chlorophyll catabolites for plant senescence.

Abstract

Chlorophyll (Chl) metabolism is pivotal to both photosynthesis and plant senescence and represents one of the most fundamental biological processes on Earth with an estimated annual turnover of 1 billion tons. During Chl degradation, only early catabolites and corresponding enzymes are well characterized, whereas for late-stage degradation products it remains often unclear if their formation involves specific enzymes. Here, we report that the ubiquitous YUCCA10 enzymes from the YUCCA flavin-containing monooxygenase (FMOs) family in land plants, normally implicated in the biosynthesis of indole-3-acetic acid (IAA) as the primary form of auxin, surprisingly catalyze the production of several predominant Chl catabolites via mechanistically distinct Baeyer–Villiger oxidation and subsequent hydrolytic γ-lactam-forming deformylation reactions. These historically postulated but hitherto undiscovered Chl degradation steps on several high molecular weight chl catabolites were verified for YUCCA10 from Vitis vinifera and Coffea arabica, while YUCCA10 from Arabidopsis thaliana lacked this activity. In contrast, all three homologs were able to catalyze the rate-limiting key step in IAA biosynthesis, akin to other YUCCA enzymes. Interestingly, Chl catabolites at physiological concentrations impaired IAA formation by YUCCA10 in vitro, suggesting a key role in leaf senescence through enzymatic feedback regulation of auxin levels.