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Catalysis is a key area of modern science and the recognition of its importance has been increasing in recent years. In view of this a Royal Society Discussion Meeting was held on 8–9 May 2017 bringing together many facets of the latest research in catalysis. The meeting was set out in four themes. The first theme Catalysis for Clean Energy was introduced by Professor Christopher Hardacre [1] (Manchester University, UK) who discussed non-thermal plasmas that enable the activation of catalysis at low temperature operation. Dr Sara Jacob [2] (Exxon Mobil, USA) described some new exciting work relevant to alkane oxidation concerning the use of dioxiranes in solution at low temperature. Professor Gianfranco Pacchioni [3] (Milan) showed the power of contemporary computational modelling techniques with new work on biomass conversion using TiO2 and ZrO2 and addressed the question of why catalyst reduction aids the process. Professor …

The first crystal structure of a class VII P450, CYP116B46 from Tepidiphilus thermophilus, has been solved at 1.9 A resolution. The structure reveals overall conservation of the P450-fold and a water conduit around the I-helix. Active site residues have been identified and sequence comparisons have been made with other class VII enzymes. A structure similarity search demonstrated that the P450-TT structure is similar to enzymes capable of oxy-functionalization of fatty acids, terpenes, macrolides, steroids and statins. The insight gained from solving this structure will provide a guideline for future engineering and modelling studies on this catalytically promiscuous class of enzymes.

Over the next decades, with the growing concern of rising atmospheric carbon dioxide (CO

The application of biocatalysis in organic synthesis is rapidly gaining popularity amongst chemists. Compared to traditional synthetic methodologies biocatalysis offers a number of advantages in terms of enhanced selectivity (chemo-, regio-, stereo-), reduced environmental impact and lower cost of starting materials. Together these advantages can contribute to more sustainable manufacturing processes across a wide range of industries ranging from pharmaceuticals to biofuels. The biocatalytic toolbox has expanded significantly in the past five years and given the current rate of development of new engineered biocatalysts it is likely that the number of available biocatalysts will double in the next few years. This textbook gives a comprehensive overview of the current biocatalytic toolbox and also establishes new guidelines or rules for “biocatalytic retrosynthesis”. Retrosynthesis is a well known and commonly used technique whereby organic chemists start with the structure of their target molecule and generate potential starting materials and intermediates through a series of retrosynthetic disconnections. These disconnections are then used to devise a forward synthesis, in this case using biocatalytic transformations in some of the key steps. Target molecules are disconnected with consideration for applying biocatalysts, as well as chemical reagents and chemocatalysts, in the forward synthesis direction. Using this textbook, students will be able to place biocatalysis within the context of other synthetic transformations that they have learned earlier in their studies. This additional awareness of biocatalysis will equip students for the modern world of organic synthesis where biocatalysts play an increasingly important role. In addition to guidelines for identifying where biocatalysts can be applied in organic synthesis, this textbook also provides examples of current applications of biocatalysis using worked examples and case studies. Tutorials enable the reader to practice disconnecting target molecules to find the ‘hidden’ biocatalytic reactions which can be applied in the synthetic direction. The book contains a complete description of the current biocatalyst classes that are available for use and also suggests areas where new enzymes are likely to be developed in the next few years. This textbook is an essential resource for lecturers and students studying synthetic organic chemistry. It also serves as a handy reference for practicing chemists who wish to embed biocatalysis into their synthetic toolbox.

The effect of extended reaction times on the regio- and enantioselectivity of the phenylalanine ammonia lyase (PAL)-catalysed amination of a subset of cinnamate derivatives was investigated. This was done using a PAL from the cyanobacterium Anabaena variabilis and incubation in a concentrated ammonia buffer. Whilst early time point analyses revealed excellent selectivities to give mostly the well-documented (S)-α-amino acid products, subsequent accumulation of other regio-/stereo- isomers was seen. For many para-substituted substrates, the β-regioisomer, a previously-unreported product with this enzyme class, was found to become more abundant than the α-, after sufficient incubation, with slight preference for the (R)-enantiomer. Although attempts to tune the selectivity of the PAL toward any of the three side products were largely unsuccessful, the results provide insight into the evolutionary history of this class of enzymes and reinforce the prominence of the toolbox of specific and selective cinnamate-aminating enzymes. Electronic supplementary material The online version of this article (10.1007/s11244-018-0898-1) contains supplementary material, which is available to authorized users.

The ability to model the activity of a protein using quantitative structure-activity relationships (QSAR) requires descriptors for the 20 naturally coded amino acids. In this work we show that by modifying some established descriptors we were able to model the activity data of 140 mutants of the enzyme epoxide hydrolase with improved accuracy. These new descriptors (referred to as physical descriptors) also gave very good results when tested against a series of four dipeptide data sets. The physical descriptors encode the amino acids using only two orthogonal scales: the first is strongly linked to hydrophilicity/hydrophobicity, and the second, to the volume of the amino acid residue. The use of these new amino acid descriptors should result in simpler and more readily interpretable models for the enzyme activity (and potentially other functions of interest, e.g., secondary and tertiary structure) of peptides and proteins.

The construction of biocatalytic cascades for the production of chemical precursors is fast becoming one of the most efficient approaches to multi-step synthesis in modern chemistry. However, despite the use of low solvent systems and renewably resourced catalysts in reported examples, many cascades are still dependent on petrochemical starting materials, which as of yet cannot be accessed in a sustainable fashion. Herein, we report the production of the versatile chemical building block cinnamyl alcohol from the primary metabolite and the fermentation product L-phenylalanine. Through the combination of three biocatalyst classes (phenylalanine ammonia lyase, carboxylic acid reductase and alcohol dehydrogenase) the target compound could be obtained in high purity, demonstrable at the 100 mg scale and achieving 53% yield using ambient temperature and pressure in an aqueous solution. This system represents a synthetic strategy in which all components present at time zero are biogenic and thus minimises damage to the environment. Furthermore we extend this biocatalytic cascade by its inclusion in an L-phenylalanine overproducing strain of Escherichia coli. This metabolically engineered strain produces cinnamyl alcohol in mineral media using glycerol and glucose as the carbon sources. This study demonstrates the potential to establish green routes to the synthesis of cinnamyl alcohol from a waste stream such as glycerol derived, for example, from lipase treated biodiesel.

Copper nanoparticles (Cu-NPs) have a wide range of applications as heterogeneous catalysts. In this study, a novel green biosynthesis route for producing Cu-NPs using the metal-reducing bacterium, Shewanella oneidensis is demonstrated. Thin section transmission electron microscopy shows that the Cu-NPs are predominantly intracellular and present in a typical size range of 20-40 nm. Serial block-face scanning electron microscopy demonstrates the Cu-NPs are well-dispersed across the 3D structure of the cells. X-ray absorption near-edge spectroscopy and extended X-ray absorption fine-structure spectroscopy analysis show the nanoparticles are Cu(0), however, atomic resolution images and electron energy loss spectroscopy suggest partial oxidation of the surface layer to Cu

Naturally occurring isoprenoid quinones mediate electron transfer in respiratory or photosynthetic chains of living organisms. Tremendous progress has been made in the elucidation of biosynthetic pathways of prenylated quinones and a number of enzymes that catalyse specific transformation steps with remarkably high regio- and stereoselectivity have been characterised. Interestingly, some of these enzymes possess broad substrate scope towards synthetic analogues, thereby enabling their application as synthetic tools. The availability of mechanistic, structural and mutagenesis information for many of the pathways enzymes presents opportunities for protein engineering, a powerful approach that may improve the suitability of these biocatalysts for industrial processes.

Cytochrome P450 monooxygenases are able to catalyse a range of synthetically challenging reactions ranging from hydroxylation and demethylation to sulfoxidation and epoxidation. As such they have great potential for biocatalytic applications but are underutilised due to often-poor expression, stability and solubility in recombinant bacterial hosts. The use of self-sufficient P450 s with fused haem and reductase domains has already contributed heavily to improving catalytic efficiency and simplifying an otherwise more complex multi-component system of P450 and redox partners. Herein, we present a new addition to the class VII family with the cloning, sequencing and characterisation of the self-sufficient CYP116B62 Hal1 from Halomonas sp. NCIMB 172, the genome of which has not yet been sequenced. Hal1 exhibits high levels of expression in a recombinant E. coli host and can be utilised from cell lysate or used in purified form. Hal1 favours NADPH as electron donor and displays a diverse range of activities including hydroxylation, demethylation and sulfoxidation. These properties make Hal1 suitable for future biocatalytic applications or as a template for optimisation through engineering.

Synthesis of the chiral amine moiety is a key challenge for synthetic organic chemistry due to its prevalence in many biologically active molecules. Imine reductase and amine oxidase enzymes have enabled the biocatalytic synthesis of a host of chiral amine compounds. In this chapter, procedures for the synthesis of chiral amines using imine reductases (IREDs), the recently discovered IRED homologues reductive aminases, and amine oxidases (AOs) are described. Amine oxidases have been the subject of mutagenesis approaches for improvement of substrate scope. The high-throughput screening method for determining active variants in amine oxidase libraries is illustrated. Finally, in an approach which takes inspiration from nature, many enzymes can be combined with each other in cascade reactions. The incorporation of imine reductase and monoamine oxidase biocatalysts into several cascade reactions, both in vitro and in vivo (where the approach moves toward synthetic biology), is reported.

Ammonia-lyases and aminomutases are mechanistically and structurally diverse enzymes which catalyze the deamination and/or isomerization of amino acids in nature by cleaving or shifting a C–N bond. Of the many protein families in which these enzyme activities are found, only a subset have been employed in the synthesis of optically pure fine chemicals or in medical applications. This review covers the natural diversity of these enzymes, highlighting particular enzyme classes that are used within industrial and medical biotechnology. These highlights detail the discovery and mechanistic investigations of these commercially relevant enzymes, along with comparisons of their various applications as stand-alone catalysts, components of artificial biosynthetic pathways and biocatalytic or chemoenzymatic cascades, and therapeutic tools for the potential treatment of various pathologies.

Heterocycles based on piperidine and pyrrolidine are key moieties in natural products and pharmaceutically active molecules. A novel multi-enzymatic approach based on the combination of a lipase with an α,ω-diamine transaminase is reported, opening up the synthesis, isolation and characterisation of a broad range of 2-substituted N-heterocycle alkaloids.

Engineered variants of phenylalanine ammonia lyase from Planctomyces brasiliensis were developed through rational design efforts focusing on the aryl binding pocket of the active site, guided by structural and phylogenetic inference. Inherent problems traditionally associated with the biocatalytic hydroamination of acrylic acids, such as low conversion and poor regioselectivity with alkyl and methoxy derivatives, could be overcome. The PbPAL variants described here represent a valuable addition to the biocatalytic toolbox, allowing previously inaccessible amino acid building blocks to be obtained regio- and enantioselectively on preparative scale.

Reductive aminases (RedAms) catalyze the asymmetric reductive amination of ketones with primary amines to give secondary amine products. RedAms have great potential for the synthesis of bioactive chiral amines; however, insights into their mechanism are currently limited. Comparative studies on reductive amination of cyclohexanone with allylamine in the presence of RedAms, imine reductases (IREDs), or NaBH3CN support the distinctive activity of RedAms in catalyzing both imine formation and reduction in the reaction. Structures of AtRedAm from Aspergillus terreus, in complex with NADPH and ketone and amine substrates, along with kinetic analysis of active-site mutants, reveal modes of substrate binding, the basis for the specificity of RedAms for reduction of imines over ketones, and the importance of domain flexibility in bringing the reactive participants together for the reaction. This information is used to propose a mechanism for their action and also to expand the substrate specificity of RedAms using protein engineering.

d-Phenylalanine derivatives are valuable chiral building blocks for a wide range of pharmaceuticals. Here, we developed stereoinversion and deracemization biocatalytic cascades to synthesize d-phenylalanine derivatives that contain electron-donating or -withdrawing substituents of various sizes and at different positions on the phenyl ring with a high enantiomeric excess (90 to >99 % ee) from commercially available racemic mixtures or l-amino acids. These whole-cell systems couple Proteus mirabilis l-amino acid deaminase with an engineered aminotransferase that displays native-like activity towards d-phenylalanine, which we generated from Bacillus sp. YM-1 d-amino acid aminotransferase. Our cascades are applicable to preparative-scale synthesis and do not require cofactor-regeneration systems or chemical reducing agents.

Biocatalytic retrosynthetic analysis of dibenz[c,e]azepines has highlighted the use of imine reductase (IRED) and ω-transaminase (ω-TA) biocatalysts to establish the key stereocentres of these molecules. Several enantiocomplementary IREDs were identified for the synthesis of (R)- and (S)-5-methyl-6,7-dihydro-5H-dibenz[c,e]azepine with excellent enantioselectivity, by reduction of the parent imines. Crystallographic evidence suggests that IREDs may be able to bind one conformer of the imine substrate such that, upon reduction, the major product conformer is generated directly. ω-TA biocatalysts were also successfully employed for the production of enantiopure 1-(2-bromophenyl)ethan-1-amine, thus enabling an orthogonal route for the installation of chirality into dibenz[c,e]azepine framework.

Here we describe a one-pot, three-enzyme, cascade involving a cytochrome P450 monooxygenase, an alcohol dehydrogenase and a reductive aminase for the synthesis of secondary amines from cycloalkanes. Amine product concentrations of up to 19.6 mM were achieved. The preparative scale amination of cyclohexane was also demonstrated with a space-time yield of 2 g L-1 d-1.

Carbon-carbon bond formation is one of the most challenging reactions in synthetic organic chemistry, and aldol reactions catalysed by dihydroxyacetone phosphate-dependent aldolases provide a powerful biocatalytic tool for combining C-C bond formation with the generation of two new stereo-centres, with access to all four possible stereoisomers of a compound. Dihydroxyacetone phosphate (DHAP) is unstable so the provision of DHAP for DHAP-dependent aldolases in biocatalytic processes remains complicated. Our research has investigated the efficiency of several different enzymatic cascades for the conversion of glycerol to DHAP, including characterising new candidate enzymes for some of the reaction steps. The most efficient cascade for DHAP production, comprising a one-pot four-enzyme reaction with glycerol kinase, acetate kinase, glycerophosphate oxidase and catalase, was coupled with a DHAP-dependent fructose-1,6-biphosphate aldolase enzyme to demonstrate the production of several rare chiral sugars. The limitation of batch biocatalysis for these reactions and the potential for improvement using kinetic modelling and flow biocatalysis systems is discussed.

Biocatalytic oxidation reactions have the potential to substitute many chemically catalyzed oxidations in the pharmaceutical and fine chemical industry due to their superior regio- and stereoselectivity and low environmental impact. Galactose oxidase (GOase) has been shown to be a promising biocatalyst for the oxidation of primary and secondary alcohols to their corresponding aldehydes and ketones, respectively. However, GOase requires a number of additives to sustain its catalytic function, such as the enzyme catalase for degradation of the byproduct hydrogen peroxide as well as single-electron oxidants to reactivate the enzyme upon loss of the amino acid radical in its active site. In this work, the addition of catalase, single-electron oxidants, and copper ions was investigated systematically in order to find the minimum concentrations required to obtain a fully active GOase. Furthermore, it was found that the concentration and type of buffer is essential for the activity of GOase, which was significantly more active in sodium phosphate buffer than in other buffers investigated. Enzyme stability and oxygen requirements are of crucial importance for the implementation of oxidase based processes. GOase was shown to be completely stable for 120 h in buffer with stirring at 25 °C, and the activity even increased 30% if the enzyme solution was also aerated in a similar experiment. The high Km for oxygen of GOase (>5 mM) relative to the solubility of oxygen in water reveals a trade-off between supplying oxygen at a sufficiently high rate and ensuring a high degree of enzyme utilization (i.e., ensuring the highest possible specific rate of reaction). Nevertheless, the good stability and high activity of GOase bode well for its future application as an industrial biocatalyst.

A novel amine oxidase (AO)/imine reductase (IRED) system was developed for the deracemization of racemic amines. By combining (R)-6-hydroxy-d-nicotine oxidase (6-HDNO) with an (R)-IRED, a panel of racemic 2-substituted piperidines and pyrrolidines were deracemized to yield the (S)-amines in high yields and enantiomeric excess values. Other N-heterocycles were deracemized with monoamine oxidase (MAO-N) or 6-HDNO in combination with ammonia borane, which allowed comparison of the two enzyme deracemization approaches with that involving a chemical reducing agent.

Enzymes of the class I lyase-like family catalyze the asymmetric addition of ammonia to arylacrylates, yielding high value amino acids as products. Recent examples include the use of phenylalanine ammonia lyases (PALs), either alone or as a gateway to deracemization cascades (giving (S)- or (R)-α-phenylalanine derivatives, respectively), and also eukaryotic phenylalanine aminomutases (PAMs) for the synthesis of the (R)-β-products. Herein, we present the investigation of another family member, EncP from Streptomyces maritimus, thereby expanding the biocatalytic toolbox and enabling the production of the missing (S)-β-isomer. EncP was found to convert a range of arylacrylates to a mixture of (S)-α- and (S)-β-arylalanines, with regioselectivity correlating to the strength of electron-withdrawing/-donating groups on the ring of each substrate. The low regioselectivity of the wild-type enzyme was addressed via structure-based rational design to generate three variants with altered preference for either α- or β-products. By examining various biocatalyst/substrate combinations, it was demonstrated that the amination pattern of the reaction could be tuned to achieve selectivities between 99:1 and 1:99 for β:α-product ratios as desired.

Cytochrome P450 monooxygenases are useful biocatalysts for C–H activation, and there is a need to expand the range of these enzymes beyond what is naturally available. A panel of 93 variants of active self-sufficient P450cam[Tyr96Phe]-RhFRed fusion enzymes with a broad diversity in active site amino acids was developed by screening a large mutant library of 16,500 clones using a simple, highly sensitive colony-based colorimetric screen against indole. These mutants showed distinct fingerprints of activity not only when screened in oxidations of substituted indoles but also for unrelated oxidations such as benzylic hydroxylations.

A chemoenzymatic approach was developed and optimized for the synthesis of a range of N-protected nonnatural l- and d-biarylalanine derivatives. Starting from 4-bromocinnamic acid and 4-bromophenylpyruvic acid using a phenylalanine ammonia lyase (PAL) and an evolved d-amino acid dehydrogenase (DAADH), respectively, both enantiomers of 4-bromophenylalanine were obtained and subsequently coupled with a panel of arylboronic acids to give the target compounds in high yield and optical purity under mild aqueous conditions.

The use of selected engineered galactose oxidase (GOase) variants for the oxidation of amino alcohols to aldehydes under mild conditions in aqueous systems is reported. GOase variant F-2 catalyses the regioselective oxidation of N-carbobenzyloxy (Cbz)-protected 3-amino-1,2-propanediol to the corresponding -hydroxyaldehyde which was then used in an aldolase reaction. Another variant, M3-5, was found to exhibit activity towards free and N-Cbz-protected aliphatic and aromatic amino alcohols allowing the synthesis of lactams such as 3,4-dihydronaphthalen-1(2H)-one, 2-pyrrolidone and valerolactam in one-pot tandem reactions with xanthine dehydrogenase (XDH) or aldehyde oxidase (PaoABC).

Significant advancements in protein engineering and DNA technology have seen biocatalytic transformations take the place of traditional chemical manipulations in both academia and industry for the preparation of active pharmaceutical ingredients (APIs) and other medicinally relevant compounds. However, despite the large repertoire of commercially available biocatalysts that are readily accessible, enzymes which mediate the formation of C–C bonds and those that enable convergent synthesis remain largely undeveloped. To expand the scope of biocatalytic retrosynthesis and enable it to complement traditional chemical retrosynthesis it is essential to develop a ‘toolbox’ of biocatalysts which build molecular complexity. Of particular interest is the development of one-pot enzymatic cascades for the synthesis of functionalised, chiral building blocks without the need for protecting group manipulations or harsh reaction conditions. Highly regio- and stereoselective chemoenzymatic cascades have been developed for the synthesis of a range of chiral amines employing ω-transaminases and monoamine oxidase variants.

A clean and green approach to amines Enzymes evolved to operate in water and to modify their substrates using comparatively nontoxic reagents. Thus, a major advantage of applying enzymes to synthetic chemistry is their compatibility with environmentally benign conditions. Mutti et al. report that two enzymes—alcohol and amine dehydrogenases—can operate in tandem to convert alcohols to amines. The reaction proceeds with ammonium as the only input and water as the only byproduct. The mechanism relies on consecutive oxidation and reduction steps, with hydrogen shuttled by a nicotinamide coenzyme. Science , this issue p. 1525

The concurrent operation of multiple catalysts can lead to enhanced reaction features including (i) simultaneous linear multi-step transformations in a single reaction flask (ii) the control of intermediate equilibria (iii) stereoconvergent transformations (iv) rapid processing of labile reaction products. Enzymes occupy a prominent position for the development of such processes, due to their high potential compatibility with other biocatalysts. Genes for different enzymes can be co-expressed to reconstruct natural or construct artificial pathways and applied in the form of engineered whole cell biocatalysts to carry out complex transformations or, alternatively, the enzymes can be combined in vitro after isolation. Moreover, enzyme variants provide a wider substrate scope for a given reaction and often display altered selectivities and specificities. Man-made transition metal catalysts and engineered or artificial metalloenzymes also widen the range of reactivities and catalysed reactions that are potentially employable. Cascades for simultaneous cofactor or co-substrate regeneration or co-product removal are now firmly established. Many applications of more ambitious concurrent cascade catalysis are only just beginning to appear in the literature. The current review presents some of the most recent examples, with an emphasis on the combination of transition metal with enzymatic catalysis and aims to encourage researchers to contribute to this emerging field.

Process analytical technologies (PAT) are used within industry to give real-time measurements of critical quality parameters, ultimately improving the quality by design (QbD) of the final product and reducing manufacturing costs. Spectroscopic and spectrophotometric methods are readily employed within PAT due to their ease of use, compatibility toward a range of sample types, robustness, and multiplexing capabilities. We have developed a UV resonance Raman (UVRR) spectroscopy approach to quantify industrially relevant biotransformations accurately, focusing on nitrile metabolizing enzymes: nitrile hydratase (NHase) and amidase versus nitrilase activity. Sensitive detection of the amide intermediate by UVRR spectroscopy enabled discrimination between the two nitrile-hydrolyzing pathways. Development of a flow-cell apparatus further exemplifies its suitability toward PAT measurements, incorporating in situ analysis within a closed system. Multivariate curve resolution-alternating least-squares (MCR-ALS) was applied to the UVRR spectra, as well as off-line HPLC measurements, to enable absolute quantification of substrate, intermediate, and product. Further application of hard modeling to MCR-ALS deconvolved concentration profiles enabled accurate kinetic determinations, thus removing the requirement for comparative off-line HPLC. Finally, successful quantitative measurements of in vivo activity using whole-cell biotransformations, where two Escherichia coli strains expressing either NHase (transforming benzonitrile to benzamide) or amidase (further conversion of benzamide to benzoic acid), illustrate the power, practicality, and sensitivity of this novel approach of multistep and, with further refinement, we believe, multiple micro-organism biotransformations.

A universal multi-stage cascade CSTR has been developed which is suitable for a wide range of continuous-flow processes. Coined by our group the ‘Freactor’ (free-to-access reactor), the new reactor integrates the efficiency of pipe-flow processing with the advanced mixing of a CSTR, delivering a general ‘plug-and-play’ reactor platform which is well-suited to multiphasic continuous-flow chemistry. Importantly, the reactor geometry is easily customized to accommodate reactions requiring long residence times (≥ 3 hours tested).

In this study a batch reactor process is compared to a flow chemistry approach for lipase-catalyzed resolution of the cyclopropanecarboxylate ester (±)-3. (1R,2R)-3 is a precursor of the amine (1R,2S)-2 which is a key building block of the API ticagrelor. For both flow and batch operation, the biocatalyst could be recycled several times, whereas in the case of the flow process the reaction time was significantly reduced.

Reductive amination is one of the most important methods for the synthesis of chiral amines. Here we report the discovery of an NADP(H)-dependent reductive aminase from Aspergillus oryzae (AspRedAm, Uniprot code Q2TW47) that can catalyse the reductive coupling of a broad set of carbonyl compounds with a variety of primary and secondary amines with up to >98% conversion and with up to >98% enantiomeric excess. In cases where both carbonyl and amine show high reactivity, it is possible to employ a 1:1 ratio of the substrates, forming amine products with up to 94% conversion. Steady-state kinetic studies establish that the enzyme is capable of catalysing imine formation as well as reduction. Crystal structures of AspRedAm in complex with NADP(H) and also with both NADP(H) and the pharmaceutical ingredient (R)-rasagiline are reported. We also demonstrate preparative scale reductive aminations with wild-type and Q240A variant biocatalysts displaying total turnover numbers of up to 32,000 and space time yields up to 3.73 g l−1 d−1. An enzyme (AspRedAm) capable of coupling carbonyls with a variety of amines in a reductive amination has now been discovered. Kinetic studies revealed that the enzyme catalysed both the imine formation step, as well as the reduction step. Structure and mutagenesis studies have highlighted essential catalytic residues and preparative scale examples have demonstrated total turnover numbers of up to 32,000.

Summary Synthetic biology applies the principles of engineering to biology in order to create biological functionalities not seen before in nature. One of the most exciting applications of synthetic biology is the design of new organisms with the ability to produce valuable chemicals including pharmaceuticals and biomaterials in a greener; sustainable fashion. Selecting the right enzymes to catalyze each reaction step in order to produce a desired target compound is, however, not trivial. Here, we present Selenzyme, a free online enzyme selection tool for metabolic pathway design. The user is guided through several decision steps in order to shortlist the best candidates for a given pathway step. The tool graphically presents key information about enzymes based on existing databases and tools such as: similarity of sequences and of catalyzed reactions; phylogenetic distance between source organism and intended host species; multiple alignment highlighting conserved regions, predicted catalytic site, and active regions and relevant properties such as predicted solubility and transmembrane regions. Selenzyme provides bespoke sequence selection for automated workflows in biofoundries. Availability and implementation The tool is integrated as part of the pathway design stage into the design-build-test-learn SYNBIOCHEM pipeline. The Selenzyme web server is available at http://selenzyme.synbiochem.co.uk. Supplementary information Supplementary data are available at Bioinformatics online.

Carboxylic acid reductases (CARs) catalyze the reduction of a broad range of carboxylic acids to aldehydes using the cofactors adenosine triphosphate and nicotinamide adenine dinucleotide phosphate, and have become attractive biocatalysts for organic synthesis. Mechanistic understanding of CARs was used to expand reaction scope, generating biocatalysts for amide bond formation from carboxylic acid and amine. CARs demonstrated amidation activity for various acids and amines. Optimization of reaction conditions, with respect to pH and temperature, allowed for the synthesis of the anticonvulsant ilepcimide with up to 96 % conversion. Mechanistic studies using site-directed mutagenesis suggest that, following initial enzymatic adenylation of substrates, amidation of the carboxylic acid proceeds by direct reaction of the acyl adenylate with amine nucleophiles.

Imine reductases (IREDs) have emerged as a valuable new set of biocatalysts for the asymmetric synthesis of optically active amines. The development of bioinformatics tools and searchable databases has led to the identification of a diverse range of new IRED biocatalysts that have been characterised and employed in different synthetic processes. This review describes the latest developments in the structural and mechanistic aspects of IREDs, together with synthetic applications of these enzymes, and identifies ongoing and future challenges in the field.

For enzyme‐catalysed biotransformations, continuous in situ detection methods minimise the need for sample manipulation, ultimately leading to more accurate real‐time kinetic determinations of substrate(s) and product(s). We have established for the first time an on‐line, real‐time quantitative approach to monitor simultaneously multiple biotransformations based on UV resonance Raman (UVRR) spectroscopy. To exemplify the generality and versatility of this approach, multiple substrates and enzyme systems were used involving nitrile hydratase (NHase) and xanthine oxidase (XO), both of which are of industrial and biological significance, and incorporate multistep enzymatic conversions. Multivariate data analysis of the UVRR spectra, involving multivariate curve resolution‐alternating least squares (MCR‐ALS), was employed to effect absolute quantification of substrate(s) and product(s); repeated benchmarking of UVRR combined with MCR‐ALS by HPLC confirmed excellent reproducibility.

A library of 132 racemic chiral amines (α-substituted methylbenzylamines, benzhydrylamines, 1,2,3,4-tetrahydronaphthylamines (THNs), indanylamines, allylic and homoallylic amines, propargyl amines) was screened against the most versatile monoamine oxidase (MAO-N) variants D5, D9 and D11. MAO-N D9 exhibited the highest activity for most substrates and was applied to the deracemisation of a comprehensive set of selected primary amines. In all cases, excellent enantioselectivity was achieved (e.e. >99%) with moderate to good yields (55-80%). Conditions for the deracemisation of primary amines using a MAO-N/borane system were further optimised using THN as a template addressing substrate load, nature of the enzyme preparation, buffer systems, borane sources, and organic co-solvents.

High levels of uric acid in urine and serum can be indicative of hypertension and the pregnancy related condition, preeclampsia. We have developed a simple, cost-effective, portable surface enhanced Raman scattering (SERS) approach for the routine analysis of uric acid at clinically relevant levels in urine patient samples. This approach, combined with the standard addition method (SAM), allows for the absolute quantification of uric acid directly in a complex matrix such as that from human urine. Results are highly comparable and in very good agreement with HPLC results, with an average

Screening of bacterial colonies to identify new biocatalytic activities is a widely adopted tool in biotechnology, but is constrained by the requirements for colorimetric or tag-based detection methods. Herein we report a label-free screening platform for biotransformations in live colonies using desorption electrospray ionization coupled with ion mobility mass spectrometry imaging (DiBT-IMMS). The screening method is demonstrated for both ammonia lyases and P450 monooxygenases expressed within live bacterial colonies and is shown to enable multiplexing of enzyme variants and substrate libraries simultaneously.

The chemical synthesis of ketoisophorone, a valuable building block of vitamins and pharmaceuticals, suffers from a number of drawbacks in terms of reaction conditions and selectivity. Herein, the first biocatalytic one-pot double oxidation of the readily available α-isophorone to ketoisophorone is described. Variants of the self-sufficient P450cam-RhFRed with improved activity have been identified to carry out the first step of the designed cascade (regio- and enantioselective allylic oxidation of α-isophorone to 4-hydroxy-α-isophorone). For the second step, the screening of a broad panel of alcohol dehydrogenases (ADHs) led to the identification of Cm-ADH10 from Candida magnoliae. The crystal structure of Cm-ADH10 was solved and docking experiments confirmed the preferred position and geometry of the substrate for catalysis. The synthesis of ketoisophorone was demonstrated both as a one-pot two-step process and as a cascade process employing designer cells co-expressing the two biocatalysts, with a productivity of up to 1.4 g L-1 d-1.

The increasing diversity of reactions mediated by biocatalysts has led to development of multistep in vitro enzyme cascades, taking advantage of generally compatible reaction conditions. The construction of pathways within single whole cell systems is much less explored, yet has many advantages. Herein we report the generation of a successful whole cell de novo enzyme cascade for the diastereoselective and/or enantioselective conversion of simple, linear keto acids into valuable cyclic amine products. The pathway starts with carboxylic acid reduction that triggers a transamination, imine formation, and subsequent imine reduction. Construction and optimization of the system was achieved by standard genetic manipulation and the cascade required only starting material, amine donor, and whole cell catalyst with cofactors provided internally by glucose metabolism. A panel of synthetic keto acids provided access to piperidines in high conversions (up to 93%) and enantiomeric excess (up to 93%).

Carboxylic acid reductase (CAR) catalyzes the ATP- and NADPH-dependent reduction of carboxylic acids to the corresponding aldehydes. The enzyme is related to the nonribosomal peptide synthetases, consisting of an adenylation domain fused via a peptidyl carrier protein (PCP) to a reductase termination domain. Crystal structures of the CAR adenylation-PCP didomain demonstrate that large-scale domain motions occur between the adenylation and thiolation states. Crystal structures of the PCP-reductase didomain reveal that phosphopantetheine binding alters the orientation of a key Asp, resulting in a productive orientation of the bound nicotinamide. This ensures that further reduction of the aldehyde product does not occur. Combining crystallography with small-angle X-ray scattering (SAXS), we propose that molecular interactions between initiation and termination domains are limited to competing PCP docking sites. This theory is supported by the fact that (R)-pantetheine can support CAR activity for mixtures of the isolated domains. Our model suggests directions for further development of CAR as a biocatalyst.

By manipulating the reaction conditions, furandicarboxylic acid (FDCA) was prepared by the biooxidation of hydroxymethyl furfural (HMF) in a continuous one-pot reaction using galactose oxidase M3-5, periplasmic aldehyde oxidase (PaoABC), catalase and horseradish peroxidase. In addition, PaoABC was successfully entrapped in a SiO2 hydrogel and recycled 14 times without loss of activity. The catalyst was able to tolerate up to 200 mM DFF concentration giving FDCA in full conversion with very promising TOF and TON values.

The discovery and characterisation of enzymes with both monoamine and diamine transaminase activity is reported, allowing conversion of a wide range of target ketone substrates with just a small excess of amine donor. The diamine co-substrates (putrescine, cadaverine or spermidine) are bio-derived and the enzyme system results in very little waste, making it a greener strategy for the production of valuable amine fine chemicals and pharmaceuticals.

The suite of biological catalysts found in Nature has the potential to contribute immensely to scientific advancements, ranging from industrial biotechnology to innovations in bioenergy and medical intervention. The endeavour to obtain a catalyst of choice is, however, wrought with challenges. Herein we report the design of a structure-based annotation system for the identification of functionally similar enzymes from diverse sequence backgrounds. Focusing on an enzymatic activity with demonstrated synthetic and therapeutic relevance, five new phenylalanine ammonia lyase (PAL) enzymes were discovered and characterised with respect to their potential applications. The variation and novelty of various desirable traits seen in these previously uncharacterised enzymes demonstrates the importance of effective sequence annotation in unlocking the potential diversity that Nature provides in the search for tailored biological tools. This new method has commercial relevance as a strategy for assaying the ‘evolvability’ of certain enzyme features, thus streamlining and informing protein engineering efforts.

Atropisomeric biaryls carrying ortho-hydroxymethyl and formyl groups were made enantioselectively by desymmetrisation of dialdehyde or diol substrates. The oxidation of the symmetrical diol substrates was achieved using a variant of galactose oxidase (GOase), and the reduction of the dialdehydes using a panel of ketoreductases. Either M or P enantiomers of the products could be formed, with absolute configurations assigned by time-dependent DFT calculations of circular dichroism spectra. The differing selectivities observed with different biaryl structures offer an insight into the detailed structure of the active site of the GOase enzyme.

The widespread application of ω‐transaminases as biocatalysts for chiral amine synthesis has been hampered by fundamental challenges, including unfavorable equilibrium positions and product inhibition. Herein, an efficient process that allows reactions to proceed in high conversion in the absence of by‐product removal using only one equivalent of a diamine donor (ortho‐xylylenediamine) is reported. This operationally simple method is compatible with the most widely used (R)‐ and (S)‐selective ω‐TAs and is particularly suitable for the conversion of substrates with unfavorable equilibrium positions (e.g., 1‐indanone). Significantly, spontaneous polymerization of the isoindole by‐product generates colored derivatives, providing a high‐throughput screening platform to identify desired ω‐TA activity.