Experimental studies support that protein engineering based on ancestral sequence reconstruction often leads to variants with biotechnologically useful biomolecular properties. These may include high stability, enhanced conformational flexibility and a modified catalysis range. Carbohydrate-active enzymes have numerous applications related to the degradation and synthesis of carbohydrates and glycoconjugates. Herein, we explore how ancestral reconstruction impacts the substrate scope of glycosidases, highly diverse enzymes that catalyze the hydrolysis of glycosidic bonds in all living cells and find applications as catalysts of the synthetic reaction. To this end, we screened 291 potential glycosidase substrates for degradation by both a modern family-1 glycosidase from the halothermophilic bacterium Halothermothrix orenii and a putative ancestral family-1 glycosidase derived from sequence reconstruction of a bacterial-eukaryotic common ancestor. The modern enzyme is the better catalyst for many substrates, but the ancestral glycosidase is more efficient with flavonoid glycosides bearing large-aglycone moieties. Analysis of the catalytic parameters for a selected set of substrates, alongside analysis of the library data using a supervised learning algorithm, supports the hypothesis that the modern enzyme tends to become less catalytically efficient with increasing substrate size, while this trend is not observed for the ancestral glycosidase. Molecular modeling supports that the ancestral catalysis pattern is linked to the existence of a highly flexible region of the protein and a cavity capable of accommodating large aglycones. Our results and analyses provide guidelines for the engineering of enzymes for the synthesis and hydrolysis of large glycoconjugates.

Covering: up to 2026Over the past decades, research on S-adenosyl-L-methionine (SAM)-dependent methyltransferases (MTs) has led to their emergence as highly selective biocatalysts for late-stage methylation reactions, serving as new tools within the chemoenzymatic synthesis of complex natural products and chemical scaffolds. Studies focussing on the identification of novel MTs, their engineering and process optimisation, have not only expanded the accessible chemical space for biocatalytic methylations but have also significantly improved their applicability for reactions conducted at preparative scale. This includes innovations in enzyme-coupled SAM recycling systems and SAM analogue generation which have additionally enabled alkylation beyond methyl transfer. Structure-guided MT mutagenesis campaigns, together with the development of novel activity assays, have contributed to the expansion of the catalytic repertoire of MTs, thereby unlocking new reactivities. This highlight article provides a brief overview of recent developments in MT biocatalysis, with particular focus on their application in the chemoenzymatic synthesis of natural products at preparative scale. Current limitations associated with MT biocatalysis are examined, and future opportunities for the implementation of MT-catalysed transformations in industrial applications are discussed.

Chlorothricin is a polyketide-derived natural product isolated from Streptomyces antibioticus. It possesses an elaborate pentacyclic aglycone core which incorporates a spirotetronic acid moiety, linked to a trans-decalin system, embedded within a macrocycle. Using synthetic substrate analogues and purified recombinant proteins, here we demonstrate that assembly of this scaffold proceeds via sequential biocatalytic Diels-Alder reactions, promoted by the enzymes ChlE3 and ChlL. Both Diels-Alderases exhibit sufficiently relaxed substrate selectivity to facilitate access to non-natural chlorothricin analogues via biotransformations. The X-ray crystal structure of ChlE3 reveals the molecular basis of decalin formation by this enzyme. Harnessing this enzymatic cascade in biocatalysis could provide a valuable biomimetic route to both natural and non-natural spirotetronates, and the work described herein lays the foundation for application of these enzymes in chemoenzymatic syntheses of complex products.

Enzymes are generally believed to evolve from promiscuous ancestors to more specialized descendants under some selection pressure related to their function. However, enzymes whose function depends on substrate promiscuity have not been studied. Here, we show that a group of highly diverse, xenobiotic-metabolizing enzymes, responsible for defense against a constantly changing battery of xenobiotic chemicals, evolved from highly thermostable ancestors. Thermostability declined in parallel with the accumulation of sequence diversity through evolution. The major lineages differed in their relative diversification, with the more stable lineage leading to greater extant sequence diversity. Thermostability was associated with a trend towards better sequestration of hydrophobic residues within the core of the protein and increased exposure of polar residues in solvent-accessible parts of the structure. Resurrected ancestral forms were active towards typical substrates and exhibited ligand-binding promiscuity comparable to, or greater than, their extant descendants. This work supports the hypothesis that robust ancestors facilitate evolutionary diversification and highlights features responsible for enhancing thermostability in a protein fold.

High-throughput screening underpins modern enzyme engineering, synthetic biology and reaction discovery, yet current workflows remain constrained by an analytical compromise between throughput and molecular specificity. Optical and labelled assays provide rapid screening but limited chemical information, whereas chromatography-coupled mass spectrometry (MS) offers molecular confidence at the expense of speed, sample consumption and solvent demand. Here we present Echo-DESI-MS, an automated nanolitre-scale MS workflow that enables label-free ultrahigh-throughput molecular screening by coupling acoustic liquid transfer with desorption electrospray ionisation MS imaging. Using contactless acoustic droplet ejection, the platform enables deposition in two minutes of up to 1536 discrete reaction aliquots onto a single microscope slide which can be analysed directly by ambient surface ionisation at approximately 3 s per sample. To support extraction of these high-density imaging datasets, we developed SpotExtractor, a dedicated software environment that converts a single MS imaging file into reaction-resolved quantitative outputs, integrating automated data extraction, sample labelling and visualisations. We validate the performance of this workflow using a 23-member cytochrome P450 BM3 hydroxylation library, achieving reaction conversion readouts with average standard deviations of 2.2%, negligible spot-to-spot carryover and approximately 120-fold faster analysis than equivalent LC-MS screening while using over three orders of magnitude less sample. The platform is compatible with repeat interrogation of deposited libraries by tandem MS and ion mobility and provides a generalisable route to sustainable high-throughput analytical screening across biocatalysis, directed evolution and high-density chemical assay workflows.

l-Piperazic acid (l-Piz) is a noncanonical, α-hydrazino acid characterized by a 1,2-diazinane heterocycle containing an N─N bond. It occurs in numerous natural products with potent biological activities and represents a key pharmaceutical building block. In nature, l-Piz is biosynthesized from l-ornithine via the intermediate N5-hydroxy-l-ornithine in a two-enzyme cascade comprising a flavin adenine dinucleotide (FAD)-dependent N-hydroxylating monooxygenase (NMO) and a heme-dependent piperazate synthase (PZS). The NMO selectively hydroxylates the δ-amino group of l-ornithine, while PZS catalyzes intramolecular N─N bond formation to generate the six-membered cyclic hydrazine scaffold of l-Piz. Here, we report the crystal structure, Piz-forming activity, and molecular dynamics (MD) analysis of SbPZS, a representative PZS from Streptomyces sp. B93. High-resolution structural analysis enabled a detailed comparison with previously characterized PZS homologs. To further delineate the molecular basis of catalysis, we performed MD simulations in combination with sequence-based bioinformatic analyses. These studies provide insight into protein-substrate interactions, conformational dynamics, and the residues that contribute to active-site organization. Moreover, we identify candidate hotspots for engineering to modulate substrate scope and catalytic efficiency. Collectively, our results establish a structural framework for understanding enzymatic N─N bond formation in Piz biosynthesis and lay the groundwork for future biocatalytic applications of PZSs.

The tetrodecamycins are tetracyclic natural products that exhibit potent antimicrobial activity against a multitude of drug-resistant pathogens. These compounds are structurally distinguished by the presence of a tetronate ring and trans-decalin with six contiguous asymmetric centres united by a seven-membered oxygen heterocycle. Herein we describe the first total synthesis of the antibiotic (-)-13-deoxytetrodecamycin. Our strategy is predicated on an enantioselective [4 + 2]-cycloaddition catalysed by the FAD-dependent Diels-Alderase TedJ, forming the trans-decalin with concomitant creation of two rings and four contiguous stereocenters with exquisite selectivity under mild conditions. In complementary studies, in vitro enzyme assays, X-ray crystallography and computational modelling are used to provide molecular insights into the TedJ catalysed reaction. These studies illustrate the power of adopting a chemoenzymatic approach for the enantioselective synthesis of a target compound which would be difficult to achieve using non-biological methods and provide a practical demonstration of the use of Diels-Alder biocatalysts in total synthesis. This approach has potentially widespread value in the global challenge of discovery and development of new antibiotics.

Here, we report the resequencing, assembly, and annotation of two actinomycete genomes containing abyssomicin gene clusters. Kutzneria buriramensis DSM 45791 with a circular chromosome of 11,681,598 bp and 4 circular plasmids (14,175-207,548 bp) and Streptomyces sp. NL15-2K with a 12,368,159 bp linear genome and circular plasmid (11,584 bp).

Amide bond formation is a basal transformation in synthetic chemistry and the pharmaceutical industry that is traditionally performed under harsh conditions, using excess amounts of amine and relying on coupling agents. Biocatalysis shows great potential in contributing to milder and more sustainable amide bond formation in water, in particular using the emerging family of amide bond synthetase (ABS) enzymes. Here, we use molecular dynamics, biocatalysis, and enzyme engineering to study amide bond formation in extant and ancestral ABS from Marinactinospora thermotolerans (McbA). Our results show that while being more thermostable, the C‐terminal domain that delivers the amine substrate to the adenylated acid intermediate is more flexible in ancestral McbA, presumably leading to an extended amine scope as observed experimentally from a small panel of aliphatic and aromatic substrates. An engineered ancestor of McbA harboring a single mutation that presumptively represent a catalytic shift residue when going from ancestral to modern biocatalyst, show two to ten‐fold improved conversions over its ancestral template while maintaining high thermostability, highlighting ancestral sequence reconstruction as a potent method in protein engineering. Kinetic experiments showed that the engineered ancestral enzyme had 2‐fold higher apparent kcat values in amide formation compared to extant enzyme, concomitant with relaxed substrate inhibition and loss‐of‐dependency on magnesium. Finally, we optimize ATP recycling utilizing a single polyphosphate kinase to showcase how engineered ancestral McbA together with reaction optimization is amenable for pharmacophore synthesis at a preparative scale.

Radical hydrofunctionalizations of electronically unbiased dienes are challenging to render regioselective, because the products are nearly identical in energy. Here, we report two engineered FMN-dependent "ene"-reductases (EREDs) that catalyze regiodivergent hydroalkylations of cyclic and linear dienes. While previous studies focused exclusively on the stereoselectivity of alkene hydroalkylation, this work highlights that EREDs can control the regioselectivity of hydrogen atom transfer, providing a method for selectively preparing constitutional isomers that would be challenging to prepare using traditional synthetic methods. Engineering the ERED from Gluconabacter sp. (GluER) furnished a variant that favors the gamma,delta-unsaturated ketone, while an engineered variant from a commercial ERED panel favors the delta,epsilon-unsaturated ketone. The effect of beneficial mutations has been investigated using substrate docking studies and the mechanism probed by isotope labeling experiments. A variety of alpha-bromo ketones can be coupled with cyclic and linear dienes. These interesting building blocks can also be further modified to generate difficult-to-access heterocyclic compounds.

In the ever-growing demand for sustainable ways to produce high-value small molecules, biocatalysis has come to the forefront of greener routes to these chemicals. As such, the need to constantly find and optimise suitable biocatalysts for specific transformations has never been greater. Metagenome mining has been shown to rapidly expand the toolkit of promiscuous enzymes needed for new transformations, without requiring protein engineering steps. If protein engineering is needed, the metagenomic candidate can often provide a better starting point for engineering than a previously discovered enzyme on the open database or from literature, for instance. In this review, we highlight where metagenomics has made substantial impact on the area of biocatalysis in recent years. We review the discovery of enzymes in previously unexplored or 'hidden' sequence space, leading to the characterisation of enzymes with enhanced properties that originate from natural selection pressures in native environments.

Amide bond synthesis is ranked as the second most important challenge in key green chemistry research areas identified by the ACS Green Chemistry Institute. While developing more sustainable amide bond forming reactions has been in focus, significantly less attention has been given to human toxicity and environmental aspects of the underlying amine and acid substrates and their corresponding coupled products, a potentially important contribution to the overall sustainability of the amide-bond-forming reactions. Here, we explore biocatalytic amide bond formation from a safer-and-more-sustainable-by-design perspective in which commercially available amines and acids as well as their corresponding amide products were evaluated in silico based on potential human toxicity and environmental fate and exposure. This in silico filtering resulted in a panel of 188 amine and 54 acid building blocks that could be classified as safe, referred to herein as "safechems". To enable couplings of safechems, we generated a panel of robust and promiscuous ancestral ATP-dependent amide bond synthetases (ABS) using McbA from Marinactinospora thermotolerans SCSIO 00652 as a template. Ancestral ABS enzymes exhibited complementary specificities in the coupling of a representative safechem subset of 17 amines and 16 acids while showing an increased thermostability of up to 20 degrees C compared to the extant biocatalyst. Finally, the pool of safechems and their corresponding amides were evaluated by USEtox (the UNEP-SETAC toxicity model), analysing not only the intrinsic properties of the compounds but evaluating their complete impact pathway including fate, exposure and effects. The amides were in general predicted as more toxic compared to the starting acids and amines through non-additive effects, emphasising that focusing on the toxicity of the building blocks alone is not sufficient to strive towards low human and ecotoxicity impact. Pursuing a safer and more sustainable by design perspective in the implementation of safechems did not prevent us from generating an array of novel products with potentially potent applications as exemplified here by enzymatic synthesis of substructures that are part of drug candidates for e.g. cancer treatment. We describe an exploratory approach to a concept of safer and more sustainable by design in biocatalytic amide bond synthesis.

The molecular origins of stereoselectivity in enzyme catalysed Diels–Alder reactions in abyssomicin biosynthesis are determined and spirotetronates prepared with the creation of 3 new stereocentres.

Nucleosides functionalized at the 2 '-position play a crucial role in therapeutics, serving as both small-molecule drugs and modifications in therapeutic oligonucleotides. However, the synthesis of these molecules often presents substantial synthetic challenges. Here we present an approach to the synthesis of 2 '-functionalized nucleosides based on enzymes from the purine nucleoside salvage pathway. Initially, active-site variants of deoxyribose-5-phosphate aldolase were generated for the highly stereoselective synthesis of d-ribose-5-phosphate analogues with a broad range of functional groups at the 2-position. Thereafter, these 2-modified pentose phosphates were converted into 2 '-modified purine analogues by construction of one-pot multienzyme cascade reactions, leading to the synthesis of guanosine (2 '-OH) and adenosine (2 '-OH, 2 '-Me, 2 '-F) analogues. This cascade allows for the control of the 2 '-functional group alongside 2-stereochemistry. Our findings demonstrate the capability of these biocatalytic cascades to efficiently generate 2 '-functionalized nucleosides, starting from simple starting materials. The chemical synthesis of nucleoside analogues with modifications at the 2-position often requires multiple steps and the extensive use of protecting groups. Now, biocatalytic cascades are reported for the synthesis of 2-functionalized sugars and 2 '-functionalized nucleosides, using enzymes derived from those of the purine nucleoside salvage pathway.

Phytochemicals have a long and successful history in drug discovery. With recent advancements in analytical techniques and methodologies, discovering bioactive leads from natural compounds has become easier. Computational techniques like molecular docking, QSAR modelling and machine learning, and network pharmacology are among the most promising new tools that allow researchers to make predictions concerning natural products' potential targets, thereby guiding experimental validation efforts. Additionally, approaches like LC-MS or LC-NMR speed up compound identification by streamlining analytical processes. Integrating structural and computational biology aids in lead identification, thus providing invaluable information to understand how phytochemicals interact with potential targets in the body. An emerging computational approach is machine learning involving QSAR modelling and deep neural networks that interrelate phytochemical properties with diverse physiological activities such as antimicrobial or anticancer effects.

Selective, one-step C-H activation of fatty acids from biomass is an attractive concept in sustainable chemistry. Biocatalysis has shown promise for generating high-value hydroxy acids but to date enzyme discovery has relied on laborious screening and produced limited hits, which predominantly oxidise the sub-terminal positions of fatty acids. Here we show that ancestral sequence reconstruction (ASR) is an effective tool to explore the sequence-activity landscape of a family of multi-domain, self-sufficient P450 monooxygenases. We resurrected eleven catalytically active CYP116B ancestors, each with a unique regioselectivity fingerprint that varied from sub-terminal in the older ancestors to mid-chain in the lineage leading to the extant, P450-TT. In lineages leading to extant enzymes in thermophiles, thermostability increased from ancestral to extant forms, as expected if thermophily had arisen de novo. Our studies show that ASR can be applied to multi-domain enzymes to develop active, self- sufficient monooxygenases as regioselective biocatalysts for fatty acid hydroxylation.

Promiscuity, the capability of catalyzing a diversity of chemical reactions, is a desirable feature in many enzymes intended for biotechnological applications. Promiscuous activities, however, are typically depressed in specialized modern enzymes. On the other hand, several ancestral reconstruction studies have reported enzymes with enhanced promiscuity. Glycosidases catalyze the hydrolysis of glycosidic bonds in all living cells but find practical applications in the synthesis of glycoconjugates as catalysts of the reverse reaction. Here, we use a library of ∼ 500 possible substrates to compare the catalytic scope of the modern family-1 glycosidase from Halothermothrix orenii with that of a putative ancestral family-1 glycosidase derived from sequence reconstruction at a bacterial-eukaryotic common ancestor. The modern enzyme is the better catalyst for the hydrolysis of small substrates typically used to assess the activity of family-1 glycosidases. We identify, however, a trend for the modern enzyme to become less catalytically efficient with increasing substrate size. Such trend is not apparent in the conformationally-flexible ancestral glycosidase, which is in fact the better catalyst for the hydrolysis of flavonoid glycosides. Our results support that ancestral sequence reconstruction may provide a basis for enzyme engineering for the synthesis of glycoconjugates with large glycosyl acceptors. ### Competing Interest Statement Leonardo De Maria, Martin Hayes and Francesco Falcioni are current employees and/or shareholders of AstraZeneca. The authors declare no competing interest.

The Diels-Alder reaction is one of the most effective methods for the synthesis of substituted cyclohexenes. The development of protein catalysts for this reaction remains a major priority, affording new sustainable routes to high value target molecules. Whilst a small number of natural enzymes have been shown capable of catalysing [4+2] cycloadditions, there is a need for significant mechanistic understanding of how these prospective Diels-Alderases promote catalysis to underpin their development as biocatalysts for use in synthesis. Here we present a molecular description of the complete reaction cycle of the bona fide natural Diels-Alderase AbyU, which catalyses formation of the spirotetronate skeleton of the antibiotic abyssomicin C. This description is derived from X-ray crystallographic studies of AbyU in complex with a non-transformable synthetic substrate analogue, together with transient kinetic analyses of the AbyU catalysed reaction and computational reaction simulations. These studies reveal the mechanistic intricacies of this enzyme system and establish a foundation for the informed reengineering of AbyU and related biocatalysts.