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Journal articleSaha P, Micklefield J, 2026,
Alternative enzymatic pathways to penicillinantibiotics
, Nature Communications, ISSN: 2041-1723The discovery of penicillin, more than a century ago, has been one of the most significant advances in medicine. Despite the growing threat of antimicrobialresistance, which has rendered many other antibiotics ineffective, penicillin derivatives remain among the most widely prescribed antibiotics. Penicillin isbiosynthesised by a large nonribosomal peptide synthetase (NRPS) enzyme, which assembles a tripeptide precursor ACV. This intermediate is subsequently cyclised by isopenicillin N synthase (IPNS) to form penicillin. ACV is similar in structure to glutathione, a ubiquitous, tripeptide antioxidant essential for aerobic life forms. Unlike ACV, glutathione is assembled using simpler ligase enzymes rather than complex NRPS machinery. In this paper, we describe an alternative pathway to penicillins that uses stand-alone ligase and epimeras eenzymes to generate peptide precursors, which can be transformed to penicillin derivatives using an engineered IPNS enzyme. Unlike the native NRPS assembly line, the ligase pathway provides direct access to therapeutically relevant penicillin G, penicillin V and ampicillin, which are currently produced by semi-synthesis.
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Journal articleAngiolini S, Bruton I, Bering L, et al., 2026, , ChemCatChem, Vol: 18, ISSN: 1867-3880
Fluorinated molecules are widely used as pharmaceuticals, agrochemicals and as various functional materials. Traditional synthetic methods for introducing fluorine substituents into organic molecules involve deleterious chemicals and lack selectivity. Enzymes have evolved in nature which can halogenate a diverse range of substrates with high selectivity under aqueous conditions, using benign inorganic halides as the halogen source. Although there are many halogenase enzymes that can chlorinate or brominate diverse substrates, only one fluorinase enzyme has been discovered to date that produces a single fluorinated adenosine derivative in nature. Herein, we complement the lack of biocatalytic fluorination protocols and address the need for cleaner and more selective fluorination methods by merging chemo and biocatalysis to selectively fluorinate compounds in a single integrated reaction. Our approach relies on combining nitrilase enzymes with photoredox catalysis to transform cheap and abundant organonitrile compounds into highly sought鈥恆fter fluorinated, trifluoromethylated, and perfluoroalkylated compounds.
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Journal articleZhao B, Micklefield J, Wang Y, et al., 2025, , Applied Biochemistry and Biotechnology, Vol: 197, Pages: 4528-4546, ISSN: 0273-2289
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Journal articleTorri D, Bering L, Yates LRL, et al., 2025, , Angewandte Chemie, Vol: 137, ISSN: 0044-8249
<jats:title>Abstract</jats:title><jats:p>Amide bond formation is fundamental in nature and is widely used in the synthesis of pharmaceuticals and other valuable products. Current methods for amide synthesis are often step and atom inefficient, requiring the use of protecting groups, deleterious reagents and organic solvents that create significant waste. The development of cleaner and more efficient catalytic methods for amide synthesis remains an urgent unmet need. Herein, we present novel biocatalytic cascade reactions for synthesising various amides under mild aqueous conditions from readily available organic nitriles combining nitrile hydrolysing enzymes and amide bond synthetase enzymes. These cooperative biocatalytic cascades enable kinetic resolution of racemic nitriles and provide a highly enantioselective biocatalytic extension of the Strecker reaction. The regioselective non鈥恉irected C−H bond amidation of simple arenes was demonstrated through the incorporation of photoredox catalysis to the front end of the cascade. C−H bond amidation of simple aromatic precursors was also achieved via a CO<jats:sub>2</jats:sub> fixation cascade combining enzymatic carboxylation and amide bond synthesis in one鈥恜ot.</jats:p>
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Journal articleTorri D, Bering L, Yates LRL, et al., 2025, , Angewandte Chemie International Edition, Vol: 64, ISSN: 1433-7851
Amide bond formation is fundamental in nature and is widely used in the synthesis of pharmaceuticals and other valuable products. Current methods for amide synthesis are often step and atom inefficient, requiring the use of protecting groups, deleterious reagents and organic solvents that create significant waste. The development of cleaner and more efficient catalytic methods for amide synthesis remains an urgent unmet need. Herein, we present novel biocatalytic cascade reactions for synthesising various amides under mild aqueous conditions from readily available organic nitriles combining nitrile hydrolysing enzymes and amide bond synthetase enzymes. These cooperative biocatalytic cascades enable kinetic resolution of racemic nitriles and provide a highly enantioselective biocatalytic extension of the Strecker reaction. The regioselective non-directed C−H bond amidation of simple arenes was demonstrated through the incorporation of photoredox catalysis to the front end of the cascade. C−H bond amidation of simple aromatic precursors was also achieved via a CO2 fixation cascade combining enzymatic carboxylation and amide bond synthesis in one-pot.
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Journal articleXu G, Torri D, Cuesta-Hoyos S, et al., 2024, , NATURE CHEMICAL BIOLOGY, Vol: 20, ISSN: 1552-4450
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Journal articleXu G, Micklefield J, 2024, , NATURE CHEMICAL BIOLOGY, Vol: 20, Pages: 1256-1257, ISSN: 1552-4450
Peptides are fundamental in life and are widely used as therapeutic agents; however, they are problematic to produce by chemical synthesis. The discovery of unusual biosynthetic pathways that produce peptide natural products reveals an alternative approach for peptide synthesis that uses ‘standalone’ ligase enzymes rather than multimodular enzymes.
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Journal articleOConnor E, Micklefield J, Cai Y, 2024, , CURRENT OPINION IN BIOTECHNOLOGY, Vol: 87, ISSN: 0958-1669
High -throughput screening technologies have been lacking in comparison to the plethora of high -throughput genetic diversification techniques developed in biotechnology. This review explores the challenges and advancements in highthroughput screening for high -value natural products, focusing on the critical need to expand ligand targets for biosensors and increase the throughput of analytical techniques in screening microbial cell libraries for optimal strain performance. The engineering techniques to broaden the scope of ligands for biosensors, such as transcription factors, G protein-coupled receptors and riboswitches are discussed. On the other hand, integration of microfluidics with traditional analytical methods is explored, covering fluorescence -activated cell sorting, Raman -activated cell sorting and mass spectrometry, emphasising recent developments in maximising throughput.
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Journal articleBering L, Thompson J, Micklefield J, 2022, , TRENDS IN CHEMISTRY, Vol: 4, Pages: 392-408
The combination of chemo-and biocatalysis in one pot (integrated catalysis) is a powerful approach to develop new routes towards important products under mild and environmentally benign reaction conditions. Integrated catalysis can improve overall synthetic efficiency and, due to the complementary nature of chemo-and biocatalysts, transformations can be performed, which would be otherwise challenging using a single catalyst. In this review, we highlight recent trends for the combination of enzymes with chemocatalysts. Transition-metal catalysis, organocatalysis, and photoredox catalysis have been combined with different biocatalysts and are discussed accordingly. We highlight further how integrated catalysis not only delivers benign substitutes for known transformations but moreover enables transformations that would be otherwise impossible.
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Journal articleBering L, Craven EJ, Thomas SAS, et al., 2022, , NATURE COMMUNICATIONS, Vol: 13
Amides are one of the most fundamental chemical bonds in nature. In addition to proteins and other metabolites, many valuable synthetic products comprise amide bonds. Despite this, there is a need for more sustainable amide synthesis. Herein, we report an integrated next generation multi-catalytic system, merging nitrile hydratase enzymes with a Cu-catalysed N-arylation reaction in a single reaction vessel, for the construction of ubiquitous amide bonds. This synergistic one-pot combination of chemo- and biocatalysis provides an amide bond disconnection to precursors, that are orthogonal to those in classical amide synthesis, obviating the need for protecting groups and delivering amides in a manner unachievable using existing catalytic regimes. Our integrated approach also affords broad scope, very high (molar) substrate loading, and has excellent functional group tolerance, telescoping routes to natural product derivatives, drug molecules, and challenging chiral amides under environmentally friendly conditions at scale. Proteins, other metabolites and many valuable synthetic products contain amide bonds and there is a need for more sustainable amide synthesis routes. Here the authors show an integrated next generation multi-catalytic system, merging nitrile hydratase enzymes with a Cu-catalysed N-arylation reaction in a single reaction vessel, for the construction of ubiquitous amide bonds.
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Journal articleThong WL, Zhang Y, Zhuo Y, et al., 2021, , NATURE COMMUNICATIONS, Vol: 12
Engineering biosynthetic assembly lines is a powerful path to new natural products but is challenging with current methods. Here the authors use CRISPR-Cas9 to exchange subdomains within NRPS to alter substrate selectivity. Re-engineering biosynthetic assembly lines, including nonribosomal peptide synthetases (NRPS) and related megasynthase enzymes, is a powerful route to new antibiotics and other bioactive natural products that are too complex for chemical synthesis. However, engineering megasynthases is very challenging using current methods. Here, we describe how CRISPR-Cas9 gene editing can be exploited to rapidly engineer one of the most complex megasynthase assembly lines in nature, the 2.0 MDa NRPS enzymes that deliver the lipopeptide antibiotic enduracidin. Gene editing was used to exchange subdomains within the NRPS, altering substrate selectivity, leading to ten new lipopeptide variants in good yields. In contrast, attempts to engineer the same NRPS using a conventional homologous recombination-mediated gene knockout and complementation approach resulted in only traces of new enduracidin variants. In addition to exchanging subdomains within the enduracidin NRPS, subdomains from a range of NRPS enzymes of diverse bacterial origins were also successfully utilized.
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Journal articleGomez-Escribano JP, Gallardo LA, Bozhuyuk KAJ, et al., 2021, , MICROBIAL GENOMICS, Vol: 7, ISSN: 2057-5858
Streptomyces clavuligerus is an industrially important actinomycete whose genetic manipulation is limited by low transformation and conjugation efficiencies, low levels of recombination of introduced DNA, and difficulty in obtaining consistent sporulation. We describe the construction and application of versatile vectors for Cas9-mediated genome editing of this strain. To design spacer sequences with confidence, we derived a highly accurate genome assembly for an isolate of the type strain (ATCC 27064). This yielded a chromosome assembly (6.75 Mb) plus assemblies for pSCL4 (1795 kb) and pSCL2 (149 kb). The strain also carries pSCL1 (12 kb), but its small size resulted in only partial sequence coverage. The previously described pSCL3 (444 kb) is not present in this isolate. Using our Cas9 vectors, we cured pSCL4 with high efficiency by targeting the plasmid’s parB gene. Five of the resulting pSCL4- cured isolates were characterized and all showed impaired sporulation. Shotgun genome sequencing of each of these derivatives revealed large deletions at the ends of the chromosomes in all of them, and for two clones sufficient sequence data was obtained to show that the chromosome had circularized. Taken together, these data indicate that pSCL4 is essential for the structural stability of the linear chromosome.
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Journal articleWinn M, Rowlinson M, Wang F, et al., 2021, , NATURE, Vol: 593, Pages: 391+-391+, ISSN: 0028-0836
Coronatine and related bacterial phytotoxins are mimics of the hormone jasmonyI-l-isoleucine (JA-Ile), which mediates physiologically important plant signalling pathways(1-4). Coronatine-like phytotoxins disrupt these essential pathways and have potential in the development of safer, more selective herbicides. Although the biosynthesis of coronatine has been investigated previously, the nature of the enzyme that catalyses the crucial coupling of coronafacic acid to amino acids remains unknown(1,2). Here we characterize a family of enzymes, coronafacic acid ligases (CfaLs), and resolve their structures. We found that CfaL can also produce JA-Ile, despite low similarity with the Jar1 enzyme that is responsible for ligation of JA and l-Ile in plants(5). This suggests that Jar1 and CfaL evolved independently to catalyse similar reactions-Jar1 producing a compound essential for plant development(4,5), and the bacterial ligases producing analogues toxic to plants. We further demonstrate how CfaL enzymes can be used to synthesize a diverse array of amides, obviating the need for protecting groups. Highly selective kinetic resolutions of racemic donor or acceptor substrates were achieved, affording homochiral products. We also used structure-guided mutagenesis to engineer improved CfaL variants. Together, these results show that CfaLs can deliver a wide range of amides for agrochemical, pharmaceutical and other applications.
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Journal articleCraven EJ, Latham J, Shepherd SA, et al., 2021, , NATURE CATALYSIS, Vol: 4, Pages: 385-394, ISSN: 2520-1158
New chemo- and biocatalytic methodology is important for the future sustainable synthesis of essential molecules. Transition metal catalysis enables the late-stage C-H functionalization of some complex molecular scaffolds, providing rapid routes to valuable products, although this is largely dependent on the availability of electronically or sterically predisposed C-H bonds for selective metalation, leaving certain regioselectivities inaccessible. Unlike metal chemocatalysis, enzymes can catalyse C-H bond functionalization, discriminating between near-identical, non-activated C-H bonds, delivering products with exquisite regioselectivity. However, enzymes typically provide access to fewer functionalities than more divergent chemocatalysis. Here we report programmable, regioselective C-H bond functionalization methodologies for the installation of versatile nitrile, amide and carboxylic acid moieties through integration of halogenase enzymes with palladium-catalysed cyanation and subsequent incorporation of nitrile hydratase or nitrilase enzymes. Using two- or three-component chemobiocatalytic systems, the regioselective synthesis of complex target molecules, including pharmaceuticals, can be achieved in a one-pot process operable on a gram scale.
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Journal articleHerbert AJ, Shepherd SA, Cronin VA, et al., 2020, , ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, Vol: 59, Pages: 14950-14956, ISSN: 1433-7851
S-adenosyl-l-methionine (SAM)-dependent methyltransferases (MTs) catalyse the methylation of a vast array of small metabolites and biomacromolecules. Recently, rare carboxymethylation pathways have been discovered, including carboxymethyltransferase enzymes that utilise a carboxy-SAM (cxSAM) cofactor generated from SAM by a cxSAM synthase (CmoA). We show how MT enzymes can utilise cxSAM to catalyse carboxymethylation of tetrahydroisoquinoline (THIQ) and catechol substrates. Site-directed mutagenesis was used to create orthogonal MTs possessing improved catalytic activity and selectivity for cxSAM, with subsequent coupling to CmoA resulting in more efficient and selective carboxymethylation. An enzymatic approach was also developed to generate a previously undescribed co-factor, carboxy-S-adenosyl-l-ethionine (cxSAE), thereby enabling the stereoselective transfer of a chiral 1-carboxyethyl group to the substrate.
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Journal articleRobinson CJ, Carbonell P, Jervis AJ, et al., 2020, , METABOLIC ENGINEERING, Vol: 60, Pages: 168-182, ISSN: 1096-7176
Bio-based production of industrial chemicals using synthetic biology can provide alternative green routes from renewable resources, allowing for cleaner production processes. To efficiently produce chemicals on-demand through microbial strain engineering, biomanufacturing foundries have developed automated pipelines that are largely compound agnostic in their time to delivery. Here we benchmark the capabilities of a biomanufacturing pipeline to enable rapid prototyping of microbial cell factories for the production of chemically diverse industrially relevant material building blocks. Over 85 days the pipeline was able to produce 17 potential material monomers and key intermediates by combining 160 genetic parts into 115 unique biosynthetic pathways. To explore the scale-up potential of our prototype production strains, we optimized the enantioselective production of mandelic acid and hydroxymandelic acid, achieving gram-scale production in fed-batch fermenters. The high success rate in the rapid design and prototyping of microbially-produced material building blocks reveals the potential role of biofoundries in leading the transition to sustainable materials production.
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Journal articleCampopiano DJ, Micklefield J, 2020, , CURRENT OPINION IN CHEMICAL BIOLOGY, Vol: 55, Pages: A1-A3, ISSN: 1367-5931
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Journal articleWinn M, Richardson SM, Campopiano DJ, et al., 2020, , CURRENT OPINION IN CHEMICAL BIOLOGY, Vol: 55, Pages: 77-85, ISSN: 1367-5931
The amide functional group is ubiquitous in nature and one of the most important motifs in pharmaceuticals, agrochemicals, and other valuable products. While coupling amides and carboxylic acids is a trivial synthetic transformation, it often requires protective group manipulation, along with stoichiometric quantities of expensive and deleterious coupling reagents. Nature has evolved a range of enzymes to construct amide bonds, the vast majority of which utilize adenosine triphosphate to activate the carboxylic acid substrate for amine coupling. Despite the fact that these enzymes operate under mild conditions, as well as possessing chemoselectivity and regioselectivity that obviates the need for protecting groups, their synthetic potential has been largely unexplored. In this review, we discuss recent research into the discovery, characterization, and development of amide bond forming enzymes, with an emphasis on stand-alone ligase enzymes that can generate amides directly from simple carboxylic acid and amine substrates.
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Journal articleDunstan MS, Robinson CJ, Jervis AJ, et al., 2020, , SYNTHETIC BIOLOGY, Vol: 5
Natural plant-based flavonoids have drawn significant attention as dietary supplements due to their potential health benefits, including anti-cancer, anti-oxidant and anti-asthmatic activities. Naringenin, pinocembrin, eriodictyol and homoeriodictyol are classified as (2S)-flavanones, an important sub-group of naturally occurring flavonoids, with wide-reaching applications in human health and nutrition. These four compounds occupy a central position as branch point intermediates towards a broad spectrum of naturally occurring flavonoids. Here, we report the development of Escherichia coli production chassis for each of these key gatekeeper flavonoids. Selection of key enzymes, genetic construct design and the optimization of process conditions resulted in the highest reported titers for naringenin (484 mg/l), improved production of pinocembrin (198 mg/l) and eriodictyol (55 mg/l from caffeic acid), and provided the first example of in vivo production of homoeriodictyol directly from glycerol (17 mg/l). This work provides a springboard for future production of diverse downstream natural and non-natural flavonoid targets.
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Journal articleBozhuyuk KAJ, Micklefield J, Wilkinson B, 2019, , CURRENT OPINION IN MICROBIOLOGY, Vol: 51, Pages: 88-96, ISSN: 1369-5274
Numerous important therapeutic agents, including widely-used antibiotics, anti-cancer drugs, immunosuppressants, agrochemicals and other valuable compounds, are produced by microorganisms. Many of these are biosynthesised by modular enzymatic assembly line polyketide synthases, non ribosomal peptide synthetases, and hybrids thereof. To alter the backbone structure of these valuable but difficult to modify compounds, the respective enzymatic machineries can be engineered to create even more valuable molecules with improved properties and/or to bypass resistance mechanisms. In the past, many attempts to achieve assembly line pathway engineering failed or led to enzymes with compromised activity. Recently our understanding of assembly line structural biology, including an appreciation of the conformational changes that occur during the catalytic cycle, have improved hugely. This has proven to be a driving force for new approaches and several recent examples have demonstrated the production of new-to-nature molecules, including anti-infectives. We discuss the developments of the last few years and highlight selected, illuminating examples of assembly line engineering.
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