Biocatalysis@TUDelft

Nature Biotechnology, Published online: 29 April 2025; doi:10.1038/s41587-025-02660-6

Overcoming key hurdles for the development of next-generation anaerobic cultivation methods could have major impacts on microbial community research and applications.

In this study, we engineered the loop connecting the FAD- and NADP-binding domains of Baeyer-Villiger monooxygenases to improve the catalytic efficiency of nicotinamide cofactors, even reduced nicotinamide cofactor biomimetics. The loop modification strategy represents a universal method applicable to improving nicotinamide cofactor catalytic efficiency in oxidoreductases and designing next-generation biocatalysts endowed with tailored catalytic performance.

Nitrogenase catalyzes the reduction of N2 to NH3 at its active site cofactor. Catalysis by the homologous V- and Mo-nitrogenases involves the same dynamic belt-S mobilization that occurs asymmetrically in the two cofactors, although V-nitrogenase differs from its Mo-counterpart in its inability to capture N2 under sulfur- and reductant-depleted conditions. The shared feature between the two nitrogenases in belt-S turnover points to nitrogenase as a sulfite reductase, whereas their distinct abilities in N2 capture could facilitate future mechanistic explorations of nitrogenase.

We developed a sustainable lab-scale method for producing bio-indigo using an engineered Synechocystis sp. PCC6803 strain expressing the enzyme mFMO and light. Optimised growth conditions yielded 112 mg/L of bio-indigo. A new purification technique using polyamide nets allows efficient recovery from the culture medium, reducing environmental impact.

ABSTRACT

Cyanobacteria are emerging as interesting cell factories, offering the significant advantage of their in-built photosynthetic machinery, which generates NADPH to support redox biocatalysis. In this study, we assessed the potential of the cyanobacterium Synechocystis sp. PCC6803 in producing the dye indigo by light-driven whole-cell biotransformation using indole as a starting compound. A stable transgenic strain expressing a flavin-containing monooxygenase from Methylophaga aminisulfidivorans (mFMO) was engineered, enabling light-dependent indigo production. Upon optimising conditions, effective biotransformations could be performed, resulting in 112 mg/L indigo (86% conversion of the furnished indole). Additionally, we present a method for the recovery of the secreted dye directly from the growth medium through solid-phase absorption on polyamide nets. Overall, the effectiveness and sustainability of the biotransformation in Synechocystis sp. PCC6803 performed at the laboratory scale provide a strong basis for further exploring the applicability of the process.

This review covers alginate lyases that depolymerise alginate into AOS (alginate oligosaccharides) and DEH (4-deoxy-L-erythro-5-hexoseulose uronate). It discusses the industrial applications of AOS and DEH in food, agriculture, and biorefinery processes. Additionally, it explores recent advancements in enzyme engineering, including chimeric enzyme construction, truncation, computer-aided design, and directed evolution, to enhance alginate lyase efficiency.

ABSTRACT

Alginate lyases depolymerize alginate and generate alginate oligosaccharides (AOS) and eventually 4-deoxy-L-erythro-5-hexoseulose uronate (DEH), a monosaccharide. Recently, alginate lyases have garnered significant attention due to the increasing demand for AOS, which exhibit bioactivities beneficial to human health, livestock productivity, and agricultural efficiency. Additionally, these enzymes play a crucial role in producing DEH, essential in alginate catabolism in bacteria. This review explains the industrial value of AOS and DEH, which contribute broadly to industries ranging from the food industry to biorefinery processes. This review also highlights recent advances in alginate lyase applications and engineering, including domain truncation, chimeric enzyme design, rational mutagenesis, and directed evolution. These approaches have enhanced enzyme performance for efficient AOS and DEH production. We also discuss current challenges and future directions toward industrial-scale bioconversion of alginate-rich biomass.

Highly selective light-driven CO₂ conversion to CO is achieved using an [FeFe]-hydrogenase-inspired molecular dyad (PS-CAT) acting simultaneously as photosensitizer and catalyst. The presence of water as a solvent is important for such selectivity. Assembling the PS-CAT in the hydrophobic part of lipid bilayers of liposomes allows the use of water as a solvent, static excited state quenching and high turnover numbers.

Abstract

We report a biomimetic system for light-driven CO₂ conversion in lipid bilayers using a [FeFe]-hydrogenase-inspired molecular dyad (PS-CAT) acting simultaneously as photosensitizer and catalyst. The molecular design of PS-CAT consists of both light-harvesting and catalytically active moieties in a single molecule. This structure allows for efficient charge transfer between these two moieties. The PS-CAT consists of only an organic chromophore and an iron complex. Photocatalytic reduction of CO₂ to CO (CO₂RR) as well as H₂ production (HER) was traced over 56 h to determine CO₂RR/HER selectivity. The presence of water in the lipid bilayer system allows for CO₂RR selectivity >99 % over the competitive HER, outperforming previous molecular, and liposome-based systems for light-driven CO₂ to CO conversion. The product selectivity in CO₂RR (e.g., CO, HCOO⁻, and CH₄) was determined via gas chromatography and NMR spectroscopy with ¹³C-labeled carbon sources. The Stern–Volmer quenching studies on the initial light-driven electron-transfer revealed static quenching and indicated a preassembly of the PS-CAT with the electron donor at the membrane-water interface.

QM/MM simulations reveal that the structural reorganization of the active site, particularly the alternation of the hydrogen bonding network and coordination switch of the HONO intermediate, is critical for the reductive cleavage of the N─O bond and NO formation in the nonheme diiron YtfE. This mechanistic insight advances understanding of nitrite reduction in nonheme diiron oxygenases and their biological roles.

Abstract

Escherichia coli (E. coli) repair of iron center protein (also known as YtfE) is a nonheme diiron protein that plays a pivotal role in bacterial responses to nitrosative stress, particularly in nitrite reduction and nitric oxide metabolism. Although previous studies have explored its potential as a nitrite reductase, the detailed catalytic mechanism, especially the role of its diiron center in nitrite reduction, remains poorly understood. In this study, we employed multiscale computational approaches, including quantum mechanics/molecular mechanics (QM/MM), quantum mechanics/molecular mechanics metadynamics (QM/MM MD), and quantum mechanics (QM) calculations, to elucidate the molecular mechanism of nitrite reduction by YtfE. Our findings reveal that structural reorganization of the active site, particularly the alternation of the hydrogen bonding network and coordination switch of the HONO intermediate, is critical for modulating the nitrite reduction mechanism. This work provides significant insights into the catalytic mechanism of YtfE, offering a theoretical foundation for understanding its role in bacterial stress responses and potential applications in synthetic biology and environmental remediation.

Nature Chemical Biology, Published online: 01 May 2025; doi:10.1038/s41589-025-01898-0

Negamycin is a decades-old antibiotic that can promote readthrough of premature stop codons and possesses a structure that contains an unusual N–N bond. Now, the long-mysterious negamycin gene cluster has been identified and characterized to reveal a heme-dependent enzyme that directly couples glycine and nitrite to form the N–N linkage.

Designing artificial enzymes remains challenging due to the need for precise active site control. Here, we present a simple strategy to create metal-free artificial enzymes by engineering Histidine clusters inside a ferritin cage. The resulting catalyst mimics peroxidase activity, and its stable cage structure offers new insights into supramolecular catalysis, opening avenues for bioinspired catalyst development.

Abstract

Developing artificial enzymes is challenging because it requires precise design of active sites with well-arranged amino acid residues. Histidine-rich oligopeptides have been recently shown to exhibit peroxidase-mimetic activities, but their catalytic function relies on maintaining unique supramolecular structures. This work demonstrates the design of a specific array of histidine residues on the internal surface of the ferritin cage to function as an active center for catalysis. The crystal structures of the ferritin mutants revealed histidine–histidine interactions, forming well-defined histidine clusters (His-clusters). These mutants exhibit peroxidase-mimetic activities by oxidizing 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide. Molecular dynamics simulations further highlight the co-localization of TMB and hydrogen peroxide at the histidine-rich clusters, indicating that the confined environment of the ferritin cage enhances their interactions. This study presents a simple yet effective approach to design metal-free artificial enzymes, paving the way for innovations in bioinspired catalysis.

A biocatalytic platform for aniline synthesis, based on the oxidative amination of readily available cyclohexanones, has been reported. Engineered variants exhibit broad substrate compatibility, enabling the synthesis of 40 structurally diverse secondary and tertiary anilines with conversions of up to 91%. Mechanistic studies revealed that directed evolution enhanced enzyme performance in imine desaturation while suppressing phenol formation.

Abstract

Aniline motifs are commonly found in natural products and synthetic molecules. While chemists have developed numerous methods for constructing C(sp²)─N bonds, their biocatalytic counterparts in nature are primarily limited to P450-based protein machineries. To address this limitation, we developed a biocatalytic platform for aniline synthesis based on oxidative amination of cyclohexanones. Through directed evolution of a flavin-dependent enzyme PtOYE, we identified several protein catalysts (e.g., OYE_G3 and OYE_M3) that exhibited activity across a broad array of substrates, enabling the preparation of 40 different secondary and tertiary anilines with various substitution patterns in up to 91% GC conversion. Mechanistic investigations revealed the improved kinetic performance of the evolved variants on the desaturation of imines. Additionally, mutations introduced through protein engineering further reduced the propensity for phenol formation. This enzymatic platform represents a highly promising application of flavin-dependent enzymes, showcasing their great potential in organic synthesis and drug development.

The near-universal genetic code of living organisms uses 64 codons to encode the 20 canonical amino acids in protein synthesis. Here we design and generate a variant of Escherichia coli with a 4 Mb synthetic genome in which we replace every known occurrence of six sense codons and a stop codon with synonymous codons. We thereby recode 105 codons to create an organism with a 57-codon genetic code; this organism - which we name Syn57 - uses 55 codons to encode the 20 canonical amino acids.

Enzymes are involved in the biosynthesis of various secondary metabolites found in nature. The catalytic mechanism is regulated by the three-dimensional structure of the enzyme especially at the catalytic site, resulting in the natural products with complicated conformation derived from a regioselective, chemoselective, and stereoselective fashion of the enzyme reaction. Prenyltransferase (PT), which belongs to the prenylsynthase (PS) family, catalyzes the condensation of isoprene to an aromatic compound, consequently producing a terpenoid scaffold structure. Therefore, it plays an important role in which expands the chemical diversity of terpenoids. Although the three-dimensional structures of PS which categorized in the same family were resolved, the catalytic mechanism of the PT has been vailed. In this study, we determined the X-ray crystal structure of a novel prenyltransferase SxPT1 derived from marine Streptomyces. Here we described that SxPT1 analyzed the enzyme reactions and discussed its catalytic mechanism.

Aminoacyl-tRNA deacylases safeguard the accurate translation of the genetic code by hydrolyzing incorrectly synthesized aminoacyl-tRNAs. Canavanyl-tRNA deacylase (CtdA) was recently shown to protect cells against the toxicity of the non-proteinogenic amino acid canavanine, which is synthesized and accumulated by various plants. In most organisms, canavanine is ligated to tRNAArg, causing translation of arginine codons with canavanine. CtdA prevents canavanine toxicity by hydrolyzing canavanyl-tRNAArg. Here, we investigated the function, structure, substrate specificity, phylogenetic distribution, and evolution of CtdA. We show that CtdA is essential to prevent canavanine cytotoxicity in Salmonella enterica, and its heterologous expression can also rescue Escherichia coli. By determining the structure of CtdA, we identified its putative binding pocket and residues that modulate enzymatic activity and specificity. We also found that CtdA displays a relaxed specificity for the aminoacyl moiety substrate as it hydrolyzes arginyl-tRNAArg. Finally, we showed that despite their structural homology, CtdA and the aminoacyl-tRNA hydrolytic domain of phenylalanyl-tRNA synthetase are functionally and evolutionarily divergent. Collectively, these results substantially expand our understanding of the CtdA family, providing new insights into its structure, function, and evolution. Moreover, this work highlights the diverse mechanisms unique to each organism to ensure faithful translation of the genetic code.

This study describes the successful identification of ancestral sequences N50 and N49, which have a longer half-life of approximately four and two times the wild-type Sp-amine transaminase, respectively.. This approach effectively enhances the thermal stability of the transaminase.

Amine transaminases (ATAs), belonging to the class III transaminases within the superfamily of pyridoxal-5′-phosphate-dependent enzymes, catalyze transamination reactions between amino donors and amino acceptors. These enzymes are particularly appealing for their role in stereospecific synthesis of chiral amines. However, the stability of most ATAs is not satisfying, limiting their suitability for industrial applications. Among them, the amine transaminase from Silicibacter pomeroyi (Sp-ATA) has drawn attention due to its high activity and broad substrate scope under mild conditions and high pH. Nevertheless, maintaining the activity at higher temperatures is a challenge. Previous studies to enhance enzyme function through directed evolution have shown promising results, yet predicting the cooperative effects of individual stabilizing mutations remains challenging. An alternative strategy is ancestral sequence reconstruction (ASR), which is based on gene sequences to create a more or less artificial phylogenetic tree. This study aims to leverage ASR techniques to explore the thermostability, solvent tolerance, and substrate profile of Sp-ATA, to find more stable transaminases. By using Sp-ATA as a template and incorporating insights from ancestral sequences, this strategy offers a promising approach for developing robust biocatalysts suitable for industrial applications.

Designing artificial metal sites in proteins is challenging due to the need to place the metal site in precise positions and to tailor the coordination environment of the metal. A modular approach based on the SpyTag/SpyCatcher technology is used to provide the Spy construct with a Cu²⁺/ATCUN site. This strategy enables versatile metalloprotein design by shifting complexity from a protein to a peptide.

Designing artificial metal binding sites within a protein is challenging since amino acid residues need to be placed in desired positions in the final construct and the use of non-natural amino acids is difficult. The alternative approach of directing the insertion of artificial metal coordination systems presents the difficulty of grafting such site in a single desired position. Spy protein is composed of a protein component (SpyCatcher) which binds spontaneously an oligopeptide (SpyTag) with formation of an isopeptide bond. A SpyTag peptide equipped with an ATCUN (amino terminal copper and nickel) binding site is designed to bind copper(II) with high femtomolar affinity both in the absence of SpyCatcher and in the reconstituted Spy construct. The Cu²⁺ ATCUN site in the reconstituted Spy protein presents a catalytic activity in reactive oxygen species production, higher than that of the SpyTag peptide alone. This method offers a novel approach for constructing artificial metalloproteins by incorporating functional metal binding sites into a peptide, which can then be clicked onto its protein counterpart. The small size and modularity of this construct make it versatile for integration into other protein systems, eventually moving the complexity from a protein to a peptide and highlighting its potential for protein design.

An alternative pathway to ephedra alkaloids via (S)–cathinone is investigated in plants. This pathway circumvents the formation of 1-phenylpropane-1,2-dione as an intermediate, which has been part of the predominantly postulated biosynthetic pathway for the last decades. This alternative pathway involves an enzymatic pyridoxal phosphate (PLP)-dependent carboligation of benzoyl-CoA and L-alanine in a single step.

Ephedra alkaloids possess some of the most basic structures of alkaloids. Despite their importance for human use and their commercial relevance, the biosynthesis of ephedra alkaloids has remained enigmatic. The predominant biosynthetic pathway in the literature proposes a thiamin-dependent carboligation followed by a transaminase, although no candidate enzymes have yet been identified in ephedra alkaloid producers. In this work, an alternative pathway in plants to ephedra alkaloids via (S)–cathinone is investigated that circumvents the formation of 1-phenylpropane-1,2-dione as an intermediate and is in full agreement with previous biosynthetic studies. This alternative pathway involves the pyridoxal phosphate (PLP)-dependent carboligation of –benzoyl-CoA– and L-alanine in a single step. The PLP-dependent formation of labeled and unlabeled (S)–cathinone is detected in the plant lysate of young stem tissue of various Ephedra species that contain Ephedra alkaloids, as well as in young leaf tissue of Catha edulis. The incorporation of labeled nitrogen from L-alanine into (S)-cathinone supports the hypothesis that an α-oxoamine synthase (AOS) catalyzes the formation of (S)-cathinone, bypassing the dione as an intermediate. These results demonstrate the involvement of a PLP-dependent AOS as a pivotal step in the biosynthesis of ephedra alkaloids.

The Psilocybe cubensis terpene synthases, CubB-CubE, were investigated. Product formation assays identified CubB as a (3R,6E)-(-)-nerolidol or linalool synthase, whereas CubC is a multiproduct synthase catalyzing the formation of β-caryophyllene and other linear and cyclic sesquiterpenes/-terpenoids. CubD and CubE catalyze predominantly sterpurene formation. The results help to explore the true natural product diversity of this iconic mushroom genus.

Psilocybe “magic mushrooms” are best known for their indolethylamine psilocybin, yet they encode enzymes for a much more diverse arsenal of small and potentially bioactive molecules. Herein, four Psilocybe cubensis clade III sesquiterpene synthases, CubB-CubE, whose genes are differently expressed in fruiting bodies compared to vegetative mycelium are reported. CubB-CubE were functionally characterized in vitro by product formation assays with heterologously produced enzymes and in vivo by transgene expression in Aspergillus niger, followed by extensive gas chromatography-mass spectrometry analyzes. CubB was identified as a single product (3R,6E)-(-)-nerolidol synthase. CubC is a multiproduct enzyme producing β-caryophyllene, β-elemene, α-humulene, and β-farnesene. CubD and CubE catalyze (near-)exclusively sterpurene formation. P. cubensis young fruiting bodies and vegetative mycelium were analyzed for sesquiterpenes, which verified the presence of the CubB product α-(3R,6E)-(-)-nerolidol. As various Psilocybe species encode highly similar enzymes, this study contributes generally to the as-yet little-understood secondary metabolome of the genus.

Xenobiotic cytochrome P450 enzymes have been shown to hydroxylate testosterone at various positions in the steroid backbone including 1beta-hydroxytestosterone. Despite the potential application to study the biochemistry of these enzymes, 1beta-hydroxytestosterone is not commercially available. A synthesis of 1beta-hydroxytestosterone from commercially available androsta-1,4-diene-3,17-dione was developed. The key step to functionalize C1 was a borylation reaction catalyzed by an in situ generated copper carbene complex. To test the versatility of the borylation reaction, androsta-2,4-dien-1-one was also used as the substrate, which underwent conjugate addition at C3. The synthetic strategy reported will be used to access other biologically relevant C1-hydroxylated steroids to explore the biochemistry of drug metabolizing P450 enzymes.

The effects of the osmolytes TMAO and sorbitol on the catalytic activity of Candida boidinii formate dehydrogenase are investigated using computational and experimental studies. The osmolytes exert different effects on the inter-domain fluctuations, solvation, and active site dynamics of the enzyme as well as on the substrate residence times. Routes for water trafficking and substrate exit are also proposed.

Abstract

The mechanisms by which osmolytes increase the biocatalytic efficiency of enzymes are still not well understood. In a joint computational and experimental study, we identified different effects, depending on the type of osmolyte, on the catalytic activity of formate dehydrogenase from Candida boidinii (CbFDH), an industrially relevant enzyme. These effects are related to enzyme inter-domain fluctuations, solvation, and active site dynamics as well as substrate residence times. The combination of these factors could explain the experimental behavior of the k _cat of CbFDH in different osmolyte solutions, compared to buffer. Our computational studies also allowed us to propose six routes, located at the interface between both domains of CbFDH, for water trafficking and formate exit between the active site of CbFDH and the solvent. The results suggest that solvent-induced changes might affect the route's dynamics and, potentially, the catalytic activity of CbFDH. Our comprehensive analysis of protein solvation, structure, and dynamics thus provides a molecular understanding of the role of these osmolytes on enzymatic activity.