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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., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Biocatalysis is becoming an indispensable tool in organic synthesis due to high enzymatic catalytic efficiency as well as exquisite chemo- and stereoselectivity. Some biocatalysts display great promiscuity including a broad substrate scope as well as the ability to catalyze more than one type of transformation. These promiscuous activities have been applied individually to efficiently access numerous valuable target molecules. However, systems in which enzymes possessing multiple different catalytic activities are applied in the synthesis are less well developed. Such multifunctional biocatalysts (MFBs) would simplify chemical synthesis by reducing the number of operational steps and enzyme count, as well as simplifying the sequence space that needs to be engineered to develop an efficient biocatalyst. In this Perspective, we highlight recently reported MFBs focusing on their synthetic utility and mechanism. We also offer insight into their origin as well as comment on potential strategies for their discovery and engineering.

Ene-reductases (EREDs) catalyze the reduction of electron-deficient C═C bonds. Herein, we report the first example of ERED-catalyzed net reduction of C═C bonds of enimines (α,β-unsaturated imines). Preliminary studies suggest their hydrolyzed ring-open ω-amino enones are the likely substrates for this step. When combined with imine reductase (IRED)-mediated C═N reduction, the result is an efficient telescoped sequence for the preparation of diastereomerically enriched 2-substituted saturated amine heterocycles.

three complementary biocatalytic routes were examined for the synthesis of the cyclopropyl amine (1R,2S)-2, which is a key building block for the anti-thrombotic agent ticagrelor 1. By employing either a ketoreductase, amidase or lipase biocatalyst, the key building blocks for synthesis of the amine 2 were obtained in 99.9, 92.5 and 46.3 ee, respectively.

Biocatalyst discovery and directed evolution are central to many pharmaceutical research programs, yet the lack of robust high-throughput screening methods for large libraries of enzyme variants generated (typically 10(6)-10(8)) has hampered progress and slowed enzyme optimization. We have developed a label-free generally applicable approach based on Raman spectroscopy which results in significant reductions in acquisition times (>30-fold). Surface enhanced Raman scattering (SERS) is employed to monitor the enzyme-catalyzed conversion by xanthine oxidase of hypoxanthine to xanthine to uric acid. This approach measures the substrates and products directly and does not require chromogenic substrates or lengthy chromatography, was successfully benchmarked against HPLC, and shows high levels of accuracy and reproducibility. Furthermore, we demonstrate that this SERS approach has utility in monitoring enzyme inhibition illustrating additional medical significance to this high-throughput screening method.

The combination of stable isotope labelling with direct infusion ion mobility mass spectrometry (IM-MS) enabled qualitative and quantitative monitoring of biocatalytic reactions with reduced analysis times, enhanced sensitivity and μL-level assay volumes. The new approach was demonstrated by applying to both lipase and monooxygenase enzymes, including multi-substrate screening.

α-Chiral amines are key intermediates for the synthesis of a plethora of chemical compounds at industrial scale. We present a biocatalytic hydrogen-borrowing amination of primary and secondary alcohols that allows for the efficient and environmentally benign production of enantiopure amines. The method relies on a combination of two enzymes: an alcohol dehydrogenase (from Aromatoleum sp., Lactobacillus sp., or Bacillus sp.) operating in tandem with an amine dehydrogenase (engineered from Bacillus sp.) to aminate a structurally diverse range of aromatic and aliphatic alcohols, yielding up to 96% conversion and 99% enantiomeric excess. Primary alcohols were aminated with high conversion (up to 99%). This redox self-sufficient cascade possesses high atom efficiency, sourcing nitrogen from ammonium and generating water as the sole by-product., (Copyright © 2015, American Association for the Advancement of Science.)

Oxidoreductases from Streptomyces sp. GF3546 [3546-IRED], Bacillus cereus BAG3X2 (BcIRED) and Nocardiopsis halophila (NhIRED) each reduce prochiral 2-methylpyrroline (2MPN) to (S)-2-methylpyrrolidine with >95 % ee and also a number of other imine substrates with good selectivity. Structures of BcIRED and NhIRED have helped to identify conserved active site residues within this subgroup of imine reductases that have S selectivity towards 2MPN, including a tyrosine residue that has a possible role in catalysis and superimposes with an aspartate in related enzymes that display R selectivity towards the same substrate. Mutation of this tyrosine residue-Tyr169-in 3546-IRED to Phe resulted in a mutant of negligible activity. The data together provide structural evidence for the location and significance of the Tyr residue in this group of imine reductases, and permit a comparison of the active sites of enzymes that reduce 2MPN with either R or S selectivity., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

Ene-reductases (ERs) are flavin dependent enzymes that catalyze the asymmetric reduction of activated carbon-carbon double bonds. In particular, α,β-unsaturated carbonyl compounds (e.g. enals and enones) as well as nitroalkenes are rapidly reduced. Conversely, α,β-unsaturated esters are poorly accepted substrates whereas free carboxylic acids are not converted at all. The only exceptions are α,β-unsaturated diacids, diesters as well as esters bearing an electron-withdrawing group in α- or β-position. Here, we present an alternative approach that has a general applicability for directly obtaining diverse chiral α-substituted carboxylic acids. This approach combines two enzyme classes, namely ERs and aldehyde dehydrogenases (Ald-DHs), in a concurrent reductive-oxidative biocatalytic cascade. This strategy has several advantages as the starting material is an α-substituted α,β-unsaturated aldehyde, a class of compounds extremely reactive for the reduction of the alkene moiety. Furthermore no external hydride source from a sacrificial substrate (e.g. glucose, formate) is required since the hydride for the first reductive step is liberated in the second oxidative step. Such a process is defined as a hydrogen-borrowing cascade. This methodology has wide applicability as it was successfully applied to the synthesis of chiral substituted hydrocinnamic acids, aliphatic acids, heterocycles and even acetylated amino acids with elevated yield, chemo- and stereo-selectivity. A systematic methodology for optimizing the hydrogen-borrowing two-enzyme synthesis of α-chiral substituted carboxylic acids was developed. This systematic methodology has general applicability for the development of diverse hydrogen-borrowing processes that possess the highest atom efficiency and the lowest environmental impact.

Phenylalanine ammonia lyases (PALs) catalyse the regio- and stereoselective hydroamination of cinnamic acid analogues to yield optically enriched α-amino acids. Herein, we demonstrate that a bacterial PAL from Anabaena variabilis (AvPAL) displays significantly higher activity towards a series of non-natural substrates than previously described eukaryotic PALs. Biotransformations performed on a preparative scale led to the synthesis of the 2-chloro- and 4-trifluoromethyl-phenylalanine derivatives in excellent ee, highlighting the enormous potential of bacterial PALs as biocatalysts for the synthesis of high value, non-natural amino acids., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Ammonia lyases catalyse the reversible addition of ammonia to cinnamic acid (1: R=H) and p-hydroxycinnamic (1: R=OH) to generate L-phenylalanine (2: R=H) and L-tyrosine (2: R=OH) respectively (Figure 1a). Both phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase (TAL) are widely distributed in plants, fungi and prokaryotes. Recently there has been interest in the use of these enzymes for the synthesis of a broader range of L-arylalanines. Aminomutases catalyse a related reaction, namely the interconversion of α-amino acids to β-amino acids (Figure 1b). In the case of L-phenylalanine, this reaction is catalysed by phenylalanine aminomutase (PAM) and proceeds stereospecifically via the intermediate cinnamic acid to generate β-Phe 3. Ammonia lyases and aminomutases are related in sequence and structure and share the same active site cofactor 4-methylideneimidazole-5-one (MIO). There is currently interest in the possibility of using these biocatalysts to prepare a wide range of enantiomerically pure l-configured α-amino and β-amino acids. Recent reviews have focused on the mechanism of these MIO containing enzymes. The aim of this review is to review recent progress in the application of ammonia lyase and aminomutase enzymes to prepare enantiomerically pure α-amino and β-amino acids., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

We have combined the biocatalytic desymmetrization of 3,4-cis-substituted meso-pyrrolidines with an Ugi-type multicomponent reaction followed in situ by a Pictet-Spengler-type cyclization reaction sequence for the rapid asymmetric synthesis of alkaloid-like polycyclic compounds.

New methods continue to be developed for the dynamic kinetic resolution (DKR) and deracemisation of racemic chiral compounds, in particular alcohols, amines and amino acids. Many of the DKR processes involve the combination of an enantioselective enzyme, often a lipase or protease, with a metal racemisation catalyst. A greater range of ruthenium-based racemisation catalysts is now available with some showing good activity for the racemisation of amines that are more difficult to epimerise than the corresponding alcohols. In terms of deracemisation processes, further improvements have been achieved with the deracemisation of alcohols, using combinations of stereocomplementary ketoreductases, and additionally transaminases have been applied to the deracemisation of racemic amines., (Copyright 2009 Elsevier Ltd. All rights reserved.)

A micro-scale technique has been developed for the process development of transaminase catalysed reactions. This pH indicator based, colorimetric assay can be used to investigate and optimise reaction conditions at 100 microL scale. Enzyme activity and stability as a function of various reaction parameters, including temperature, pH and co-solvent concentration, have been determined. Additionally, reactions have been scaled up from 100 microL to 25 mL under the optimal reaction conditions identified by the micro-scale process development activities. Excellent conversion (>99%) and enantioselectivity (>99% ee) were obtained.

Enzymes are increasingly being used as biocatalysts in the generation of products that have until now been derived using traditional chemical processes. Such products range from pharmaceutical and agrochemical building blocks to fine and bulk chemicals and, more recently, components of biofuels. For a biocatalyst to be effective in an industrial process, it must be subjected to improvement and optimization, and in this respect the directed evolution of enzymes has emerged as a powerful enabling technology. Directed evolution involves repeated rounds of (i) random gene library generation, (ii) expression of genes in a suitable host and (iii) screening of libraries of variant enzymes for the property of interest. Both in vitro screening-based methods and in vivo selection-based methods have been applied to the evolution of enzyme function and properties. Significant developments have occurred recently, particularly with respect to library design, screening methodology, applications in synthetic transformations and strategies for the generation of new enzyme function.

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. [ABSTRACT FROM AUTHOR]

Reductive aminases (RedAms) have recently emerged as promising biocatalysts for the synthesis of chiral secondary amines by coupling primary amines with aldehydes/ketones. However, access to tertiary amines remains more problematic, particularly when coupling ketones with secondary amines. Here we show that the scope of these enzymes can be extended to allow selective reductive aminations of cyclic secondary amines, such as piperidines and morpholines, with both aldehydes and ketones. These biotransformations provide access to important motifs found in active pharmaceutical ingredients and other bioactive molecules. RedAm‐361, discovered from a metagenomic library, was engineered via directed evolution to allow efficient coupling of cyclic amines with carbonyl partners, including dynamic kinetic resolutions of α‐functionalized aldehydes and enantioselective amination of ketones. These RedAms now serve as valuable scaffolds for the engineering of industrial biocatalysts to produce key pharmaceutical intermediates. [ABSTRACT FROM AUTHOR]

In 2017, two companies, Novartis AG and Syngenta AG, joined forces with the group of Prof. Buller, head of the Competence Center for Biocatalysis (CCBIO), to tackle the challenge of enzymatic late‐stage halogenation. This biocatalytic method was considered to provide a more sustainable approach to late‐stage halogenation of complex molecules than traditional synthetic approaches. Using machine‐learning guided protein engineering, α‐ketoglutarate dependent halogenases were evolved into versatile catalysts capable of selectively chlorinating inactivated C−H bonds. Structurally diverse molecules, namely an analogue of martinelline as well as two members of the soraphen natural product family, were enzymatically chlorinated at two distinct positions in a regio‐ and stereoselective manner, thus demonstrating the synthetic usefulness of such a strategy. As part of our three‐year collaboration, flavin‐dependent halogenases were also studied. [ABSTRACT FROM AUTHOR]

N‐alkanoyl‐N‐methylglucamides (MEGAs) are non‐toxic surfactants widely used as commercial ingredients, but more sustainable syntheses towards these compounds are highly desirable. Here, we present a biocatalytic route towards MEGAs and analogues using a truncated carboxylic acid reductase construct tailored for amide bond formation (CARmm‐A). CARmm‐A is capable of selective amide bond formation without the competing esterification reaction observed in lipase catalysed reactions. A kinase was implemented to regenerate ATP from polyphosphate and by thorough reaction optimisation using design of experiments, the amine concentration needed for amidation was significantly reduced. The wide substrate scope of CARmm‐A was exemplified by the synthesis of 24 commercially relevant amides, including selected examples on a preparative scale. This work establishes acyl‐phosphate mediated chemistry as a highly selective strategy for biocatalytic amide bond formation in the presence of multiple competing alcohol functionalities. [ABSTRACT FROM AUTHOR]

Electron‐rich phenolic substrates can be derived from the depolymerisation of lignin feedstocks. Direct biotransformations of the hydroxycinnamic acid monomers obtained can be exploited to produce high‐value chemicals, such as α‐amino acids, however the reaction is often hampered by the chemical autooxidation in alkaline or harsh reaction media. Regioselective O‐methyltransferases (OMTs) are ubiquitous enzymes in natural secondary metabolic pathways utilising an expensive co‐substrate S‐adenosyl‐l‐methionine (SAM) as the methylating reagent altering the physicochemical properties of the hydroxycinnamic acids. In this study, we engineered an OMT to accept a variety of electron‐rich phenolic substrates, modified a commercial E. coli strain BL21 (DE3) to regenerate SAM in vivo, and combined it with an engineered ammonia lyase to partake in a one‐pot, two whole cell enzyme cascade to produce the l‐DOPA precursor l‐veratrylglycine from lignin‐derived ferulic acid. [ABSTRACT FROM AUTHOR]

Covering: 2015 up to July 2021 The market for cosmetics is consumer driven and the desire for green, sustainable and natural ingredients is increasing. The use of isolated enzymes and whole-cell organisms to synthesise these products is congruent with these values, especially when combined with the use of renewable, recyclable or waste feedstocks. The literature of biocatalysis for the synthesis of ingredients in cosmetics in the past five years is herein reviewed. [ABSTRACT FROM AUTHOR]

A key aim of biocatalysis is to mimic the ability of eukaryotic cells to carry out multistep cascades in a controlled and selective way. As biocatalytic cascades get more complex, reactions become unattainable under typical batch conditions. Here a number of continuous flow systems were used to overcome batch incompatibility, thus allowing for successful biocatalytic cascades. As proof‐of‐principle, reactive carbonyl intermediates were generated in situ using alcohol oxidases, then passed directly to a series of packed‐bed modules containing different aminating biocatalysts which accordingly produced a range of structurally distinct amines. The method was expanded to employ a batch incompatible sequential amination cascade via an oxidase/transaminase/imine reductase sequence, introducing different amine reagents at each step without cross‐reactivity. The combined approaches allowed for the biocatalytic synthesis of the natural product 4O‐methylnorbelladine. [ABSTRACT FROM AUTHOR]

Enzyme catalysis is gaining increasing importance in synthetic chemistry. Nowadays, the growing number of biocatalysts accessible by means of bioinformatics and enzyme engineering opens up an immense variety of selective reactions. Biocatalysis especially provides excellent opportunities for late‐stage modification often superior to conventional de novo synthesis. Enzymes have proven to be useful for direct introduction of functional groups into complex scaffolds, as well as for rapid diversification of compound libraries. Particularly important and highly topical are enzyme‐catalysed oxyfunctionalisations, halogenations, methylations, reductions, and amide bond formations due to the high prevalence of these motifs in pharmaceuticals. This Review gives an overview of the strengths and limitations of enzymatic late‐stage modifications using native and engineered enzymes in synthesis while focusing on important examples in drug development. [ABSTRACT FROM AUTHOR]

N‐Substituted α‐amino esters are widely used as chiral intermediates in a range of pharmaceuticals. Here we report the enantioselective biocatalyic synthesis of N‐substituted α‐amino esters through the direct reductive coupling of α‐ketoesters and amines employing sequence diverse metagenomic imine reductases (IREDs). Both enantiomers of N‐substituted α‐amino esters were obtained with high conversion and excellent enantioselectivity under mild reaction conditions. In addition >20 different preparative scale transformations were performed highlighting the scalability of this system. [ABSTRACT FROM AUTHOR]

The integration of electro and bio‐catalysis offers new ways of making molecules under very mild, environmentally benign conditions. We show that TEMPO mediated electro‐catalytic oxidation of alcohols can be adapted to work in aqueous buffers, with minimal organic co‐solvent, enabling integration with biocatalytic reductive amination using the AdRedAm enzyme. The combined process offers a new approach to amine alkylation with native alcohols, a key bond formation in the chemical economy that is currently achieved via precious metal‐catalyzed hydrogen‐borrowing technologies. The electrobio transformation is effective for primary and secondary alcohols undergoing coupling with allyl, propargyl, benzyl, and cyclopropyl amines, and has been adapted for use with solid‐supported AdRedAm for ease of operation. [ABSTRACT FROM AUTHOR]

Enantiomerically enriched chiral secondary alcohols serve as valuable building blocks for drug intermediates and fine chemicals. In this study the deracemisation of secondary alcohols to generate enantiomeric pure chiral alcohols has been achieved by combining enantio‐selective enzymatic oxidation of a secondary alcohol, by a variant of GOase (GOase M3‐5), with either non‐selective ketone reduction via transfer hydrogenation (TH) or enantio‐selective asymmetric transfer hydrogenation (ATH). Both the enzymatic oxidation system and the transition‐metal mediated reduction system were optimised to ensure compatibility with each other resulting in a homogeneous reaction system. 1‐(4‐nitrophenyl)ethanol was generated with 99 % conversion and 98 % ee by the deracemisation method, and it has been extended to a series of other secondary alcohols with comparable results. [ABSTRACT FROM AUTHOR]

Objectives: Formate dehydrogenases (FDHs) are NAD(P)H-dependent enzymes that catalyse the reversible oxidation of formate to CO2. The main goal was to use directed evolution to obtain variants of the FDH from Chaetomium thermophilum (CtFDH) with enhanced reduction activity in the conversion of CO2 into formic acid. Results: Four libraries were constructed targeting five residues in the active site. We identified two variants (G93H/I94Y and R259C) with enhanced reduction activity which were characterised in the presence of both aqueous CO2(g) and HCO3−. The A1 variant (G93H/I94Y) showed a 5.4-fold increase in catalytic efficiency (kcat/KM) compared to that of the wild-type for HCO3− reduction. The improved biocatalysts were also applied as a coupled cofactor recycling system in the enantioselective oxidation of 4-phenyl-2-propanol catalysed by the alcohol dehydrogenase from Streptomyces coelicolor A3 (ScADH). Conversions in these reactions increased from 56 to 91% when the A1 variant was used instead of wild-type CtFDH. Conclusions: Two variants presenting up to five-fold increase in catalytic efficiency and kcat were obtained and characterised. They constitute a promising enzymatic alternative for CO2 utilization and will serve as scaffolds to be further developed in order to meet industrial requirements. [ABSTRACT FROM AUTHOR]

Process intensification is crucial for industrial implementation of biocatalysis and can be achieved by continuous process operation in miniaturized reactors with efficiently immobilized biocatalysts, enabling their long-term use. Due to their extremely large surface-to-volume ratio, nanomaterials are promising supports for enzyme immobilization. In this work, different functionalized nanofibrous nonwoven membranes were embedded in a two-plate microreactor to enable immobilization of hexahistidine (His 6)-tagged amine transaminases (ATAs) in flow. A membrane coated with Cu2+ ions gave the best results regarding His 6 -tagged ATAs immobilization among the membranes tested yielding an immobilization yield of up to 95.3 % for the purified N -His 6 -ATA-wt enzyme. Moreover, an efficient one-step enzyme immobilization process from overproduced enzyme in Escherichia coli cell lysate was developed and yielded enzyme loads up to 1088 U mL−1. High enzyme loads resulted in up to 80 % yields of acetophenone produced from 40 mM (S)-α-methylbenzylamine in less than 4 min using a continuously operated microreactor. Up to 81 % of the initial activity was maintained in a 5-day continuous microreactor operation with immobilized His 6 -tagged ATA constructs. The highest turnover number within the indicated time was 7.23·106, which indicates that this immobilization approach using advanced material and reactor system is highly relevant for industrial implementation. [Display omitted] • A microreactor with nonwoven nanofiber membrane was developed. • Nanofibers with various functional groups were tested for enzyme immobilization. • His6-tagged amine transaminase was immobilized on membranes with metal ions. • One-step purification and immobilization with very high enzyme loads was reached. • Long-term stability of microreactor system and high turnover numbers were obtained. [ABSTRACT FROM AUTHOR]

The combination of biocatalysis and chemo‐catalysis increasingly offers chemists access to more diverse chemical architectures. Here, we describe the combination of a toolbox of chiral‐amine‐producing biocatalysts with a Buchwald–Hartwig cross‐coupling reaction, affording a variety of α‐chiral aniline derivatives. The use of a surfactant allowed reactions to be performed sequentially in the same flask, preventing the palladium catalyst from being inhibited by the high concentrations of ammonia, salts, or buffers present in the aqueous media in most cases. The methodology was further extended by combining with a dual‐enzyme biocatalytic hydrogen‐borrowing cascade in one pot to allow for the conversion of a racemic alcohol to a chiral aniline. [ABSTRACT FROM AUTHOR]

The conversion of carboxylic acids, such as acrylic acids, to amines is a transformation that remains challenging in synthetic organic chemistry. Despite the ubiquity of similar moieties in natural metabolic pathways, biocatalytic routes seem to have been overlooked for this purpose. Herein we present the conception and optimisation of a two‐enzyme system, allowing the synthesis of β‐phenylethylamine derivatives from readily‐available ring‐substituted cinnamic acids. After characterisation of both parts of the reaction in a two‐step approach, a set of conditions allowing the one‐pot biotransformation was optimised. This combination of a reversible deaminating and irreversible decarboxylating enzyme, both specific for the amino acid intermediate in tandem, represents a general method by which new strategies for the conversion of carboxylic acids to amines could be designed. [ABSTRACT FROM AUTHOR]

In living cells, the overall rates of catalytic reaction chains (cascades) are massively enhanced by nanoconfinement of enzymes in tiny enclosed volumes: presented in such a way, interdependent catalysts are highly concentrated, and distances (active site‐to‐active site) across which intermediates and cofactors must diffuse, may be tiny. In a parallel technology exploiting this principle, enzyme cascades are powered, amplified, and monitored in real time as they work in concert, being nanoconfined within the pores of an electrically conductive metal oxide electrode. The technology, nicknamed the electrochemical leaf, mimics chloroplast biosynthesis by exploiting nicotinamide adenine dinucleotide (NADP(H)) recycling catalysed by the flavoenzyme, ferredoxin NADP+ reductase (FNR). Adsorbed on the inner walls of the nanopores, FNR rapidly transfers electrons between the electrode and NADP(H). This activity is coupled to an oxidoreductase enzyme, also nanoconfined within the pores, which recycles the cofactor and selectively synthesises a desired product or senses an analyte. Use of catalyst and cofactor is very efficient. The technology is simple, inexpensive and adaptable, and scalable to both microscopic levels (diagnostics) and macroscopic levels (organic synthesis). Whereas native FNR is specific for NADP(H), the technology can be extended by genetic engineering to include NAD(H) as recycling cofactor. [ABSTRACT FROM AUTHOR]

• Carboxylic acid reductases (CARs) chemoselectively reduce carboxylic acids to aldehydes. • CARs are Mg2+, ATP and NADPH dependent and can be applied in vivo or in vitro with cofactor recycling. • CARs have been applied in a variety of biocatalytic cascades to date. • CARs are promising industrial biocatalysts although more development is required for robustness and scalability. Carboxylic acid reductases (CARs) are an emerging biocatalyst platform for the synthesis of a diverse array of aldehydes from carboxylic acids, operating chemoselectively and under mild aqueous conditions. As such, there is growing interest in the industrial application of these enzymes, both for the synthesis of aldehyde end-products, which are particularly prevalent in the flavor and fragrance industries, and aldehyde intermediates in multi-enzyme cascades. This perspective aims to review recent developments in the applications of CARs with a focus on the challenges and considerations involved in their implementation, as well as potential solutions with a view to increased industrial utility. [ABSTRACT FROM AUTHOR]

No Abstract [ABSTRACT FROM AUTHOR]

The conversion of saturated fatty acids to high value chiral hydroxy‐acids and lactones poses a number of synthetic challenges: the activation of unreactive C−H bonds and the need for regio‐ and stereoselectivity. Here the first example of a wild‐type cytochrome P450 monooxygenase (CYP116B46 from Tepidiphilus thermophilus) capable of enantio‐ and regioselective C5 hydroxylation of decanoic acid 1 to (S)‐5‐hydroxydecanoic acid 2 is reported. Subsequent lactonization yields (S)‐δ‐decalactone 3, a high value fragrance compound, with greater than 90 % ee. Docking studies provide a rationale for the high regio‐ and enantioselectivity of the reaction. The cytochrome P450 monooxygenase, CYP116B46, from Tepidiphilus thermophilus is capable of enantio‐ and regioselective C5 hydroxylation of decanoic acid 1 to (S)‐5‐hydroxydecanoic acid 2. Subsequent lactonization yields (S)‐δ‐decalactone 3, a high value fragrance compound, with greater than 90 % ee. Docking studies provide a rationale for the high regio‐ and enantioselectivity of the reaction. [ABSTRACT FROM AUTHOR]

Herein we report the biocatalytic synthesis of substituted pyrazines and pyrroles using a transaminase (ATA) to mediate the key amination step of the ketone precursors. Treatment of α‐diketones with ATA‐113 in the presence of a suitable amine donor yielded the corresponding α‐amino ketones which underwent oxidative dimerization to the pyrazines. Selective amination of α‐diketones in the presence of β‐keto esters afforded substituted pyrroles in a biocatalytic equivalent of the classical Knorr pyrrole synthesis. Finally we have shown that pyrroles can be prepared by internal amine transfer catalyzed by a transaminase in which no external amine donor is required. The key amination step of the ketone precursors in a biocatalytic synthesis of substituted pyrazines and pyrroles is mediated using a transaminase (ATA). Treatment of α‐diketones with ATA‐113 in the presence of an amine donor yielded α‐amino ketones which underwent oxidative dimerization to the pyrazines. Selective amination of α‐diketones in the presence of β‐keto esters afforded substituted pyrroles in a biocatalytic equivalent of the classical Knorr pyrrole synthesis. [ABSTRACT FROM AUTHOR]

A multi‐enzymatic cascade process involving transaminases (TAs) and reductive aminases (RedAms) to produce enantiomerically pure 2,5‐disubstituted pyrrolidine alkaloids from their respective 1,4‐diketones is reported. Several TAs were screened and the best results for diketone monoamination were obtained with an R‐selective TA from Mycobacterium chlorophenicum and with an S‐selective TA from Bacillus megaterium. Pyrroline reduction was best performed by a reductive aminase from Ajellomyces dermatitidis (AdRedAm). Finally, a biocatalytic one‐pot cascade was implemented using the aforementioned enzymes and a variety of 2‐methyl‐5‐alkylpyrrolidines were produced with high (>99 %) conversion, diastereomeric and enantiomeric excess values. Cascades get antsy! A one‐pot enzymatic cascade using transaminase and reductive aminase biocatalysts has been successfully implemented for the regio‐ and stereoselective synthesis of a panel of 2‐methyl‐5‐alkylpyrrolidines from the corresponding 1,4‐diketones. [ABSTRACT FROM AUTHOR]

Abstract: We report a simple, mild, and synthetically clean approach to accelerate the rate of enzymatic oxidation reactions by a factor of up to 100 when compared to conventional batch gas/liquid systems. Biocatalytic decomposition of H2O2 is used to produce a soluble source of O2 directly in reaction media, thereby enabling the concentration of aqueous O2 to be increased beyond equilibrium solubility under safe and practical conditions. To best exploit this method, a novel flow reactor was developed to maximize productivity (g product L−1 h−1). This scalable benchtop method provides a distinct advantage over conventional bio‐oxidation in that no pressurized gas or specialist equipment is employed. The method is general across different oxidase enzymes and compatible with a variety of functional groups. These results culminate in record space‐time yields for bio‐oxidation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: The ability of cytochrome P450 monooxygenases to catalyse a wide variety of synthetically challenging C−H activation reactions makes them highly desirable biocatalysts both for the synthesis of chiral intermediates and for late‐stage functionalisations. However, P450s are plagued by issues associated with poor expression, solubility and stability. Catalytically self‐sufficient P450s, in which the haem and reductase domains are fused in a single protein, obviate the need for additional redox partners and are attractive as biocatalysts. Here we present a panel of natural self‐sufficient P450s from thermophilic organisms (CYP116B65 from A. thermoflava, CYP116B64 from A. xiamenense, CYP116B63 from J. thermophila, CYP116B29 from T. bispora and CYP116B46 from T. thermophilus). These P450s display enhanced expression and stability over their mesophilic homologues. Activity profiling of these enzymes revealed that each P450 displayed a different fingerprint in terms of substrate range and reactivity that cover reactions as diverse as hydroxylation, demethylation, epoxidation and sulfoxidation. The productivity of the bio‐transformation of diclofenac to produce the 5‐hydroxy metabolite increased 42‐fold using the thermostable P450‐AX (>0.5 g L−1 h−1) compared to the P450‐RhF system reported previously. In conclusion, we have generated a toolkit of thermostable self‐sufficient P450 biocatalysts with a broad substrate range and reactivity. [ABSTRACT FROM AUTHOR]

Abstract: The NADP(H)‐dependent reductive aminase from Aspergillus oryzae (AspRedAm) was combined with an NADPH oxidase (NOX) to develop a redox system that recycles the co‐factor. The AspRedAm‐NOX system was applied initially for the kinetic resolution of a variety of racemic secondary and primary amines to yield S‐configured amines with enantiomeric excess (ee) values up to 99 %. The addition of ammonia borane to this system enabled the efficient deracemization of racemic amines, including the pharmaceutical drug rasagiline and the natural product salsolidine, with conversions up to >98 % and >99 % ee Furthermore, by using the AspRedAm W210A variant it was possible to generate the opposite R enantiomers with efficiency comparable to, or even better than, the wildtype AspRedAm. [ABSTRACT FROM AUTHOR]