Biocatalysis@TUDelft

Nature Chemical Biology, Published online: 24 April 2026; doi:10.1038/s41589-026-02209-x

Decarboxylative condensation is essential for polyketide and fatty acid biosynthesis, yet the mechanism underlying nonelongating modules without this step remains elusive. Here the authors report a condensation-independent intramodular translocation mechanism in trans-acyltransferase polyketide synthases.

Nature Synthesis, Published online: 23 April 2026; doi:10.1038/s44160-026-01058-9

The biosynthetic pathway for dimeric cyclotryptamine alkaloids—(−)-chimonanthine, meso-chimonanthine, (−)-folicanthine and (+)-sinodamine B—has been reported in Calycanthaceae plants. The pathway features two cytochrome P450 enzymes, which catalyse the enantioselective dimerization of tryptamine and four N-methyltransferases, which mediate precise regio- and stereoselective methylation.

Nature Chemistry, Published online: 23 April 2026; doi:10.1038/s41557-026-02139-0

Non-canonical amino acids are important building blocks for natural products but finding them is nontrivial. Now, transfer RNA deacylases have proved their utility as markers for biosynthetic gene clusters producing non-canonical amino acids, thereby accelerating the discovery of atypical natural products.

The fatal mushroom toxin l-(+)-muscarine is one of the most prominent natural products. We elucidated its biosynthetic origin in the sweating mushroom Collybia rivulosa based on incorporation of stable isotope-labeled compounds and extensive mass spectrometric analyses. We identified l-lysine and l-alanine/pyruvate as primary building blocks, thereby fundamentally revising the established biosynthetic model.

ABSTRACT

l-(+)-Muscarine is a widespread fatal toxin produced by various mushrooms that pose a severe threat to human health when they are mistaken for edible species. Apart from a single 1970s study that assumed l-glutamate and pyruvate were the building blocks of this unusual quaternary amine, surprisingly little is known about the toxin's biogenesis. We used Collybia rivulosa (syn. Clitocybe rivulosa), a mushroom notorious for producing muscarine, as our model for stable isotope incorporation experiments and subsequent extensive mass spectrometric analysis. Our results provide unambiguous evidence that the backbone of muscarine is assembled from two amino acids, l-lysine and l-alanine. Furthermore, we found that iterative ε-methylation of non-protein-bound l-lysine is the biosynthetic gateway step that yields ε-N,N,N-trimethyl-l-lysine. This methylation is specific to fungi that produce muscarine. Despite a substrate overlap with the biosynthesis of l-carnitine, we demonstrate that these two pathways are distinct. Our results provide compelling insight into the biogenetic origin of muscarine and fundamentally revise the previous biosynthetic model for this infamous toxin. The revised biosynthesis model lays the foundation to discover as yet unknown muscarine-like metabolites that are potentially toxic as well or pharmacologically relevant.

Understanding stereoselective biotransformations has implications for predicting drug disposition and response and may also inspire novel biocatalytic and biomimetic strategies to address challenges in metabolite and API synthesis.

ABSTRACT

Chirality is an important determinant of drug action, as enantiomers can exhibit markedly different pharmacological and toxicological profiles. Although the importance of stereochemistry in drug efficacy is well established, its role in drug metabolism and disposition remains comparatively underexplored, despite the inherently stereoselective nature of drug metabolizing enzymes. Given the high prevalence of chiral drugs in clinical use and among newly approved drugs, a systematic evaluation of stereoselective drug metabolism is needed. Understanding stereoselective biotransformations has important implications for predicting drug disposition and response and may also inspire novel biocatalytic and biomimetic strategies to address challenges in enantioselective synthesis of chiral active pharmaceutical ingredients and their metabolites. In this Systematic Review, we examine current trends and practices in the investigation of stereoselectivity in drug metabolism, the key factors influencing stereoselective metabolism, and the associated challenges and opportunities. We highlight how biocatalytic approaches can improve stereoselective access to chiral metabolites, and how insights from drug metabolism and pharmacokinetics (DMPK) studies can inspire the development of novel biocatalytic and biomimetic synthesis routes. Transfer of learning and cross‑disciplinary collaboration between biocatalysis and DMPK scientists will be critical for accelerating progress in these areas and for addressing shared challenges, including stereoselectivity prediction.

Bioelectrode platform for sustainable CO₂-to-formate conversion, combining electrochemical cofactor regeneration with enzyme immobilization. Cu-modified electrodes enhanced NADH recycling compared to SnO₂, while coupling with affinity-immobilized formate dehydrogenase improved enzymatic turnover and stability while maintaining faradaic efficiency. The system demonstrates provides a practical route for scalable electroenzymatic CO₂ reduction.

This study reports scalable bioelectrodes for sustainable CO₂-to-formate conversion that integrate on-electrode cofactor regeneration with enzyme immobilization. Carbon felt (CF) supports were coated with copper (Cu) or tin oxide (SnO₂) nanoparticles, allowing for reproducible and straightforward fabrication. Electrochemical characterization revealed that Cu-modified electrodes (CF–NpCu) outperformed SnO₂-modified ones (CF-NpSnO₂) in NADH regeneration, achieving nearly double the faradaic efficiency (FE) toward formate and conversion yield. Coupling CF-NpCu electrodes with affinity-immobilized formate dehydrogenase (FDH) produced 4.4 mM formate after 5 h, a threefold increase compared to the free enzyme system. Although the free enzyme displayed higher intrinsic kinetics, immobilization positioned FDH proximal to the electrode, mitigating diffusional limitations, accelerating NADH turnover, and improving stability. The integrated system achieved a productivity of 43 µmol h⁻¹ cm⁻² and demonstrated reusability, highlighting its practical applicability. Despite moderate efficiency losses due to side reactions such as hydrogen evolution, this work establishes a scalable bioelectrode platform that effectively combines cofactor regeneration with enzymatic CO₂ reduction, providing a promising route toward sustainable and industrially relevant electroenzymatic processes.

Engineered Escherichia coli produces six enzymes (UMPK, PPK3, GALK, NAHK, GALU, PPA) that are purified and used in a cell-free cascade to synthesize UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-galactose (UDP-Gal) from inexpensive substrates. The cascades can be coupled with glycosyltransferases for in situ nucleotide sugar regeneration, enabling oligosaccharide synthesis, including human milk oligosaccharides.

Naturally occurring in breast milk, human milk oligosaccharides (HMOs) are of great interest as an ingredient for infant nutrition due to numerous associated health benefits. Current commercial production relies mainly on microbial fermentation, while enzymatic synthesis is used to produce milligram scales for scientific studies. Enzymatic synthesis using glycosyltransferases and nucleotide sugars is especially promising due to high reaction yields, but is limited by low activity of glycosyltransferases and the high cost of nucleotide sugars. This study presents a novel approach that uses a single engineered E. coli BL21(DE3) strain to simultaneously express six recombinant enzymes (UMPK, PPK3, GALK, NAHK, GALU, and PPA). This enables dual-nucleotide sugar synthesis through two integrated multienzyme cascades. The system uses cost-effective substrates, including uridine 5′-monophosphate (UMP), N-acetylglucosamine (GlcNAc), galactose (Gal), and ATP. In situ ATP regeneration is achieved through polyphosphate (PolyP _n ) breakdown. Comparative studies of three different expression strain configurations demonstrated that crude cell lysate could serve as an effective biocatalyst. This eliminates the need for expensive enzyme purification while maintaining high catalytic activity. Using crude cell lysate, conversion yields approaching 100% were obtained. Both UDP-GlcNAc and UDP-Gal were successfully purified using anion-exchange chromatography. Based on the UV spectrum, purities of 85–99% and recovery yields exceeding 90%, respectively, were achieved. The practical application of this system was demonstrated by the successful synthesis of two HMOs: Lacto-N-triose II (LNTII) and lacto-N-neotetraose (LNnT), which demonstrates an effective nucleotide sugar recycling in coupled enzymatic reactions and paves the way toward larger scale production.

Whole-cell, light-driven biotransformations in photoautotrophic microorganisms offer the potential for higher atom economy by directly harnessing reducing equivalents and oxygen generated through natural photosynthesis. Current advances include the development of strains with improved growth rates and reactor designs that mitigate self-shading, particularly at larger scales. However, these benefits must be weighed against the volume of wastewater generated during processing to ensure that the overall approach remains sustainable for industrial application.

Photobiocatalysis with photoautotrophic whole cells has demonstrated strong potential for producing chiral molecules and platform chemicals using sustainable inputs such as light, water and CO₂ under mild reaction conditions. Coupling enzymatic transformations directly to natural photosynthesis enables higher atom efficiency compared with heterotrophic systems. However, large-scale application remains challenging, particularly due to light attenuation in photobioreactors. In this review, we summarize recent advances in whole-cell photobiotransformations with emphasis on process conditions. We also discuss strategies for intensifying photobiocatalysis through improved reactor design and new immobilization materials, along with developments in fast-growing photoautotrophic strains. Sustainability analyses indicate that organic electron donors represent only one factor influencing environmental performance, and simply replacing them with photosynthetic water splitting does not inherently yield a carbon-negative process. Nonetheless, our calculations show that when high substrate loadings are combined with wastewater use and optimized downstream processing, photosynthesis-driven biotechnology can offer substantial reductions in CO₂ emissions.

Several point mutations were introduced into the α-l-fucosidase iso1 from Paenibacillus thiaminolyticus (α-l-f1Pth-wt) to enhance its transfucosylation activity. The S237V variant (α-l-f1Pth-S237V) demonstrated superior regioselectivity in the synthesis of 3′-and 6′-fucosyllactose and functionalized 3′-fucosyllactose bearing a Boc-protected ethylthioureidyl linker. The isolated products show potential in biosensor development and HMO-based diagnostics.

ABSTRACT

To enhance the yield of l-fucosylated molecules synthesized via transfucosylation, we employed α-l-transfucosidases, enzymes engineered from α-l-fucosidases to favour transglycosylation over hydrolysis. This study investigated the transglycosylation potential of mutated variants of α-l-fucosidase iso1 from Paenibacillus thiaminolyticus designed by structural comparison with Thermotoga maritima α-l-transfucosidase. Using site-directed mutagenesis, point mutations (S237A, S237G, S237P, S237V, Y189F) were introduced, and the corresponding recombinant proteins were successfully expressed and purified. Among them, the S237V variant achieved the highest overall yield of fucosylated lactose and increased the transglycosylation/hydrolysis ratio more than 20-fold compared to the wild-type enzyme. This variant enabled regioselective synthesis of 3′-and 6′-fucosyllactose, as well as a functionalized 3′-fucosyllactose bearing a Boc-protected ethylthioureidyl linker (3′-FucLac-tBoc), with all structures confirmed by NMR spectroscopy. The engineered α-l-transfucosidase iso1 from Paenibacillus thiaminolyticus catalysed the regioselective synthesis of structurally defined fucosyllactose derivatives, including C-1 functionalized analogs suitable for immobilization on biosensor surfaces or macromolecular scaffolds, thus expanding the biocatalytic toolbox and demonstrating the potential of semi-rational enzyme design for targeted HMO-based glycoengineering applications.

Adaptive evolution of Dehalobacter restrictus strain UNSWDHB under a ratio of 1:9 chloroform:1,1-DCA regime increased 1,1-DCA dechlorination. Sequencing and molecular dynamics simulation reveal high-entropy tmrA mutations that improved 1,1-DCA binding energy and stabilize the catalytic pocket, outlining a route to tune reductive dehalogenases for bioremediation.

ABSTRACT

Organohalide-respiring bacteria capable of metabolizing multiple organohalogens represent valuable tools for bioremediation and offer intriguing evolutionary potential for adaptation to non-native substrates. In the present study, a chloroform respiring Dehalobacter restrictus strain was examined for its adaptability to a low affinity substrate (1,1-dichloroethane) over five subcultures (~28 generations). We obtained an enhanced 11,1-dichloroethane dechlorination rate by culturing with a 1:9 ratio of native (chloroform) to non-native (1,1-dichloroethane) substrates. We identified mutations corresponding to amino acids located in high-entropy regions of the TmrA protein sequence, suggesting mutational plasticity at these sites. We revealed that the mutated TmrA structure showed increased binding affinity for 1,1-dichloroethane by using molecular dynamics simulations and binding free energy computations. These findings provide insights into the adaptability of anaerobic organohalide-respiring bacteria toward non-native organohalogens and identify structural features of reductive dehalogenases that may be exploited for future enzyme engineering in bioremediation applications.

While enormous amounts of sequence information have become available, assignment of sequence to a particular enzymatic function has remained elusive. Here we describe a framework that drives a general protein language model to find a target reaction without specific training, using an initial bridgehead protein. At the heart of this framework is PLM-clust, an algorithm that employs k-means on top of protein language model embeddings to convert sequence space into functional reservoirs of latent space, and samples from these clusters based on accelerated zero-shot scoring. We demonstrate PLM-clust in a recursive discovery process (with enzyme hit rates quickly rising to >90%), segmenting isofunctional reservoirs and exploring them in greater detail. This approach - exemplified for glycosyl hydrolases (a xylanase, >100-fold activity increase) and for imine reductases (IREDs, >100-fold increase in catalytic promiscuity profiles) - reliably brings about novel enzymes that are proficient at the catalytic task at hand, reaching deeply into sequence space with a majority of residues exchanged.

The de novo design of enzymes remains a central challenge, requiring consideration of catalytic mechanism and optimization across biochemical and biophysical criteria. To capture these criteria, we draw on principles from evolutionary biology. Here, we present dEVA (design by EVolutionary Algorithm), a multi-objective design framework for structure-based protein design. We apply dEVA to the zero-shot, de novo design of metalloenzymes by optimizing for the coordination sphere of catalytic metals. We fully characterize one of these designs: a bi-zinc metalloenzyme exhibiting promiscuous hydrolytic activity towards both phosphomonoesters and phosphodiesters. This design achieves a catalytic efficiency (kcat/KM) of up to 1500 M-1s-1 and a rate enhancement ((kcat/KM)/kw) of up to 3 x 1013, comparable to characterized natural phosphatases. dEVA offers a general and modular strategy for the programmable design of protein function without dependence on natural templates, predefined motif, or evolutionary information.

Optimized high-cell-density cultivation in the DASGIP® multibioreactor enabled a 30-fold increase in soluble expression of arylmalonate decarboxylase (BbAMDase) from Bordetella bronchiseptica in E. coli BL21 (DE3). Linear glucose and optimized glycerol feeding strategies during induction ensured stable, scalable enzyme production.

This study develops and optimizes a high-cell-density cultivation (HCDC) of E. coli BL21 (DE3) to improve enzyme production utilizing DASGIP multibioreactor systems. By shifting from traditional shake flask methods to HCDC, we observed a 43-fold increase in biomass production, with a comparable mass-specific activity of the cell-free extract. The HCDC demonstrated a 30-fold increase in the active, soluble expression of arylmalonate decarboxylase (AMDase) from Bordetella bronchiseptica (BbAMDase) compared to conventional methodology. The protocol was designed to be readily adapted to various expression approaches and to provide a robust foundation for broader use in recombinant protein production. The HCDC was achieved with a linear glucose feed to promote robust cell growth. We optimized the glycerol feeding strategy during isopropyl β-D-1-thiogalactopyranoside (IPTG) induction to maximize AMDase production. Throughout the induction phase, we monitored both soluble expression yield and enzymatic activity, aiming to establish a process that is not only profitable but also efficient and stable.

A self-sufficient, NAD(P)H-independent enzyme cascade for the synthesis of indigo from L-tryptophan. The minimal cascade uses pyruvate, a byproduct of tryptophan cleavage, to generate hydrogen peroxide, which fuels the key hydroxylation of indole to indoxyl by an engineered tyrosine hydroxylase. Indoxyl then spontaneously dimerizes under aerobic conditions to form indigo, creating a closed-loop system for sustainable production from renewable resources.

Indigo is currently produced from petrochemical sources, which poses significant environmental challenges. Sustainable biotechnological alternatives are therefore highly desirable. Enzymatic synthesis of indigo from L-tryptophan via indole has been demonstrated, but conventional pathways based on flavin-containing monooxygenases require costly coenzymes such as NAD(P)H, limiting their practical applicability. In this study, we present a novel, self-sufficient, NAD(P)H-independent enzyme cascade for indigo biosynthesis from the renewable feedstock L-tryptophan. The cascade starts with conversion of L-tryptophan into indole and pyruvate by a tryptophanase. As next steps, the system couples an engineered bacterial tyrosine hydroxylase, which converts indole into indoxyl using hydrogen peroxide, with a pyruvate oxidase that generates the required peroxide in situ. The cascade thereby transforms a reaction byproduct into the oxidizing equivalent needed for the subsequent step, establishing a closed catalytic cycle with minimal auxiliary inputs. After optimizing cascade parameters, the system produced 0.25 mM indigo from 5 mM L-tryptophan. Although the overall yield remains moderate, this proof-of-principle demonstrates a sustainable and cost-effective enzymatic route for indigo production from biobased starting materials, providing an environmentally friendly alternative to petrochemical synthesis.

Five sesquiterpene synthases of Psilocybe cubensis were investigated in vitro and in vivo. CubF is a clade I synthase producing α-muurolol, whereas clade IV synthases CubG1 and CubG2 produce epi-isozizaene and β-duprezianene. Further clade IV synthases CubH and CubI primarily make dauca-4(11),8-diene and β-barbatene, respectively. Our results help chart the natural product metabolome of one of the most iconic mushroom genera.

Apart from the psychedelic psilocybin, the metabolite spectrum of Psilocybe “magic mushrooms” comprises sesquiterpenes, a class of natural products known to exhibit receptor-modulating bioactivities. However, the composition of the sesquiterpene profile has largely remained an open question. Here, we report the characterization of five Psilocybe cubensis sesquiterpene synthases, both in vitro using recombinantly produced enzymes and in vivo in Aspergillus niger. CubF is a clade I α-muurolol synthase. The investigated clade IV synthases were the near-identical CubG1 and CubG2 synthases, which catalyze mainly epi-isozizaene and β-duprezianene formation. Furthermore, CubH and CubI were identified as primarily making dauca-4(11),8-diene and β-barbatene, respectively. Gas chromatographic analyses of the headspaces of P. cubensis vegetative mycelium and fruiting bodies showed qualitative and quantitative differences, with sterpurene being among the major compounds in mycelium and dauca-4(11),8-diene in fruiting bodies. This fundamental knowledge of the P. cubensis terpenome may help distinguish the pharmacological effects of magic mushrooms versus pure psilocybin.

The genetic incorporation of noncanonical amino acids (ncAAs) into proteins represents an effective approach to endow enzymes with novel functions. Here, we highlight the novel concept of integration of ncAAs biosynthesis and incorporation firmly into the realm of enzyme design, establishing an efficient strategy for creating artificial enzymes with in situ biosynthesized ncAAs featuring non-natural catalytic activity.

Artificial enzymes engineered by site-specific incorporation of catalytically active noncanonical amino acids (ncAAs) into protein scaffolds represent a rapidly advancing class of biocatalysts, particularly for chemical transformations lacking natural enzymatic counterparts. Integrating biosynthesis and genetic incorporation of ncAAs has rapidly expanded the toolkit for enzyme design and catalysis. This review surveys engineered metabolic routes and precursor feeding strategies that supply diverse ncAAs in vivo, and contrasts cell-free and cellular approaches for their production. We examine advances in orthogonal translation systems and site-specific incorporation that enable installation of catalytic, redox, and spectroscopic functionalities, and highlight applications where ncAA-bearing proteins catalyze new-to-nature reactions, and improve selectivity for directed evolution. Recent work on in-cell biosynthesis of ncAAs coupled to on-demand incorporation is discussed for its potential to streamline workflows and enable enzyme design and catalysis in one-pot. Finally, we identify remaining challenges and outline opportunities for coupling metabolic engineering and protein engineering to create next-generation artificial enzymes.

Adenylation enzymes selectively transfer acyl substrates to their cognate carrier proteins (CPs). To capture their transient interactions, cross-linking reactions were conducted using pantetheine probes with varying linker lengths. Structural analysis reveals that changing the linker length does not affect the binding interface interactions between the adenylation enzyme and the CP.

Adenylation enzymes transfer acyl substrates selectively onto carrier proteins (CPs) in natural product biosynthesis. Despite the importance of adenylation enzyme–CP interactions, structural information on these transient complexes remains limited. Previously, we developed a pantetheine cross-linking probe (named C2Br), which contains an ethylenediamine linker with a reactive bromoacetamide group, and determined the structure of the cross–linked complex of the adenylation enzyme HitB with the CP HitD. Here, we investigated the linker-length effects of pantetheine probes in the cross-linking reactions of two adenylation enzymes, HitB and EntE, with CPs using probes with different diamine linkers, such as C2Br and C4Br, the latter containing a longer butanediamine linker moiety. Both adenylation enzymes formed cross-linked complexes with CPs irrespective of the probe used, but the reaction efficiencies depended on the linker length. Crystal structural analysis showed that the HitB–HitD interface interactions in the HitB–C4Br-HitD complex are essentially identical to those in the HitB–C2Br-HitD complex. In contrast, the diamine moieties of probes adopt different interaction modes, accounting for the observed variations in cross-linking efficiencies. A repertoire of pantetheine probes with varying linker lengths will facilitate structural studies on adenylation enzyme–CP interactions by enabling optimization for each adenylation enzyme.

A novel multienzyme cascade converts plant-derived salicin into salicylic acid. Overcoming matrix challenges in crude extracts, this green biocatalytic route delivers 95% yield. It offers a sustainable path to high-value bioingredients of natural origin for preservative applications.

This study presents a novel multienzymatic strategy for the efficient and green production of valuable salicylic acid from plant-derived salicin. The biocatalytic route couples β-glucosidase for deglycosylation with laccase and xanthine oxidase (XO) for subsequent oxidation. The primary objective of this approach was to assess the feasibility of enzyme coupling within a single one-pot reaction. The results demonstrate that this strategy successfully enabled the transformation of salicylic alcohol into salicylic acid, mediated by a compatible laccase/TEMPO and XO system in a one-step process at pH 7 and 25°C. However, the integration of β-glucosidase into a one-pot system for direct salicin conversion was found to reduce the oxidative efficiency of XO, thus necessitating a sequential approach. Applying this optimized enzymatic cascade to a crude Salix purpurea bark extract initially encountered limitations in oxidation due to matrix complexity. Subsequent optimization of biocatalyst dosage delivered complete substrate oxidation and a 95% salicylic acid yield. This research significantly broadens the applicability of multienzymatic biocatalytic cascades, offering a promising and sustainable route for transforming natural compounds within complex plant extracts into high-value bioactive ingredients.

The ability of SAM-dependent enzymes to accept S-adenosyl-D-methionine [D-SAM, (SS,RC)-SAM] instead of the native cofactor S-adenosyl-L-methionine [L-SAM, (SS,SC)-SAM] remains largely unexplored. Challenging the stereochemical preference of SAM-dependent enzymes, we investigated the ability of different enzyme classes to accept D-SAM. Contrary to common assumptions, the tested N- and O-methyl transferases (MTs), as well as one of the examined C-MTs accepted D-SAM. Docking studies suggest that acceptance of D-SAM by C-MTs may be influenced by the angle between the transferable methyl group of SAM and the nucleophilic carbon of the substrate, along with enzyme and substrate flexibility. In addition to conventional MTs, the radical SAM glutamine C-MT QCMT showed low but detectable methylation activity with D-SAM. Furthermore, the azetidine-2-carboxylic acid synthase AzeJ not only uses D-SAM but also incorporates the stereocentre of D-methionine into the cyclic amino acid product. The pyridoxal 5'-phosphate (PLP)-dependent enzyme 1-aminocyclopropyl-1-carboxylic acid synthase (ACCS) also showed detectable turnover with D-SAM. These findings broaden the understanding of enzyme stereoselectivity, provide an overview of D-SAM-utilising enzymes, and identify first enzyme systems that may serve as starting points for engineering efforts aimed at shifting cofactor preference towards D-SAM.

A whole-cell deracemization cascade combining L-amino acid deaminase (LAAD), D-amino acid transaminase (DAAT), and R-selective amine transaminase (RATA) enables efficient D-amino acid synthesis. RATA-driven D-alanine recycling, using α-methylbenzylamine as a cosubstrate, serves as a robust equilibrium shifter that overcomes the thermodynamic limitation of DAAT reactions and suppresses metabolic pyruvate depletion. The modular catalytic construct allows independent activity tuning of each module.

Thermodynamically neutral equilibria often limit the synthetic utility of amino acid transaminases. An effective strategy to overcome this limitation is to design a catalytic cascade that circumvents the thermodynamic barrier by recycling the coproduct into the cosubstrate. Here, we present a whole-cell deracemization cascade for the synthesis of D-amino acids, in which the D-amino acid transaminase (DAAT) reaction is thermodynamically driven by an R-selective amine transaminase (RATA). The cascade consists of three compartmentalized modules, each implemented in a distinct Escherichia coli whole-cell biocatalyst: (i) an acceptor-supply module (ASM) that converts the L-enantiomer of racemic amino acid to α-keto acid using L-amino acid deaminase (LAAD); (ii) an amine-transfer module (ATM) that produces D-amino acid from the α-keto acid using DAAT with D-alanine; and (iii) a donor-recycling module (DRM) that regenerates D-alanine using RATA with α-methylbenzylamine (α-MBA). Integration of the three modules enabled one-pot deracemization of amino acids within the overlapping substrate scope of LAAD and DAAT, affording the desired D-amino acids in yields exceeding 90%. This study demonstrates a whole-cell cascade system that leverages highly exergonic α-MBA-mediated conversion of pyruvate to D-alanine to overcome the unfavorable DAAT equilibrium, providing an efficient route for synthesizing D-amino acids from inexpensive racemic precursors.

Selective bioconjugation reactions enable important drug delivery strategies, relying on taggable moieties incorporated into bioactive compounds. To address a pressing need for biosynthetic access to reactive, bioactive natural products, a customizable approach to the de novo biosynthesis of Diels-Alder-taggable natural product analogs was established, and its applicability was demonstrated in bioconjugation reactions. The developed biosynthetic platform enables the broader implementation of site-selective modifications in peptide therapeutics.

Per- and polyfluoroalkyl substances (PFAS) are highly resistant to enzymatic C-F bond cleavage, and hydrolytic defluorination of long-chain PFAS has rarely been demonstrated. Here, we report selective hydrolytic defluorination of branched perfluorooctanoic acid (PFOA) isomers by a haloacid dehalogenase (4A) from Delftia acidovorans strain D4B. A fluoride-specific riboswitch biosensor was used for initial substrate screening, followed by scaled-up assays in which fluoride release was quantified using a fluoride ion-selective electrode. Defluorination products were subsequently identified by liquid chromatography-mass spectrometry (LC-MS). Although purified 4A (10 M) readily catalyzed hydrolytic defluorination of fluoroacetic acid, incubation of PFOA (0.5 mM) with purified 4A resulted in a statistically significant increase in fluoride release at elevated enzyme loading (500 M). High-resolution LC-MS/MS analysis revealed that defluorination products originated from minor branched PFOA isomers rather than linear PFOA. Molecular docking analyses supported catalytically plausible binding geometries for branched PFOA isomers, positioning the substrate -carbon within [~]4 [A] of the catalytic aspartate residue. These findings demonstrate previously unrecognized hydrolytic reactivity of a haloacid dehalogenase toward branched PFAS isomers and expand the known catalytic scope of the haloacid dehalogenase family. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/719434v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@d4a9aeorg.highwire.dtl.DTLVardef@1d00b0borg.highwire.dtl.DTLVardef@185118borg.highwire.dtl.DTLVardef@142d50c_HPS_FORMAT_FIGEXP M_FIG C_FIG SYNOPSISEnzymatic defluorination of PFAS is rarely observed in environmental systems. This study identifies hydrolytic defluorination of branched PFOA isomers, improving understanding of PFAS defluorination at the enzyme level.

Protein stabilization is a "Holy Grail" of biocatalysis, and stability design is an area of intense research interest. While it is increasingly feasible to effectively increase enzyme thermostability, optimization without compromising activity or selectivity remains a significant challenge. Here, we use full-atom protein sequence design with sidechain conditioning (FAMPNN) to engineer thermostable variants of the borneol dehydrogenase from Salvia rosmarinus (SrBDH1), an enzyme from a family where unselective enzymes dominate, and selectivity is determined by dynamical considerations. By combining FAMPNN design with residue conservation analysis and avoiding active site residues, we were able to computationally design SrBDH1 variants with up to 10 {degrees}C enhanced thermostability and strongly increased half-life time at elevated temperature, while retaining selectivity towards (+)-borneol. This design framework, integrating de novo and physics-based protein design tools, demonstrates that stability can be enhanced without disrupting functionally relevant dynamics, providing a route to engineer robust and selective biocatalysts. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/719482v1_ufig1.gif" ALT="Figure 1"> View larger version (97K): org.highwire.dtl.DTLVardef@19b2dd2org.highwire.dtl.DTLVardef@dd39e4org.highwire.dtl.DTLVardef@3d75dorg.highwire.dtl.DTLVardef@303b80_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG