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.

One-Pot Biocatalytic In Vivo Methylation-Hydroamination of Bioderived Lignin Monomers to Generate a Key Precursor to L-DOPA.

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.

One-Pot Biocatalytic In Vivo Methylation-Hydroamination of Bioderived Lignin Monomers to Generate a Key Precursor to L-DOPA.

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.

Toward scalable biocatalytic conversion of 5-hydroxymethylfurfural by galactose oxidase using coordinated reaction and enzyme engineering.

5-Hydroxymethylfurfural (HMF) has emerged as a crucial bio-based chemical building block in the drive towards developing materials from renewable resources, due to its direct preparation from sugars and its readily diversifiable scaffold. A key obstacle in transitioning to bio-based plastic production lies in meeting the necessary industrial production efficiency, particularly in the cost-effective conversion of HMF to valuable intermediates. Toward addressing the challenge of developing scalable technology for oxidizing crude HMF to more valuable chemicals, here we report coordinated reaction and enzyme engineering to provide a galactose oxidase (GOase) variant with remarkably high activity toward HMF, improved O2 binding and excellent productivity (>1,000,000 TTN). The biocatalyst and reaction conditions presented here for GOase catalysed selective oxidation of HMF to 2,5-diformylfuran offers a productive blueprint for further development, giving hope for the creation of a biocatalytic route to scalable production of furan-based chemical building blocks from sustainable feedstocks.

Synthesis of protected 3-aminopiperidine and 3-aminoazepane derivatives using enzyme cascades.

Multi-enzyme cascades utilising variants of galactose oxidase and imine reductase led to the successful conversion of N-Cbz-protected l-ornithinol and l-lysinol to l-3-N-Cbz-aminopiperidine and l-3-N-Cbz-aminoazepane respectively, in up to 54% isolated yield. Streamlining the reactions into one-pot prevented potential racemisation of key labile intermediates and led to products with high enantiopurity.

Engineered formate dehydrogenase from Chaetomium thermophilum, a promising enzymatic solution for biotechnical CO2 fixation.

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.

Natural heterogeneous catalysis with immobilised oxidase biocatalysts.

The generation of immobilised oxidase biocatalysts allowing multifunctional oxidation of valuable chemicals using molecular oxygen is described. Engineered galactose oxidase (GOase) variants M1 and M3-5, an engineered choline oxidase (AcCO6) and monoamine oxidase (MAO-N D9) displayed long-term stability and reusability over several weeks when covalently attached on a solid support, outperforming their free counterparts in terms of stability (more than 20 fold), resistance to heat at 60 °C, and tolerance to neat organic solvents such as hexane and toluene. These robust heterogenous oxidation catalysts can be recovered after each reaction and be reused multiple times for the oxidation of different substrates.

An Engineered Alcohol Oxidase for the Oxidation of Primary Alcohols.

Structure-guided directed evolution of choline oxidase has been carried out by using the oxidation of hexan-1-ol to hexanal as the target reaction. A six-amino-acid variant was identified with a 20-fold increased kcat compared to that of the wild-type enzyme. This variant enabled the oxidation of 10 mm hexanol to hexanal in less than 24 h with 100 % conversion. Furthermore, this variant showed a marked increase in thermostability with a corresponding increase in Tm of 20 °C. Improved solvent tolerance was demonstrated with organic solvents including ethyl acetate, heptane and cyclohexane, thereby enabling improved conversions to the aldehyde by up to 30 % above conversion for the solvent-free system. Despite the evolution of choline oxidase towards hexan-1-ol, this new variant also showed increased specific activities (by up to 100-fold) for around 50 primary aliphatic, unsaturated, branched, cyclic, benzylic and halogenated alcohols.

Discovery of a new metal and NAD+-dependent formate dehydrogenase from Clostridium ljungdahlii.

Over the next decades, with the growing concern of rising atmospheric carbon dioxide (CO2) levels, the importance of investigating new approaches for its reduction becomes crucial. Reclamation of CO2 for conversion into biofuels represents an alternative and attractive production method that has been studied in recent years, now with enzymatic methods gaining more attention. Formate dehydrogenases (FDHs) are NAD(P)H-dependent oxidoreductases that catalyze the conversion of formate into CO2 and have been extensively used for cofactor recycling in chemoenzymatic processes. A new FDH from Clostridium ljungdahlii (ClFDH) has been recently shown to possess activity in the reverse reaction: the mineralization of CO2 into formate. In this study, we show the successful homologous expression of ClFDH in Escherichia coli. Biochemical and kinetic characterization of the enzyme revealed that this homologue also demonstrates activity toward CO2 reduction. Structural analysis of the enzyme through homology modeling is also presented.

Characterisation of a Bacterial Galactokinase with High Activity and Broad Substrate Tolerance for Chemoenzymatic Synthesis of 6-Aminogalactose-1-Phosphate and Analogues.

Glycosyl phosphates are important intermediates in many metabolic pathways and are substrates for diverse carbohydrate-active enzymes. Thus, there is a need to develop libraries of structurally similar analogues that can be used as selective chemical probes in glycomics. Here, we explore chemoenzymatic cascades for the fast generation of glycosyl phosphate libraries without protecting-group strategies. The key enzyme is a new bacterial galactokinase (LgGalK) cloned from Leminorella grimontii, which was produced in Escherichia coli and shown to catalyse 1-phosphorylation of galactose. LgGalK displayed a broad substrate tolerance, being able to catalyse the 1-phosphorylation of a number of galactose analogues, including 3-deoxy-3-fluorogalactose and 4-deoxy-4-fluorogalactose, which were first reported to be substrates for wild-type galactokinase. LgGalK and galactose oxidase variant M1 were combined in a one-pot, two-step system to synthesise 6-oxogalactose-1-phosphate and 6-oxo-2-fluorogalactose-1-phosphate, which were subsequently used to produce a panel of 30 substituted 6-aminogalactose-1-phosphate derivatives by chemical reductive amination in a one-pot, three-step chemoenzymatic process.

Application of Biocatalysis to on-DNA Carbohydrate Library Synthesis.

DNA-encoded libraries are increasingly used for the discovery of bioactive lead compounds in high-throughput screening programs against specific biological targets. Although a number of libraries are now available, they cover limited chemical space due to bias in ease of synthesis and the lack of chemical reactions that are compatible with DNA tagging. For example, compound libraries rarely contain complex biomolecules such as carbohydrates with high levels of functionality, stereochemistry, and hydrophilicity. By using biocatalysis in combination with chemical methods, we aimed to significantly expand chemical space and generate generic libraries with potentially better biocompatibility. For DNA-encoded libraries, biocatalysis is particularly advantageous, as it is highly selective and can be performed in aqueous environments, which is an essential feature for this split-and-mix library technology. In this work, we demonstrated the application of biocatalysis for the on-DNA synthesis of carbohydrate-based libraries by using enzymatic oxidation and glycosylation in combination with traditional organic chemistry.

Semi-Rational Design of Geobacillus stearothermophilus L-Lactate Dehydrogenase to Access Various Chiral α-Hydroxy Acids.

Chiral α-hydroxy acids (AHAs) are rapidly becoming important synthetic building blocks, in particular for the production of pharmaceuticals and other fine chemicals. Chiral compounds of a variety of functionalities are now often derived using enzymes, and L-lactate dehydrogenase from the thermophilic organism Geobacillus stearothermophilus (bsLDH) has the potential to be employed for the industrial synthesis of chiral α-hydroxy acids. Despite the thorough characterization of this enzyme, generation of variants with high activity on non-natural substrates has remained difficult and therefore limits the use of bsLDH in industry. Here, we present the engineering of bsLDH using semi-rational design as a method of focusing screening in a small and smart library for novel biocatalysts. In this study, six mutant libraries were designed in an effort to expand the substrate range of bsLDH. The eight variants identified as having enhanced activity toward the selected α-keto acids belonged to the same library, which targeted two positions simultaneously. These new variants now may be useful biocatalysts for chiral synthesis of α-hydroxy acids.

Action at a distance: mutations of peripheral residues transform rapid reversible inhibitors to slow, tight binders of cyclooxygenase-2.

Cyclooxygenase enzymes (COX-1 and COX-2) catalyze the conversion of arachidonic acid to prostaglandin G2. The inhibitory activity of rapid, reversible COX inhibitors (ibuprofen, naproxen, mefenamic acid, and lumiracoxib) demonstrated a significant increase in potency and time dependence of inhibition against double tryptophan murine COX-2 mutants at the 89/90 and 89/119 positions. In contrast, the slow, time-dependent COX inhibitors (diclofenac, indomethacin, and flurbiprofen) were unaffected by those mutations. Further mutagenesis studies suggested that mutation at position 89 was principally responsible for the changes in inhibitory potency of rapid, reversible inhibitors, whereas mutation at position 90 may exert some effect on the potency of COX-2-selective diarylheterocycle inhibitors; no effect was observed with mutation at position 119. Several crystal structures with or without NSAIDs indicated that placement of a bulky residue at position 89 caused a closure of a gap at the lobby, and alteration of histidine to tryptophan at position 90 changed the electrostatic profile of the side pocket of COX-2. Thus, these two residues, especially Val-89 at the lobby region, are crucial for the entrance and exit of some NSAIDs from the COX active site.

Bioretrosynthetic construction of a didanosine biosynthetic pathway.

Concatenation of engineered biocatalysts into multistep pathways markedly increases their utility, but the development of generalizable assembly methods remains a major challenge. Herein we evaluate 'bioretrosynthesis', which is an application of the retrograde evolution hypothesis, for biosynthetic pathway construction. To test bioretrosynthesis, we engineered a pathway for synthesis of the antiretroviral nucleoside analog didanosine (2',3'-dideoxyinosine). Applying both directed evolution- and structure-based approaches, we began pathway construction with a retro-extension from an engineered purine nucleoside phosphorylase and evolved 1,5-phosphopentomutase to accept the substrate 2,3-dideoxyribose 5-phosphate with a 700-fold change in substrate selectivity and threefold increased turnover in cell lysate. A subsequent retrograde pathway extension, via ribokinase engineering, resulted in a didanosine pathway with a 9,500-fold change in nucleoside production selectivity and 50-fold increase in didanosine production. Unexpectedly, the result of this bioretrosynthetic step was not a retro-extension from phosphopentomutase but rather the discovery of a fortuitous pathway-shortening bypass via the engineered ribokinase.

Molecular differences between a mutase and a phosphatase: investigations of the activation step in Bacillus cereus phosphopentomutase.

Prokaryotic phosphopentomutases (PPMs) are di-Mn(2+) enzymes that catalyze the interconversion of α-D-ribose 5-phosphate and α-D-ribose 1-phosphate at an active site located between two independently folded domains. These prokaryotic PPMs belong to the alkaline phosphatase superfamily, but previous studies of Bacillus cereus PPM suggested adaptations of the conserved alkaline phosphatase catalytic cycle. Notably, B. cereus PPM engages substrates when the active site nucleophile, Thr-85, is phosphorylated. Further, the phosphoenzyme is stable throughout purification and crystallization. In contrast, alkaline phosphatase engages substrates when the active site nucleophile is dephosphorylated, and the phosphoenzyme reaction intermediate is only stably trapped in a catalytically compromised enzyme. Studies were undertaken to understand the divergence of these mechanisms. Crystallographic and biochemical investigations of the PPM(T85E) phosphomimetic variant and the neutral corollary PPM(T85Q) determined that the side chain of Lys-240 underwent a change in conformation in response to active site charge, which modestly influenced the affinity for the small molecule activator α-D-glucose 1,6-bisphosphate. More strikingly, the structure of unphosphorylated B. cereus PPM revealed a dramatic change in the interdomain angle and a new hydrogen bonding interaction between the side chain of Asp-156 and the active site nucleophile, Thr-85. This hydrogen bonding interaction is predicted to align and activate Thr-85 for nucleophilic addition to α-D-glucose 1,6-bisphosphate, favoring the observed equilibrium phosphorylated state. Indeed, phosphorylation of Thr-85 is severely impaired in the PPM(D156A) variant even under stringent activation conditions. These results permit a proposal for activation of PPM and explain some of the essential features that distinguish between the catalytic cycles of PPM and alkaline phosphatase.

Assessing directed evolution methods for the generation of biosynthetic enzymes with potential in drug biosynthesis.

To address the synthesis of increasingly structurally diverse small-molecule drugs, methods for the generation of efficient and selective biological catalysts are becoming increasingly important. 'Directed evolution' is an umbrella term referring to a variety of methods for improving or altering the function of enzymes using a nature-inspired twofold strategy of mutagenesis followed by selection. This article provides an objective assessment of the effectiveness of directed evolution campaigns in generating enzymes with improved catalytic parameters for new substrates from the last decade, excluding studies that aimed to select for only improved physical properties and those that lack kinetic characterization. An analysis of the trends of methodologies and their success rates from 81 qualifying examples in the literature reveals the average fold improvement for k (cat) (or V (max)), K (m) and k (cat)/K (m) to be 366-, 12- and 2548-fold, respectively, whereas the median fold improvements are 5.4, 3 and 15.6. Further analysis by enzyme class, library-generation methodology and screening methodology explores relationships between successful campaigns and the methodologies employed.

Evolution of the antibiotic resistance protein, FosA, is linked to a catalytically promiscuous progenitor.

The fosfomycin (1) resistance proteins FosA and FosX in pathogenic microorganisms are related to a catalytically promiscuous progenitor encoded in a phn operon in Mesorhizobium loti. The mlr3345 gene product (FosX(Ml)) from M. loti has a very low epoxide hydrolase activity and even lower glutathione transferase activity toward 1 and does not confer resistance to the antibiotic. In vitro homologous recombination of the mlr3345 and pa1129 genes (a fosA gene from Pseudomonas aeruginosa that does confer robust resistance to 1) produces recombinant proteins that confer resistance to 1 and indicate that the FosA resistance proteins are functionally and genetically related to mlr3345.