Biocatalysis@TUDelft

Nature Chemical Biology, Published online: 16 July 2025; doi:10.1038/s41589-025-01970-9

Monoterpene indole alkaloids are formed via a 3S stereoselective condensation between secologanin and tryptamine. Here the authors uncover the mechanism of epimerization behind uncommon 3R-containing alkaloids in Mitragyna speciosa (kratom) and study their inclusion in downstream biosynthesis.

Nature Chemical Biology, Published online: 17 July 2025; doi:10.1038/s41589-025-01990-5

Genes for Taxol

Scaling beyond the laboratory exposes enzymes to new stresses. Exposure experiments reveal that dynamic gas-liquid interfaces introduced by sparging or air entrainment during agitation are the main drivers of HMFO deactivation. Replacing bubbles with a membrane-based bubble-free aeration system dramatically enhances stability. These experiments provide valuable insights into the underlying deactivation mechanism, guiding the design of more robust biocatalytic processes.

Abstract

The impact of agitation on protein aggregation is often misattributed to shear stress rather than related phenomena such as cavitation and gas entrainment from the surface. For some time now, it has been known that shear is unlikely to harm most proteins directly. Rather, interfacial phenomena, particularly those involving dynamic gas-liquid interfaces are critical contributors to protein damage, which leads to aggregation and compromises stability. This work investigated the kinetic stability of 5-hydroxymethylfurfural oxidase (HMFO; EC: 1.1.3.47) in a 2 L stirred tank reactor. Exposure experiments revealed that the leading cause of enzyme deactivation was exposure to the gas-liquid interface, either produced deliberately when sparging gas into the system or by accidental air entrainment from the overhead space due to mechanical stirring. This was further proven by experiments using the Bio Thrust membrane module, which enabled bubble-free aeration thus, confirming that exposure to the gas-liquid interface is the leading cause of deactivation.

Nature Biotechnology, Published online: 15 July 2025; doi:10.1038/s41587-025-02751-4

Computationally designed enzymes show potent catalytic activity

We present EnzyMS, a Python-based LC-MS analysis pipeline tailored for biocatalysis. Applying it to Fe(II)/αKG-dependent halogenase WelO5∗, we discovered a previously unreported oxidative demethylation of soraphen A. Guided by computational design, a WelO5∗ variant with 3-fold improved activity was identified by testing just three variants. This work highlights how data-driven analysis can uncover new biocatalytic transformations and streamline enzyme discovery.

A photo(bio)electrochemical cell comprising an inorganic photoanode paired with a biocathode introduces a versatile platform for NADPH regeneration. The cell performs reductive biocatalysis, producing highly pure chiral products. The system operates using either isolated enzymes or whole cells as biocatalysts, harnessing light as its sole source of energy.

The widespread use of organofluorides in modern society has inadvertently led to the bioaccumulation of harmful pollutants, most prominently per- and polyfluorinated alkyl substances (PFAS). In principle, tailored biocatalysts able to cleave C--F bonds represent an attractive strategy to combat this (emerging) environmental crisis. However, Nature is largely impartial to C--F bonds, with fluoroacetate dehalogenases (FAcDs) standing out as a notable exception, catalyzing the hydrolysis of single C--F bonds in fluoroacetate at high turnover rates. To expand the substrate scope of FAcDs and harness its catalytic prowess for non-natural organofluorides, we designed and applied a robust growth-based selection strategy for large-scale FAcD engineering. Specifically, we demonstrate that FAcD-catalyzed C--F bond cleavage of (natural and) synthetic organofluorides generates metabolizable carbon sources for bacteria, enabling in vivo enrichment of active FAcD variants. By forcing populations expressing diverse FAcD-libraries to utilize various organofluorides as sole carbon source, we elicited a broad panel of FAcD variants that displayed improved activities and drastically altered substrate profiles. In these efforts, we also identified a previously overlooked inhibition pathway, which largely impedes the conversion of gem-difluoride compounds. Overall, our study presents the first large-scale engineering campaign of FAcDs and introduces an operationally simple selection platform that paves the way toward adapting these enzymes for the sustainable degradation of contaminating organofluorides.

Control over mutational library diversity is an essential consideration when engineering proteins, but is often fraught with trade-offs between diversity, specificity, and affordability. Contemporary library assembly approaches often incorporate oligonucleotide pool synthesis to achieve affordable, precise mutagenesis; however, these oligos are often reliant on complex designs to facilitate downstream PCR and/or restriction digests. Direct hybridisation of oligo pools is an overlooked strategy to simplify mutagenesis, especially when paired with a type IIS restriction cloning approach. We validate this approach by designing, hybridising, and deep sequencing single and dual substitution CDR region parts derived from nanobody GA10. Assembly of these parts into a full-length nanobody CDS facilitated the phage display of variant libraries for affinity maturation against its cyclic peptide target. Variants identified through enrichment analysis were expressed in isolation and yielded improved affinities by more than 100-fold. Recent advances in machine learning have successfully inferred improved variants outside of screened library space, but require controlled, multi-mutant libraries. The library assembly approach outlined in this research is well-suited for such approaches.

Ferulic acid decarboxylases (FDC) are promising enzymes for synthetically useful biocatalytic decarboxylation and carboxylation reactions with their broad substrate scope. However, temperature and light instability of the enzyme is a major concern. Moreover, unfavorable thermodynamic barrier towards carboxylation direction limits enzyme’s ability for CO2 fixation reactions. Here, by in situ immobilization of FDC from Capronia coronata (CcFDC) inside ZIF-type metal-organic frameworks (MOFs), we obtained catalytic biocomposites, i.e. CcFDC@MOFs. Among the three types of CcFDC@MOFs tested, CcFDC@ZIF-67 exhibited excellent advantages compared to the free enzyme, with impressive thermal and light stability, higher turnover numbers, a broad temperature optimum extending into 75 °C as well as high reusability in decarboxylation reactions. Moreover, CcFDC@ZIF-67 can catalyze carboxylation reactions more efficiently than the free enzyme, which indicates that CcFDC@ZIF-67 has the potential to act as both a bicarbonate/CO2 capturing material and a CO2 fixation catalyst. We further improved carboxylation yields by sequential batch and semiflow reaction designs, with the semiflow design exhibiting 13-fold increase in total turnover numbers and 15-fold increase in space-time yields over the batch reaction, demonstrating CcFDC@ZIF-67’s ability as a bi-functional biocomposite for CO2 capture and conversion.

Recently, the number of ML-based tools for protein design has greatly expanded. Although there have been many successful uses of these tools for improved stability, solubility, and ligand binding, there have been fewer uses of these tools for designing proteins that have intrinsic allosteric mechanisms. In this regard, allosteric transcription factors (aTFs) are a class of regulatory proteins that includes repressors and activators that respond to environmental signals by allosteric communication to regulate their binding with DNA elements. The data exist for evaluating design algorithms for their ability to take allostery into account, as many aTFs have previously been engineered to respond to new ligands, enabling their use as biosensors. In particular, previous work from our lab used directed evolution to change the effector specificity of the transcriptional repressor, RamR, from cholic acids to each of five benzylisoquinoline alkaloids (BIAs). We wanted to see to what extent we could recapitulate these results by instead using LigandMPNN to design the ligand binding pocket. The wild-type RamR structure was predicted in complex with the five BIAs, and the binding pocket was then targeted for computational redesign. However, there was little overlap between the results of directed evolution and computational redesign, and in fact the nine redesigned protein variants tested proved not to be functional in Escherichia coli. Overall, these and other results suggest that different protein design methods may be needed to advance the computational design of allosteric or conformationally flexible proteins.

The convergent, stereoselective, and protecting-group-free synthesis of non-canonical amino acids, particularly those bearing a tetrasubstituted α-stereogenic center, remains a challenging task. By leveraging pyridoxal radical biocatalysis, we developed photobiocatalytic oxidative coupling methods for the assembly of α-tetrasubstituted amino acids, including β-branched variants, in a diastereo- and enantioselective fashion. Through repurposing and directed evolution of Thermotoga maritima threonine aldolases, we transformed a traditionally two-electron pyridoxal phosphate (PLP) dependent enzyme into a highly active and stereoselective biocatalyst for single-electron C–C bond formation, enabling enantioconvergent conversion of a broad range of racemic organoboron substrates. Our evolved radical C–C bond forming PLP enzymes achieved a total turnover number (TTN) of 3,100 under dual photobiocatalytic conditions, the highest TTN reported to date for an unnatural photobiocatalytic transformation. Mechanistic studies using radical clock probes and electron paramagnetic resonance (EPR) spectroscopy uncovered an unexpected role of free PLP in oxidative radical generation under photochemical conditions. Molecular dynamics (MD) simulations further elucidated the origin of enantio- and diastereoselectivity in the key radical addition step between the enzymatic quinonoid and the carbon-centered radical. Collectively, these results underscore the power of pyridoxal radical biocatalysis to access a broad spectrum of valuable non-canonical amino acid products via intermolecular, enzyme-controlled asymmetric radical chemistry, a transformation that remains elusive to both state-of-the-art small-molecule catalysis and native enzymology.

Paired electrolysis enables the simultaneous coupling of the CO2 reduction reaction (CO2RR) with anodic waste valorisation to form valuable products, but achieving selective, efficient and stable product formation remains a challenge. In this study, W-containing formate dehydrogenase (FDH) from Nitratidesulfovibrio vulgaris Hildenborough is immobilised onto an electrode made from carbon felt coated with porous TiO2 and paired with commercial Ni foam to demonstrate the first enzymatic flow electrolyser for the simultaneous conversion of CO2 and waste (plastic and biomass) to the single product formate. The enzymatic flow electrolyser achieved a combined faradaic efficiency (FE) towards formate of almost 200% for both electrodes and can operate at an unprecedently low full-cell voltage of −1.5 V for 122 h. The aqueous formate produced in the semiartificial electrolyser was further utilised downstream as a C1 building block in the photocatalytic hydrocarboxylation of alkenes, providing a path for the domino valorisation of CO2 and waste towards bulk and fine chemical synthesis.

Synthetic metabolism has the potential to transform carbon capture, bioremediation, or bioproduction strategies. To transfer metabolic designs from an in vitro context to living model systems such as the bacterium Escherichia coli, metabolic engineers incentivize the maintenance and use of the introduced metabolic module by making cell survival dependent on it (growth-coupled selection). However, creating and characterizing appropriately rewired selection strains is nontrivial and requires labor-intensive growth phenotyping in various conditions. To enhance the community use of extant selection strains, we compiled designs covering the central, amino acid, and energy metabolism of E. coli for this review, and we revisit the key concepts of growth-coupled selection.

Nature Catalysis, Published online: 11 July 2025; doi:10.1038/s41929-025-01365-y

The CO dehydrogenase–acetyl-coenzyme A synthase complex produces acetyl-coenzyme A from CO2, but its structural dynamics during catalysis remain unresolved. Now cryo-EM maps of six intermediate states reveal how ligand binding to a Ni–Fe cluster orchestrates the conformational changes of the complex during catalysis.

The thaumarchaeal 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle represents one of the most efficient mechanisms for CO2 fixation discovered to date. Within this cycle, the enzyme encoded by Nmar_1308 from Nitrosopumilus maritimus SCM1 plays a crucial role due to its dual functionality as both a crotonyl-CoA hydratase (CCAH) and a 3-hydroxypropionyl-CoA dehydratase (3HPD). Although the importance of a bifunctional enzyme for lowering the cost of biosynthesis, the details of structural dynamics are still missing. Here, in addition to our cryogenic temperature structures, we determined the first ambient temperature structures of the Nmar_1308 protein by Serial Femtosecond X-ray Crystallography (SFX). The determined structures capture previously unobserved conformational dynamics of the Nmar_1308 protein, providing invaluable information for future synthetic biology applications.