Biocatalysis@TUDelft

Standard enzyme screening often overlooks industrial stress. We propose integrating conditions like pH shifts, low oxygen, interfacial deactivation, and high substrate/product levels into early high-throughput assays. By measuring stress-relevant metrics such as turnover under stress, cofactor efficiency, and solvent stability, the enzyme selection becomes more predictive, improving success rates during scale-up and process development.

ABSTRACT

The selection of enzymes for use as catalysts in chemical processes is typically carried out under optimal buffered conditions. However, under industrial conditions, at large scale, the environment usually includes pH gradients, oxygen limitation, interfacial deactivation, and high substrate or product concentrations. As a result, enzymes that perform well during initial laboratory screening often fail to maintain performance in large-scale bioreactors. In this perspective, we propose the integration of process-relevant stress conditions into the early stages of enzyme screening. We outline a framework in which parameters such as total turnover number (TTN) under stress, cofactor coupling efficiency, and solvent induced unfolding thresholds are measured alongside activity. These can be implemented in high-throughput formats with minimal methodological change. We also present recent experimental studies where similar approaches have been used, although not always explicitly framed as stress-based screening. The goal is to better align enzyme selection with real process demands, reducing uncertainty in early-stage development. By shifting stress factors from test conditions to reportable and reproducible selection pressure, screening outcomes can be more predictive, and the overall efficiency of biocatalyst development for scale-up can be improved.

Efficient engineering of nonribosomal peptide synthetases (NRPSs) is a key strategy for expanding valuable peptide natural products. Current bioinformatics-guided approaches are often constrained by a set of predefined fusion sites, remaining experimentally challenging in most NRPS systems. Here we present the Recombineering Accelerated Evolution (RACE), which harnesses Red/ET recombineering mediated partially matched homologous recombination to recapitulate recombination-driven NRPS evolution on a highly accelerated timescale. Application of RACE to six known NRPS gene clusters generated 830 recombinants and yielded over 600 novel peptides including novel bis-lipopeptides. These recombinants reveal 112 previously unrecognized recombination fusion sites, providing an extensive landscape for NRPS evolution and more useful resources for guiding NRPS engineering. The RACE establishes a new paradigm for programmable NRPS evolution and enables rapid discovery of bioactive peptides.

Polyurethanes (PURs) represent a significant challenge in plastic waste management due to their chemical resilience and limited recycling options. In this study, we report the identification and characterization of six novel bacterial urethanases, expanding the enzymatic repertoire for targeted PUR depolymerization. These enzymes demonstrated carbamate-cleaving activity optimally under alkaline conditions, maintaining stability across a pH range of 7 to 10 and varying thermal and solvent tolerances. Among the candidate enzymes, u17, u10, and u15 collectively exhibited high activity, catalytic efficiency, and thermostability, establishing a strong foundation for further optimization. Building on these results, u15 emerged as particularly notable for its catalytic efficiency on the carbamate model substrate di-urethane ethylene methylenedianiline, DUE-MDA, with a kcat/KM of 51.8 {+/-} 0.1 (s-1mM-1). and this motivated its selection for detailed structural analysis. High-resolution crystallography of u15 revealed key active-site architecture, including the conserved amidase signature catalytic triad and flexible loop regions that influence substrate binding and specificity. Molecular docking and molecular dynamics simulations further elucidated substrate binding determinants of u15 during urethane bond hydrolysis. Docking of DUE-MDA revealed two distinct substrate orientations (Pose A and Pose B) differing in the positioning of the carbamate group relative to Ser177. Pose A was more stable and catalytically competent, maintaining the substrate within the oxyanion hole and sustaining optimal geometry for nucleophilic attack by Ser177. Comparable behavior was observed for the partially hydrolyzed intermediate mono-urethane ethylene methylenedianiline, MUE-MDA, indicating a conserved binding mode across substrates. Collectively, these findings highlight amidase signature urethanases as valuable scaffolds for advancing sustainable and scalable biocatalytic recycling of polyurethanes. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/705263v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@b74003org.highwire.dtl.DTLVardef@cb8760org.highwire.dtl.DTLVardef@24bd5eorg.highwire.dtl.DTLVardef@c7ae9_HPS_FORMAT_FIGEXP M_FIG C_FIG

The mechanism of pacifigorgiadiene synthase from Burkholderia gladioli was investigated through isotopic labeling experiments and DFT calculations. The results revealed an unexpected labeling pattern which appeared to be different in the main and the side products, requiring unprecedented mechanistic explanations, including a “break-flip-cyclise” sequence to set the correct stereochemistry.

ABSTRACT

The sesquiterpene synthase BgPgS from Burkholderia gladioli produces the main product (–)-1-epi-pacifigorgia-6,10-diene, besides a few structurally related compounds. The enzyme mechanism of BgPgS was addressed through isotopic labeling experiments, revealing several intricate mechanistic problems. Unexpectedly, the isotopic labelings for the Me groups C12 and C13 occurred in different positions for the main and the side products. Moreover, as demonstrated in this study, two hydrogen atoms must change from the bottom to the top hemisphere, which is not possible through standard terpene biosynthesis routines with suprafacial hydrogen migrations. Our rational solution involves two mechanistic explanations: First, a key rearrangement may be associated with a conformational change that rotates one hydrogen from bottom to top. Second, a “break-flip-cyclize” sequence explains the change of side by the other hydrogen. DFT calculations show that the proposed terpene cyclization cascades are energetically feasible; only one problematic activation barrier (>25 kcal/mol) remains. However, several mechanistic alternatives either failed to explain the experimental results of the isotopic labeling experiments or were associated with even higher activation barriers. Our biosynthetic proposal for pacifigorgiadiene biosynthesis can be understood as a contribution that awaits further investigation and scientific debate for its ultimate resolution.

Kaitocephalin (KCP) is a neuroprotective fungal metabolite with a unique scaffold of amino acids linked via C–C bonds. Genome-transcriptome analyses identified its biosynthetic gene cluster (kpb cluster) in Eupenicillium shearii. LC-MS/MS profiling identified four new KCP-related compounds. In vitro assays using 2(S)-dechlorokaito lactate, one of the four metabolites as a substrate revealed the functions of KpbI, KpbM, and KpbB, showing that KpbI catalyzes an unprecedented two-step oxidation.

ABSTRACT

Kaitocephalin (KCP) is a neuroprotective natural product that acts as an antagonist of ionotropic glutamate receptors, making it a highly promising lead for drug discovery. It possesses a unique scaffold composed of three amino acids connected via C─C bonds, which appears peptide-like but is formed without peptide bonds. In this study, we identified the KCP biosynthetic gene cluster (kpb cluster) in the producing fungus Eupenicillium shearii through integrated genomic and transcriptomic analyses. LC-MS/MS profiling and chemical derivatization of E. shearii extracts led to the discovery of four novel pathway-related metabolites. In vitro enzymatic assays with 2(S)-dechlorokaito lactate, one of the four identified metabolites, as a substrate enabled functional characterization of KpbI, KpbM, and KpbB involved in KCP formation. Among them, the dioxygenase KpbI was found to catalyze an unprecedented two-step oxidation to form the d-serine moiety. In addition, isotope tracing experiments provided new insights into the origin of the l-proline moiety. These findings establish a foundation for future studies aimed at elucidating the complete biosynthetic mechanism of KCP.

A two-step process was transferred from batch to flow mode for the synthesis of (R)-2-fluoro-1-phenylethanol as a benchmark, consisting of a decarboxylative fluorination in aqueous media, followed by an enantioselective biocatalytic reduction of the prochiral intermediate with RrADH immobilized on silica supraparticles. The biocatalyst achieves an excellent enantiomeric excess of >99.9% with an overall yield of 91% for the complete process.

ABSTRACT

The growing demand for sustainable and efficient methods for synthesizing fine chemicals has increased interest in innovative approaches for accessing high-quality chiral building blocks, particularly fluoroalcohols, which are relevant for the production of active pharmaceutical ingredients (APIs). This study presents the complete integration of a two-step process in a continuous flow reactor system for the synthesis of (R)-2-fluoro-1-phenylethanol as a reference molecule. To this end, the individual reaction steps and technologies for the decarboxylative fluorination of 3-oxo-3-phenylpropanoic acid in aqueous media, followed by an enantioselective biocatalytic reduction of the prochiral intermediate phenacyl fluoride, were adapted and implemented in a compact laboratory system for performance demonstration. Alcohol dehydrogenase (ADH) from Rhodococcus ruber (RrADH) produced in a plant-derived BY2 cell-free expression system was used as the biocatalyst, which was immobilized via an imine bond on glutaraldehyde-modified silica supraparticles. The immobilized enzymes were used in batch mode for comprehensive kinetic studies of the enantioselective reduction, including evaluations of their operational and storage stability. Excellent enantiomeric excess (> 99.9%) and overall yields of up to 91% were achieved for both synthesis steps. These results are a prerequisite for the targeted and stable use of the enzyme in a continuously operated two-step process, which was achieved by using a serial micro batch reactor (SMBR) setup with a capillary reactor for precise temperature control. This study demonstrates the advantages of combining immobilized biocatalysts with continuous-flow operation for achieving high efficiency and selectivity in the synthesis of chiral fluoroalcohols. The integrated process provides a sustainable and versatile basis for future developments in the green synthesis of fluorinated building blocks relevant to pharmaceutical applications.

p-Benzoyl-l-phenylalanine (pBzF) integrates genetic code expansion with benzophenone photochemistry to enable proximity-based labeling, programmable biocatalysis, and biosafe microbial engineering, all from a single, site-specifically encoded amino acid.

ABSTRACT

p-Benzoyl-l-phenylalanine (pBzF) is a widely used noncanonical amino acid (ncAA) that expands the chemical repertoire of proteins. Its benzophenone (BP) chromophore undergoes near-quantitative intersystem crossing (ISC) to a triplet state, furnishing a highly efficient, site-addressable photoreactive handle. Beyond photochemistry, the bulky, hydrophobic side chain introduces distinct steric and electronic effects that enable new reactivity in protein active sites. Genetic incorporation of pBzF in vivo, including directed evolution, has unlocked applications ranging from site-specific photo-crosslinking for interaction mapping to engineering antibody fragments, sharpening monoclonal antibody (mAb) epitope recognition, and creating protein-based photocatalysts. pBzF has also proved powerful for mechanistic studies by stabilizing short-lived intermediates. More recently, pBzF-containing proteins have been leveraged in light-driven transformations, including [2+2] photocycloadditions, deracemizations, and dehalogenations, and in the construction of artificial photosynthetic systems. This review critically discusses these advances and establishes pBzF as a versatile photochemical and structural motif for building proteins with non-natural, light-responsive, and catalytically competent functions.

The class-defining monoterpenoid indole alkaloid (MIA) scaffold strictosidine is generated by condensation of tryptamine and secologanin by the Pictet-Spenglerase strictosidine synthase (STR). All previously characterized STR orthologs are strictly 3S-stereoselective. Here, we report that the medicinal plant species Pogonopus speciosus (Rubiaceae) accumulates the 3R epimer vincosidic acid produced by a an ortholog of STR. This ortholog, named here Epi-STR, exclusively produces the 3R configuration and is capable of accepting both secologanic acid and the methylester secologanin as aldehyde substrates. Using comparative phylogenetic and structural analyses, we determine the amino acid residues that confer stereoselectivity. Through rational design of reciprocal amino acid substitutions, we achieved switches in stereoselectivity in a canonical STR and in Epi-STR. The stereoselectivity of the engineered mutants is also dependent on the identity of the aldehyde substrate. Notably, previous and extensive engineering efforts have failed to switch the stereo-selectivity of STR. Therefore, this discovery now allows cost-effective epimer-pure access to the R-epimer, and offers mechanistic insights into the enzyme stereoselectivity of this important reaction. This work also highlights the importance of phytochemical analyses of poorly described plant species.

Nature Communications, Published online: 07 February 2026; doi:10.1038/s41467-026-69265-8

Butenolides are important features of many bioactive compounds but known butenolide biosynthetic pathways are complex and challenging to harness. In this study, the authors report that avenolide, a 4-alkylbutenolide that regulates avermectin production in Streptomyces avermitilis, is assembled from a fatty acyl thioester by a multifunctional flavoenzyme and an iterative cytochrome P450.

Nature Communications, Published online: 07 February 2026; doi:10.1038/s41467-026-69211-8

Despite the success of engineering cytochrome P450 enzymes as peroxygenases to utilize cost-effective H2O2, their applications are limited due to poor H2O2 tolerance. Here, the authors report a cytochrome P450 enzyme, P450stri, from Streptomyces triculaminicus and its engineering as a peroxygenase with strong H2O2 tolerance for regioselective hydroxylation of steroids.

Mammalian phosphatidylserine synthase-1 and -2 synthesize phosphatidylserine (PS) by replacing the headgroup of either phosphatidylcholine (PC, PTDSS1) or phosphatidylethanolamine (PE, PTDSS2) with a serine. We determined structures of PTDSS2 from Equus caballus in complex with either PE or serine substrates to resolutions of 2.8-3.2 [A]. The structures define substrate binding sites and reveal that the phosphate group of PE is coordinated by two Ca2+. In addition, we found that PTDSS2 has significant phospholipase D (PLD) activity in the absence of serine, which was not reported previously, and that Ca2+ is required for the PLD activity. These discoveries enrich our knowledge in the mechanism of mammalian PTDSS.

Long-chain acyl-CoA carboxylase (LCC) is an essential enzyme complex in mycobacteria that supplies acyl-CoA precursors for the biosynthesis of mycolic acids and complex lipids, yet its architecture, substrate selectivity, and conformational dynamics have remained poorly defined. Here we determine pre- and post-reaction states of the endogenous 868-kDa LCC complex from Mycobacterium smegmatis by cryo-electron microscopy at 2.1-3.7 Angstrom resolution. These structures visualize ATP-dependent redistribution of the biotin carboxyl carrier protein and capture multiple substrate-free and substrate-engaged carboxyltransferase intermediates. LCC assembles into an asymmetric 8:2:4:2 organization of AccA3, AccD4, AccD5, and AccE5, with two biotin carboxylase modules flexibly tethered to a heterohexameric carboxyltransferase core. We define the structural basis of substrate selectivity within the CT core: AccD5 selectively binds the short-chain substrate C3-CoA, whereas AccD4 accommodates the long-chain substrate C16-CoA only in an open conformation. Unexpectedly, AccD4 in its closed conformation also engages and positions C3-CoA, revealing previously unrecognized conformational plasticity in substrate recognition. Together, these structural intermediates establish a mechanistic framework for LCC function, explaining how chemically distinct acyl-CoA substrates are processed within a single complex and revealing features that may contribute to the regulation of carbon flux into mycobacterial lipid biosynthesis.

Mycobacterium tuberculosis topoisomerase I (MtbTOP1) is essential for the viability of the causative agent of TB. There are still significant unanswered questions regarding the dynamic conformations during catalysis of relaxation of negatively supercoiled DNA by MtbTOP1. We aim to study the flexible hinge residues that control the dynamics of inter-domain rearrangements involved in the enzyme conformational changes that allow the opening-closing of the topoisomerase gate. We used the online server PACKMAN to predict possible hinges from the MtbTOP1 crystal structure. The predicted region "PRO506 to LEU526" at the border between domains D2 and D4 with a p-value <0.05 was then studied as a potential hinge. The highly conserved ARG516 from this region interacts with the DNA inside the protein toroidal cavity. This arginine maintains inter-domain interaction with GLU207 of D4 and ASP691 of D5 domains. After introducing alanine substitutions, we further studied the mutant topoisomerases in biochemical experiments. The results showed a significant loss in DNA relaxation activity without affecting DNA binding and cleavage after mutating GLU207 and ARG516, consistent with their role as hinge residues in domain rearrangements.