The Impact of Metagenomics on Biocatalysis.

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.

In Search of Complementary Extraction Methods for Comprehensive Coverage of the Escherichia coli Metabolome.

Escherichia coli is an invaluable research tool for many fields of biology, in particular for the production of recombinant enzymes. However, the activity of many such recombinant enzymes cannot be determined using standard biochemical assays, as often, the relevant substrates are not known, or the products produced are not detectable. Today, the biochemical footprints of such unknown enzyme activities can be revealed via the analysis of the metabolomes of the recombinant E. coli clones in which they are expressed, using sensitive technologies such as mass spectrometry. However, before any metabolites can be identified, it is necessary to achieve as high a coverage of the potential metabolites present within E. coli as possible. We have therefore analyzed a wide range of different extraction methods against the cell free extracts of various recombinant E. coli clones. The results were analyzed to determine the minimum number of extractions that achieved high recovery and coverage of metabolites. Two methods were selected for further analysis due to their ability to produce not only high numbers of ions, but also wide mass coverage and a high degree of complementarity. One extraction method uses acetonitrile and water, in a 4:1 ratio, which is then dried down and reconstituted in the chromatography running buffer prior to injection onto the chromatography column, and the other extraction method uses a combination of methanol, water and chloroform, in a 3:1:1 ratio, which is injected directly onto the chromatography column. These two extraction methods were shown to be complementary to each other, as regards the respective metabolites extracted, and to cover a large range of metabolites.

Regioselective Radical Alkylation of Arenes Using Evolved Photoenzymes.

Substituted arenes are ubiquitous in molecules with medicinal functions, making their synthesis a critical consideration when designing synthetic routes. Regioselective C-H functionalization reactions are attractive for preparing alkylated arenes; however, the selectivity of existing methods is modest and primarily governed by the substrate's electronic properties. Here, we demonstrate a biocatalyst-controlled method for the regioselective alkylation of electron-rich and electron-deficient heteroarenes. Starting from an unselective "ene"-reductase (ERED) (GluER-T36A), we evolved a variant that selectively alkylates the C4 position of indole, an elusive position using prior technologies. Mechanistic studies across the evolutionary series indicate that changes to the protein active site alter the electronic character of the charge transfer (CT) complex responsible for radical formation. This resulted in a variant with a significant degree of ground-state CT in the CT complex. Mechanistic studies on a C2-selective ERED suggest that the evolution of GluER-T36A helps disfavor a competing mechanistic pathway. Additional protein engineering campaigns were carried out for a C8-selective quinoline alkylation. This study highlights the opportunity to use enzymes for regioselective radical reactions, where small molecule catalysts struggle to alter selectivity.

Regioselective Radical Alkylation of Arenes Using Evolved Photoenzymes.

Substituted arenes are ubiquitous in molecules with medicinal functions, making their synthesis a critical consideration when designing synthetic routes. 1,2 Regioselective C-H functionalization reactions are attractive for preparing alkylated arenes, 3-5 however, the selectivity of existing methods is modest and primarily governed by substrate electronic properties. 6,7 Here, we demonstrate a biocatalyst-controlled method for the regioselective alkylation of electron-rich and electron-deficient heteroarenes. Starting from an unselective 'ene'-reductase (ERED) (GluER-T36A), we evolved a variant that selectively alkylates the C4 position of indole, an elusive position using prior technologies. Mechanistic studies across the evolutionary series indicate that changes to the protein active site alter the electronic character of the charge transfer (CT) complex responsible for radical formation. This resulted in a variant with a significant degree of ground state change transfer in the CT complex. Mechanistic studies on a C2 selective ERED suggest that the evolution of GluER-T36A helps disfavor a competing mechanistic pathway. Additional protein engineering campaigns were carried out for a C8 selective quinoline alkylation. This study highlights the opportunity to use enzymes for regioselective reactions where small molecule catalysts struggle to alter selectivity.

Synthesis of Stereoenriched Piperidines via Chemo-Enzymatic Dearomatization of Activated Pyridines.

The development of efficient and sustainable methods for the synthesis of nitrogen heterocycles is an important goal for the chemical industry. In particular, substituted chiral piperidines are prominent targets due to their prevalence in medicinally relevant compounds and their precursors. A potential biocatalytic approach to the synthesis of this privileged scaffold would be the asymmetric dearomatization of readily assembled activated pyridines. However, nature is yet to yield a suitable biocatalyst specifically for this reaction. Here, by combining chemical synthesis and biocatalysis, we present a general chemo-enzymatic approach for the asymmetric dearomatization of activated pyridines for the preparation of substituted piperidines with precise stereochemistry. The key step involves a stereoselective one-pot amine oxidase/ene imine reductase cascade to convert N-substituted tetrahydropyridines to stereo-defined 3- and 3,4-substituted piperidines. This chemo-enzymatic approach has proved useful for key transformations in the syntheses of antipsychotic drugs Preclamol and OSU-6162, as well as for the preparation of two important intermediates in synthetic routes of the ovarian cancer monotherapeutic Niraparib.

An asymmetric sp3-sp3 cross-electrophile coupling using 'ene'-reductases.

The catalytic asymmetric construction of Csp3-Csp3 bonds remains one of the foremost challenges in organic synthesis1. Metal-catalysed cross-electrophile couplings (XECs) have emerged as a powerful tool for C-C bond formation2-5. However, coupling two distinct Csp3 electrophiles with high cross-selectivity and stereoselectivity continues as an unmet challenge. Here we report a highly chemoselective and enantioselective Csp3-Csp3 XEC between alkyl halides and nitroalkanes catalysed by flavin-dependent 'ene'-reductases (EREDs). Photoexcitation of the enzyme-templated charge-transfer complex between an alkyl halide and a flavin cofactor enables the chemoselective reduction of alkyl halide over the thermodynamically favoured nitroalkane partner. The key C-C bond-forming step occurs by means of the reaction of an alkyl radical with an in situ-generated nitronate to form a nitro radical anion that collapses to form nitrite and an alkyl radical. An enzyme-controlled hydrogen atom transfer (HAT) affords high levels of enantioselectivity. This reactivity is unknown in small-molecule catalysis and highlights the potential for enzymes to use new mechanisms to address long-standing synthetic challenges.

Chemoenzymatic Cascades for the Enantioselective Synthesis of ß-Hydroxysulfides Bearing a Stereocentre at the C-O or C-S Bond by Ketoreductases.

Chiral ß-hydroxysulfides are an important class of organic compounds which find broad application in organic and pharmaceutical chemistry. Herein we describe the development of novel biocatalytic and chemoenzymatic methods for the enantioselective synthesis of ß-hydroxysulfides by exploiting ketoreductase (KRED) enzymes. Four KREDs were discovered from a pool of 384 enzymes identified and isolated through a metagenomic approach. KRED311 and KRED349 catalysed the synthesis of ß-hydroxysulfides bearing a stereocentre at the C-O bond with opposite absolute configurations and excellent ee values by novel chemoenzymatic and biocatalytic-chemical-biocatalytic (bio-chem-bio) cascades starting from commercially available thiophenols/thiols and α-haloketones/alcohols. KRED253 and KRED384 catalysed the enantioselective synthesis of ß-hydroxysulfides bearing a stereocentre at the C-S bond with opposite enantioselectivities by dynamic kinetic resolution (DKR) of racemic α-thioaldehydes.

Multifunctional biocatalyst for conjugate reduction and reductive amination.

Chiral amine diastereomers are ubiquitous in pharmaceuticals and agrochemicals1, yet their preparation often relies on low-efficiency multi-step synthesis2. These valuable compounds must be manufactured asymmetrically, as their biochemical properties can differ based on the chirality of the molecule. Herein we characterize a multifunctional biocatalyst for amine synthesis, which operates using a mechanism that is, to our knowledge, previously unreported. This enzyme (EneIRED), identified within a metagenomic imine reductase (IRED) collection3 and originating from an unclassified Pseudomonas species, possesses an unusual active site architecture that facilitates amine-activated conjugate alkene reduction followed by reductive amination. This enzyme can couple a broad selection of α,ß-unsaturated carbonyls with amines for the efficient preparation of chiral amine diastereomers bearing up to three stereocentres. Mechanistic and structural studies have been carried out to delineate the order of individual steps catalysed by EneIRED, which have led to a proposal for the overall catalytic cycle. This work shows that the IRED family can serve as a platform for facilitating the discovery of further enzymatic activities for application in synthetic biology and organic synthesis.

A Coupled Ketoreductase-Diaphorase Assay for the Detection of Polyethylene Terephthalate-Hydrolyzing Activity.

In the last two decades, several PET-degrading enzymes from already known microorganisms or metagenomic sources have been discovered to face the growing environmental concern of polyethylene terephthalate (PET) accumulation. However, there is a limited number of high-throughput screening protocols for PET-hydrolyzing activity that avoid the use of surrogate substrates. Herein, a microplate fluorescence screening assay was described. It was based on the coupled activity of ketoreductases (KREDs) and diaphorase to release resorufin in the presence of the products of PET degradation. Six KREDs were identified in a commercial panel that were able to use the PET building block, ethylene glycol, as substrate. The most efficient KRED, KRED61, was combined with the diaphorase from Clostridium kluyveri to monitor the PET degradation reaction catalyzed by the thermostable variant of the cutinase-type polyesterase from Saccharomonospora viridis AHK190. The PET degradation products were measured both fluorimetrically and by HPLC, with excellent correlation between both methods.

An Engineered Cytidine Deaminase for Biocatalytic Production of a Key Intermediate of the Covid-19 Antiviral Molnupiravir.

The Covid-19 pandemic highlights the urgent need for cost-effective processes to rapidly manufacture antiviral drugs at scale. Here we report a concise biocatalytic process for Molnupiravir, a nucleoside analogue recently approved as an orally available treatment for SARS-CoV-2. Key to the success of this process was the development of an efficient biocatalyst for the production of N-hydroxy-cytidine through evolutionary adaption of the hydrolytic enzyme cytidine deaminase. This engineered biocatalyst performs >85 000 turnovers in less than 3 h, operates at 180 g/L substrate loading, and benefits from in situ crystallization of the N-hydroxy-cytidine product (85% yield), which can be converted to Molnupiravir by a selective 5'-acylation using Novozym 435.

Synthesis of Pharmaceutically Relevant 2-Aminotetralin and 3-Aminochroman Derivatives via Enzymatic Reductive Amination.

2-Aminotetralin and 3-aminochroman derivatives are key structural motifs present in a wide range of pharmaceutically important molecules. Herein, we report an effective biocatalytic approach towards these molecules through the enantioselective reductive coupling of 2-tetralones and 3-chromanones with a diverse range of primary amine partners. Metagenomic imine reductases (IREDs) were employed as the biocatalysts, obtaining high yields and enantiocomplementary selectivity for >15 examples at preparative scale, including the precursors to Ebalzotan, Robalzotan, Alnespirone and 5-OH-DPAT. We also present a convergent chemo-enzymatic total synthesis of the Parkinson's disease therapy Rotigotine in 63 % overall yield and 92 % ee.

Rapid Screening of Diverse Biotransformations for Enzyme Evolution.

The lack of label-free high-throughput screening technologies presents a major bottleneck in the identification of active and selective biocatalysts, with the number of variants often exceeding the capacity of traditional analytical platforms to assess their activity in a practical time scale. Here, we show the application of direct infusion of biotransformations to the mass spectrometer (DiBT-MS) screening to a variety of enzymes, in different formats, achieving sample throughputs equivalent to ∼40 s per sample. The heat map output allows rapid selection of active enzymes within 96-well plates facilitating identification of industrially relevant biocatalysts. This DiBT-MS screening workflow has been applied to the directed evolution of a phenylalanine ammonia lyase (PAL) as a case study, enhancing its activity toward electron-rich cinnamic acid derivatives which are relevant to lignocellulosic biomass degradation. Additional benefits of the screening platform include the discovery of biocatalysts (kinases, imine reductases) with novel activities and the incorporation of ion mobility technology for the identification of product hits with increased confidence.

Asymmetric Synthesis of N-Substituted α-Amino Esters from α-Ketoesters via Imine Reductase-Catalyzed Reductive Amination.

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.

Screening and characterization of a diverse panel of metagenomic imine reductases for biocatalytic reductive amination.

Finding faster and simpler ways to screen protein sequence space to enable the identification of new biocatalysts for asymmetric synthesis remains both a challenge and a rate-limiting step in enzyme discovery. Biocatalytic strategies for the synthesis of chiral amines are increasingly attractive and include enzymatic asymmetric reductive amination, which offers an efficient route to many of these high-value compounds. Here we report the discovery of over 300 new imine reductases and the production of a large (384 enzymes) and sequence-diverse panel of imine reductases available for screening. We also report the development of a facile high-throughput screen to interrogate their activity. Through this approach we identified imine reductase biocatalysts capable of accepting structurally demanding ketones and amines, which include the preparative synthesis of N-substituted ß-amino ester derivatives via a dynamic kinetic resolution process, with excellent yields and stereochemical purities.

Cytochromes P450 (P450s): A review of the class system with a focus on prokaryotic P450s.

Cytochromes P450 (P450s) are a large superfamily of heme-containing monooxygenases. P450s are found in all Kingdoms of life and exhibit incredible diversity, both at sequence level and also on a biochemical basis. In the majority of cases, P450s can be assigned into one of ten classes based on their associated redox partners, domain architecture and cellular localization. Prokaryotic P450s now represent a large diverse collection of annotated/known enzymes, of which many have great potential biocatalytic potential. The self-sufficient P450 classes (Class VII/VIII) have been explored significantly over the past decade, with many annotated and biochemically characterized members. It is clear that the prokaryotic P450 world is expanding rapidly, as the number of published genomes and metagenome studies increases, and more P450 families are identified and annotated (CYP families).

Profiling Substrate Promiscuity of Wild-Type Sugar Kinases for Multi-fluorinated Monosaccharides.

Fluorinated sugar-1-phosphates are of emerging importance as intermediates in the chemical and biocatalytic synthesis of modified oligosaccharides, as well as probes for chemical biology. Here we present a systematic study of the activity of a wide range of anomeric sugar kinases (galacto- and N-acetylhexosamine kinases) against a panel of fluorinated monosaccharides, leading to the first examples of polyfluorinated substrates accepted by this class of enzymes. We have discovered four new N-acetylhexosamine kinases with a different substrate scope, thus expanding the number of homologs available in this subclass of kinases. Lastly, we have solved the crystal structure of a galactokinase in complex with 2-deoxy-2-fluorogalactose, giving insight into changes in the active site that may account for the specificity of the enzyme toward certain substrate analogs.

Biochemical characterisation of an α1,4 galactosyltransferase from Neisseria weaveri for the synthesis of α1,4-linked galactosides.

The human cell surface trisaccharide motifs globotriose and P1 antigen play key roles in infections by pathogenic bacteria, which makes them important synthetic targets as antibacterial agents. Enzymatic strategies to install the terminal α1,4-galactosidic linkage are very attractive but have only been demonstrated for a limited set of analogues. Herein, a new bacterial α1,4 galactosyltransferase from N. weaveri was cloned and produced recombinantly in E. coli BL21 (DE3) cells, followed by investigation of its substrate specificity. We demonstrate that the enzyme can tolerate galactosamine (GalN) and also 6-deoxygalactose and 6-deoxy-6-fluorogalactose as donors, and lactose and N-acetyllactosamine as acceptors, leading directly to analogues of Gb3 and P1 that are valuable chemical probes and showcase how biocatalysis can provide fast access to a number of unnatural carbohydrate analogues.

Enantioselective Synthesis of α-Thiocarboxylic Acids by Nitrilase Biocatalysed Dynamic Kinetic Resolution of α-Thionitriles.

The enantioselective synthesis of α-thiocarboxylic acids by biocatalytic dynamic kinetic resolution (DKR) of nitrile precursors exploiting nitrilase enzymes is described. A panel of 35 nitrilase biocatalysts were screened and enzymes Nit27 and Nit34 were found to catalyse the DKR of racemic α-thionitriles under mild conditions, affording the corresponding carboxylic acids with high conversions and good-to-excellent ee. The ammonia produced in situ during the biocatalytic transformation favours the racemization of the nitrile enantiomers and, in turn, the DKR without the need of any external additive base.

Enantioselective Aldol Addition of Acetaldehyde to Aromatic Aldehydes Catalyzed by Proline-Based Carboligases.

Aromatic ß-hydroxyaldehydes, 1,3-diols, and α,ß-unsaturated aldehydes are valuable precursors to biologically active natural products and drug molecules. Herein we report the biocatalytic aldol condensation of acetaldehyde with various aromatic aldehydes to give a number of aromatic α,ß-unsaturated aldehydes using a previously engineered variant of 4-oxalocrotonate tautomerase [4-OT(M45T/F50A)] as carboligase. Moreover, an efficient one-pot two-step chemoenzymatic route toward chiral aromatic 1,3-diols has been developed. This one-pot chemoenzymatic strategy successfully combined a highly enantioselective aldol addition step catalyzed by a proline-based carboligase [4-OT(M45T/F50A) or TAUT015] with a chemical reduction step to convert enzymatically prepared aromatic ß-hydroxyaldehydes into the corresponding 1,3-diols with high optical purity (e.r. up to >99:1) and in good isolated yield (51-92%). These developed (chemo)enzymatic methodologies offer alternative synthetic choices to prepare a variety of important drug precursors.

Enantioselective Synthesis of Pharmaceutically Active γ-Aminobutyric Acids Using a Tailor-Made Artificial Michaelase in One-Pot Cascade Reactions.

Chiral γ-aminobutyric acid (GABA) analogues represent abundantly prescribed drugs, which are broadly applied as anticonvulsants, as antidepressants, and for the treatment of neuropathic pain. Here we report a one-pot two-step biocatalytic cascade route for synthesis of the pharmaceutically relevant enantiomers of γ-nitrobutyric acids, starting from simple precursors (acetaldehyde and nitroalkenes), using a tailor-made highly enantioselective artificial "Michaelase" (4-oxalocrotonate tautomerase mutant L8Y/M45Y/F50A), an aldehyde dehydrogenase with a broad non-natural substrate scope, and a cofactor recycling system. We also report a three-step chemoenzymatic cascade route for the efficient chemical reduction of enzymatically prepared γ-nitrobutyric acids into GABA analogues in one pot, achieving high enantiopurity (e.r. up to 99:1) and high overall yields (up to 70%). This chemoenzymatic methodology offers a step-economic alternative route to important pharmaceutically active GABA analogues, and highlights the exciting opportunities available for combining chemocatalysts, natural enzymes, and designed artificial biocatalysts in multistep syntheses.