Biocatalysis@TUDelft

Unspecific peroxygenases (UPOs) are shown to perform nitrene chemistry, utilizing the inexpensive commodity chemical hydroxylamine as cosubstrate. 1,2,3,4-tetrahydronaphthalene is investigated as model substrate using a diverse panel of UPOs, reaching turnover numbers up to 745 and enantiomeric excess values up to 62%. Overall, the first account of UPO activity regarding non-natural nitrene transfer reactivities is reported.

Unspecific peroxygenases (UPOs) perform challenging oxyfunctionalization chemistry with high catalytic efficiency and reaction stability. In this report, nitrene chemistry to the current repertoire of UPO chemistry is added, utilizing the inexpensive commodity chemical hydroxylamine as cosubstrate. 1,2,3,4-tetrahydronaphthalene is investigated as model substrate using a diverse panel of UPOs, reaching turnover numbers up to 745 and enantiomeric excess values up to 62%. Overall, the first account of UPO activity regarding non-natural nitrene activities is reported.

3-Halochromones are useful intermediates that enable access to bioactive chromone derivatives. A chemoenzymatic method is reported to synthesize 3-halochromones catalyzed by an engineered vanadium-dependent chloroperoxidase from Curvularia inaequalis. The reactions are performed in an aqueous buffer containing a surfactant TPGS-750-M and 10% acetone with moderate to excellent yields over a range of substrates.

Synthesis strategies of chromones have been widely investigated due to the abundance of chromone moiety in bioactive compounds and natural products. Of which, 3-halochromones are a versatile set of precursors to synthetically access valuable compounds with chromone frameworks. Generally, 3-halochromones are synthesized from o-hydroxy enaminones through oxidative α-halogenation, a process that often uses toxic and corrosive chemicals. Herein, an alternative strategy is presented for oxidative α-halogenation catalyzed by vanadium-dependent chloroperoxidase from Curvularia inaequalis (CiVCPO) with H₂O₂/KX (X = Cl, Br, and I) in an aqueous medium. With a micellar system from a surfactant TPGS-750-M, substrate concentration can be increased to 50 mM without compromising the yield, thereby, significantly reducing the use of organic solvents. Substrate scope investigation reveals that bromination and chlorination processes prefer electron-donating substituents although moderate electron-withdrawing groups are tolerated (20 examples). Additionally, iodination processes can be performed under the optimized condition. However, slow conversion indicates that further optimization is needed. It is also found that iodination can occur without CiVCPO, albeit at a lower conversion. Further investigation suggests that such a conversion took place via I₂ generated in situ. Overall, this chemoenzymatic method can offer an environmentally friendly approach to access a variety of 3-bromo or 3-chlorochromones.

Enzymatic D-acylase/N-succinyl-amino acid racemase tandem for the production of optically pure D-amino acids and structural basis for D-acylase substrate recognition.

ABSTRACT

N-Acyl-D-amino acid deacylases (EC 3.5.1.81, also known as D-acylases) have been studied for decades for their utility in the kinetic resolution of N-acetyl-D,L-amino acids (NAAs) due to a marked stereospecificity. In conjunction with an N-succinyl-amino acid racemase (NSAR), they impulse the dynamic kinetic resolution (DKR) of different NAAs until the corresponding enantiomerically pure D-amino acids. Besides the clear interest in this enzyme cascade, the application of D-acylase/NSAR tandems has been only briefly described outside the industrial field. In this work, we revisit D-acylases for the DKR of NAAs, reporting the characterisation of two new recombinant D-acylases belonging to Bordetella petrii and Klebsiella pneumoniae. The enzymes were successfully coupled with the recombinant NSAR from Geobacillus stearothermophilus for the biosynthesis of D-methionine or D-aminobutyric acid. We also carried out the structural characterisation of the D-acylase from Klebsiella pneumoniae (KleDacyl), providing the second experimental 3-D structure of a member of this family of enzymes. The structural model shows a highly dynamic character of this amidohydrolase superfamily member, supplying a snapshot of an open conformation of the enzyme most likely preceding substrate entrance into the catalytic cleft. Our results confirm for the first time the importance of an α/β mobile domain in the substrate specificity of D-acylases (region 282–341 in KleDacyl), opening up new strategies for structural-based protein engineering strategies.

Applied and Environmental Microbiology, Volume 91, Issue 7, July 2025.

The cover art symbolizes the transformation of the mesophilic amylase psA (red) into its cold-adapted variant psA121 (cyan) through computational linker engineering. The central swirling motif reflects a shift in temperature adaptation, driven by increased interdomain separation between the catalytic domain and the carbohydrate-binding module. This visual captures how a simple change in linker sequence enables enhanced enzymatic activity at low temperatures by modifying the conformational dynamics of bidomain enzyme amylase, as described by Zhongyue J. Yang et al. in their Research Article (e202505991).

Nature, Published online: 10 June 2025; doi:10.1038/d41586-025-01826-1

An open collection of tips and tools could help researchers and publishers to pick up on problematic research.

Nitrile groups are vital components of diverse bioactive molecules, however, their enzymatic origins in fungal natural products remain largely unknown. Here, we report the discovery of an argininosuccinate synthetase (AsS)-like enzyme, ArtA, which plays a key role in the biosynthesis of a nitrile-containing nonribosomal peptide, auranthine. Structural and mechanistic studies, including X-ray crystallography, site-directed mutagenesis, docking, and molecular dynamics simulations, reveal that ArtA retains core AsS features, but possesses a remodeled active site that accommodates glutamine and facilitates nitrile formation through a unique mechanism. Furthermore, we demonstrated that ArtA homologs are prevalent across fungi and bacteria, and also demonstrated nitrile synthetase activity of representative homologs from both fungi and bacteria, suggesting a broader distribution of this function. The identification of ArtA and its homologs opens new avenues for genome mining and biotechnological applications targeting nitrile-containing natural products.

Chemically reactive microbial natural products have enabled therapeutic development1,2 via their well-established bioactivities including anticancer,3 antibiotic,4,5 and antioxidant6 activities. However, discovery of reactive metabolites is particularly challenging because they may not tolerate traditional bioactivity-guided isolation workflows.7 Diazo-containing natural products are a subset of highly reactive microbial metabolites that display potent bioactivity8-11 and enable powerful (bio)synthetic transformations;12,13 however, instability of the diazo group to light,14,15 heat,16,17 mild acid,18 and mechanical shock19 has precluded their efficient discovery and application. Here, we develop a reactivity-based screening approach to capture diazo-containing metabolites and facilitate their discovery by mass spectrometry. This workflow revealed two novel diazo-containing natural products, 4-diazo-3-oxo-butanoic acid and diazoacetone, from the human lung pathogen Nocardia ninae. Biosynthetic investigations revealed a distinct enzymatic logic for diazo formation involving hydrazone oxidation catalyzed by the metalloenzyme Dob3, and biochemical characterization of Dob3 suggests promising future applications in biocatalysis. Overall, our work highlights the power of reactivity-guided strategies for identifying reactive metabolites and facilitating the discovery of unique enzymatic transformations.

The use of single carbon (C1) molecules, such as carbon dioxide or formate, is crucial in the transition from a linear, petroleum-based economy to a circular bioeconomy. Formate can serve as both a carbon and energy source, further enhancing its attractiveness as a feedstock. Cupriavidus necator, a lithoautotrophic microbial chassis strain, provides an opportunity to leverage formate for the synthesis of valuable products. However, its ability to grow on formate and the subsequent coupling of that process to recombinantly produced redox enzymes for the efficient production of high-value-added products in a biotransformation has not yet been established. Here, we report the development of a formate-driven C. necator whole-cell chassis that recombinantly produces Rieske oxygenases (ROs) and elaborate on possible stress responses of the cells during formatotrophic cultivation. Our whole-cell chassis efficiently catalyzes the oxyfunctionalization of olefins fueled by formate oxidation. For instance, styrene was dihydroxylated to (R)-1-phenylethane-1,2-diol in an excellent 95% yield and with good enantioselectivity (74% ee) under formatotrophic conditions. The product yield and optical purity obtained demonstrate the synthetic usefulness of formate-fueled whole-cell biotransformations in C. necator.

Nature Synthesis, Published online: 10 June 2025; doi:10.1038/s44160-025-00819-2

Lignin valorization is hampered by the requirement for expensive cofactors and low conversions. Now a chemoenzymatic platform with coordinated cofactor self-circulation for realizing efficient lignin-to-molecule conversion is reported, facilitating the advancement of sustainable biorefineries.

An unspecific peroxygenase from Aspergillus niger (AniUPO) has been successfully engineered to catalyze the enantioselective α-hydroxylation of β-ketoesters. This biocatalytic process demonstrates high efficiency and selectivity with scalability to preparative levels, expanding the catalytic repertoire and synthetic applicability of UPOs in selective oxyfunctionalization.

Abstract

Unspecific peroxygenases (UPOs) are promising biocatalysts for selective oxyfunctionalization. Compared to cytochrome P450 enzymes (P450s), the catalytic potential of UPOs has been less investigated, largely due to their limited natural diversity and the challenges associated with their optimization through enzyme engineering. In this study, we engineered an UPO from Aspergillus niger (AniUPO) to catalyze the enantioselective α-hydroxylation of β-ketoesters, a valuable transformation yet to be realized in biocatalysis. Through enzyme engineering, two AniUPO variants, AniUPO-M3 and AniUPO-M6, were developed to produce a wide range of enantioenriched α-hydroxy-β-ketoesters, achieving up to 97% yield, 4140 total turnover number (TTN), and >99:1 enantiomeric ratio (er). The biocatalytic process operates under mild conditions and is scalable for preparative applications. This study broadens the catalytic repertoire of UPOs and enhances their potential for industrial applications.