Biocatalysis@TUDelft

Engineering an acyl-CoA ligase shifts thiol specificity toward inexpensive synthetic thiols such as N-acetylcysteamine (SNAC), enabling enzymatic generation of acyl-SNAC starter units that support polyketide biosynthesis.

Fatty acyl-coenzyme A (CoA) thioesters are indispensable intermediates in primary metabolism and essential building blocks for biosynthetic pathways yielding fuels, fine chemicals, and pharmaceuticals. However, large-scale production of acyl-CoAs is frequently constrained by the intracellular availability and high cost of CoA, motivating the development of alternative strategies for precursor generation. Here, we report the engineering of the acylCoA ligase AcsA _Pc to preferentially utilize inexpensive, membrane-permeable synthetic CoA surrogates, with a focus on N-acetylcysteamine (SNAC). Building on a broad specificity variant (D449E), structure-guided saturation mutagenesis and random mutagenesis, coupled with high-throughput colorimetric screening, progressively shifted thiol specificity from CoA to pantetheine and SNAC. The resulting double mutant, F430W/D449E, exhibits a 26-fold improvement in SNAC utilization relative to wildtype while concomitantly reducing CoA activity. Kinetic analyses reveal that these gains arise from increased reaction rates with synthetic thiols and altered substrate preferences across a representative acid panel. The functional relevance of AcsA-generated acyl-SNACs was demonstrated in a reconstituted polyketide system, where enhanced availability of SNAC-linked starter units drove an ∼8-fold increase in pyrone formation. This work establishes AcsA as a tunable platform for orthogonal generation of polyketide precursors and a general framework for alleviating CoA-dependent biosynthetic bottlenecks.

Amic acid-based decoy molecules were developed by simple mixing of acid anhydrides and amines. The newly identified phthalic acid-based decoy molecules enabled whole-cell benzene hydroxylation by cytochrome P450BM3, achieving phenol production about 3 mM, without the need for prior complex chemical synthesis and purification of the decoy molecules.

ABSTRACT

Decoy molecules, which are substrate analogues, serve as enabling additives that unlock the catalytic potential of cytochrome P450BM3 toward non-native substrates; however, their broader application has been limited by the requirement for prior chemical synthesis. In this study, we report amic acids as a new scaffold for decoy molecules, inspired by their structural similarity to the native P450BM3 substrate, N-palmitoylglycine. Amic acid-based decoy molecules were rapidly prepared by simple mixing of cyclic anhydrides and amines and were directly evaluated without purification, enabling rapid screening. Using this approach, phthalic acid-based decoy molecules were identified that promoted not only benzene hydroxylation but also toluene hydroxylation and styrene epoxidation, while also expanding the substrate scope to a broader range of aromatic and aliphatic compounds. X-ray crystallographic analysis further revealed that the obtained decoy molecules closely mimic the native substrate within the active site of P450BM3. Notably, in whole-cell benzene hydroxylation, phenol production nearly 3 mM was achieved, and comparable activity was observed even when unpurified decoy molecule solutions prepared within tens of minutes were used. Owing to their facile preparation and ease of use, amic acid-based decoy molecules offer a generally applicable approach for expanding P450BM3-catalyzed reactions.

This study introduces a combined mechanical and biological process for converting cotton textile waste into fermentable sugars. Textiles are preprocessed in a cutting mill and subsequently subjected to mechanoenzymatic treatment in a wet rotor mill. Iterative milling cycles enhance glucose yield, while extended milling reduces particle size without further improvement.

With the continuously increasing volume of textile waste and the limitations of current recycling strategies, there is a growing need for the development of environmentally sustainable and efficient processing methods. Cellulose derived from post-consumer textile waste represents a promising and cost-effective substrate for enzymatic hydrolysis due to its abundance and low market value. This study investigates the synergistic effect of a cellulase enzyme mixture combined with wet rotor milling to enhance glucose yields during the enzymatic hydrolysis of cotton-based textile waste. The impact of mechanical energy input is assessed by varying milling durations in the presence and absence of enzymes. Enzyme-assisted milling enables a streamlined, single-step process, increasing glucose yield by approximately 12% compared to conventional hydrolysis for 6 h. Two iterative cycles of milling followed by incubation in a feed tank are evaluated. The highest glucose conversion (38%) is achieved by combining a premilling step with iterative cycles of milling performed with minimal milling time and subsequent enzymatic hydrolysis. Extended milling times reduce enzymatic activity, suggesting potential inhibitory effects under certain conditions. Overall, the findings support that integrating enzymatic hydrolysis into milling operations is a viable strategy for the partial recycling and valorization of textile waste.

This study establishes a multidimensionally engineered Escherichia coli platform for efficient gadusol biosynthesis. By integrating high-throughput quantitative analysis, regulatory optimization, and downstream processing, we enable scalable production of this natural UV-protective and antioxidant cyclohexenone, advancing its industrial and functional applications.

Proceedings of the National Academy of Sciences, Volume 123, Issue 19, May 2026.

The immobilization of PLP on porous supports functionalized with phenylboronic groups reduces its photodegradation and enables co-immobilized transaminases to operate under light irradiation without the need for exogenous cofactor addition.

ABSTRACT

Recent studies reveal that PLP-dependent amine transaminases are prone to light-induced inactivation due to cofactor-triggered conformational changes. To overcome this limitation, we herein develop a robust and broadly applicable strategy to prevent photoinactivation of PLP-dependent transaminases. Our results show that both HEPES buffer and light exposure are the most detrimental factors affecting the stability of the transaminase from Pseudomonas fluorescens (PfATA), leading to enzyme inactivation across all tested PLP concentrations. To overcome this limitation, we explored enzyme immobilization on both translucent and opaque supports functionalized with boronic groups. This support functionalization absorbs PLP and acts as a light quencher, avoiding PLP photodegradation and extending the PfATA operational half-life by a factor of four compared to its free counterpart. Finally, by utilizing this immobilization chemistry, we developed a self-sufficient heterogeneous biocatalyst (ssHB) by co-immobilizing PfATA, formate dehydrogenase, and L-alanine dehydrogenase, as well as the PLP and NADH cofactors, to aminate benzaldehyde with ammonium via a transamination/reductive amination cascade. This ssHB enables quantitative aldehyde amination in the first reaction cycle without the need for exogenous cofactor supply under ambient light conditions. Recycling of the three enzymes and the two cofactors was, however, limited, encouraging the further optimization of the multi-enzyme/cofactor immobilized system.

This review highlights advances in engineering Saccharomyces cerevisiae for the sustainable biocatalytic production of pharmaceutically relevant amines, amino alcohols, amino acids, and complex alkaloids. It focuses on strategies to increase productivity, including enhanced enzyme expression, cofactor regeneration, precursor channeling, pathway balancing, and the removal of interfering enzymes, all aimed at enabling scalable industrial applications.

Whole-cell biocatalysis offers a sustainable alternative to traditional chemical synthesis for producing pharmaceutically relevant, often chiral, amines and amino acids. Saccharomyces cerevisiae has emerged as a privileged microbial chassis due to its robustness, ease of genetic manipulation, and GRAS status. This concise review summarizes recent advances in metabolic and genetic engineering of S. cerevisiae for amine biocatalysis, focusing on strategies to overcome bottlenecks such as enzyme gene expression, cofactor regeneration, and precursor channeling. The first section covers state-of-the-art methods for engineered strain construction, including genomic editing, optimization of gene expression (copy number, promoters, terminators, codon usage), and metabolic engineering (pathway balancing, compartmentalization, cofactor supply, transport proteins, auxiliary enzymes, and enzyme targeting via signal peptides), all enhancing product yields and enabling complex amine synthesis. The central section critically discusses compound families accessible via engineered S. cerevisiae, including various amines, amino alcohols, and amino acids such as l-carnitine, ergothioneine, halogenated tryptamine, serotonin, psilocybin, spermidine, l-ornithine, and mycosporine derivatives. Bioproduction of complex alkaloids, such as tropine derivatives (hyoscyamine and scopolamine) and ergot alkaloids, is also reviewed. Finally, current challenges and future perspectives are outlined, highlighting the integration of systems and synthetic biology tools to establish S. cerevisiae as a scalable platform for industrial amine production.

Ene-reductases (EREDs) efficiently reduce activated C═C bonds. Expanding the current application, they catalyze the selective reduction of highly substituted cyclohexenones. Immobilized, they were scaled to 200 mL, achieving full substrate conversion without enzyme degradation.

ABSTRACT

Ene-reductases (EREDs) efficiently reduce activated C═C bonds, but their activity toward tetra-substituted alkenes has been largely unexplored. Here, we report the first systematic study demonstrating that several EREDs catalyze the stereoselective reduction of tri- and tetra-substituted cyclohexenones while investigating the influence of different cofactors. To enable a scalable application, the best enzyme (YqjM) was co-immobilized with glucose dehydrogenase on Ni–NTA cellulose beads, achieving >99% immobilization efficiency and high operational stability. The immobilized system was successfully scaled to 200 mL, reaching full substrate conversion without enzyme degradation over 9 days.

Cytochrome P450s catalyze a diverse array of reactions including crosslinking of aromatic side chains in the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs). ApyO is a cytochrome P450 enzyme that forms a C-C bond between two tyrosines in a YLY motif in the substrate ApyA, the precursor peptide of the RiPP aminopyruvatide. We utilized cell-free translation to generate ApyA variants and probe the substrate tolerance of ApyO. Through Alphafold-based modelling and in vitro assays, we show that ApyO accepts the 10 C-terminal residues of ApyA and requires a conserved Arg/Lys in the substrate peptide. Inspired by substrate sequences found in orthologous biosynthetic gene clusters, we substituted one of the tyrosine residues with a tryptophan and observed that ApyO catalyzed the formation of an N-C bond between the indole of Trp and the C{varepsilon}2 of Tyr. ApyO unexpectedly catalyzed formation of a C-O bond between the two tyrosine residues when we substituted the leucine residue in the YLY motif with tyrosine and tryptophan. We also show that a peptide containing a biaryl linkage and the C-terminal aminopyruvate displayed sub-nanomolar inhibitory activity against selected proteases. Overall, this study demonstrates plasticity in the manner of macrocyclization catalyzed by the P450 ApyO and provides a starting point for chemoenzymatic approaches towards producing diverse macrocyclic scaffolds.

Chemoenzymatic reactions using the siderophore synthetase DesD from Salinispora tropica and hydroxamic acid substrates (native, non-native) with different multiplicities (monomer, dimer) and components (homo-/heterodimer) followed different reaction trajectories, which biased the architectures of the chelator major products and indicated DesD contains a site that governs the binding of dimeric substrates.

ABSTRACT

The siderophore synthetase DesD generates hydroxamic acid chelators with applications in metal-based radiopharmaceuticals and sequestering toxic or commodity metals. DesD has been used in chemoenzymatic syntheses with native substrates, with less focus on non-native substrates. This study investigated masking the modest activity of the non-native substrate N-hydroxy-N-glutarylcadaverine (2) by forming a heterodimer with the native DesD substrate N-hydroxy-N-succinylcadaverine (1). Recombinant DesD from Salinispora tropica (StDesD) was evaluated with combinations of homo- and heterodimers of 1 and 2 (3–6) as substrates, including N-to-C positional isomers. Chemoenzymatic reactions using heterodimers of 1 and 2 (5, 6) showed similar substrate consumption and product types to the native 1 homodimer (3), with substrate consumption about three times greater than 2 alone, demonstrating the success of the masking approach. Furthermore, dimeric substrates ablated the ability of StDesD to generate the odd-numbered trimeric hexadentate macrocycle desferrioxamine E (DFOE) as its native major product, instead generating the even-numbered tetrameric macrocycles of dimeric substrates (3, 5, 6) as major products. While the StDesD upper limit of iterations per substrate was similar for monomeric and dimeric substrates, the latter generated chelators with unprecedented cavity sizes and denticities, including an icosadentate chelator that formed a 3:1 metal:ligand complex with Ga(III).

Genetic code expansion introduces new-to-nature chemical moieties into ribosomally synthesized proteins. In practice, the scope of functional groups that can be accessed using this method is often limited by noncanonical amino acid (ncAA) availability. Producing ncAAs directly in cells can circumvent poor ncAA uptake or commercial unavailability, but limited enzymes suitable for this application exist. In vitro evolution campaigns have been remarkably successful in yielding synthetically useful "ncAA synthases." However, these enzymes are optimized for preparative-scale synthesis and their activities often do not translate well to cellular biosynthesis. Thus, expanding strategies to engineer enzymes specifically for ncAA production within cells will benefit further implementation of genetic code expansion. Here, we use phage-assisted noncontinuous and continuous evolution to evolve enzymes for improved synthesis of non-canonical tyrosine derivatives in E. coli. Using simple serial passaging, we uncovered mutations that doubled the production of an expensive ncAA, 3-methoxytyrosine, by tyrosine phenol lyase, and furthermore evolved variants that enable 3-iodotyrosine biosynthesis, a transformation the parent enzyme is unable to catalyze. Additionally, we evolved a recently reported tyrosine synthase for improved production of 3-halogenated tyrosines, identifying variants that exhibit high activity even at low substrate concentrations owing to a [~]8-fold reduction in KM. Our results demonstrate that phage assisted evolution can be used to rapidly improve the activity of enzymes for ncAA production in cells.

Modern protein design methods based on deep learning allow generation of customized protein scaffolds with diverse geometries and functionalities. Here, we capitalize on these recent advances to develop hyper-thermostable de novo CO2 reductases featuring a cobalt porphyrin IX cofactor (CoPPIX). CoPPIX containing enzymes were assembled in vivo through media supplementation with cobalt salts and assessed for photocatalytic CO2 reductase activity. We identified two cysteine-ligated designs that exhibit high activity (>1000 turnovers at rates of up to 25 min-1) while suppressing competing hydrogen evolution pathways. A 2.1 [A] crystal structure shows close agreement to the design model with the Co-Cys bond programmed as intended. This study showcases the power of computational protein design in developing artificial enzymes to activate challenging molecules such as CO2.

Arylmalonate decarboxylase (AMDase) stereoselectively converts disubstituted malonates to chiral carboxylic acids, but its substrate spectrum is very limited regarding the size of the smaller substituent. Inspired by the observation that (S)-selective AMDase variants also convert larger substrates, we unlocked the synthesis of the (R)-enantiomers of -aryl and -alkenyl n-butanoic and n-pentanoic acids, respectively, in exquisite enantiopurity.

In vegetarian diets, phytate is known to disrupt the adsorption of minerals. Fortifying foods with phytase, a therapeutic enzyme known to mitigate phytate, might increase the uptake of important nutrients. Phytase is susceptible to environmental stress such as heat and acidic conditions encountered during food processing. Therefore, we developed and optimized a core-shell microparticle composed of a phytase-chitosan core and a shell consisting of cross-linked alginate-{kappa}-carrageenan. Ethanol was used to precipitate the microparticles, and the ethanol concentration was optimized along with the chitosan and phytase ratio and the alginate-carrageenan concentration, to form stable core-shell microparticles. The optimized core-shell microparticles have a loading capacity of 32.7% with a high encapsulation efficiency of 80.3% and uniform micro-size with a diameter of 3.2 {micro}m and a poly-dispersity index of 0.178. Loaded phytase retained 62.7% enzymatic activity after heat treatment and digestion conditions. These results indicate that core-shell microparticles are suitable for retaining enzyme activity within the food matrix under typical food processing conditions. HighlightsO_LIDevelopment of size-controlled core-shell microparticles to protect phytase C_LIO_LIPhytase-chitosan microparticles are surrounded by an alginate-{kappa}-carrageenan shell C_LIO_LIOptimization achieved 32.7% loading capacity with a uniform size of 3.2 {micro}m C_LIO_LICore-shell microparticles retained 62.7% enzyme activity after heat and digestion C_LIO_LIPhytase powder (2 mg) is required for a single maize meal C_LI

Isoflavone hydroxylases (IFHs, CYP81E) convert isoflavone aglycones into their respective hydroxylated intermediates, which direct legume isoflavones into specialized defense pathways. In soybean, their functions have been studied mostly in the context of the daidzein-derived glyceollin biosynthesis. Here we combine metabolomics-guided feature mining, phylogenetic analysis, heterologous enzymology, structural elucidation, and in planta metabolite validation to determine the functional landscape of the soybean IFH family. Analysis of a soybean isoflavonoid-enriched metabolomic dataset revealed unidentified hydroxyisoflavone features that co-accumulated with glyceollins, indicating branch chemistry that is not well-recognized. The systematic characterization of the repertoire of soybean CYP81E has demonstrated that 9 out of 11 GmIFHs are catalytically active and collectively span both 2'- and 3'-hydroxylation of the major soybean isoflavone aglycones. Among them, GmIFH9A showed broad substrate scope and regioselectivity, yielding canonical and previously unknown hydroxylated isoflavone products. NMR and LC-MS/MS were used to identify and validate the hydroxylated isoflavone products as 2'-hydroxyglycitein and 2'-hydroxyformononetin, whose presence was also confirmed in soybean roots, thus confirming two of the hidden soybean isoflavonoid network metabolites. Kinetic studies also indicated that, although the majority of GmIFHs prefer daidzein and genistein as substrates, a few isoforms are active towards methoxylated isoflavones as well, indicating functional divergence in this expanded family. Our findings collectively redefine soybean IFHs as a multi-functional enzyme module that expands the hydroxyisoflavone chemical space and reveals new biosynthetic entry points beyond canonical glyceollin pathway.

Fungal unspecific peroxygenases (UPOs) are promiscuous heme-containing enzymes that selectively oxyfuntionalize C-H bonds. Structural and enzymatic reaction studies of an evolved UPO mutant from Candolleomyces (Psathyrella) aberdarensis (PabUPO-I) reveal how the heme channel architecture dictates the regioselectivity towards alkanes, fatty acids and norisoprenoids, providing insights for future engineering of this enzyme.

Fungal unspecific peroxygenases (UPOs) are versatile biocatalysts capable of inserting oxygen into nonactivated CH bonds. Despite their significance in organic synthesis, only a few UPOs have been characterized structurally and biochemically. In the current study, we provide insights into the structure and reaction profile of an unusual, acidic, long UPO from Candolleomyces aberdarensis that was evolved for heterologous expression in yeast. The crystal structure of the enzyme was resolved at a resolution of 1.8 Å, which helped to reveal several catalytic determinants that differ from those of canonical long UPOs. Cocrystallization experiments with alkanes, fatty acids, and the norisoprenoids α-damascone and α-ionone, were carried out together with the analysis of enzymatic reactions. With alkanes and fatty acids, the biocatalyst performs oxygenations at the ω−1 and ω−2 positions, producing diols and ketones. Conversely, the enzyme acts on the α-ionone ring and the vinylic aliphatic chain of α-damascone, generating 3-hydroxy-α-ionone and 10-hydroxy-α-damascone, respectively. The substrate preferences of this biocatalyst are marked by an intensely hydrophobic heme channel, with some residues protruding into the tunnel, and a characteristic catalytic tripod comprising a Met residue in a dual conformation. Molecular dynamics simulations revealed that enzyme catalysis is primarily driven by dynamic channel gating, ligand-specific preorganization within the access tunnel, and complex diffusion pathways leading to the active site.

Science, Volume 392, Issue 6798, Page 643-647, May 2026.

Science, Volume 392, Issue 6798, Page 582-583, May 2026.