Biocatalysis@TUDelft

Science, Volume 389, Issue 6760, Page 618-622, August 2025.

A novel PET hydrolase, bbPET0069, was identified from a soil microbial genome. bbPET0069 and CALB showed remarkable synergy in PET degradation. Using surface feature analysis, PET degradation activity of bbPET0069 was significantly improved. This combination of a two-enzyme system and surface feature analysis holds promise for enhancing emerging PET-degradation enzymes.

Abstract

We here report a novel PET hydrolase originating from a soil microbial genome sequence. This enzyme, bbPET0069, exhibits characteristics resembling a cutinase-like Type I PET-degrading enzyme but lacks disulfide bonds. Notably, bbPET0069 displayed remarkable synergy with Candida antarctica lipase B (CALB), demonstrating rapid and efficient PET degradation. To improve the PET degradation activity of bbPET0069, we employed a 3D structural modeling to identify mutation sites around its substrate binding domain combined with a protein language model for effective mutation prediction. Through three initial rounds of directed evolution, we achieved a significant enhancement in PET degradation with CALB, resulting in a 12.6-fold increase compared to wild-type bbPET0069 without CALB. We confirmed its PET degradation activity in PET nanoparticles and films, and our proposed approach enabled efficient PET degradation to terephthalic acid monomers up to 95.5%. Our approach, which integrates a two-enzyme system with protein engineering, demonstrates the potential for enhancing the activity of emerging PET-degradation enzymes, which may possess unique attributes.

Discovering and optimizing reactions is central to synthetic chemistry. However, chemical reactions are traditionally screened using relatively low-throughput methods, prohibiting exploration of diverse chemical space, particularly for reactions involving multiple components with millions of potential combinations. State-of-the-art technologies designed to increase screening throughput are limited to thousands of reactions and often require weeks to months for data collection. Here we report a DNA-encoded combinatorial screening platform, enabling large matrices of reactions to be performed in a single test tube followed by simultaneous reaction analysis using DNA sequencing. Using this platform, a single researcher performed 504,000 reactions in < 3 days, encompassing a variety of reaction conditions and times. We combined this platform with data science tools to design a targeted library covering chemical space broadly, then used the resulting dataset to train a machine learning model for both prediction and interpretation tasks, demonstrating utility for reaction development and mechanistic studies. Overall, this technology discloses fundamentally new opportunities in data-driven discovery, optimization, and mechanistic understanding of synergistic catalytic systems.

Science Advances, Volume 11, Issue 33, August 2025.

Several luciferases have been developed for imaging and biosensing, and the collection continues to grow as new applications are pursued. The current workflow for luciferase optimization, while successful, remains laborious and inefficient. Mutant libraries are generated in vitro and screened, "winning" mutants are picked by hand, and the isolated sequences are subjected to additional rounds of mutagenesis and screening. Here, we present a streamlined platform for luciferase engineering that removes the need for manual library generation during each cycle. We purposed an orthogonal DNA replication (OrthoRep) system for continuous hypermutation of a well-known luciferase (GeNL). Short cycles of culturing and screening were sufficient to evolve the enzyme, with no repetitive manual library generation necessary. New GeNL variants were identified that exhibit improved light outputs with a non-cognate and inexpensive luciferin. We further characterized the novel luciferases in cell models. Collectively this work establishes OrthoRep and continuous hypermutation as a viable method to engineer luciferases, and sets the stage for more rapid development of bioluminescent reporters. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=57 SRC="FIGDIR/small/669707v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@121475aorg.highwire.dtl.DTLVardef@1d376b4org.highwire.dtl.DTLVardef@162b8aorg.highwire.dtl.DTLVardef@184731e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Mass spectrometry-based untargeted analysis, or metabolomics, typically requires chromatography to separate charged ions from different compounds. However, analyzing a large number of samples using serial injections limits throughput and can introduce batch effects or retention time shifts during extended data acquisition. To address these limitations, we propose single file injections (SFI), a method for parallel data acquisition from multiple samples without instrument modifications. In SFI, pooled samples are initially injected to serve as reference peaks for subsequent analyses. Repeated injections of individual samples are then performed at fixed time intervals using isocratic elution. This approach significantly increases throughput, potentially allowing for the analysis of 1000 samples per day, with a marginal time cost per sample equivalent to the fixed injection interval. To ensure compatibility with conventional analysis techniques, an open source software is developed to analyze data from SFI, which can recover peaks back to their original samples and is available online (https://github.com/yufree/sfi).

Nature Catalysis, Published online: 12 August 2025; doi:10.1038/s41929-025-01390-x

Expanding the methods for constructing artificial enzymes is of high interest. Now a photoactive cofactor is designed that mimics NAD+, allowing its insertion into a range of NAD+-binding protein scaffolds to catalyse inter- and intramolecular [2 + 2] cycloaddition reactions.

Nonheme iron enzyme, 4-hydroxymandelate synthase from Amycolatopsis orientalis (AoHMS), was engineered to catalyze enantioselective amino azidation via a stepwise nitrene addition and radical azide transfer.

Abstract

Alkene difunctionalization represents an important category of reactions in organic synthesis, with a diverse array of transformations developed over the past decades for various synthetic applications. Nevertheless, the scope and diversity of biocatalytic alkene difunctionalization have been limited, constraining its synthetic utility. In this study, we repurposed nonheme iron enzymes to generate iron nitrene intermediates for alkene difunctionalization. 4-hydroxymandelate synthase from Amycolatopsis orientalis (AoHMS) was successfully engineered for direct alkene aminoazidation to produce chiral 2-azidoamines. Directed evolution was performed on AoHMS to provide evolved variants that could utilize O-pivaloylhydroxylamine triflic acid as the nitrene precursor and produced various primary aminoazidation products with up to 44% yield, 44 total turnover number (TTN), and 98.5:1.5 enantiomeric ratio (e.r.). Mechanistic studies indicated that this new biocatalytic transformation proceeds through a stepwise radical addition and azide recombination pathway. This work expands the catalytic toolbox of metalloenzymes and opens up new opportunities for biosynthesis by introducing nonnatural olefin difunctionalization reactions into biocatalysis.

The discovery of urethanases shows an opportunity to access biotechnological recycling of polyurethane-based plastics (PURs), widely used in the manufacture of everyday materials. However, the mechanistic understanding of these enzymes remains under debate. In this work, we report a QM/MM-based mechanistic study of the metagenome-derived urethanase UMG-SP-2 catalyzing the degradation of a urethane-like model compound, 4-nitrophenyl benzylcarbamate (pNC). A high-quality structural model generated with AlphaFold2, prior to the availability of the crystal structure, accurately captured the Ser-Ser-Lys catalytic triad characteristic of amidase signature enzymes. Highly accurate constant-pH non-equilibrium molecular dynamics and Monte Carlo (neMD/MC) simulations provided the full titration curve of active site Lys, explaining the need for alkaline media for the enzyme to be active. The generation of the free energy landscape, obtained by means of Free Energy Perturbation methods with the M06-2X DFT functional describing the QM region of the full system, reveals an esterase-like three-step mechanism of UMG-SP-2, acylation, hydrolysis, and decarboxylation, with all steps being kinetically feasible. Our computational results show very good agreement with experimental kinetic data, with a calculated free energy barrier of 21.2 kcal·mol⁻¹ for the rate-determining step, compared to 22.9 kcal·mol⁻¹ derived from the experimentally measured TOF. The present results also open the door for the final decarboxylation occurring in solution after release of the product of the hydrolysis step, or within the active site. These findings provide atomistic insight into urethanase function and establish a robust framework for the future design of biocatalysts targeting polyurethane degradation.

“The most important future application of my research would be the integration of designer enzymes into biocatalytic processes in industry… If I were a piece of lab equipment, I would be a plate reader, trying to deal with 96 things in parallel…”

Find out more about Cathleen Zeymer in her Introducing… Profile.

Recent advances in de novo protein design have greatly outpaced standard protein biochemistry workflows, and experimental testing has been a bottleneck in the validation of new designs and methodologies. Here, we describe experimental and computational workflows to address the issues of scale, speed and reproducibility of common in vitro protein testing methods, enabling at least an order of magnitude increase in throughput while reducing wetlab time. Semi-Automated Protein Production (SAPP) is a rapid, modular, scalable and cost-effective protocol, enabling up to milligram-scale protein production, and standardized characterization including yield, dispersity, and oligomeric state of hundreds of designs per day, at the cost-equivalent of a few DNA oligos per construct. End-to-end protocol execution takes 48 hours, with about 6 hours spent benchside using mostly standard laboratory equipment. This protocol has become the standard at our institute, providing critical experimental validation for dozens of projects spanning tens of thousands of designs. Since at least 80% of SAPPs total cost comes from synthetic DNA, we also developed a scalable demultiplexing protocol (DMX) to leverage oligo pools as input DNA, providing a further 5-fold reduction in costs, enabling >1000 designs to be purified and characterized in arrayed, clonal format at a cost of $5 per construct. By reframing standard molecular biology practices and orchestrating wetlab workflows with partial automation instead of complex end-to-end robotics, these protocols should be widely adoptable, accelerating protein design.

Doxorubicin, a glycosylated type II PKS-derived natural product first isolated from Streptomyces peucetius is a widely prescribed anticancer medicine and cytotoxic agent. It is also the final oxidation product of DoxA, a cytochrome P450 that catalyzes three sequential C–H oxidation reactions at the C13 and C14 positions on deoxy-daunorubicin leading to doxorubicin. A key limitation of in vitro P450 biocatalysis with anthracycline substrates is the rapid reductive de-glycosylation in the presence of electron donors like NADPH and ferredoxin reductase leading to inactive aglycones. This undesired reaction derails late-stage oxidative functionalization of anthracycline derivatives for clinical development. Recently, we demonstrated that DnrV, a previously uncharacterized vicinal oxygen chelate (VOC) protein in the doxorubicin BGC, prevents reductive activation thereby mitigating the cellular redox stress and metabolite destruction associated with redox cycling. In this study, we employed biochemical, biophysical, and structural methods to elucidate the molecular basis of DnrV function. Our analyses revealed that DnrV is a multifunctional protein that simultaneously modulates the redox characteristics of the bound anthracycline and induces catalytic rate enhancement of the iterative cytochrome P450 DoxA. These are novel functions of the ancient VOC protein family, representing a new functional class we term REdox-Associated CytoToxin binding protein (REACT). Homologues of DnrV from non-anthracycline BGCs display similar redox-protective activity, and some extend this function to the bacterial metabolite menadione. These findings establish REACTs as a conserved family of multifunctional proteins that modulate quinone redox behavior and promote oxidative tailoring of redox-active natural products.

Functional group migration (FGM) reactions represent a fundamental class of transformations in organic chemistry, enabling the repositioning of functional moieties in non-obvious ways. However, catalytic asymmetric radical-mediated FGMs re-main rare, due to the inherent challenges of achieving catalyst-controlled enantioselectivity over free radical intermediates. Herein, we repurpose imine reductases (IREDs), a class of biotechnologically important enzymes known for their substrate promiscuity, to enable the first examples of catalytic asymmetric cyano group migration via a radical mechanism. An orthog-onal set of radical enzymes, including PbaIREDCym and SmiIREDCym, were engineered, allowing both 1,4- and 1,5-cyano group migrations reactions to occur in an enantiodivergent fashion. The use of nonionic surfactant TPGS-1000 was found to improve both the yield and enantioselectivity of these cyano migration reactions. This biocatalytic process exhibited a broad substrate scope and is readily scalable, affording a rare example of chiral non-amine product assembly with imine reductases. More broadly, stereoselective radical biocatalysis with engineered IREDs and other versatile enzymes provide a potentially general solution to challenging asymmetric FGM reactions.

Alkene hydrogenation is a cornerstone of chemical synthesis, yet enzymatic strategies remain limited to electron deficient substrates via hydride transfer. With heme enzymes, we unlock an unprecedented hydrogenation pathway – termed biocatalytic cooperative metal hydrogen atom transfer – for the asymmetric reduction of unactivated olefins. A silane promoted, heme-cysteine redox cycle in the active site catalyzes sequential hydrogen atom transfer to challenging scaffolds including 1,1-disubstituted as well as tri- and tetrasubstituted alkenes. The evolved enzymes are promiscuous, oxygen-tolerant, utilize earth-abundant iron, and can operate on gram scale under ambient conditions. Orthogonal hydrogen atom sources enable site-divergent asymmetric isotope labeling. Mechanistic and computational studies support a stepwise radical process, highlighting the potential for independent stereocontrol during the delivery of each hydrogen atom. Our work introduces a fundamentally new biochemical logic for stereoselective olefin reduction and provides a platform for next-generation biocatalytic hydrogenation.

We have engineered the heme biosynthetic pathway in E. coli to develop an efficient cofactor supplementation system for hemoproteins. Through the incorporation of the heterogeneous C4 pathway, the engineered cells significantly increase the expression levels of active holohemoproteins and enhance the efficiency of whole-cell and lysate-based biocatalysis promoted by a variety of hemoproteins.

Abstract

Biocatalysis using heme-dependent enzymes provides a powerful synthetic platform to facilitate a variety of chemical transformations required for organic synthesis. Despite recent advances in biocatalysis, recombinant expression systems for hemoproteins leave much room for improvement due to the strict regulation of heme biosynthesis in the host organism. To develop an efficient cofactor supplementation system for the expression of active holohemoproteins, we describe metabolic engineering of the heme biosynthetic pathway in E. coli. Through incorporation of a heterogeneous C4 pathway involving 5-aminolevulinic acid synthase of Paracoccus denitrificans, it was found that the concentrations of 5-aminolevulinic acid and heme in the engineered cells are increased during cultivation, and the expression level of the holohemoproteins is significantly improved. Notably, the heme content in the engineered cells is even higher than that produced by conventional cultivation methods, which add 5-aminolevulinic acid into the culture medium. Furthermore, we also demonstrate the application of this engineered E. coli cells in whole-cell and lysate-based biocatalysis using various types of heme-dependent enzymes. Considering the recent demand for biocatalysis, the system developed in this study will serve as a new practical and versatile platform for hemoprotein-based biocatalysis.