DESC:DESCRIPTION
FIELD OF THE INVENTION
[0001] The present invention relates to the field of biocatalysis, biochemistry, and molecular biology. More specifically, the present invention relates to engineered Formate Dehydrogenase for biocatalysis and cofactor recycling.
BACKGROUND OF THE INVENTION
[0002] Extensive research of formate dehydrogenase (FDH) on cofactor recycling of NAD(P)+ to NAD(P)H for improved recycling ability. However, FDH weren’t explored as a functional biocatalyst for the reduction of bulky 2-oxo-acids. In the present invention, we report an engineered artificial formate dehydrogenase (arFDH) to effectively facilitate the ketone reductions of the substrates having 2-oxo-acid group converting into 2-hydroxy-acid, Fig. 1 where “R” is benzyl or 2-phenylethyl or (4-hydroxyphenyl) methyl or methyl.
[0003] Enzymatic reductions are widely used in various industries, including pharmaceuticals and specialty chemicals to synthesize specific compounds with high selectivity and purity. The oxidation-reduction (redox) reactions are mainly catalysed by a diverse group of enzymes called Oxidoreductases. They play a crucial role in biocatalysis and biotransformation for their ability to catalyze selective reductions with high specificity, by transferring electrons between molecules. This conversion is facilitated by enzyme cofactor (a non-protein molecule) like Nicotinamide adenine dinucleotide (NAD) and its analogue, or Nicotinamide adenine dinucleotide phosphate (NADP) as high-potential electron donors (Fischer & Pietruszka, 2010; Matsuda et. al., 2009; Torres Pazmiño et. al., 2010; Wei et. al., 2017; Wohlgemuth, 2010). Enzymatic synthesis of hydroxy-acid containing compounds is an established process for synthesis of pharmaceutically important intermediates. For example, Ketoreductase (KRED), D-Lactate dehydrogenase (D-LDH), and alcohol dehydrogenase (ADH) are reported to convert 2-oxo-acids into 2-hydroxy-acids in the presence of Glucose Dehydrogenase or Formate dehydrogenase as a cofactor recycling system (Zheng et. al., 2013). However, one of the limitations of this process is the lack of ability to convert compounds with bulky groups with for commercial viability.
[0004] The bulky substrates namely, 2-oxo-4-phenylbutyric acid can be used to synthesize (2R)-2-hydroxy-4-phenylbutanoic acid, a key intermediate in the production of pharmaceuticals such as Benazepril, Enalapril, Ramipril, Lisinopril, and Quinapril, which are drugs used for the treatment of high blood pressure. 4-hydroxyphenylpyruvic acid can be used in the synthesis of (2S)-2-hydroxy-3-(4-hydroxyphenyl)propanoic acid which is a key intermediate for the synthesis of saroglitazar, a drug used to treat non-cirrhotic non-alcoholic steatohepatitis and diabetic dyslipidemia. Phenylpyruvic acid was used in the synthesis of (R)-phenyllactic acid which is a key intermediate in the synthesis of scopolamine, which is used to prevent symptoms of motion sickness, irritable bowel syndrome, and Parkinson's disease. The biocatalytic reduction of the substrates have been previously reported (Shen et. al., 2014, Yi et. al., 2021, Busto et. al., 2016). However, in all the reported cases, the biocatalytic synthesis of these substrates involves the use of a two enzyme-one pot solution coupled with the use of a co-factor recycling system and yet the reported activity is not up to the scale of commercial viability. Co-factor recycling helps reduce the overall cost of the process and minimize the need for large quantities of expensive cofactors (van der Donk & Zhao, 2003; Wichmann & Vasic-Racki, 2005). The more widely used co-factor recycling system is Glucose dehydrogenase (GDH) because of its high enzymatic activity, and glucose, which is used as a co-substrate, is relatively inexpensive, but the major drawback is the formation gluconate as a byproduct that can interfere with the reaction by significantly altering the pH which can be detrimental to the processes. Whereas, in case of formate dehydrogenase (FDH) the reaction is irreversible, broad pH optimum (pH 6.0-9.0) and the byproduct released is gaseous CO2 which helps drive the reaction towards the product formation, the inert nature of the CO2 does not inactivate enzymes or cause significant pH shifts. Thus, FDH is the enzyme of choice for reactions requiring NAD(P)H (Tishkov & Popov, 2006).
[0005] The incorporation of strategic engineering via various means for FDH was facilitated through the implementation of modifications in the active site and peripheral residues. The successful fitting of bulky 2-oxo-acid group substrates was accomplished by engineering the active site, where bulky amino acids were replaced with smaller amino acids. The activity for transforming the bulky substrate containing a 2-oxo-acid group into a 2-hydroxy-acid has been accomplished by altering the electrostatic nature of the active site through the substitution of with a negatively charged residue. Formate dehydrogenases (FDHs, EC 1.17.1.9) reduces NAD(P)+ to NAD(P)H and oxidizes formate-ion to carbon dioxide (CO2) in the presence of formic acid and water. They belong to the superfamily of D-specific dehydrogenases that acts on 2-oxo-acid group with the D-configuration (Vinals et al., 1993).
[0006] The difficulty in using FDH is that it loses its ability to function due to a lack of stability, which can be caused by a variety of factors like pH, temperature, the ions being used, mechanical stress, etc (Alekseeva et al., 2015; Andreadeli et al., 2008; Gul-Karaguler et al., 2001; Kazuya Mitsuhashi, 2004). Most of the FDHs show low specific activity of about 5-7 U/mg of protein at 30 ? (Popov & Lamzin, 1994). Many engineering strategies such as random mutagenesis, rational designs, in-silico design to improve the activity of FDHs are often used so that expensive making-up of enzyme can be reduced and a small of enzyme can be used in the industrial process. For example, in 2012 the Alekseeva et al. prepared single point mutants of formate dehydrogenase from soya Glycine max (SoyFDH) using site-directed mutagenesis to improve the catalytic activity and thermal stability simultaneously. All the substitutions at F290 introduced into SoyFDH showed increase in KmHCOO- from 1.5 to 5.0 mM, and no change in the KmNAD+. The mutations F290S, F290N and F290D increased the Tm values by 2.9 °C, 4.3 °C and 7.8 °C, respectively. F290D and F290S showed 2-fold increase in the catalytic efficiency toward NAD+ whereas F290A showed 2-fold higher catalytic efficient towards both NAD+ and HCOOH (Kargov et al., 2015). The incorporation of the mutant C23S/F285S into Candida boidinii Formate Dehydrogenase (CboFDH) resulted in a 1.7-fold increase in the catalytic constant, raising it from 3.7 s-1 to 6.2 s-1. Also, the specific activity of the enzyme was improved to 9.1 U/mg from 5.5 U/mg (Heike Slusarczyk, 2003). Similary, the Jiang et al., 2016 demonstrated the mutant V120S exhibited the highest catalytic efficiency i.e., it improved Kcat by 3.48-fold and Kcat/Km by 1.60-fold towards HCOONH4, when compared to wild. Conversely, the V120D, N187D displayed 1.50-fold increase in Kcat/Km towards NAD+.
[0007] The currently known FDHs have been shown to strongly prefer NAD+ over NADP+, because of which they have poor NADPH regenerating system (Tishkov & Popov, 2004). The mutations were introduced to modify the FDH from Mycobacterium vaccae N10 (MycFDH) to alter it and make it stable to a-haloketone ethyl 4-chloroacetoacetate (ECAA) and capable of NADPH regeneration. The variant A198G/D221Q increased the catalytic efficiency (Kcat/Km) towards NADP+ and C145S/C255V further improved efficiency by 6-fold and resistance to ECAA. The mutant C145S/A198G/D221Q/C255V demonstrated a specific activity of 4?±0.13 U/mg and reduced Km to 0.147 mM from 40 mM (Hoelsch et al., 2013), whereas Hatrongjit & Packdibamrung, 2010 successfully designed mutants of the FDH from Burkholderia stabilis (BstFDH) to shift the preference from NADP+ to NAD+. The mutant Q223D decreased the Km for NAD+ to 0.06 mM and decreased the efficiency with NADP+ by 185-fold, improving by 18-fold towards NAD+.
OBJECTS OF THE INVENTION
[0008] The primary objective of the invention is to create an engineered formate dehydrogenase biocatalyst for the conversion of bulky substrates having 2-oxo-acid group to 2-hydroxy-acid group. The engineered arFDH enzyme to convert 2-oxo-4-phenylbutyric acid to (2R)-2-hydroxy-4-phenylbutanoic acid, 4-hydroxyphenylpyruvic acid to (2S)-2-hydroxy-3-(4-hydroxyphenyl)propanoic acid and phenylpyruvic acid to (R)-phenyllactic acid. In addition to its function as reductase, another scope was to enhance its ability to recycle the cofactor NAD(P)+ and simultaneously enhance the enzyme stability in a broader temperature range. Furthermore, the polynucleotide that encodes arFDH is operably linked to one or more promoter sequences that promote the production of recombinant arFDH in a recombinant host cell using an expression vector and expressed in a recombinant host cell.
SUMMARY OF THE INVENTION
[0009] The invention introduces a novel capability of engineered formate dehydrogenase biocatalysts, which can reduce bulky substrates containing 2-oxo-acid functional groups to 2-hydroxy-acids. In addition to its function as reductase, the enzyme was engineered to enhance its ability to recycle the cofactor NAD(P)+.
[0010] During the conventional process, oxidoreductases (SDRs), convert ketones into alcohols, during which, the coenzyme NAD(P)H gets oxidized to form NAD(P)+ and must be recycled for the next step of the reaction, which typically requires additional enzymes such as FDH to reduce NAD(P)+ to NAD(P)H. The engineered arFDH given in SEQ ID NO: 01 converts bulky substrates having 2-oxo-acid group to 2-hydroxy-acid (Fig. 1).
[0011] The incorporation of strategic engineering via various means for FDH was facilitated through the implementation of modifications in the active site and peripheral residues. The active site of arFDH was engineered to accommodate the bulky 2-oxo-acid substrate by increasing the active site volume. The incorporated mutations in positions which contained bulky amino acids were mutated with smaller amino acid of the same class in order to maintain functionality. Some of the positions which were mutated are X98G, X99G, and X100A which enabled the fitting of substrates, 2-oxo-4-phenylbutyric acid, 4-hydroxyphenylpyruvic acid and phenylpyruvic acid.
[0012] The activity for bulky substrate comprised of a 2-oxo-acid group into a 2-hydroxy-acid has been achieved through the modification of the electrostatic nature of the active site by substituting with a negatively charged residue in arFDH.
[0013] The arFDH was developed for the conversion of 2-oxo-4-phenylbutyric acid to (2R)-2-hydroxy-4-phenylbutanoic acid, 4-hydroxyphenylpyruvic acid to (2S)-2-hydroxy-3-(4-hydroxyphenyl)propanoic acid and phenylpyruvic acid to (R)-phenyllactic acid wherein, the arFDH showed conversion of >85%, >88% and >92% for the above mentioned substrates, respectively. The synthesized (2R)-2-hydroxy-4-phenylbutanoic acid is efficiently used in the synthesis of pharmaceutical drugs like Benazepril, Enalapril, Ramipril, Lisinopril and Quinapril. The other synthesized intermediates (2S)-2-hydroxy-3-(4-hydroxyphenyl)propanoic acid, and (R)-phenyllactic acid is used in the synthesis of saroglitazar, and scopolamine respectively. Pril-suffix-based drugs are widely used as angiotensin-converting enzyme (ACE) inhibitors and are a commonly prescribed class of medications for high blood pressure and other heart-related conditions. Saroglitazar is widely used in the treatment of non-cirrhotic non-alcoholic steatohepatitis and diabetic dyslipidemia, whereas scopolamine is widely used to prevent symptoms of motion sickness, irritable bowel syndrome, and Parkinson's disease. The invention also relates to designed arFDH which showed 3-fold enhancement in FDH activity as a part of the cofactor recycling as compared to previously reported FDHs. The invention addresses the limitation of using a two-enzyme system in an industrial process condition by reducing the number of operational steps and enzymes required. Additionally, the arFDH gene was constructed in the expression plasmid operably linked to a promoter sequence that promotes the production of the recombinant engineered formate dehydrogenase (Fig. 2) which is consecutively expressed in a host cell.
[0014] The present disclosure provides an engineered formate dehydrogenase (FDH) enzyme that are capable of stereoselectivity converting the 2-oxo-acid group to 2-hydroxy-acid group and having an improved property. It is shown in the present disclosure that naturally occurring formate dehydrogenase from organisms doesn’t reduces the 2-oxo-acid group to 2-hydroxy-acid group. Since the wild-type formate dehydrogenases are generally selective for the conversion of formate to carbon dioxide and vice-versa along with the recycling of NAD(P)+ to NAD(P)H. For substrate like phenylpyruvic acid, 2-oxo-4-phenylbutyric acid, 4-hydroxyphenylpyruvic acid and pyruvate, the wild type of formate dehydrogenases, enzymes display insignificant activity towards the 2-oxo-acid group of the substrate. However, the engineered formate dehydrogenase enzyme of the present disclosure, which are derived from the computational method are capable of reducing phenylpyruvic acid, 2-oxo-4-phenylbutyric acid, 4-hydroxyphenylpyruvic acid and pyruvate into (R)-phenyllactic acid, (R)-2-Hydroxy-4-phenylbutyric acid, (2S)-2-hydroxy-3-(4-hydroxyphenyl)propanoic acid, and (S)-lactate respectively. Hence, the formate dehydrogenase described herein are characterized by induced activity as compared to the wild-type formate dehydrogenase for the reduction of 2-oxo-acid groups. These polypeptides of the disclosure are consequently referred to as engineered formate dehydrogenase. The induced activity is based on the mutating the residues at positions X24 is Ala, X79 is Leu, X81 is Thr, X147 is Ser, X314 is Glu, X336 is Ser; X386 is Asp; and X389 is Arg;
BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1: Conversion of 2-oxo-acid group to 2-hydroxy-acid group using an engineered arFDH with a self-cofactor recycling system using formate as a co-substrate. The substrate scope of engineered arFDH, where “R” is namely benzyl, 2-phenylethyl, (4-hydroxyphenyl)methyl and Methyl, which is indicative of the substrate scope of the engineered arFDH for bulky 2-oxo-acid molecules.

[0016] Figure 2: A schematic diagram showing the plasmid construction of arFDH/pET28b(+) where arFDH construct is inserted in pET28b(+) plasmid between NcoI and XhoI restriction site.

[0017] Figure 3: The depicted structures of enzyme-substrate complexes highlight the interaction between the enzyme's active site residues and the substrates. It was observed that the substrate adopts energetically favourable near-attack conformations by forming various interactions with the residues lining the active site pocket. The interactions between the active site residues of the arFDH and substrates are highlighted using dotted lines to depict the nature of interactions. A) Represents the energetically favourable conformation of phenylpyruvic acid to yield (R)-phenyllactic acid, the active site of arFDH where H333 and G124 form conventional hydrogen bonds with the phenylpyruvic acid, and H333 also stabilizes the substrate by forming a p- p stacking interaction. B) Represents the energetically favourable conformation of 2-oxo-4-phenylbutyric acid to yield (2R)-2-hydroxy-4-phenylbutanoic acid, the active site of arFDH where I123 formed Amide-p interaction with the substrate, R285, G336, N147 and H333 are formed conventional hydrogen bonds with the (2R)-2-hydroxy-4-phenylbutanoic acid. C) Represents the energetically favourable conformation of 4-hydroxyphenylpyruvic acid to yield (2S)-2-hydroxy-3-(4-hydroxyphenyl)propanoic acid, active site of arFDH where H333 and R285 formed conventional hydrogen bonds with 4-hydroxyphenylpyruvic acid D) Represents the energetically favourable conformation of Pyruvic acid to yield D-lactic acid, the active site of arFDH enzyme where F99 formed a p-s interaction, P98 formed a p-alkyl interaction, N147 and I123 formed conventional hydrogen bonds, I123 also formed hydrophobic interactions, and R285 stabilized the carboxylic group of the substrate by forming the charge interactions with Pyruvic acid.

[0018] Figure 4: Reaction coordinates for the conversion of 2-oxo-4-phenylbutyric acid into (2R)-2-hydroxy-4-phenylbutanoic acid derived using Quantum Molecular Dynamics (QMD) simulation, depicting (A) The near attack conformation of the substrate within the enzyme's active site was considered as the starting point for QM calculation (Ground state, GS). At this stage, the distance between the oxygen atom of the reactive keto group within the substrate and the hydrogen atom of the catalytic His residue was 1.76 Å. At the same time, the distance separating the NAD(P)H coenzyme from the carbonyl carbon of the reactive keto group was 2.23 Å. These specific atomic distances characterize the stable configuration of the substrate and enzyme complex at the initial stage of the reaction, commonly referred to as the ground state. (B) Transition state 1 (TS1): The reactive keto oxygen of the substrate abstracts the proton from the catalytic His. The distance between the keto oxygen of the substrate and the proton of the His was quantitatively measured to be approximately 1.06 Å. (C) Intermediate state (IS): In this step the carbonyl carbon of the substrate with a partial positive charge attracts the hydride of the NAD(P)H. The distance between the NAD(P)H hydride and the carbonyl carbon was measured to be 2.48 Å. (D) Transition state 2 (TS2): In this transition state the hydride was transferred to the carbonyl carbon and the distance between the carbon and hydride is 1.58 Å. (E) Product (P): the stable product with a proton transfer from the catalytic His and the hydride from NAD(P)H.

[0019] Figure 5: Relative free energy profile (kcal/mol) obtained from the QMD simulations for conversion of (1) phenylpyruvic acid to (R)-phenyllactic acid, (2) 2-oxo-4-phenylbutyric acid to 2-hydroxy-4-phenylbutanoic acid, (3) 4-hydroxyphenylpyruvic acid to (2S)-2-hydroxy-3-(4-hydroxyphenyl) propanoic acid and (4) pyruvate into lactate . Energy required for the carbonyl oxygen of the substrate to abstract the hydrogen from catalytic His was determined to be rate limiting step in the reaction as the energy required to attain TS1 state in all substrates was higher compared to energy required to attain TS2 state. The non-native substrates namely, phenylpyruvic acid, 2-oxo-4-phenylbutyric acid and 4-hydroxyphenylpyruvic acid have demonstrated similar activation energy profiles for the engineered arFDH which indicates feasibility for conversion to their respective products. The active site substitutions and mutations that altered the electrostatics of the active site enabled the conversion of the bulky substrates.

[0020] Figure 6: Schematic presentation of the workflow for the development of engineered artificial Formate dehydrogenase of the present invention.

[0021] Figure 7: The expression of engineered formate dehydrogenase in cell lysate assessed using SDS-PAGE. The bands are given for the screened variants such as SEQ ID NO: 1, 3, 26, 58, 103, 143, 178, 198 and 200.

[0022] Figure 8: The schematic flowchart of STEPS 3-5 in Figure. 6 for deriving the arFDH of the present invention. A) Top 4 base template sequences were derived from curated FDH database. B) potential energy of the residues across different regions of the structures from the top four sequences were calculated and the energies were deposited on the residues. C) A weight matrix was generated based on the information of per residue potential energy values. D) Motif information was derived from each of the top 4 templates T1-T4 (Represented as grey spheres encircled in the structures in (B)). E) An average FDH sequence was derived from the top four FDH sequences and, motifs from the template sequences were iteratively transferred onto the average sequence by an AI/ML based sequence generation algorithm that takes information of the weighted score for each motif, derived from the potential energies of the corresponding residues to derive the artificial formate dehydrogenase sequences as described in SEQ ID NO: 1, 202, 203, 204, 205, 206, 207, 208, 209, 210. F) Computational assessment of generated artificial formate dehydrogenase sequences to derive the arFDH described in SEQ ID NO: 1
BRIEF DESCRIPTION OF THE INVENTION
TERMINOLOGIES EXPLAINED / ABBREVIATIONS
[0023] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein and the laboratory procedure of mutagenesis, cell culture, microbiology, biocatalysis, enzyme and analytical chemistry described below are those well-known and commonly employed in the art. Such techniques and methods are well-known and described in numerous texts and reference works well known to those of skill in the art.
[0024] “Encoding,” “encode” herein refers to transforming coding polynucleotide sequence into polypeptide sequence or protein or enzyme to perform the reactions.
[0025] “Protein,” “polypeptide”, and “peptide” are used interchangeably herein to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification.
[0026] “Amino acids” are referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by IUPAC-IUB biochemical nomenclature commission.
[0027] “Polynucleotide”, “nucleic acid” refers to two or more nucleosides that are covalently linked together. The polynucleotide may be wholly comprised of ribonucleotides (i.e., RNA), wholly comprised of 2’-deoxyribonucleotides (i.e., DNA). While the nucleosides will typically be linked together via standard phosphodiester linkages. The polynucleotide may be single-stranded or double-stranded or may include both single-stranded regions and double-stranded regions. Moreover, while a polynucleotide will typically be composed of the naturally occurring encoding nucleobases (i.e., adenine, guanine, uracil, thymine, and cytosine).
[0028] “Reference Sequence” refers to a defined sequence used as a basis for a sequence comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotide or amino acids residues in length, at least 25 residues in length, at least 50 residues in length, or the full length of the nucleic acid or polypeptide. Since two polynucleotides or polypeptides may each (1) comprise a sequence, (i.e., a portion of the complete sequence), that is similar between the two sequences, and (2) may further comprise a sequence that is divergent between the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity.
[0029] In some embodiments, a “reference sequence” can be based on a primary amino acid sequence, where the reference sequence is a sequence that can have one or more changes in the primary sequence. For instance, a reference sequence “based on SEQ ID NO: 1 having at the residue corresponding to X24 is Ala refers to a reference sequence in which the corresponding residue at X24 in SEQ ID NO:1 has be changed to Ile.
[0030] “Comparison window” refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acids residues wherein a sequence may be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids and wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of two sequences. The comparison window can be longer than 20 contiguous residues, and includes, optionally 30, 40, 50 100, or longer windows.
[0031] “artificial formate dehydrogenase (arFDH)”, “engineered formate dehydrogenase”, herein to denote the mutated polypeptide sequence of formate dehydrogenase as claimed in this invention.
[0032] “Recombinant” when used with reference to e.g., a cell, nucleic acid, or polypeptide, refers to a material, or a material corresponding to the natural or native form of the material, that has been modified in a manner that would not otherwise exist in nature, or is identical thereto but produced or derived from synthetic materials and/or by manipulation using recombinant techniques. Non-limiting examples include, among other, recombinant cells expressing genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise expressed at a different level.
[0033] “Recombinant engineered formate dehydrogenase”, herein refers to denote the engineered formate dehydrogenase derived using the method mentioned in the embodiment. The engineered formate dehydrogenase variants with the mutations are incorporated in a vector system and transformed using a heterologous system for expression and the conversion of the substrates and co-substrates mentioned in the embodiment. These contain the plurality of all the engineered sequences mentioned in the embodiment.
[0034] “Partial de novo”, herein to denote an approach to introduce non-canonical substitution at a given hotspots to improve vdw based interactions.
[0035] “phylogenetic based substitutions” herein to denote to introduce a canonical substitution at a given hotspots to improve catalytic efficiency for the substrates.
[0036] “Biocatalytic processes” herein also known as enzymatic processes, refer to chemical reactions or transformations that are catalysed by enzymes from biological sources or whole cells.
[0037] “Stereoselectivity” herein refers to the preferential formation in a chemical or enzymatic reaction of one stereoisomer over another. Stereoselectivity can be partial, where the formation of one stereoisomer is favoured over the other, or it may be complete where only one stereoisomer is formed. When the stereoisomer are enantiomers, the stereoselectivity is referred to as enantioselectivity, the fraction (typically reported as a percentage) of one enantiomer in the sum of both. It is commonly alternatively, reported in the art (typically as a percentage) as the enantiomeric excess (e.e) calculated therefrom according to the formula [major enantiomer -minor enantiomer]/[major enantiomer + minor enantiomer].
[0038] “Site-directed mutagenesis” herein refers to a molecular biology technique to make specific nucleotide mutations to the DNA sequence at desired locations to investigate the functional significance of particular amino acids in a protein.
[0039] "Stoichiometric amounts" herein refers to the specific quantities of reactants that must be present in precise proportions to enable the progression of the biocatalytic reaction.
[0040] “Percentage of sequence identity” herein to refer to comparison among polynucleotides or polypeptides and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise addition or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. Those of skill in the art uses established algorithms available to align two sequences. Optimal alignment of sequences for comparison can be conducted with algorithms like Smith and Waterman (Smith et. al., 1981), Needleman and Wunsch (Needleman et. al., 1970) and Pearson and Lipman (Pearson et. al., 1988).
[0041] An “expression vector” herein refers to an expression plasmid or expression construct, a type of DNA molecule used in molecular biology and genetic engineering to produce a specific protein of interest in a host organism, typically a bacterium, yeast, or mammalian cell.
[0042] “Cofactor recycling” In this context, cofactor recycling refers to the process of recycling or regenerating and reusing cofactors in enzymatic reactions. Cofactors recycling is an important process as they are expensive and uneasy processes to produce in large quantities.
[0043] “Curated FDH sequences” in this context refers to a manually reviewed collection of protein sequences related to the Formate dehydrogenase, with an annotation of sequence variation, functional sites, and structural information’s.
[0044] “Acidic, Basic, Polar, and Non-Polar amino acids”
[0045] L-Glu (E) and L-Asp (D) are among the acidic amino acids or residues. L-Arg (R) and L-Lys (K) are among the basic amino acids or residues. L-Asn (N), L-Gln (Q), L-Ser (S), and L-Thr (T) are among the polar amino acids or residues. L-Gly (G), L-Leu (L), L-Val (V), L (A) are non-polar amino acids or residues.
[0046] “Hydrophilic, hydrophobic, aromatic, aliphatic amino acids”
[0047] hydrophilic amino acids or residues include L-Thr (T), L-Ser (S), L-His (H), L-Glu (E), L-Asn (N), L-Gln (Q), L-Asp (D), L-Lys (K) and L-Arg (R), hydrophobic amino acids or residues include L-Pro (P), L-Ile (I), L-Phe (F), L-Val (V), L-Leu (L), L-Trp (W), L-Met (M), L-Ala (A) and L-Tyr (Y), aromatic amino acids or residues include L-Phe (F), L-Tyr (Y) and L-Trp (W) and aliphatic amino acids or residues include L-Ala (A), L-Val (V), L-Leu (L) and L-Ile (I). Although owing to the pKa of its heteroaromatic nitrogen atom L-His (H) it is sometimes classified as a basic residue, or as an aromatic residue as its side chain includes a heteroaromatic ring.
[0048] “Amino acid difference or residue difference” refers to a change in the residue at a specified position of a polypeptide sequence when compared to a reference sequence. For example, a residue difference at position X36, where the reference sequence has an Isoleucine, refers to a change of the residue at position X62 to any residue other than Isoleucine. As disclosed herein, an enzyme can include one or more residue differences relative to a reference sequence, where multiple residue differences typically are indicated by a list of the specified positions where changes are made relative to the reference sequence.
[0049] “Improved enzyme property” herein refers to a ketoreductase polypeptide that exhibits an improvement in any enzyme property as compared to a arFDH. For the engineered formate dehydrogenase polypeptides described herein, the comparison is generally made to the arFDH enzyme. Enzyme properties for which improvements are desirable include but are not limited to, enzymatic activity (which can be expressed in terms of percent conversion of the substrate), thermal stability, pH activity profile, cofactor requirements, refractoriness to inhibitors (e.g., product inhibition), stereospecificity, stereoselectivity, higher substrate load tolerance, organic solvent tolerance and improved substrate scope.
[0050] “Derived from” as used herein in the context of engineered formate dehydrogenase enzymes, identifies the artificial formate dehydrogenase derived computationally, upon which the engineering was based. For example, the engineered formate dehydrogenase enzyme of SEQ ID NO: 3 was obtained by artificially evolving, over multiple generations the gene encoding the Artificial formate dehydrogenase (arFDH) enzyme of SEQ ID NO: 1. Thus, this engineered formate dehydrogenase enzyme is “derived from” the arFDH of SEQ ID NO: 1.
[0051] “Near Attack Conformation” refers to the specific arrangement of the substrate in the enzyme active site which promotes the catalysis. The near-attack conformation reflects the geometry that allows the substrate to react efficiently with the enzyme, facilitating the transformation into the product.
[0052] “Increased enzymatic activity, “improved co-factor recycling ability” refers to an improved property of the engineered formate dehydrogenase polypeptides, which can be represented by an increase in activity, or increase in percentage conversion of the substrate to the product or increase in residual activity as compared to the reference formate dehydrogenase enzyme.
[0053] “Conversion” refers to the enzymatic conversion of the substrate to corresponding product. “Percentage conversion” refers to the percent of the substrate that is converted to the product within a period under specified conditions.
[0054] “Residual activity” activity refers to the remaining enzymatic activity after subjecting the enzyme to specific conditions that might denature the enzyme. Here, specific conditions refer to the change in pH and temperature. For example, 100% indicating no loss of activity and lower percentage represents reduced activity due to change in conditions.
[0055] “pH stable” refers to a formate dehydrogenase polypeptide that maintains similar activity (60 to 80%) after exposure to broad range of pH (5-9) for a period (0.5 -24 hour) compared to the wild-type enzyme exposed to the same range of pH (5-9).
[0056] “Thermostable” refers to a formate dehydrogenase polypeptide that maintains similar activity (60% to 80%) after exposure to elevated temperatures (40-65°C) for a period (0.5 -24 hour) compared to the wild -type enzyme exposed to the same elevated temperature.
[0057] “Codon optimized” refers to changes in the codons of the polynucleotide encoding a protein to those preferentially used in a particular organism such that the encoded protein is efficiently expressed in the organism of the interest.
[0058] “Operably linked” is defined herein as a configuration in which a control sequence is appropriately placed (i.e., in a functional relationship) at a position relative to a polynucleotide of interest such that the control sequence directs or regulates the expression of the polynucleotide and/or polypeptide of interest.
[0059] “Promoter sequence” refers to a nucleic acid sequence that is recognized by a host cell for expression of polynucleotide of interest.
[0060] “Substrate” in the context of a biocatalyst mediated process refers to the compound or molecule acted on by the biocatalyst. For example, a formate dehydrogenase biocatalyst used in the process disclosed herein there is a formic acid (HCOOH or Sodium formate (HCOONa), or Ammonium formate (NH4HCOO).
[0061] “Co-substrate” in the context of a engineered formate dehydrogenase is a small molecule that temporarily binds to an enzyme during a reaction, assisting in the conversion of substrates. Here the co-substrates are formate or their corresponding salts Sodium formate (HCOONa) or Ammonium formate (NH4HCOO) or formic acid (HCOOH).
[0062] “Product” in the context of a biocatalyst mediated process refers to the compound or molecule resulting from the action of the biocatalyst. For example, an exemplary product for a formate dehydrogenase biocatalyst used in a process disclosed herein is a carbon dioxide.
[0063] “Suitable reaction conditions” refers to those conditions in the biocatalytic reaction solution (e.g., ranges of enzyme loading, substrate loading, co-factor loading, temperature, pH, buffers, co-solvent etc.,) under which a formate dehydrogenase polypeptide of the present invention can convert one or more substrate to a product. For example, conversion of formic acid to carbon dioxide. Exemplary “suitable reaction conditions” are provided in the present invention and illustrated by the examples.
[0064] “Biotransformation” refers to the chemical reactions that are catalysed by microorganisms in terms of growing or resting cells or that are catalysed by isolated enzymes. It has high stereo- or regioselectivity combined with high product purity and high enantiomeric excesses, biotransformation’s can be technically superior to traditional chemical synthesis.
[0065] “Promoter” refers to a specific region of DNA upstream of a gene where relevant proteins (such as RNA polymerase and transcription factors) bind to initiate transcription of that gene. The resulting transcription produces an RNA molecule (such as mRNA).
[0066] “Substitution” refers to replacing native amino acids residue in a polypeptide sequence with desired amino acid for a specific characteristic.
[0067] “Multiple sequence alignment (MSA)” methods refer to identify the evolutionary relationship and common patterns between proteins. Precisely, it refers to the sequence alignment of three or more biological sequences usually DNA, RNA, or protein.
[0068] "Inducible promoters" in this context refers to a regulated promoter which becomes on or active in the cell in response to specific stimuli.
[0069] “Expression vector” in this context refers to a plasmid containing the gene of interests.
[0070] The present invention provides an engineered arFDH capable of converting bulky substrates having 2-oxo-acid group to product containing 2-hydroxy-acid group, specifically converting phenylpyruvic acid, 2-oxo-4-phenylbutyric acid, 4-hydroxyphenylpyruvic acid and pyruvate to (R)-phenyllactic acid, 2-hydroxy-4-phenylbutanoic acid, (2S)-2-hydroxy-3-(4-hydroxyphenyl)propanoic acid and lactate, respectively with an improved enzyme property.
[0071] The present invention details engineered arFDH polypeptides that show 90% sequence identity to SEQ ID No. 1 and contains the features of X24 is Ala, X79 is Leu, X81 is Thr, X147 is Ser, X314 is Glu, X336 is Ser; X386 is Asp; and X389 is Arg;
[0072] In some embodiments, the engineered arFDH polypeptides also contains one or more of the following residue differences when compared to the polypeptide of SEQ ID No. 1: X5 is Leu or Ala or Ile; X13 is Val or Glu or Thr; X16 is Tyr or Met or Gln; X19 is Thr or Lys or Ala; X21 is Cys or Pro or Ile; X27 is Lys or Thr or Gln; X28 is Ile, Val or Pro; X29 is Asp or Glu or Lys; X30 is His or Ala; X33 is Ser or Asp; X36 is Ile or Ala or Ser; X37 is Leu, Ala or Val; X38 is Cys; X49 is Gln or Thr or Ala; X62 is Glu or Pro or Arg; X67 is Asn or Gln or Leu; X72 is Lys or Ile or Ala; X84 is Phe; X93 is Lys or Ile; X94 is Val, Ala or Leu; X106 is Pro or Ala; X131 is Glu or Asp; X132 is Ser or Thr; X134 is Lys or Met or Asn; X138 is Val or Met or Leu; X139 is Arg or Ile; X149 is Asp or Asn or His; X161 is Ser, Thr or Ala; X167 is Leu or Val or Thr; X171 is Glu or Gln or Asn; X173 is Ala or Ile; X174 is Arg; X186 is His or Gly or Gln; X190 is Leu, Val or Ala; X194 is Asp or Gln or Asn; X212 is Ala, Gly or Val; X216 is Val or Ala; X217 is Arg or Lys; X219 is Arg or Lys; X228 is Glu or Asp or Leu; X229 is Ser or Ala or Asp; X232 is Lys or Arg or Leu; X235 is Asn or His or Asp; X236 is Lys or Val or Ala; X239 is Glu or Asp; X240 is Asp or Pro or Thr; X270 is Ser or Met or Leu; X302 is Arg or Gln or His; X318 is Lys or Ala or Gln; X320 is Glu or Asp; X347 is Cys; X357 is Glu or Leu; X370 is Asp or Glu or Ser; X375 is Arg or Val; X385 is Glu or Asn or Lys; X388 is Lys or Arg or Gly; X389 is Arg; X395 is Leu or Glu or Ser; X396 is Lys or Arg or His; X397 is Phe; X398 is Lys or Arg or Glu;
[0073] Additionally, the active site of arFDH is mutated at one or more positions to accommodate phenylpyruvic acid and the mutations are X98 is Gly; X99 is Gly; X100 is Ala; X123 is Val, X126 is Glu or Ala or Gly; X142 is Gln or Asn; X345 is His or Ala or Gly or Phe or Thr; X337 is Ser or Asn or Ala or Gly; X335 is Ala or Gly; and X310 is Ala or Gly.
[0074] Furthermore, the active site of arFDH is mutated at one or more positions to accommodate 2-oxo-4-phenylbutyric acid and 4-hydroxyphenylpyruvic acid are X106 is Gly; X140 is Asn or Gln, X345 is Ala; X98 is Gly; X99 is Gly; X100 is Ala; X283 is Ser or Asn or Ala or Gly or Val; X284 is Gly or Val or Ala or Leu; X382 is Ile or Phe or Gln; X122 is Ala or Ser; X121 is Gly; X124 is Ala or Ser; and X127 is Val or Ala or Leu.
[0075] In some embodiment, the engineered arFDH polypeptide also contains one or more of the following residue differences for having the cofactor specificity of NAD+ and NADP+ to recycle into NADH and NADPH respectively, when compared to the polypeptide of SEQ ID No. 1: X224 is Arg; X223 is Asn; and X380 is Gln;
[0076] In some embodiments, the engineered formate dehydrogenase is capable of converting 2-oxo-acid of the structural formula (II) to corresponding to 2-hydroxy-acid of structural formula (I): having the indicated stereochemical configuration at the stereogenic center marked with an *;

[0077] wherein R is benzyl, 2-phenylethyl, (4-hydroxyphenyl)methyl or Methyl, having an enantiomeric excess of at least 90% over the opposite enantiomer.

[0078] In some embodiments, the engineered formate dehydrogenase that comprises an amino acid sequence that has a substitution or a plurality of amino acids substitutions, as described previously, with respect to the amino acid sequence shown in SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 and having catalytic activity for the conversion of bulky substrates having 2-oxo-acid group to 2-hydroxy-acid group under suitable reaction conditions such as 300 mM substrate, in 0.1 M phosphate buffer (pH 7.0) containing sodium formate as a co-substrate.
[0079] In addition to its function as reductase, the enzyme was engineered to enhance its ability to recycle the cofactors NADP+ to NADPH or NAD+ to NADH.
[0080] In some embodiments, the engineered formate dehydrogenase is expressed using pET28a(+) vector and transformed using CaCl2 into a competent Escherichia coli BL21 (DE3) cells. The cultures were grown in agitation at 37 °C. A 100-ml over-night pre-culture of transformed cells were inoculated in 2 L of LB medium, 50 µg/ml kanamycin. Once the culture had reached OD600 = 1, protein expression was induced by 0.1 mM isopropyl ß-d-1-thiogalactopyranoside (IPTG) and cells were further grown at 18 °C for 20 h. The cells were harvested by centrifugation at 6000g for 10 min.
[0081] In some embodiments, the engineered formate dehydrogenase is purified using Ni-NTA column, where the pellet from the centrifuged process was washed twice in the washing buffer (20 mM phosphate buffer, 0.5 M NaCl pH 7.4) and resuspended in 30 ml of the same buffer. Cells were lysed using a French press set at 1.35 KPa; debris were removed by centrifugation at 20,000g for 20 min and the supernatant was loaded onto a 10-ml Ni-NTA column, pre-equilibrated with the washing buffer. Elution was performed in the elution buffer (20 mM phosphate buffer, 0.5 M NaCl, 0.250 M imidazole pH 7.4) and 1-ml fractions were harvested. Fractions with Abs280 higher than 0.4 were combined and loaded on a pre-equilibrated gel filtration Superdex 200 column.
[0082] In some embodiments, the mutation of formate dehydrogenase is carried out using the QuikChange site-directed mutagenesis kit according to the manufacturer’s instructions. A pET-28a(+)-arFDH vector generated was used as template DNA from the first rounds of mutagenic PCR. The mutated gene is cloned between NcoI and XhoI restriction sites. The mutations were combined by consecutive rounds of site-directed mutagenesis and the incorporation of mutations were confirmed by sequencing.
[0083] In some embodiment, the screening of the mutant library was done with the colonies which were randomly picked into the primary 96-deep well plates containing LB Medium (300uL) with ampicillin (50µg/mL) per well. After incubated at 37°C , 800rpm overnight, the preculture (20µL) was transferred from the primary plate to the secondary 96-deep well plate containing LB medium (430µL) with ampicillin (50µg/mL) and was further incubated at 37°C, 800 rpm for 2 to 3 h, then the isopropyl-ß-D-thiogalactopyranoside (IPTG) was added to the culture plates with the final concentration of 0.2 mM to induce protein expression at 16°C for 24 h. Next, the cells were harvested by centrifugation at 2500 x g, 4°C for 15 min and then were resuspended in 200 µL lysis buffer (lysozyme, 750 mg/L, DNase,10 mg/L, potassium phosphate, 100 mM, pH 7.5), the suspension was further incubated at 30°C, 800 rpm for 2 h. A clear cell-free extract was obtained by centrifugation at 2500 x g, 4°C for 10 min. An aliquot (100 µL) of the supernatant was used to evaluate the mutant’s reactivity with NAD(P)+ by mixing with 100 µL of screening buffer (sodium formate, 0.2 M; NAD(P)+, 2 mM; potassium phosphate, 100 mM, pH 7.5). The concentration of the NAD(P)+ in the screening buffer and the addition amount of cell-free extract was stepwise decreased to 0.5 mM and 20 µL with the increased activity of the engineered FDH variants, respectively. The NAD(P)+ reduction was monitored at 340 nm, 30°C for 5 min. Positive hits with higher 340 nm absorbance change were sequenced, cultured by shake flask fermentation, and subjected to Ni-affinity chromatography for further characterization.
[0084] Furthermore, a method for producing arFDH gene comprises preparing a host organism E. coli BL21 (DE3) and introducing a plasmid pET28b(+), in which arFDH gene is arranged under the control of inducible promoter (Fig. 2). Followed by culturing the host, inducing the expression of the arFDH gene after the logarithmic growth phase, culturing the host at a temperature that is lower than the optimum temperature for the growth of the host cells and allows the survival of the host, and thus causing the expression of the arFDH within the host.
[0085] In some embodiment, the activity assay for the conversion of substrate Phenylpyruvic acid to (R)-phenyllactic acid was performed in 0.1 M phosphate buffer (pH 7.0) containing 300 mM phenylpyruvic acid, 0.1mM NAD(P)+ sodium formate as a co-substrate (1.5 eq. of substrate), at 37°C. By altering the quantities of one substrate at a fixed and saturating concentration of the second, kinetic constants were determined from duplicate or triple measurements of starting rates.
[0086] In some embodiment, the activity assay for the conversion of substrate 2-oxo-4-phenylbutyric acid to (R)-2-Hydroxy-4-phenylbutyric acid was performed in 0.1 M phosphate buffer (pH 7.0) containing 300 mM 2-oxo-4-phenylbutyric acid, 0.1mM NAD(P)+ sodium formate as a co-substrate (1.5 eq. of substrate), at 37°C. By altering the quantities of one substrate at a fixed and saturating concentration of the second, kinetic constants were determined from duplicate or triple measurements of starting rates.
[0087] In some embodiment, the activity assay for the conversion of substrate 4- hydroxyphenylpyruvic acid to (2S)-2-hydroxy-3-(4-hydroxyphenyl)propanoic acid was performed in 0.1 M phosphate buffer (pH 7.0) containing 300 mM 4-hydroxyphenylpyruvic acid, 0.1mM NAD(P)+ sodium formate as a co-substrate (1.5 eq. of substrate), at 37°C. By altering the quantities of one substrate at a fixed and saturating concentration of the second, kinetic constants were determined from duplicate or triple measurements of starting rates.
[0088] In some embodiment, the activity assay for the conversion of substrate pyruvate to (S)-lactate was performed in 0.1 M phosphate buffer (pH 7.0) containing 300 mM 4-hydroxyphenylpyruvic acid, 0.1mM NAD(P)+ sodium formate as a co-substrate (1.5 eq. of substrate), at 37°C. By altering the quantities of one substrate at a fixed and saturating concentration of the second, kinetic constants were determined from duplicate or triple measurements of starting rates.
[0089] In some embodiment, the co-factor recycling activity assay for the conversion of Formate to carbon dioxide was performed with the engineered formate dehydrogenase (arFDH). Where the activity was measured in a 1.4-mL quartz cuvette containing 1 mL of the assay mixture at 30°C by monitoring the change of absorbance at 340 nm within 1 min on a UV-spectrometer. The reaction mixture (1 mL) contained potassium phosphate buffer (100mM, pH 7.5), sodium formate (500mM), NAD(P)+ (0.5mM), and appropriately diluted purified formate dehydrogenase variants. One unit of enzyme activity was defined as the amount of FDH required for the formation of 1µM NAD(P)H in 1 min. The concentration of sodium formate for Km determination were varied from 10mM to 800mM in a potassium phosphate buffer (100 mM, pH 7.5) containing 1mM NAD(P)+. Similarly, the concentrations of NAD(P)+ for the measurement of Km were varied from 0.1 mM to 10 mM in potassium phosphate buffer (100 mM, pH 7.5) containing 500 mM sodium formate. The reaction mixture was incubated at 30°C, then monitored the increase of the absorbance at 340 nm.
[0090] In some embodiment, the biocatalytic co-factor recycling using engineered formate dehydrogenase for NAD(P)H regeneration in combination with Ketoreductase (KRED) was carried out in 2-mL Eppendorf tubes containing 1 mL reaction mixture and agitated at 30°C for 24 hr. The reaction mixture contained 100 mM acetophenone, 5% (v/v) ethanol, 100 Units of KRED, 2.4 units of engineered formate dehydrogenase, potassium phosphate buffer (100mM, pH 6.0) and 150mM sodium formate. After 24 hours of incubation, the reaction mixtures were extracted with 1mL EtOAc, the organic layer was dried over anhydrous Na2SO4 analysed by chiral GC.
[0091] In some embodiment, the biocatalytic co-factor recycling using engineered formate dehydrogenase for NAD(P)H regeneration in combination with Imine reductase (IRED) was carried out in 2-mL Eppendorf tubes containing 1 mL reaction mixture and agitated at 30°C for 24 hr. The reaction mixture contained 100mM of piperideine, 0.1 mM NAD(P)+, 15% (v/v) DMSO, 2U IRED, 1.8 U engineered formate dehydrogenase, potassium phosphate buffer (100mM, pH 6.0) and 150mM sodium formate. After 24 hours of incubation, the reaction mixtures were extracted with 1mL EtOAc, the organic layer was dried over anhydrous Na2SO4 analysed by chiral GC.
[0092] In some embodiment, the biocatalytic co-factor recycling using engineered formate dehydrogenase for NAD(P)H regeneration in combination with Baeyer-Villiger Monoxygenase (BVMO) was carried out in 2-mL Eppendorf tubes containing 1 mL reaction mixture and agitated at 30°C for 24 hr. The reaction mixture contained 50mM cyclohexanone, 0.1 mM NAD(P)+, 2% (v/v) Ethanol, 1.3 U BVMO enzyme, 1.3 U engineered formate dehydrogenase, potassium phosphate buffer (100 mM, pH 7.0), 75mM sodium formate. After 24 hours of incubation, the reaction mixtures were extracted with 1mL EtOAc, the organic layer was dried over anhydrous Na2SO4 analysed by chiral GC.
[0093] In some embodiment, the biocatalytic co-factor recycling using engineered formate dehydrogenase for NAD(P)H regeneration in combination with Leucine Dehydrogenase (LeuDH) was carried out in 10mL scale in 50 mL flask employing LeuDH enzyme. Th reaction mixture contains 500 mM NH4Cl-NH3.H2O buffer (pH 9.5), 100 mM trimethylpyruvic acid (TMP), 200 mM sodium formate and 0.1 g of LeuDH enzyme and engineered formate dehydrogenase enzyme. The reaction mixture was incubated at 30°C and 200 rpm for 35h, and 100µL samples were periodically removed for high-performance liquid chromatography (HPLC) analysis.
[0094] In some embodiment, the biocatalytic co-factor recycling using engineered formate dehydrogenase for NAD(P)H regeneration in combination with Amine Dehydrogenase (AmDH) was carried out by adding 20µL of ketone (Cyclohexanone) to 580 µL ethyl acetate. The methylamine-HCl and cyclohexanone stock solutions were made up in water and DMSO respectively. DMSO concentrations was kept to a constant final concentration of 1% in the total reaction mixtures. The reactions were incubated at 25°C with shaking at 150 r.p.m. Reaction conditions where ammonia was the amine donor contained the following in a 3 mL total reaction volume: 10mM cyclohexanone, 12 mM ammonium formate, 3 U/mL engineered formate dehydrogenase, 0.5 mM NAD(P)H with 1mg/mL of AmDH, made up to 3 mL total volume with 2M ammonium formate buffer pH 8.0. Aliquots of 200 µL were taken every 1 h between t=0-8h and then t=24h, with t=0 time points being taken directly after the addition of enzyme. Aliquots were quenched with 20 µL of 10 M NaOH and then extracted with 600 µL ethyl acetate after which the organic layer was dried using MgSO4 and then analysed using GC.
[0095] In some embodiment, the biocatalytic co-factor recycling using engineered formate dehydrogenase for NAD(P)H regeneration in combination with Ene-Reductase (ERED) was carried out using ene-reductase and engineered formate dehydrogenase present in a conical tube together with 700µL of Tris-HCl buffer (50mM, pH 7.5) and 2-cyclohexene-1-one stock solution (100mM, 150µL). The reaction was started by adding 150µL of NAD(P)H stock solution (6.0 mM) and carried out at 40°C in a water bath with shaking. After centrifugation, 100µL samples of the supernatant were removed from the mixture and added in 900 µL of Tris-HCl buffer (50 mM, pH 7.5) to perform the activity assay. The activity was measured by high-performance liquid chromatograph (HPLC) with 100µL samples were periodically removed for analysis.
[0096] In some embodiment, the biocatalytic co-factor recycling using engineered formate dehydrogenase for NAD(P)H regeneration in combination with Alcohol Dehydrogenase (ADH) was carried out in 2-mL Eppendorf tubes containing 1 mL reaction mixture and agitated at 35°C for 12 hr. The reaction mixture contained 100 mM acetophenone, 5% (v/v) ethanol, 50 Units of ADH, 2.0 units of engineered formate dehydrogenase, potassium phosphate buffer (100mM, pH 7.0) and 150mM sodium formate. After 24 hours of incubation, the reaction mixtures were extracted with 1mL EtOAc, the organic layer was dried over anhydrous Na2SO4 analysed by chiral GC.
[0097] In some embodiment, the biocatalytic co-factor recycling using engineered formate dehydrogenase for NAD(P)H regeneration in combination with Glucose Dehydrogenase (GDH) was carried out in in 2-mL Eppendorf tubes containing 1 mL reaction mixture and agitated at 30°C for 24 hr. The reaction mixture contained 100 mM acetophenone, 5% (v/v) ethanol, 50 Units of GDH, 2.5 units of engineered formate dehydrogenase, potassium phosphate buffer (100mM, pH 7.0) and 150mM sodium formate. After 24 hours of incubation, the reaction mixtures were extracted with 1mL EtOAc, the organic layer was dried over anhydrous Na2SO4 analysed by chiral GC.
[0098] In some embodiment, the engineered formate dehydrogenase is immobilized using CLEA method, where the engineered formate dehydrogenase (50 U) was mixed in Tris-HCl buffer (50mM, pH 7.5, 1.5mL) and then BSA (20 mg) was added in the mixture under stirring. The ammonium sulphate stock solution (5.3 M) was prepared by dissolving solid ammonium sulphate in Tris-HCl buffer (50mM, pH 7.5), and then the stock solution was added in above enzyme solution drop by drop to reach final ammonium sulphate concentration of 4.0 M. After keeping the mixture for 60 min at 4 °C for complete precipitation of enzymes, glutaraldehyde stock solution (4.7 M) or oxidized dextran stock solution (0.4mM) was added to a final concentration of 15% (v/v). The mixture was kept at 4°C for 3h with shaking. The immobilized FDH were collected by centrifugation at 10,614 x g for 5 min and washed three times with Tris-HCl buffer (50 mM, pH 7.5). The resulting FDH-CLEAs were stored at 4°C for further use.
[0099] In some embodiment, the engineered formate dehydrogenase is immobilized using Biomimetic immobilization (BI) method, where the engineered FDH (50 U) in a sodium phosphate buffer (50mM, pH 7.0, 1.8 mL) was mixed with 628 µL of a hydrolysed tetramethyl orthosilicate (TMOS) solution. TMOS was hydrolysed with hydrochloric acid (HCl, 1 mM) to a final concentration of 1.0 M. The mixture was agitated for 3 h at 4 °C. The immobilized engineered FDH was collected by centrifuging at 6,793 x g for 5 min and washed three times with a sodium phosphate buffer (50 mM, pH 7.0). The resulting immobilized engineered FDH were used in the subsequent experiments. Control experiments, wherein silica was prepared without adding the enzymes, were then performed.
[0100] In some embodiment, the engineered formate dehydrogenase is co-immobilized with KRED by Biomimetic immobilization (BI) method, where engineered formate dehydrogenase is used as cofactor recycling enzyme for conversion of NAD(P)+ to NAD(P)H. The engineered formate dehydrogenase (50U) and KRED (14 U) in a sodium phosphate buffer (50mM, pH 7.0, 1.8 mL) was mixed with 628 µL of a hydrolyzed tetramethyl orthosilicate (TMOS) solution. TMOS was hydrolyzed with hydrochloric acid (HCl, 1 mM) to a final concentration of 1.0 M. The mixture was agitated for 3 h at 4 °C. The co-immobilized KRED and FDH were collected by centrifuging at 6,793 x g for 5 min and washed three times with a sodium phosphate buffer (50 mM, pH 7.0). The resulting KRED-FDH were used in the subsequent experiments. Control experiments, wherein silica was prepared without adding the enzymes, were then performed.
[0101] In some embodiment, the engineered formate dehydrogenase is immobilized by Calcium Alginate, where the immobilization of engineered formate dehydrogenase was prepared by dissolving 9 g of sodium alginate in 300 ml of growth medium, and stirring until complete dissolution of the sodium alginate was achieved. The resulting solution contained 3% alginate by weight. To prevent premature gel formation, the phosphate concentration in the medium was adjusted to less than 100µM. Approximately 250 g of wet cells were thoroughly suspended in the alginate solution, and the mixture was allowed to stand to facilitate the escape of air bubbles. The yeast-alginate mixture was then dripped from a height of 20 cm into 1000 ml of crosslinking solution. (The crosslinking solution was prepared by adding an additional 0.05M of CaCl2 to the growth media. The calcium crosslinking solution was agitated using a magnetic stirrer. Gel formation occurred at room temperature upon direct contact between the sodium alginate drops and the calcium solution. Relatively small alginate beads were preferred to minimize mass transfer resistance. A diameter of 0.5-2 mm was readily achieved using a syringe and needle. The beads were fully hardened within 1-2 hours. The concentration of CaCl2 was approximately one-fourth of the strength used for enzyme immobilization, and the beads were subsequently washed with fresh calcium crosslinking solution and stored in 4°C for further use.
[0102] In some embodiment, the engineered formate dehydrogenase is immobilized by Chitosan beads, where immobilization of engineered formate dehydrogenase is prepared by hydrogel chitosan beads (HGBs), 20 g of chitosan was dissolved in 1 L of a mixed acid solution containing 2% (w/v) of acetic acid, 1% (w/v) of lactic acid, and 1% (w/v) of citric acid. The resulting chitosan solution was introduced dropwise into 0.8 N NaOH by a peristaltic pump. The resultant HGBs were washed with deionized water until pH became neutral. dried chitosan beads (DBs) were prepared by drying the HGBs obtained above at 60 °C for 24 h. For core–shell chitosan beads (CSB) preparation, DBs were soaked in a 0.5% (w/v) acetic acid solution for 60 s and then neutralized by the addition of an equal molar amount of NaOH. To attain high efficiency of engineered formate dehydrogenase biocatalyst, the immobilization conditions, such as pH and glutaraldehyde and engineered formate dehydrogenase concentrations, were optimized. HGBs and CSBs were activated with 0.05–8.0% (w/v) glutaraldehyde in a buffer with a pH range of 5.0–8.0 (acetate buffer pH 5.0–6.0; phosphate buffer pH 7.0–8.0) at 4 °C for 24 h. After that, the activated chitosan beads were incubated with an engineered formate dehydrogenase solution (25–800 U per g of beads; or 0.97–30.3 mg per g of beads) at 4 °C for 24 h with mild agitation. The immobilized engineered formate dehydrogenase enzymes were washed with 1 M NaCl to remove an electrostatically adsorbed enzyme and then were washed 3 times with 50 mM acetate buffer pH 5.5. The resultant immobilized engineered formate dehydrogenase enzyme was kept at 4 °C for further enzyme reactions.
[0103] In some embodiment, the engineered formate dehydrogenase is immobilized by Polymeric matrix, where the immobilization performed using the Dilbeads and Polyethyleneimine (PEI). The Dilbeads (10g) were washed with methanol and treated with a solution of Polyethyleneimine (PEI) (mol. wt. 70000, 100 g/L water; 100 mL) overnight. The supernatant was decanted, and the polymer was washed with distilled water till the pH of washings reached neutrality. The polymer was dried at 60°C in oven. This polymer was further used for engineered formate dehydrogenase immobilization. The PEI modified polymer (10g) was mixed with engineered formate dehydrogenase enzyme solution (100 mL), pH of the medium was adjusted to 7.0 and shaken overnight at room temperature at 150 rpm on an orbital shaker. The supernatant was collected, and the polymer was washed with deionized water (3x 100 mL). The engineered formate dehydrogenase enzyme bound polymer was further crosslinked with dextran aldehyde. The dextran aldehyde was prepared by dissolving dextran (1.7 g) in deionized water (50 mL). Sodium periodate (3.5 g) was added while stirring. After 3h, the oxidised dextran was dialyzed in cellulose acetate membrane tube (mol. wt. cutoff 10,000) against distilled water at 4°C for 24 hr. The dialyzed solution (100 mL). The dextran aldehyde solution (10 mL) was diluted with distilled water to 100 mL and the polymer beads bearing engineered formate dehydrogenase were added. The contents were shaken at room temperature overnight at 150 rpm. The polymer was then washed with deionized water (3 x 100 mL), air dried and stored in 4 °C for further enzymatic reactions.
[0104] In some embodiment, the engineered formate dehydrogenase is immobilized by mesoporous silica, where the immobilization of engineered formate dehydrogenase was performed in aqueous solutions. Where the Mesoporous silica (MPS) particles were prepared by dispersing 5 mg of dry mesoporous silica particles in 1ml phosphate-citrate buffer, using vortexing for 10 min at 10 rpm followed by sonication (at a power of 70 W) for 20 min in order to dissolve any particle aggregates, and a final step of vortexing for 5min. Engineered formate dehydrogenase-particle samples were prepared by mixing 20µl of engineered formate dehydrogenase solution (20mg/ml in phosphate-citrate buffer) with 200 µl of MPS solutions (5mg/ml) diluted to a final volume of 500 µl with phosphate-citrate buffer. Each sample contained a total amount of 400 µg enzyme/mg MPS. Reference samples of free Engineered formate dehydrogenase were prepared in the same way but replacing the 200 µl MPS solution with buffer. In the latter case the final protein concentration 0.8mg/ml corresponds to a volume fraction of about 0.1%, whereas the accumulation in the particle pores leads to much higher local volume fractions of 20-60%. The samples were incubated at 25ºC for 48 h during gentle stirring, and then centrifuged for 6 min. The pelleted protein-particles complexes were re-suspended and washed three times with 500 µl of phosphate citrate buffer by repeated centrifugation and resuspension. The purified MPS particles with the immobilized engineered formate dehydrogenase were finally re-suspended by adding 100 µl of buffer and vortexing for a few minutes until homogenous samples were obtained for the spectroscopic measurements and further conduction of the reaction assay.
[0105] In some embodiment, the engineered formate dehydrogenase is immobilized by Zeolite, where the immobilization of engineered formate dehydrogenase is performed using ZSM-5 zeolites catalysts as follows. First, 100 mg of engineered formate dehydrogenase preparation was dissolved in 10 ml of acetate buffer solution (pH 5, 20 mM), and then 0.5 g of ZSM-5 zeolite was added. The mixed dispersion was continuously shaken for 12 h to ensure adequate adsorption of engineered formate dehydrogenase preparation onto the surface of the ZSM-5 support. The final engineered formate dehydrogenase preparation ZSM-5 catalyst was obtained after crosslinking the engineered formate dehydrogenase preparation on the ZSM-5 surface by immersion in a glutaraldehyde solution (0.5%) for 4 h at 25°C, with stirring at 120 r min-1. The residual un-crosslinked engineered formate dehydrogenase preparation was removed by continuous washing until no protein was detected in the washing solution. The amount of engineered formate dehydrogenase preparation immobilized on the zeolite was calculated from the difference between the initial amount of engineered formate dehydrogenase preparation and the amount of un-crosslinked engineered formate dehydrogenase preparation.
[0106] In some embodiment, the immobilized engineered formate dehydrogenase reaction assay is conducted, where the immobilized formate dehydrogenase is mixed with 700 µL of Tris-HCl buffer (50 mM, pH 7.5) and sodium formate stock solution (440 mM, 150 µL). The reaction was started by adding 150 µL of NAD(P)+ stock solution (6.0 mM) and carried out at 40°C in a water bath with shaking. After centrifugation,100 µL samples of the supernatant were removed from the mixture and added in 900 µL of Tris-HCl buffer (50 mM, pH 7.5) to perform the activity assay. The activity was determined by measuring the increased NAD(P)H concentration at 340 nm. The absorption values were limited to a range from 0.2 to 0.8. One unit of FDH activity (U) was defined as the amount of enzyme required to generate 1µmol of NAD(P)H per minute.
[0107] In some embodiment, the engineered formate dehydrogenase thermal stability is assayed in a sodium-phosphate buffer (0.1 M, pH 7.0), where several Eppendorf tubes (0.5 ml) with 100µl formate dehydrogenase enzyme solution (0.25 mg/ml) were prepared for each experiment. The tubes were incubated in a water bath at different temperatures (20–55°C, ±0.1°?). At fixed time intervals, a tube was transferred from the bath to cold water (4°C) for 5 min. Then, the solution was centrifuged for 3 min at 12 000 rpm using an Eppendorf 5415D centrifuge. The residual FDH activity was measured by monitoring the change of absorbance at 340 nm within 1 min on a UV-spectrometer.
[0108] In some embodiment, the engineered formate dehydrogenase pH stability is assayed in a sodium-phosphate buffer (0.1 M) with varying pH range from 3 to 9. Several Eppendorf tubes (0.5 ml) with 100µl formate dehydrogenase enzyme solution (0.25 mg/ml) were prepared for each experiment. The tubes were incubated in a water bath at temperatures of 30°C. At fixed time intervals, a tube was transferred from the bath to cold water (4°C) for 5 min. Then, the solution was centrifuged for 3 min at 12 000 rpm using an Eppendorf 5415D centrifuge. The residual FDH activity was measured by monitoring the change of absorbance at 340 nm within 1 min on a UV-spectrometer.
[0109] To create/facilitate the mutations of formate dehydrogenase described in the present invention, several of predetermined amino acid residues are substituted in the SEQ ID 1. The mutations have significantly improved the reduction activity for bulky substrates with 2-oxo-acid group, cofactor NAD(P)+ recycling ability and stability. Here, “improved stability” means broader thermal stability (20-55°C).
[0110] The plausible enzyme substrate (ES) complex of Phenylpyruvic acid, 2-oxo-4-phenylbutyric acid, 4-hydroxyphenylpyruvic acid, and pyruvic acid in the active site of arFDH was established with key residue interactions for promoting the catalysis (Fig. 3). It was observed that the substrates adopt energetically favourable near-attack conformations by forming various interactions with the residues lining the active site pocket. The energetically favourable conformation of phenylpyruvic acid within the active site of arFDH was derived, where H333 and G124 form conventional hydrogen bonds with the substrate, and H333 also stabilizes the substrate by forming a p- p stacking interaction (Fig. 3A). The energetically favourable conformation of 2-oxo-4-phenylbutyric acid within the active site of arFDH was derived, where I123 formed Amide-p interaction with the substrate, R285, G336, N147 and H333 are formed conventional hydrogen bonds with the substrate (Fig. 3B). The energetically favourable conformation of 4-hydroxyphenylpyruvic acid within the active site of arFDH was derived, where H333 and R285 formed conventional hydrogen bonds (Fig. 3C).The energetically favourable conformation of Pyruvic acid within the active site of arFDH enzyme was derived, where F99 formed a p-s interaction, P98 formed a p-alkyl interaction, N147 and I123 formed conventional hydrogen bonds, I123 also formed hydrophobic interactions, and R285 stabilized the carboxylic group of the substrate by forming the charge interactions (Fig. 3D).
[0111] Mechanistic feasibility of arFDH with different substrates to product conversion were explored using Quantum molecular dynamics (QMD) simulations. A relative energy profile (in kcal/mol) plotted for conversion of phenylpyruvic acid to (R)-phenyllactic acid, 2-oxo-4-phenylbutyric acid to 2-hydroxy-4-phenylbutanoic acid, 4-hydroxyphenylpyruvic acid to (2S)-2-hydroxy-3-(4-hydroxyphenyl)propanoic acid and pyruvate into lactate. Two transition states TS1 and TS2 and one intermediate state IS1 was predicted (Fig. 4). The relative energy difference of substrates in TS1, IS1 and TS2 shows different energy barriers associated with the reaction, which was the foundation stone and optimizing enzyme design and engineering strategies to accept bulky 2-oxo-acid substrate and converting into hydroxy-acids by arFDH with increased catalytic efficiency and substrate specificity. Energy required for the carbonyl oxygen of the substrate to abstract the hydrogen from catalytic His was determined to be rate limiting step in the reaction as the energy required to attain TS1 state in all substrates was higher compared to energy required to attain TS2 state (Fig. 5). The non-native substrates namely, phenylpyruvic acid, 2-oxo-4-phenylbutyric acid and 4-hydroxyphenylpyruvic acid have demonstrated similar activation energy profiles for the engineered arFDH which indicates feasibility for conversion to their respective products. The active site substitutions and mutations that altered the electrostatics of the active site enabled the conversion of the bulky substrates.
[0112] In some embodiments, the arFDH of the present invention and its variants were designed using a De novo based computational engineering method as illustrated in Fig. 6. In STEP 1, a plurality of FDH sequences from GenBank, UniProt, etc. were compiled in an internal curated database of FDH sequences (Table 12), where the curated FDH sequences were modelled to obtain three dimensional structures complexed with the cofactor (NAD(P)+) molecule. In STEP 2, Induced fit modes of the modelled FDH-cofactor complexes and the substrates: phenylpyruvic acid, 2-oxo-4-phenylbutyric acid, 4-hydroxyphenylpyruvic acid and pyruvates, were derived to determine plausible near attack conformations (Fig. 3). In STEP 3, Sequences were ranked based on interaction energies between the residues and the substrates to derive top four FDH sequences from the curated FDH database (Table 1): Mycolicibacterium vaccae (Accession ID: Q93GV1), Candida boidinii (Accession ID: O13437), Pseudomonas sp.101 (Accession ID: P33160) and Granulicella mallensis (Accession ID: AEU36496.1) (Fig. 8A). In STEP 4, potential energy of the residues across different regions of the structures from the top four sequences were calculated and the energies were deposited on the residues (Fig. 8B). An average sequence was derived from the consensus sequence of the top four FDH sequences and, motifs from the template sequences were transferred onto the average sequence using an AI/ML sequence generation algorithm based on a weighted score for each motif, derived from the potential energies of the corresponding residues (Fig. 8C-E). In STEP 5, the generated artificial FDH sequences (SEQ ID NO: 1, 202, 203, 204, 205, 206, 207, 208, 209, & 210) were subjected to computational structure validation studies and residue-residue pair interaction energy calculations (PIE). Furthermore, the generated artificial FDH sequences were computationally assessed for their ability to effectively bind NAD(P)H and NAD(P)+ cofactors and the arFDH-NAD(P)H/NAD(P)+ complex was used to derive induced fit modes of 2-oxo-acid compounds, specifically, 2-oxo-4-phenylbutyric acid, 4-hydroxyphenylpyruvic acid and phenylpyruvic acid. The derived arFDH sequences containing the substitution X24 as Ala, X79 as Leu, X81 as Thr, X147 as Ser, X314 as Glu, X336 as Ser; X386 as Asp; and X389 as Arg showed an optimal near attack conformation of the substrates 2-oxo-4-phenylbutyric acid, 4-hydroxyphenylpyruvic acid, phenylpyruvic acid and pyruvate and were consistently observed in the top ranked artificial FDH sequences. The engineered artificial formate dehydrogenase derived from the above method was described by the polypeptide given in SEQ ID NO: 1 and the residues that contributed to its substrate activity were categorised as features (Fig. 8F). In STEP 6, the enzyme-cofactor-substrate complexes derived from induced fit modes were subjected to Short Molecular Dynamics Simulations, which analyses the binding stability and flexibility of the 2-oxo-acid compounds in the active site of the arFDH. The results of the short molecular dynamics simulations were used as baseline for computational engineering studies, to derive engineered variants that show improved reduction activity against 2-oxo-acids substrates with improved co-factor recyclability and improved stability. In STEP 7, the conformational ensembles from short molecular dynamics simulations were assembled and enclosed within an interaction profiling grid, as described in Kumar P, et.al., (2023) to derive residue hotspots. Briefly in STEP 8, probes that mimic amino acid side chains, solvent molecules and formate ions were placed at each grid point to calculate Pair Interaction Energies (PIE). In STEP 9, regions of the arFDH with high cumulative PIE values were considered to be unstable regions and top two residues with high PIE values in each unstable region was considered as hotspots. In STEP 10, the generation of target mutagenesis of selected hotspots was based on substituting amino acid residues in the amino acid pairs with sub-optimal PIE. In STEP 11, substitutions were derived based on rational design, phylogenetic approaches, partial de-novo approaches and PIE energies are recalculated for the improvement in PIE for different substitution from different approaches. In STEP 12, the variants derived using these approaches were iteratively validated for improvement in PIE for each pair of amino acids hotspots. In STEP 13, the identified variants with significantly improved PIE are simulated using a Quantum Molecular Dynamics approach, wherein relative energy for the reaction was measured with respect to the arFDH (Fig. 4 and 5). In STEP 14, the variants with the lower Transition State (TS) and Intermediate State (INT) energies with respect to the arFDH were ranked based on the energy profiles (Fig. 4). In STEPS 15 and 16, the top ranked variants from the previous steps are synthesized and validated using enzymatic assay for the improvement in the activity and ranked according to the detected activity.
[0113] In some embodiments, the engineered arFDH polypeptide can also use formate and their corresponding salts are Sodium formate (HCOONa) or Ammonium formate (NH4HCOO) or formic acid (HCOOH) as a co-substrate for cofactor recycling.
[0114] In some of embodiment the suitable process conditions for arFDH comprises a temperature range of 20 °C to about 55 °C.
[0115] In some of embodiment the suitable process conditions for arFDH comprises a pH of 4.0 to about 9.0.
[0116] In some of embodiment the suitable process conditions for arFDH comprises the substrate compound at a loading of about 5g/L to about 100g/L.
[0117] In some of embodiment the suitable process conditions for arFDH comprises the formate loading of 2g/L to about 10g/L.
[0118] In some of embodiment the suitable process conditions for arFDH comprises the cofactor NAD(P)+ loading of 0.5 g/L to about 2g/L.
[0119] In some of the embodiment the suitable process conditions for arFDH comprises the engineered arFDH at a concentration from 0.5 g/L to about 5g/L.
[0120] Stability can be evaluated with the residual activity of arFDH in temperature range of 20 to 55°C. Also, the expression “stability is improved” means that residual FDH activity after mutagenesis is statistically minimum 3-fold higher than that of any reported FDH. Here, the enzyme activity of arFDH and any reported formate dehydrogenase or engineered formate can be determined by appropriately using conventionally known techniques. For example, through direct or indirect measurement of the amount of NAD(P)+ diminished or NAD(P)H generated according to the following formula during the cofactor recycling, or the amounts of the components consumed according to the following reaction formula, the enzyme activity of formate dehydrogenase contributing to the following formula can be measured.
[0121] HCOO- + NAD+/NADP+ à CO¬2 + NADH/NADPH
[0122] The present disclosure provides the arFDH that work on formate and corresponding salts thereof. Sodium formate, ammonium formate, potassium formate, and formic acid are substrates along with engineered arFDH. The arFDH polypeptide has one or more improved properties such as bulky 2-oxo-acid reducing activity, thermal stability, and enhanced cofactor recycling ability.
S. NO Organisms Accession ID
01 Mycolicibacterium vaccae Q93GV1
02 Candida boidinii O13437
03 Pseudomonas sp.101 P33160
04 Granulicella mallensis AEU36496.1
[0123] Table 1: The derived template sequences which were used for designing the artificial formate dehydrogenase (arFDH).
SEQ ID No. Residue Difference Relative to SEQ ID No: 1 Activity Stability Expression
1 NA + + ++
2 X27Q, X167L, X302R, X235N, X240D ++ + ++
3 X36A, X94A, X241S, X212Q, X93K + ++ +
4 X16M, X36S, X241D, X338S, X33D ++ ++ ++
5 X138L, X49A, X387T, X241D, X240T +++ + ++
6 X62R, X13V, X190I, X212R, X21P, X72I + ++ ++
7 X19T, X27T, X235N, X244S, X93I, X240P ++ ++ +
8 X16Y, X161N, X241D, X190V, X304C, X131E +++ ++ ++
9 X173I, X67N, X302H, X190I, X304G, X240P ++ + ++
10 X94A, X27T, X244A, X298I, X328Q, X320E, X38C + ++ ++
11 X161A, X173A, X302R, X190I, X294L, X38C, X106A ++ + ++
12 X36S, X62P, X328H, X228D, X190I, X219R, X106A ++ + +++
13 X171N, X173I, X298I, X294L, X190I, X37H, X72K ++ + +++
14 X94A, X67L, X244S, X216V, X387V, X320D, X38C +++ + +
15 X13V, X16Y, X398R, X241S, X232L, X217K, X194Q +++ ++ +++
16 X29D, X94A, X328H, X232R, X216L, X30E, X149H +++ ++ +
17 X16M, X161N, X241S, X328H, X398R, X134M, X270M +++ + +++
18 X94A, X138M, X244A, X232L, X212K, X221M, X131E, X270L + ++ ++
19 X13V, X62R, X328Q, X398R, X228E, X229D, X131E, X270M ++++ ++ +++
20 X132A, X84F, X190V, X221L, X294L, X216A, X131E, X93K ++ ++ +
21 X173I, X167L, X229D, X338S, X216L, X328Q, X30E, X37H ++ ++ +
22 X94A, X161A, X232L, X294L, X228D, X328H, X33D, X106A, X240T +++ ++ ++
23 X13V, X171E, X216A, X328Q, X338S, X186G, X304C, X21P, X33S ++++ ++ +++
24 X29D, X138L, X190V, X212Q, X229S, X338S, X37H, X217K, X106A + ++ ++
25 X16M, X67L, X328Q, X318Q, X229A, X244S, X149H, X37H, X217K +++ ++ +
26 X161N, X19A, X221L, X241S, X338S, X229S, X294L, X304C, X239D, X320D +++ ++ +++
27 X28V, X132A, X244A, X294L, X212R, X396H, X216L, X106A, X320E, X21C ++++ ++ +
28 X62R, X132A, X241D, X338S, X298M, X302R, X328H, X236K, X33D, X38C ++++ ++ +++
29 X28I, X132A, X228E, X294L, X190V, X398K, X302R, X194N, X270L, X38C +++ ++ +
30 X49Q, X16M, X216L, X241S, X221M, X186Q, X396H, X37H, X320D, X33D +++ ++ +++
31 X84F, X62R, X318K, X186G, X229A, X241D, X396H, X304S, X149N, X134M ++++ ++ ++
32 X173I, X5I, X302R, X228L, X387T, X318A, X186G, X38C, X134N, X131E +++ + +
33 X19K, X167L, X318A, X186H, X190V, X298I, X396K, X30E, X304S, X134K + + +++
34 X62P, X5I, X396H, X229S, X216L, X244S, X328Q, X235N, X239E, X33S, X72I +++ + ++
35 X36S, X49T, X318K, X190V, X241D, X294L, X229D, X232R, X194Q, X131E, X217K +++ ++ ++
36 X94A, X84F, X212K, X387V, X328H, X229D, X186H, X302R, X217K, X194D, X38C ++ + +++
37 X94A, X171Q, X396K, X338S, X398K, X302R, X228D, X216L, X194Q, X21P, X236V ++ ++ +
38 X138L, X36S, X186G, X387V, X302R, X396H, X398K, X294L, X338S, X219R, X304G, X194N ++ ++ +++
39 X67N, X13E, X294L, X221L, X318K, X298I, X216A, X328Q, X186G, X149H, X37H, X240P ++ ++ ++
40 X49Q, X36S, X244S, X387T, X294L, X241S, X298M, X221L, X235H, X131E, X106A, X236V +++ + +++
41 X171E, X13T, X294L, X235N, X221L, X318Q, X229S, X241D, X212Q, X240T, X149H, X270L + ++ +++
42 X67Q, X5L, X387V, X229D, X235N, X338S, X232R, X221L, X216T, X194N, X93I, X149D, X33S +++ + +++
43 X27K, X138V, X212Q, X235D, X328Q, X228D, X190V, X244S, X216A, X33D, X219K, X134K, X240T ++++ ++ ++
44 X62P, X29D, X232L, X302R, X221L, X398K, X190I, X212K, X186H, X21I, X304C, X30E, X320D +++ ++ +++
45 X67Q, X84F, X212K, X294L, X228L, X398K, X190V, X318K, X338S, X217K, X219R, X236K, X38C ++++ ++ ++
46 X29K, X67L, X212R, X338S, X229D, X235D, X190V, X294L, X221L, X236V, X33S, X149N, X194D ++++ ++ +++
47 X94A, X36A, X318K, X244S, X229D, X302R, X298I, X294L, X235N, X149D, X139R, X37H, X33D +++ + +++
48 X27Q, X173A, X318K, X186H, X387V, X235N, X212R, X228L, X294L, X304S, X37H, X149D, X320D +++ ++ +
49 X132A, X167V, X232K, X216A, X298I, X302R, X328H, X228D, X212K, X219K, X240P, X236K, X139R ++++ ++ ++
50 X62P, X138V, X244S, X241S, X216A, X221M, X186G, X302H, X229D, X232K, X131E, X239E, X219K, X72A ++++ ++ +
51 X84F, X138V, X186Q, X396H, X228D, X294L, X235N, X298I, X338S, X318K, X139R, X38C, X270M, X236K ++++ ++ +++
52 X5L, X94A, X387T, X318A, X398E, X241D, X221M, X396R, X298I, X235N, X149H, X134M, X93K, X139R ++++ ++ ++
53 X167L, X36S, X228L, X387V, X216A, X328H, X235H, X398R, X318A, X338S, X304C, X33S, X320D, X72I ++++ ++ +
54 X171Q, X16Y, X228D, X212R, X241S, X298M, X190V, X294L, X302H, X396H, X318K, X270S, X106A, X131D, X240P ++++ ++ +++
55 X132A, X5A, X232L, X228D, X298I, X190V, X294L, X338S, X328Q, X216A, X235N, X320E, X239E, X106A, X217K ++++ ++ ++
56 X62R, X27Q, X398K, X190V, X396H, X229D, X216V, X186H, X338S, X298M, X318Q, X304C, X320D, X30E, X38C ++ + ++
57 X49A, X62P, X398R, X216A, X338S, X244S, X302R, X241D, X235H, X298I, X228L, X72K, X37H, X217R, X38C +++ + ++
58 X132G, X13T, X216L, X298M, X396R, X398K, X328H, X244A, X241S, X387V, X232L, X219K, X21I, X217R, X304S, X131E ++ + +
59 X167V, X28I, X298I, X232L, X235H, X228D, X212Q, X302H, X396K, X294L, X229D, X240T, X304G, X239E, X33D, X217R ++ ++ ++
60 X94A, X28P, X228L, X221L, X318Q, X338S, X294L, X186Q, X328H, X298I, X387V, X320E, X270L, X139R, X72I, X134N +++ ++ +
61 X27T, X5I, X186Q, X241S, X244A, X228L, X338S, X235H, X190V, X232K, X229A, X72A, X131D, X219K, X139I, X134M ++ ++ +++
62 X49A, X161A, X398E, X396R, X232R, X318K, X228D, X298M, X244S, X387V, X338S, X217K, X149N, X33S, X37H, X93K +++ ++ +
63 X27K, X13V, X294L, X216V, X298I, X212K, X398E, X328Q, X186Q, X190V, X387T, X320D, X236K, X149D, X139R, X270L ++++ ++ +
64 X28I, X167T, X186H, X294L, X338S, X328H, X244S, X235H, X398K, X221M, X387V, X304S, X236K, X217K, X320E, X131D +++ + +++
65 X171Q, X28P, X216T, X228E, X387V, X212Q, X328Q, X302R, X232L, X241D, X338S, X139R, X304C, X134K, X106A, X194Q ++++ ++ ++
66 X29D, X5I, X318Q, X294L, X244A, X302H, X235D, X229S, X298I, X328Q, X190I, X221M, X106A, X37H, X139R, X320D, X38C ++++ ++ +++
67 X62R, X27K, X232R, X396R, X328Q, X294L, X241D, X190V, X318K, X298M, X229D, X228E, X131E, X30E, X194D, X149H, X21P +++ ++ +++
68 X28P, X84F, X190I, X387T, X328H, X338S, X186G, X294L, X302R, X232L, X221L, X229A, X38C, X33S, X37H, X21C, X304S +++ + +
69 X171E, X13V, X190V, X398E, X216L, X241D, X396H, X235N, X387V, X186G, X232L, X328Q, X236A, X131D, X106A, X21P, X219R +++ + +
70 X62P, X28V, X318K, X212K, X228E, X244A, X221L, X294L, X302R, X298I, X232R, X398R, X186Q, X30E, X106A, X139R, X38C, X270M +++ + ++
71 X138L, X161A, X387T, X235N, X216V, X232L, X244A, X338S, X221M, X302R, X398K, X318K, X294L, X30E, X320E, X139R, X240P, X270S +++ + +
72 X27K, X132G, X229A, X318A, X298M, X294L, X186Q, X232L, X190I, X398R, X302R, X235D, X221L, X106A, X134K, X37H, X270L, X149H ++++ ++ ++
73 X94A, X161A, X216L, X241S, X387V, X328H, X190V, X235N, X338S, X228L, X396K, X298I, X212R, X30E, X131E, X139I, X240D, X304C ++++ ++ ++
74 X13V, X167T, X302H, X216T, X186H, X294L, X298M, X190I, X387T, X221L, X241S, X232R, X328Q, X33S, X131E, X320D, X236V, X149D +++ + +
75 X62P, X19K, X398R, X235D, X244S, X338S, X229D, X221M, X318Q, X190V, X216L, X186G, X232K, X72K, X320D, X93I, X270M, X149N ++++ ++ ++
76 X171Q, X28V, X216A, X235N, X396H, X221L, X318A, X298I, X294L, X387T, X302R, X212Q, X229A, X219K, X194D, X38C, X139I, X304C ++ ++ +
77 X36S, X62R, X190I, X318Q, X294L, X302H, X221M, X328H, X216T, X235N, X228E, X338S, X387T, X240T, X131D, X21C, X304C, X33S ++ ++ ++
78 X84F, X16Y, X190I, X302R, X228D, X221M, X387T, X328H, X212R, X398K, X229A, X232R, X294L, X72I, X236A, X219R, X106A, X38C, X320E +++ ++ +++
79 X62R, X27K, X338S, X190V, X328H, X396H, X212Q, X398R, X241D, X387T, X235H, X221L, X318Q, X72A, X21C, X149N, X33S, X93I, X131E ++ + ++
80 X36A, X5L, X235N, X228E, X294L, X241D, X244A, X396H, X302H, X216T, X328Q, X232R, X190I, X72K, X219R, X131D, X134M, X304S, X37H ++ + +++
81 X132A, X171N, X212R, X216T, X328Q, X244A, X229D, X186Q, X221L, X294L, X387V, X398R, X235N, X239D, X219K, X72I, X194Q, X37H, X320E +++ ++ +
82 X13T, X49A, X398R, X396H, X338S, X228E, X186H, X318K, X294L, X235D, X229S, X387T, X212K, X244A, X93K, X219R, X131D, X134K, X239D, X21P ++++ ++ ++
83 X173A, X62R, X338S, X229S, X244S, X396K, X190I, X186H, X328H, X221L, X318K, X212R, X387V, X294L, X131D, X139R, X37H, X219R, X217K, X72I +++ ++ ++
84 X138V, X171Q, X338S, X302H, X241D, X221L, X318K, X244S, X235H, X387T, X294L, X186G, X398K, X228E, X239D, X194Q, X37H, X236K, X149H, X33S ++++ ++ +++
85 X49Q, X132G, X232L, X294L, X212K, X235D, X190I, X241S, X387T, X216L, X298I, X302R, X318Q, X244A, X219K, X106A, X93I, X33D, X72I, X38C ++++ ++ +
86 X16Q, X36A, X241D, X229D, X235H, X190V, X186G, X232L, X387T, X244A, X302R, X298M, X212Q, X228E, X93I, X219R, X149H, X139I, X33D, X304C +++ + +++
87 X171Q, X62R, X294L, X212R, X396R, X229A, X302H, X235D, X228E, X232R, X398R, X328Q, X216T, X387T, X149N, X219R, X194D, X304C, X72A, X38C ++ + +
88 X84F, X167V, X216V, X302R, X212Q, X294L, X328Q, X244S, X387T, X396H, X232K, X190I, X298M, X235N, X93K, X21P, X30E, X131D, X304C, X37H ++ ++ +++
89 X84F, X67L, X229A, X318Q, X190V, X221L, X244A, X387T, X232R, X241S, X298M, X302H, X328H, X294L, X219K, X239D, X38C, X270L, X106A, X21P ++ ++ +
90 X161A, X29K, X338S, X241D, X212R, X244A, X396H, X328H, X398K, X294L, X302R, X318A, X216V, X387T, X235H, X37H, X219R, X194Q, X239E, X236K, X134M ++ ++ +
91 X171Q, X16Y, X235H, X221L, X398K, X232R, X228E, X229S, X190V, X241S, X212K, X302R, X318K, X387T, X396K, X139I, X106A, X149H, X72A, X33S, X219R +++ ++ ++
92 X138L, X84F, X221M, X244S, X302R, X294L, X232K, X338S, X216A, X396R, X328H, X212R, X241D, X298M, X190V, X106A, X134M, X72I, X139R, X236V, X270M ++ + ++
93 X84F, X132G, X244S, X228L, X235D, X186Q, X216A, X229S, X387T, X302H, X190V, X232L, X318Q, X298I, X294L, X239D, X37H, X320E, X33D, X93K, X38C ++ + ++
94 X138L, X5A, X244A, X232L, X338S, X328H, X221L, X228L, X186G, X235H, X318A, X294L, X298M, X229S, X241S, X149H, X134K, X217R, X240T, X194D, X131E +++ + +
95 X19K, X167T, X302R, X186H, X396K, X228E, X229S, X387T, X398E, X235N, X216V, X212Q, X298I, X241S, X190I, X21I, X131E, X270S, X239D, X236K, X320D ++ ++ +
96 X29K, X94A, X387V, X396R, X398K, X318K, X232R, X212K, X216T, X328H, X294L, X235H, X338S, X241D, X229A, X131E, X106A, X134K, X21I, X37H, X149N ++ ++ +++
97 X161N, X16M, X212K, X235N, X229A, X228L, X318K, X298I, X241S, X186G, X398E, X221L, X396H, X338S, X294L, X131E, X134M, X320E, X38C, X37H, X106A ++++ ++ +
98 X132A, X36A, X232L, X387V, X229S, X294L, X235D, X241D, X318A, X186H, X398E, X298I, X216A, X244S, X302R, X131E, X240T, X106A, X270L, X72K, X134N ++++ ++ +
99 X19K, X62P, X216V, X228D, X221M, X387V, X298M, X212Q, X338S, X244A, X235N, X302H, X229A, X294L, X398R, X21C, X194D, X270L, X217R, X38C, X149H ++ ++ +
100 X138V, X13V, X235N, X190I, X241S, X396H, X398E, X387V, X302H, X186G, X298I, X318Q, X244A, X328Q, X232R, X294L, X131E, X21C, X194N, X149N, X236K, X106A, X304C +++ + +
101 X173I, X49T, X244A, X318K, X190I, X302R, X186H, X235D, X216L, X212Q, X294L, X387V, X232L, X338S, X298M, X396K, X37H, X236A, X194Q, X240D, X131D, X38C, X320D ++ ++ +
102 X167T, X16M, X212R, X241D, X338S, X294L, X318A, X302H, X244S, X228D, X229S, X328H, X298I, X398R, X232L, X190I, X106A, X219K, X320D, X30E, X270L, X21P, X139I ++ + +
103 X84F, X5L, X235H, X190I, X216L, X298I, X186Q, X221L, X398K, X228D, X244S, X302H, X328Q, X241S, X232L, X318Q, X320D, X270S, X239E, X106A, X149N, X72I, X38C ++ + +++
104 X36S, X16Q, X318Q, X338S, X298I, X190I, X186Q, X228D, X328H, X221M, X294L, X216L, X232L, X235H, X212Q, X398E, X219K, X270L, X93K, X217R, X139R, X240D, X38C ++++ ++ +
105 X28I, X67N, X235H, X212R, X396R, X298M, X294L, X216T, X228E, X232R, X302R, X241S, X328Q, X190V, X338S, X398K, X93I, X106A, X139R, X194N, X72I, X131D, X37H +++ ++ ++
106 X13V, X36A, X212Q, X398R, X228D, X294L, X221M, X298M, X396R, X318Q, X241S, X190V, X232L, X328Q, X338S, X235H, X217R, X139I, X149D, X236A, X240T, X320E, X106A ++++ ++ ++
107 X16M, X27Q, X190I, X241S, X216T, X318Q, X228E, X294L, X232K, X244S, X186G, X338S, X212R, X328H, X396H, X298M, X304C, X320E, X236K, X194N, X240P, X33S, X217K ++++ ++ +
108 X161N, X49T, X221M, X294L, X398K, X244A, X212K, X228D, X302H, X396H, X328Q, X338S, X190V, X318Q, X216V, X235N, X304G, X93I, X38C, X33S, X106A, X72I, X239D +++ + ++
109 X132G, X13E, X186H, X302R, X241D, X232R, X235N, X228D, X387T, X318K, X294L, X229A, X190V, X338S, X216L, X398E, X320D, X139R, X134N, X72I, X131D, X93I, X30E ++++ ++ ++
110 X84F, X161N, X241S, X328H, X186H, X232R, X318A, X387T, X212R, X235N, X190I, X221L, X216V, X229S, X338S, X244S, X38C, X33S, X304C, X106A, X21P, X93I, X139I +++ ++ ++
111 X28V, X29K, X235H, X294L, X221M, X190V, X228D, X229S, X387V, X216L, X338S, X241S, X328Q, X302R, X244S, X232R, X93K, X37H, X30E, X106A, X194Q, X236V, X38C ++ + ++
112 X138M, X171N, X398K, X396H, X338S, X298M, X229S, X212K, X318Q, X190V, X232L, X241S, X235N, X216V, X328H, X186Q, X30E, X239E, X217K, X240P, X106A, X21P, X33S +++ ++ ++
113 X28I, X132G, X396K, X228D, X186Q, X212R, X229S, X190V, X216V, X398E, X328H, X235H, X232K, X298M, X338S, X221M, X320E, X217R, X37H, X149H, X131D, X30E, X21I ++++ ++ +
114 X28P, X13E, X221M, X298I, X318Q, X216V, X212K, X396K, X387T, X398E, X232L, X338S, X328Q, X244S, X241D, X190V, X270M, X21I, X30E, X33D, X236K, X106A, X93K +++ + ++
115 X29K, X62P, X186G, X328Q, X338S, X241S, X318A, X228L, X398R, X298I, X396R, X302H, X387T, X229A, X221M, X244S, X33D, X37H, X21P, X93I, X38C, X304S, X139I ++++ ++ +++
116 X167V, X161A, X229A, X235D, X294L, X232K, X396H, X318Q, X244S, X387T, X212K, X190I, X328H, X298I, X302H, X221L, X216V, X219R, X134M, X270S, X72A, X38C, X139I, X106A +++ + +
117 X67N, X171N, X229A, X318Q, X294L, X338S, X232R, X228L, X221M, X302R, X216L, X398E, X396H, X235N, X387V, X244S, X298I, X131E, X270S, X93K, X240T, X304G, X219K, X72A ++ + ++
118 X173A, X36S, X387V, X396H, X186G, X228L, X229D, X298M, X235D, X221L, X241D, X328Q, X338S, X294L, X302R, X212R, X190V, X270S, X72I, X33D, X21C, X217R, X194Q, X239D ++ + +
119 X161N, X84F, X186Q, X228E, X302H, X387V, X298M, X294L, X396H, X328H, X229A, X212Q, X398K, X221L, X232K, X235D, X216A, X239D, X131E, X236K, X219R, X21P, X106A, X72I +++ ++ ++
120 X67N, X19T, X241S, X387T, X229A, X244A, X228D, X212K, X328H, X294L, X398R, X190I, X221M, X338S, X216L, X235H, X298M, X139R, X149H, X194Q, X30E, X72K, X217R, X106A +++ + +
121 X138M, X132G, X302H, X190I, X338S, X235H, X216T, X229D, X398E, X328H, X298M, X241S, X294L, X396H, X244A, X221M, X387T, X21P, X134N, X38C, X219K, X240D, X106A, X72I ++ ++ ++
122 X19T, X62R, X338S, X294L, X228D, X298M, X387T, X318Q, X302R, X216V, X186Q, X241D, X398E, X244S, X212Q, X396K, X221L, X240T, X270L, X149N, X139I, X194D, X239D, X219R +++ ++ +++
123 X167T, X36S, X212R, X229A, X216V, X244A, X298M, X235D, X398K, X387V, X302H, X232L, X328Q, X221L, X228E, X294L, X190V, X37H, X38C, X33S, X217K, X93K, X134K, X239D ++++ ++ +
124 X13E, X132G, X398R, X229S, X396R, X241D, X328Q, X221M, X298I, X190I, X244S, X338S, X302H, X318K, X216T, X228L, X232R, X33S, X320D, X21P, X304G, X236K, X72K, X270M ++++ ++ ++
125 X84F, X132A, X298M, X229D, X186Q, X228L, X221L, X302H, X338S, X235H, X294L, X190V, X318Q, X387T, X232L, X241S, X396H, X320E, X106A, X149D, X236K, X72K, X139I, X21I ++ + ++
126 X132G, X5I, X241D, X232R, X302H, X186H, X338S, X318K, X298M, X235H, X244S, X328Q, X221L, X387T, X190V, X216V, X212Q, X320D, X270M, X21C, X33D, X194N, X217R, X30E ++++ ++ ++
127 X29D, X67L, X235N, X244S, X294L, X387V, X232L, X318Q, X212K, X302H, X241D, X398K, X228L, X328Q, X229S, X221L, X298I, X33D, X304C, X219R, X139R, X72K, X37H, X194D +++ + +
128 X13T, X16Q, X318Q, X328H, X232L, X396H, X244S, X298M, X387V, X229S, X338S, X190I, X216A, X302H, X212Q, X221L, X235H, X134M, X33S, X72I, X236K, X304C, X37H, X38C ++ ++ ++
129 X36A, X173I, X298M, X294L, X216V, X228L, X221M, X338S, X318A, X190V, X398E, X328Q, X396H, X229A, X244A, X302R, X387T, X239D, X30E, X320E, X219K, X131E, X33D, X217R ++++ ++ +++
130 X173A, X28P, X318K, X396K, X241D, X190V, X328Q, X235N, X298I, X186H, X338S, X232R, X244S, X212R, X387T, X229D, X294L, X134M, X149N, X30E, X217K, X72A, X37H, X320E ++ + +
131 X173A, X62R, X216T, X229A, X298M, X387T, X328H, X235D, X232R, X228L, X338S, X318A, X212R, X241D, X302R, X244S, X186Q, X396R, X30E, X320E, X239E, X93K, X33S, X72A, X38C ++ ++ ++
132 X28I, X171Q, X298M, X387T, X232K, X228L, X229A, X235N, X221L, X302H, X396R, X216L, X398R, X212R, X338S, X318K, X190V, X328H, X240T, X270S, X30E, X304S, X131D, X33D, X72I +++ ++ +
133 X28P, X84F, X318Q, X216A, X241D, X387V, X235D, X294L, X229A, X338S, X398E, X302R, X190V, X244S, X228L, X221L, X232R, X298I, X21P, X106A, X38C, X30E, X139I, X320E, X72A ++++ ++ +++
134 X171E, X84F, X212R, X229S, X241D, X398K, X328H, X244A, X216T, X186Q, X318K, X221L, X190V, X387T, X228D, X232K, X302H, X235H, X33D, X320E, X236A, X106A, X93I, X30E, X38C +++ + +
135 X167L, X62R, X302R, X398E, X294L, X190V, X396R, X229S, X244A, X298M, X228L, X216L, X235D, X186G, X318Q, X387V, X338S, X221M, X37H, X236V, X93I, X38C, X21P, X217R, X270L ++ + +
136 X62R, X13T, X387T, X302R, X216T, X228L, X398K, X294L, X186G, X338S, X235D, X229A, X328H, X221L, X190I, X298M, X241D, X232R, X21P, X134M, X33D, X139I, X217R, X131E, X30E ++++ ++ +
137 X5A, X84F, X318Q, X186G, X298I, X235H, X216A, X398K, X212Q, X190I, X229A, X387V, X294L, X338S, X396K, X302H, X328Q, X228D, X194Q, X240P, X30E, X304G, X239D, X21C, X236V ++ ++ +++
138 X16M, X27T, X328H, X235H, X338S, X302R, X186Q, X396K, X190I, X228L, X229A, X398K, X241D, X216L, X232R, X298M, X244A, X318K, X270L, X219K, X38C, X134K, X320D, X131D, X304C ++++ ++ ++
139 X132G, X173I, X244A, X216A, X294L, X190V, X228D, X398K, X229D, X235D, X221M, X387T, X328Q, X396R, X298I, X318A, X232K, X338S, X131D, X149H, X320D, X21I, X72I, X194N, X93I ++++ ++ +++
140 X29K, X84F, X216L, X229A, X338S, X318K, X235N, X232R, X212Q, X396R, X387T, X302H, X228D, X294L, X398K, X328H, X186Q, X221L, X270M, X217R, X304C, X38C, X93I, X33D, X106A ++++ ++ ++
141 X161A, X29E, X229A, X235D, X302H, X216V, X241D, X398K, X232K, X212K, X328Q, X338S, X186G, X228E, X294L, X190V, X396H, X244S, X33D, X72K, X21C, X131E, X219R, X106A, X139R ++ + +++
142 X28I, X27Q, X186H, X298M, X387T, X232L, X241S, X228L, X235H, X212Q, X216L, X244A, X396K, X302R, X294L, X190I, X398R, X229S, X304C, X131E, X93K, X149D, X21C, X194Q, X270S ++++ ++ ++
143 X16M, X171Q, X235N, X387V, X212Q, X338S, X294L, X302H, X398E, X396R, X244S, X241S, X186G, X328Q, X229S, X228L, X216A, X298M, X33D, X37H, X131D, X134K, X139R, X21P, X194D ++ + ++
144 X171N, X138V, X328H, X232K, X387T, X212Q, X396K, X294L, X221L, X318Q, X298M, X216V, X186Q, X244A, X228L, X229A, X398K, X235D, X304C, X38C, X217R, X33D, X270S, X21I, X30E +++ + ++
145 X132A, X5A, X294L, X302R, X318K, X212K, X221M, X396H, X235H, X338S, X398R, X328H, X190I, X216V, X244A, X229A, X232K, X241S, X139R, X33S, X239E, X304S, X38C, X106A, X240P +++ + ++
146 X27K, X171E, X387V, X235N, X232R, X241D, X294L, X229S, X398R, X186Q, X338S, X396K, X318K, X302R, X221M, X328H, X216T, X190I, X134M, X21C, X236V, X33D, X37H, X270M, X38C ++++ ++ +
147 X5L, X16Q, X396R, X398E, X328Q, X221L, X338S, X244A, X216T, X235H, X212R, X229S, X318K, X186G, X302R, X228L, X190I, X241S, X240T, X236V, X134N, X194D, X106A, X30E, X21P ++++ ++ +
148 X36A, X94A, X338S, X294L, X212K, X232K, X235N, X229D, X190I, X216A, X228D, X328H, X398K, X318A, X396R, X186Q, X298I, X387T, X239D, X236A, X240D, X21P, X38C, X149H, X320D ++++ ++ +++
149 X173A, X29D, X186G, X396K, X229A, X328H, X244A, X228E, X221M, X398E, X387T, X318Q, X190I, X302H, X235N, X338S, X241S, X216T, X149H, X239D, X72A, X139R, X131E, X134K, X320E ++++ ++ +
150 X138M, X94A, X229A, X235H, X338S, X232L, X328Q, X186H, X221M, X298M, X244A, X212Q, X216A, X294L, X398E, X302H, X241S, X387V, X37H, X240T, X219R, X236V, X33D, X239E, X93I, X106A +++ + ++
151 X161N, X167T, X212R, X186G, X328H, X228L, X396R, X294L, X235N, X302H, X232K, X244S, X229S, X298M, X338S, X221M, X387T, X216V, X240T, X217R, X37H, X304G, X131D, X149H, X38C, X72K ++++ ++ ++
152 X171Q, X161N, X228E, X298I, X318K, X212Q, X190I, X216A, X221M, X338S, X328H, X387V, X396H, X398R, X229S, X302H, X294L, X186H, X30E, X219R, X106A, X217K, X194D, X304G, X139R, X131E ++++ ++ +++
153 X173A, X138V, X212K, X318A, X294L, X328Q, X338S, X228E, X221M, X229D, X396H, X216V, X235N, X190I, X398K, X186H, X298M, X241S, X30E, X38C, X149H, X194Q, X131D, X21P, X239E, X219K ++++ ++ ++
154 X27K, X167T, X241S, X328H, X318Q, X294L, X228D, X298I, X216T, X235H, X212Q, X190I, X398E, X302R, X396R, X244A, X186Q, X232L, X38C, X217R, X21P, X239D, X33D, X149N, X320E, X106A ++ ++ ++
155 X167V, X161N, X387V, X235N, X212K, X302H, X186H, X221M, X298I, X228E, X318K, X328Q, X232K, X216A, X396H, X294L, X190V, X244A, X37H, X240D, X72I, X93I, X270M, X236V, X320D, X219R ++ + +++
156 X5A, X62R, X212R, X221M, X338S, X387V, X190I, X241D, X298I, X216L, X302R, X396K, X328Q, X398E, X235N, X228E, X294L, X229D, X239E, X219K, X217K, X139R, X134M, X194N, X131E, X30E +++ ++ +
157 X173A, X84F, X228E, X328Q, X298M, X338S, X221L, X396K, X244S, X235N, X294L, X318A, X232K, X212R, X398R, X216V, X387T, X190V, X149N, X134K, X139I, X217R, X304G, X219K, X106A, X270M ++ ++ +
158 X62R, X19K, X228E, X318Q, X216T, X338S, X298M, X328Q, X398E, X294L, X241D, X186Q, X244A, X302R, X212Q, X221L, X229D, X387T, X131D, X93K, X270M, X236V, X30E, X320E, X134M, X21I ++ ++ ++
159 X161N, X19T, X212R, X241D, X338S, X298M, X318A, X244S, X216V, X294L, X235D, X190I, X396R, X232R, X228L, X302H, X221L, X398K, X30E, X72I, X320D, X194D, X38C, X131D, X217K, X21P ++++ ++ +++
160 X27K, X138L, X190I, X302R, X338S, X212R, X216T, X244S, X229A, X228D, X318K, X387T, X294L, X328H, X396K, X235D, X241S, X298I, X38C, X131D, X139I, X304G, X72A, X93K, X37H, X134N +++ ++ +
161 X67Q, X5L, X235N, X328Q, X294L, X221L, X186Q, X387V, X302H, X229A, X298M, X338S, X241D, X396K, X190I, X228L, X216V, X244S, X270L, X194Q, X149D, X240D, X217K, X21I, X219K, X304G ++++ ++ +++
162 X161N, X36S, X396K, X212R, X338S, X232L, X294L, X186Q, X244A, X241S, X398K, X387T, X229D, X190I, X228D, X328Q, X235H, X318K, X38C, X304G, X106A, X239E, X240D, X236V, X219R, X33S +++ + +
163 X29K, X27K, X387V, X398R, X232K, X221L, X212Q, X302H, X298I, X229A, X396H, X328H, X318Q, X186G, X228D, X216A, X294L, X244S, X320D, X194D, X217K, X72I, X134M, X240P, X21C, X270S +++ + +
164 X94A, X138L, X387T, X235N, X232K, X241S, X216A, X328Q, X338S, X221L, X186G, X398E, X212R, X229A, X298I, X396R, X294L, X302R, X304S, X33D, X149D, X219R, X139I, X320D, X134N, X131D +++ ++ +++
165 X19A, X161N, X212K, X294L, X338S, X235H, X228L, X396R, X241S, X186G, X216T, X398R, X328H, X298I, X232K, X190V, X244A, X229D, X236A, X21C, X131E, X30E, X304G, X240D, X37H, X72I +++ + ++
166 X173I, X171Q, X396K, X328Q, X294L, X318Q, X212Q, X302H, X398R, X190I, X235N, X241D, X387T, X244S, X216V, X298M, X186G, X232L, X93I, X134K, X30E, X270S, X37H, X139I, X131D, X239E ++++ ++ +
167 X173A, X16M, X298M, X398R, X232R, X387V, X186G, X212R, X229S, X338S, X190V, X396R, X244S, X241S, X216A, X228L, X302H, X221L, X318K, X21I, X149N, X72I, X194Q, X217R, X106A, X37H, X304G ++++ ++ +++
168 X28P, X36S, X338S, X294L, X190V, X318A, X298I, X244S, X396R, X387T, X328Q, X235D, X212K, X302R, X186G, X398R, X229A, X216L, X228E, X320E, X33S, X236K, X37H, X240P, X149H, X239D, X38C +++ + +
169 X29D, X161A, X235N, X302H, X398E, X221L, X228L, X190I, X212R, X229D, X241S, X244S, X328Q, X232K, X387V, X294L, X216V, X338S, X396H, X21P, X33S, X131D, X134K, X270M, X139I, X93I, X240P ++++ ++ +++
170 X19T, X173A, X302H, X398K, X186Q, X235H, X338S, X387T, X212R, X221M, X396K, X244A, X216V, X232K, X241S, X229D, X228D, X328H, X298I, X270M, X134K, X236A, X21C, X320E, X72A, X240D, X30E +++ ++ ++
171 X27T, X19K, X298I, X186H, X338S, X241S, X318A, X302H, X235D, X229A, X216L, X228E, X387T, X221M, X294L, X396K, X398K, X328Q, X212R, X72I, X236K, X134N, X320E, X37H, X139I, X270L, X239E +++ + +
172 X29E, X67Q, X396K, X302H, X232K, X318Q, X387T, X294L, X398K, X221L, X216T, X338S, X228D, X235D, X244A, X241S, X212R, X190I, X229D, X106A, X72K, X131E, X270S, X320E, X149D, X30E, X33D ++++ ++ ++
173 X94A, X62P, X235H, X294L, X318K, X216A, X338S, X186H, X244S, X302H, X190I, X298I, X387V, X228D, X241D, X221L, X398K, X232R, X328Q, X30E, X38C, X33S, X219K, X304C, X149N, X37H, X72K ++++ ++ +
174 X161A, X27K, X229S, X244A, X396H, X235N, X298I, X328H, X398E, X294L, X302R, X216T, X221L, X190I, X241S, X186Q, X212Q, X387V, X228E, X219K, X21C, X320E, X304G, X270M, X93K, X149D, X72A +++ ++ ++
175 X67Q, X94A, X396H, X298I, X190V, X228E, X229S, X235N, X232K, X302H, X216L, X387T, X328Q, X244S, X398E, X338S, X318A, X294L, X186G, X37H, X33D, X239E, X240T, X217R, X131E, X270M, X304C ++++ ++ ++
176 X19K, X173I, X318K, X186G, X298I, X216T, X302H, X190V, X235N, X229A, X221M, X387T, X232L, X328Q, X398K, X228E, X241S, X338S, X244A, X21C, X236K, X194Q, X33S, X304S, X239E, X72K, X217R +++ ++ ++
177 X28P, X36A, X398K, X235H, X190I, X396K, X387T, X228E, X221M, X232K, X298M, X212R, X244S, X216A, X241S, X229S, X294L, X328Q, X186G, X236A, X320E, X72A, X37H, X240T, X106A, X21P, X38C ++++ ++ ++
178 X16Y, X171E, X216V, X186G, X398E, X235D, X387T, X244A, X338S, X298M, X294L, X190V, X232K, X241S, X396H, X318Q, X302H, X221L, X228L, X37H, X139I, X236V, X72I, X239D, X149H, X240P, X30E ++++ ++ +++
179 X28V, X167T, X298M, X212Q, X235H, X241S, X328H, X387V, X244S, X398R, X338S, X396K, X190I, X232K, X318K, X229A, X186H, X216A, X228L, X72A, X270S, X37H, X194D, X93K, X131E, X33S, X139I ++ + +
180 X171E, X13E, X229S, X232L, X241S, X212K, X398R, X244S, X302H, X186H, X221M, X298M, X294L, X318K, X387T, X190V, X235N, X216T, X396H, X228L, X21I, X30E, X304G, X33D, X134M, X139R, X320D, X106A +++ ++ +
181 X84F, X62R, X216V, X302R, X396R, X186Q, X228L, X212Q, X241D, X294L, X235N, X338S, X298M, X398K, X244S, X328Q, X232L, X229S, X221M, X190I, X131E, X72A, X239E, X93K, X240T, X21I, X270M, X37H +++ + +++
182 X173A, X29D, X228L, X190V, X387T, X398R, X396H, X232L, X229D, X328Q, X298M, X241D, X221M, X244A, X216T, X235D, X186Q, X318A, X294L, X302R, X37H, X149D, X21I, X270L, X33S, X30E, X240T, X194N ++++ ++ +++
183 X132A, X67Q, X338S, X298M, X228L, X302H, X221L, X216L, X244S, X229A, X241D, X212K, X318Q, X186H, X235D, X328H, X294L, X387T, X232L, X190V, X33S, X106A, X21P, X139I, X38C, X320D, X194D, X236K +++ ++ +
184 X132A, X138M, X396R, X294L, X241S, X232R, X228L, X302H, X328H, X186H, X244S, X398R, X235H, X298I, X318K, X216L, X212Q, X387V, X190I, X338S, X219R, X139R, X320D, X37H, X270L, X217R, X134M, X131E +++ ++ +++
185 X5A, X132A, X235D, X190V, X302H, X244S, X398E, X318A, X338S, X232L, X294L, X328Q, X298I, X387V, X229S, X396K, X228D, X212Q, X186H, X241D, X72I, X194Q, X217R, X33S, X30E, X320D, X134K, X139I ++++ ++ +++
186 X173A, X49A, X229S, X398R, X241S, X212Q, X318A, X328Q, X232L, X221L, X190V, X186Q, X298M, X302H, X387V, X216T, X338S, X228E, X235N, X244A, X320D, X131E, X217R, X33S, X93K, X270S, X72K, X38C ++++ ++ ++
187 X49Q, X27K, X396H, X387V, X241D, X216V, X338S, X232K, X228E, X229A, X186G, X244S, X318Q, X190V, X328Q, X302R, X221M, X294L, X212K, X235N, X239D, X240T, X236A, X21C, X320D, X38C, X33D, X106A +++ ++ +
188 X138V, X19A, X229S, X294L, X228E, X398E, X241S, X318K, X235H, X221L, X244S, X396H, X216T, X338S, X190V, X298I, X387T, X328Q, X212R, X186Q, X33S, X239D, X131E, X194D, X217R, X72K, X149N, X270L +++ + +++
189 X132A, X94A, X232R, X318Q, X328Q, X294L, X298M, X244S, X338S, X190I, X302R, X216T, X212K, X229S, X398E, X241D, X186G, X221M, X235H, X387V, X139I, X72A, X320E, X236A, X149D, X219K, X240T, X33D, X106A +++ + +++
190 X13V, X16Q, X396R, X228L, X398R, X216L, X302H, X232R, X338S, X229D, X244A, X387T, X318K, X298I, X235D, X328H, X221L, X186Q, X294L, X241D, X239D, X304C, X134N, X240T, X149N, X21P, X217K, X30E, X38C +++ ++ ++
191 X36S, X84F, X235D, X318K, X241D, X229S, X244A, X212Q, X338S, X328H, X190V, X232L, X294L, X298M, X216V, X387T, X186G, X228L, X398E, X396R, X134M, X93I, X38C, X240D, X239D, X149H, X33S, X139I, X194Q +++ ++ +
192 X62R, X28P, X229D, X235H, X212K, X244A, X338S, X190I, X387T, X396R, X294L, X298M, X221M, X186H, X398K, X328H, X241D, X216A, X318A, X232L, X93I, X134M, X139R, X194D, X236A, X131E, X270S, X304G, X37H ++ + +
193 X29K, X132G, X228D, X302H, X298I, X190I, X232L, X328Q, X186H, X244S, X241S, X396R, X235N, X216L, X212R, X221M, X387T, X338S, X398R, X294L, X270L, X21P, X236K, X304G, X134N, X240D, X320D, X131D, X93I ++ + ++
194 X94A, X16M, X338S, X186H, X232K, X387T, X318A, X294L, X302H, X298I, X221M, X235N, X216L, X229S, X398E, X396R, X244A, X328H, X212Q, X228D, X240D, X30E, X194D, X131E, X219R, X33S, X304G, X270S, X93I ++ ++ +
195 X171Q, X173A, X235N, X212K, X396K, X241D, X229A, X318Q, X338S, X302R, X216A, X298I, X328H, X186H, X221M, X398E, X228L, X232R, X190I, X387V, X30E, X72K, X194N, X131E, X270L, X304S, X236A, X149D, X37H +++ + +
196 X28V, X16Y, X302R, X387T, X229A, X221M, X228D, X298I, X190I, X241D, X235N, X216V, X244A, X212R, X294L, X338S, X186Q, X232R, X396R, X328H, X318Q, X30E, X38C, X217R, X37H, X149H, X134M, X93I, X33S, X131D +++ ++ +++
197 X28P, X138L, X387T, X229A, X212R, X398K, X221M, X216V, X318K, X294L, X338S, X302R, X396K, X186G, X232L, X328Q, X298I, X244A, X241D, X190I, X228E, X217R, X219K, X106A, X38C, X239E, X37H, X139R, X30E, X240D ++ ++ +
198 X19K, X28I, X186G, X387T, X235H, X398R, X241S, X302H, X298I, X328H, X216L, X229S, X396H, X318Q, X212K, X244S, X338S, X221L, X228E, X190V, X294L, X239D, X320D, X134N, X240D, X72I, X219R, X38C, X30E, X149D, X223N ++++ ++ +++
199 X29E, X173A, X229D, X216L, X235N, X244S, X228D, X294L, X232L, X328Q, X318K, X396H, X221M, X387T, X212R, X302H, X338S, X298I, X190I, X186H, X241S, X236K, X139I, X134N, X320E, X194Q, X21I, X30E, X149H, X37H, X224R, X223N +++ ++ +
200 X27T, X62R, X338S, X241D, X229S, X228E, X232K, X298I, X294L, X235D, X216V, X328H, X190V, X396K, X318Q, X212K, X302R, X186G, X398R, X221L, X387T, X240P, X33S, X149N, X72K, X106A, X30E, X320D, X236K, X239D, X380Q ++++ ++ +++
[0124] In Table 2, above, in the activity column, single plus “+” indicates an activity improvement of 10-20% of the activity of SEQ ID NO: 1, two pluses “++” indicates an activity improvement of 60-80% of SEQ ID NO: 1, three pluses “+++” indicates an activity improvement of 100-120% of SEQ ID NO: 1 and four pluses “++++” indicates an activity improvement of 150-200% of SEQ ID NO: 1. In the stability column, a single plus “+” indicates that the polypeptide exhibits measurable activity after heat treatment of 2 hr at 55°C, two pluses “++” indicates that the polypeptide has greater than 200% improvement in activity as compared to SEQ ID NO: 1, when comparing activity for both proteins after heat treatment of 2 hours at 55°C. In the expression column, single plus “+” indicates “low expression”, two pluses “++” indicates “mild expression” and three pluses “+++” indicates “High expression” as compared to SEQ ID NO: 1.
[0125] In some embodiments, the engineered formate dehydrogenase polypeptides of the disclosure are improved as compared to an arFDH enzyme having (R) selectivity, e.g., SEQ ID NO :1, with respect to their enzymatic activity, for example, their rate of converting the substrate to product. The polypeptide having the SEQ IND NO: 1 is used herein as a reference polypeptide because the arFDH do not exhibit the appreciable conversion for converting phenylpyruvic acid to (R)-phenyllactic acid. In some embodiments, the engineered formate dehydrogenase polypeptides are capable of converting the substrate to product at a rate that is at least 5-fold, 10-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold or 250-fold over the rate of SEQ ID NO: 1. In some embodiments, the engineered formate dehydrogenases polypeptide are capable of converting the substrate to product with a conversion percentage of at least 10%, 50%, 95%, or 99.9% of the rate of SEQ ID NO: 1.
[0126] In some embodiment, the engineered formate dehydrogenase polypeptide of the disclosure is capable of converting phenylpyruvic acid to (R)-phenyllactic acid with an stereomeric excess greater than 90% and conversion is improved over the arFDH polypeptide having the sequence of SEQ ID NO: 1. Exemplary polypeptides, that are improved over SEQ ID NO: 1 with respect to enzymatic activity, include but are not limited to, polypeptides comprising amino acid sequences corresponding to SEQ ID NO: 1,3,7, 10, 18, 36, 58, 80, 87,91, 94, 100, 153, 198, and 200.
[0127] In some embodiment, the engineered formate dehydrogenase polypeptide of the disclosure is capable of converting phenylpyruvic acid to (R)-phenyllactic acid with an stereomeric excess greater than 99% and conversion is improved over the arFDH polypeptide having the sequence of SEQ ID NO: 1. Exemplary polypeptides, that are improved over SEQ ID NO: 1 with respect to enzymatic activity, include but are not limited to, polypeptides comprising amino acid sequences corresponding to SEQ ID NO: 7, 36, 80, 100, 153 and 200.
[0128] In some embodiment, the engineered formate dehydrogenase polypeptide of the disclosure is capable of converting phenylpyruvic acid to (R)-phenyllactic acid with an stereomeric excess greater than 99% and conversion of at least 95% over the arFDH polypeptide having the sequence of SEQ ID NO: 1. Exemplary polypeptides, that are improved over SEQ ID NO: 1 with respect to enzymatic activity, include but are not limited to, polypeptides comprising amino acid sequences corresponding to SEQ ID NO: 7, 36, 80, 100, 153, and 200.
[0129] In some embodiments, the engineered formate dehydrogenase polypeptides of the disclosure are improved as compared to an arFDH enzyme having (R) selectivity, e.g., SEQ ID NO :1, with respect to their enzymatic activity, for example, their rate of converting the substrate to product. The polypeptide having the SEQ IND NO: 1 is used herein as a reference polypeptide because the arFDH do not exhibit the appreciable conversion for converting 2-oxo-4-phenylbutyric acid to (R)-2-Hydroxy-4-phenylbutyric acid. In some embodiments, the engineered formate dehydrogenase polypeptides are capable of converting the substrate to product at a rate that is at least 5-fold, 10-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold or 250-fold over the rate of SEQ ID NO: 1. In some embodiments, the engineered formate dehydrogenases polypeptide are capable of converting the substrate to product with a conversion percentage of at least 10%, 50%, 95%, or 99.9% of the rate of SEQ ID NO: 1.
[0130] In some embodiment, the engineered formate dehydrogenase polypeptide of the disclosure is capable of converting 2-oxo-4-phenylbutyric acid to (R)-2-Hydroxy-4-phenylbutyric acid with an stereomeric excess greater than 90% and conversion is improved over the arFDH polypeptide having the sequence of SEQ ID NO: 1. Exemplary polypeptides, that are improved over SEQ ID NO: 1 with respect to enzymatic activity, include but are not limited to, polypeptides comprising amino acid sequences corresponding to SEQ ID NO: 1, 4, 6, 13, 21, 24, 38, 42, 68, 110, 122, 126, 142, 198, and 200.
[0131] In some embodiment, the engineered formate dehydrogenase polypeptide of the disclosure is capable of converting 2-oxo-4-phenylbutyric acid to (R)-2-Hydroxy-4-phenylbutyric acid with an stereomeric excess greater than 99% and conversion is improved over the arFDH polypeptide having the sequence of SEQ ID NO: 1. Exemplary polypeptides, that are improved over SEQ ID NO: 1 with respect to enzymatic activity, include but are not limited to, polypeptides comprising amino acid sequences corresponding to SEQ ID NO: 6, 38, 68, 126, and 200.
[0132] In some embodiment, the engineered formate dehydrogenase polypeptide of the disclosure is capable of converting 2-oxo-4-phenylbutyric acid to (R)-2-Hydroxy-4-phenylbutyric acid with an stereomeric excess greater than 99% and conversion of at least 95% over the arFDH polypeptide having the sequence of SEQ ID NO: 1. Exemplary polypeptides, that are improved over SEQ ID NO: 1 with respect to enzymatic activity, include but are not limited to, polypeptides comprising amino acid sequences corresponding to SEQ ID NO: 38, 68, 126 and 200.
[0133] In some embodiments, the engineered formate dehydrogenase polypeptides of the disclosure are improved as compared to an arFDH enzyme having (S) selectivity, e.g., SEQ ID NO :1, with respect to their enzymatic activity, for example, their rate of converting the substrate to product. The polypeptide having the SEQ IND NO: 1 is used herein as a reference polypeptide because the arFDH do not exhibit the appreciable conversion for converting 4-hydroxyphenylpyruvic acid to convert into (2S)-2-hydroxy-3-(4-hydroxyphenyl)propanoic acid. In some embodiments, the engineered formate dehydrogenase polypeptides are capable of converting the substrate to product at a rate that is at least 5-fold, 10-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold or 250-fold over the rate of SEQ ID NO: 1. In some embodiments, the engineered formate dehydrogenases polypeptide are capable of converting the substrate to product with a conversion percentage of at least 10%, 50%, 95%, or 99.9% of the rate of SEQ ID NO: 1.
[0134] In some embodiment, the engineered formate dehydrogenase polypeptide of the disclosure is capable of converting 4-hydroxyphenylpyruvic acid to convert into (2S)-2-hydroxy-3-(4-hydroxyphenyl)propanoic acid with an stereomeric excess greater than 90% and conversion is improved over the arFDH polypeptide having the sequence of SEQ ID NO: 1. Exemplary polypeptides, that are improved over SEQ ID NO: 1 with respect to enzymatic activity, include but are not limited to, polypeptides comprising amino acid sequences corresponding to SEQ ID NO: 1, 4, 5, 6, 8, 20, 24, 34, 38, 41, 86, 91, 127, 198, 199 and 200.
[0135] In some embodiment, the engineered formate dehydrogenase polypeptide of the disclosure is capable of converting 4-hydroxyphenylpyruvic acid to convert into (2S)-2-hydroxy-3-(4-hydroxyphenyl)propanoic acid with an stereomeric excess greater than 99% and conversion is improved over the arFDH polypeptide having the sequence of SEQ ID NO: 1. Exemplary polypeptides, that are improved over SEQ ID NO: 1 with respect to enzymatic activity, include but are not limited to, polypeptides comprising amino acid sequences corresponding to SEQ ID NO: 8, 24, 41, 86, 198 and 200.
[0136] In some embodiment, the engineered formate dehydrogenase polypeptide of the disclosure is capable of converting 4-hydroxyphenylpyruvic acid to convert into (2S)-2-hydroxy-3-(4-hydroxyphenyl)propanoic acid with an stereomeric excess greater than 99% and conversion of at least 95% over the arFDH polypeptide having the sequence of SEQ ID NO: 1. Exemplary polypeptides, that are improved over SEQ ID NO: 1 with respect to enzymatic activity, include but are not limited to, polypeptides comprising amino acid sequences corresponding to SEQ ID NO: 8, 86, 198 and 200.
[0137] In some embodiments, the engineered formate dehydrogenase polypeptides of the disclosure are improved as compared to an arFDH enzyme having (S) selectivity, e.g., SEQ ID NO :1, with respect to their enzymatic activity, for example, their rate of converting the substrate to product. The polypeptide having the SEQ IND NO: 1 is used herein as a reference polypeptide because the arFDH do not exhibit the appreciable conversion for converting pyruvate to convert into (S)-lactate. In some embodiments, the engineered formate dehydrogenase polypeptides are capable of converting the substrate to product at a rate that is at least 5-fold, 10-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold or 250-fold over the rate of SEQ ID NO: 1. In some embodiments, the engineered formate dehydrogenases polypeptide are capable of converting the substrate to product with a conversion percentage of at least 10%, 50%, 95%, or 99.9% of the rate of SEQ ID NO: 1.
[0138] In some embodiment, the engineered formate dehydrogenase polypeptide of the disclosure is capable of converting pyruvate to convert into (S)-lactate with an stereomeric excess greater than 90% and conversion is improved over the arFDH polypeptide having the sequence of SEQ ID NO: 1. Exemplary polypeptides, that are improved over SEQ ID NO: 1 with respect to enzymatic activity, include but are not limited to, polypeptides comprising amino acid sequences corresponding to SEQ ID NO: 1, 7, 8, 10, 13, 18, 22, 33, 36, 70, 108, 154, 182, 198, and 200.
[0139] In some embodiment, the engineered formate dehydrogenase polypeptide of the disclosure is capable of converting pyruvate to convert into (S)-lactate with an stereomeric excess greater than 99% and conversion is improved over the arFDH polypeptide having the sequence of SEQ ID NO: 1. Exemplary polypeptides, that are improved over SEQ ID NO: 1 with respect to enzymatic activity, include but are not limited to, polypeptides comprising amino acid sequences corresponding to SEQ ID NO: 10, 33, 36, 182, and 200.
[0140] In some embodiment, the engineered formate dehydrogenase polypeptide of the disclosure is capable of converting pyruvate to convert into (S)-lactate with an stereomeric excess greater than 99% and conversion of at least 95% over the arFDH polypeptide having the sequence of SEQ ID NO: 1. Exemplary polypeptides, that are improved over SEQ ID NO: 1 with respect to enzymatic activity, include but are not limited to, polypeptides comprising amino acid sequences corresponding to SEQ ID NO: : 13, 36, 182, and 200.
[0141] In some embodiments, the engineered formate dehydrogenase polypeptides of the disclosure are improved as compared to an arFDH enzyme having efficient cofactor recycling efficiency for the conversion of NADP+ to NADPH or NAD+ to NADH , with using a co-substrate sodium formate to carbon dioxide conversion, e.g., SEQ ID NO :1, with respect to their enzymatic activity, for example, their rate of converting the substrate to product. The polypeptide having the SEQ IND NO: 1 is used herein as a reference polypeptide because the arFDH do not exhibit the appreciable conversion for sodium formate to carbon dioxide for the co-factor recycling of NADP+ to NADPH or NAD+ to NADH. In some embodiments, the engineered formate dehydrogenase polypeptides are capable of converting the substrate to product at a rate that is at least 5-fold, 10-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold or 250-fold over the rate of SEQ ID NO: 1. In some embodiments, the engineered formate dehydrogenases polypeptide are capable of converting the substrate to product with a conversion percentage of at least 10%, 50%, 95%, or 99.9% of the rate of SEQ ID NO: 1. In some embodiments, the engineered formate dehydrogenases polypeptide are capable of converting the substrate to product at a rate that is at least 10-20%, 60-80%, 100-120% or 150-200% of the rate of SEQ ID NO: 1.
[0142] In some embodiment, the engineered formate dehydrogenase polypeptide of the disclosure is capable of converting sodium formate to carbon dioxide for the co-factor recycling NADP+ to NADPH or NAD+ to NADH with activity greater than 50 % over the arFDH polypeptide having the sequence of SEQ ID NO: 1. Exemplary polypeptides, that are improved over SEQ ID NO: 1 with respect to enzymatic activity, include but are not limited to, polypeptides comprising amino acid sequences corresponding to SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, and 200.
[0143] In some embodiment, the engineered formate dehydrogenase polypeptide of the disclosure is capable of converting sodium formate to carbon dioxide for the co-factor recycling NADP+ to NADPH or NAD+ to NADH with activity greater than 90% over the arFDH polypeptide having the sequence of SEQ ID NO: 1. Exemplary polypeptides, that are improved over SEQ ID NO: 1 with respect to enzymatic activity, include but are not limited to, polypeptides comprising amino acid sequences corresponding to SEQ ID NO: 15, 59, 61, 80, 87, 90, 92, 94, 97, 99, 100, 105, 120, 144, 176, 177, 180, 184, 186, 198, 199 and 200.
[0144] In some embodiment, the engineered formate dehydrogenase polypeptide of the disclosure is capable of converting sodium formate to carbon dioxide for the co-factor recycling NADP+ to NADPH or NAD+ to NADH with conversion greater than 99.0% over the arFDH polypeptide having the sequence of SEQ ID NO: 1. Exemplary polypeptides, that are improved over SEQ ID NO: 1 with respect to enzymatic activity, include but are not limited to, polypeptides comprising amino acid sequences corresponding to SEQ ID NO: 97, 177, 186, 198, 199, and 200.
[0145] In some embodiment, the engineered formate dehydrogenase polypeptide of the disclosure is capable of converting in less than about 24 hours at least about 90% of the substrates phenylpyruvic acid, 2-oxo-4-phenylbutyric acid, 4-hydroxyphenylpyruvic acid, pyruvate and sodium formate is converted into (R)-phenyllactic acid, (R)-2-Hydroxy-4-phenylbutyric acid, (2S)-2-hydroxy-3-(4-hydroxyphenyl)propanoic acid, (S)-lactate and Carbon dioxide respectively, in at least 99.9% conversion, and enantiomeric excess of at least about 99% (where it’s applicable to substrates), when carried out with the polypeptide at an amount of less than about 1% by weight with respect to the amount of substrates. Exemplary polypeptides that have this capability include, but are not limited to, polypeptide comprising amino acid sequences corresponding to SEQ ID NO: 1, 4, 5, 7, 8, 10 13, 15, 20, 21, 22, 34, 36, 38, 42, 36, 58, 59, 61, 68, 70, 80, 86, 87, 90, 91, 92, 94, 97, 99, 100, 105, 108, 110, 120, 122, 126, 127, 142, 144, 153, 154, 176, 177, 180, 182, 184, 186, 198, 199, 200,
[0146] In some embodiment, the engineered formate dehydrogenase polypeptide of the disclosure is capable of converting substrates phenylpyruvic acid, 2-oxo-4-phenylbutyric acid, 4-hydroxyphenylpyruvic acid, pyruvate and sodium formate is converted into (R)-phenyllactic acid, (R)-2-Hydroxy-4-phenylbutyric acid, (2S)-2-hydroxy-3-(4-hydroxyphenyl)propanoic acid, (S)-lactate and Carbon dioxide respectively with an enantiomeric excess of at least about 90% (where it’s applicable to substrates) and at a rate that is at least about 200% greater than the artificial formate dehydrogenase polypeptide having the SEQ ID NO: 1, wherein the polypeptide is also capable, after a heat treatment of 55°C for 2 hours, of converting the substrate to the product at a rate that is at least about 100% greater than the polypeptide having the sequence of SEQ ID NO: 1 (where the polypeptide of SEQ ID NO: 1 was also treated with the same heat treatment). Exemplary polypeptides having such properties include, but are not limited to, polypeptide comprising amino acid sequences corresponding to SEQ ID NO: 6, 10, 12, 26, 32, 42, 54, 60, 78, 84, 100, 106, 121, 129, 131, 149, 156, 160, 172, 182, 198, 199, and 200.
[0147] In some embodiment, the engineered formate dehydrogenase polypeptide of the disclosure is capable of converting substrates phenylpyruvic acid, 2-oxo-4-phenylbutyric acid, 4-hydroxyphenylpyruvic acid, pyruvate and sodium formate is converted into (R)-phenyllactic acid, (R)-2-Hydroxy-4-phenylbutyric acid, (2S)-2-hydroxy-3-(4-hydroxyphenyl)propanoic acid, (S)-lactate and Carbon dioxide respectively with an enantiomeric excess of at least about 90% (where it’s applicable to substrates) and at a rate that is at least about 200% greater than the artificial formate dehydrogenase polypeptide having the SEQ ID NO: 1, wherein the polypeptide is also capable, after a treatment of 2 hr in a pH range from 4.0-9.0, of converting the substrate to the product at a rate that is at least about 100% greater than the polypeptide having the sequence of SEQ ID NO: 1 (where the polypeptide of SEQ ID NO: 1 was also treated with the same heat treatment). Exemplary polypeptides having such properties include, but are not limited to, polypeptide comprising amino acid sequences corresponding to SEQ ID NO: 6, 10, 12, 26, 32, 42, 54, 60, 78, 84, 100, 106, 121, 129, 131, 149, 156, 160, 172, 182, 198, 199, and 200.
[0148] In some embodiments, the engineered formate dehydrogenase polypeptide is capable of stereo selectively reducing the substrate to the product with a percent e.e. that is at least about 90%, where the polypeptide that is at least about 90%, where the polypeptide comprises an amino acids sequences corresponding to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, and 200.
[0149] In another aspect, the present disclosure provides a host cell comprising a polynucleotide encoding an improved formate dehydrogenase polypeptide of the present disclosure, the polynucleotide being operatively linked to one or more control sequences for expression of the formate dehydrogenase enzyme in the host cell. Host cells for use in expressing the formate dehydrogenase polypeptides encoded by the expression vectors of the present invention are well known in the art and include but are not limited to, bacterial cells, such as E.coli, Saccharomyces cerevisiae, and Komagataella pastoris. Appropriate culture mediums and growth conditions for the above-described host cells are well known in the art.
[0150] Polynucleotides for expression of the Ketoreductases may be introduced into cells by various methods known in the art. Techniques include among others, electroporation, biolistic particle bombardment, liposome mediated transfection, calcium chloride transfection, and protoplast fusion. Various methods for introducing polynucleotides into cells will be apparent to the skilled artisan.
[0151] An exemplary host cell is E. coli BL21. The expression vector was created by operatively linking a polynucleotide encoding an improved formate dehydrogenase into the plasmid pET28b(+) operatively linked to the T7 promoter under the control of lacI repressor. The expression also contained the pBR322 origin of replication and the Kanamycin resistance gene. Cells containing the subject polynucleotide in E. coli BL21 can be isolated by subjecting the cells to kanamycin selection.
[0152] In some embodiments, to make the improved formate dehydrogenase polynucleotide and polypeptide of the present disclosure, the artificial formate dehydrogenase that catalyses the reduction reaction is obtained from computationally designed method. In some embodiments, the artificial formate dehydrogenase polynucleotide is codon optimized to enhance the expression of formate dehydrogenase in a specified host cell. As an illustration, the artificial formate dehydrogenase polynucleotide sequence encoding the formate dehydrogenase polypeptide was constructed from codon optimization methods known in the art. The artificial formate dehydrogenase polynucleotide sequence, designated as SEQ ID NO: 201, was codon optimized for expression in E. coli and the codon optimized polynucleotide sequence is cloned into an expression vector, placing the expression of the formate dehydrogenase gene under the control of the T7 promoter and lacI repressor gene. Clones expressing the active formate dehydrogenase in E. coli were identified and the gene sequenced to confirm their identity. The artificial formate dehydrogenase sequence designated (SEQ ID NO: 201) was the parent sequence utilized as the starting point for most experiments and library construction of engineered formate dehydrogenase evolved from the artificial formate dehydrogenase. The engineered formate dehydrogenase can be obtained by subjecting the polynucleotide encoding the artificial formate dehydrogenase to mutagenesis as discussed above.
[0153] The clones obtained following mutagenesis treatment are screened for engineered formate dehydrogenase having a desired improved enzyme property. Measuring enzyme activity from the expression libraries can be performed using the standard biochemistry technique of monitoring the rate of decrease (via a decrease in absorbance or fluorescence) of NAD(P)+ or NAD(P)H concentration, as it is converted into NAD(P)+ (For example, see Example 8, 9, 10, 11, 12, 13, 14, 15, 16). In this reaction, the NAD(P)H is consumed (oxidized) by the formate dehydrogenase by formate dehydrogenase as the engineered formate dehydrogenase reduces a ketone substrate to the corresponding hydroxyl group. The rate of decrease of NAD(P)H concentration, as measured by the decrease in absorbance of fluorescence, per unit time indicates the relative enzymatic activity of formate dehydrogenase polypeptide in a fixed amount of the lysate (or a lyophilized powder made therefrom). The stereochemistry of the product can be ascertained by various known techniques, and as provided in the Examples. Where the improved enzyme property desired is thermal stability, enzyme activity may be measured after subjecting the enzyme preparations to a defined temperatures and measuring the amount of enzyme activity remaining after heat treatments. Clones containing a polynucleotide encoding a ketoreductase are then isolated, sequenced to identify the nucleotides sequence changes (if any), and used to express the enzyme in a host cell.
[0154] The engineered formate dehydrogenase enzyme expressed in a host cell can be recovered from the cells and or the culture medium using any one or more of the well-known techniques for protein purification, including among others, lysozyme treatment, sonication, filtration, salting-out, ultracentrifugation, and chromatography. Suitable solutions for lysing and the high efficiency extraction of proteins from bacteria, such as E. coli, are commercially available.
[0155] In some embodiment, the arFDH polypeptide is obtained by conventionally known methods for protein production. Specifically, a method for producing arFDH gene comprises a preparation of a host organism E. coli BL21 (DE3) and introducing through a expression vector, in which arFDH gene is arranged under the control of inducible promoters between the restriction sites (Fig. 2). Followed by culturing the host, inducing the expression of the arFDH gene after the logarithmic growth phase, culturing the host at a temperature that is lower than the optimum temperature for the growth of the host cells and allows the survival of the host, and thus causing the expression of the arFDH within the host. As an inducible promoter to be used in the method for producing the engineered arFDH, any other conventional promoters can also be used without limitations. As an illustration, when the expression host E. coli is utilized, an inducible promoter that can be activated by the presence of isopropyl-D-1-thiogalactopyranoside (IPTG) can activate transcription. The promoters T7, Trp, Lac, Trc, and Tac are a few examples of this type.
[0156] Chromatographic techniques for isolation of the formate dehydrogenase polypeptide include, among other, reverse phase chromatography, high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, and affinity chromatography. Conditions for purifying a particular enzyme will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., and will be apparent to those having skill in the art.
[0157] In some embodiments, the formate dehydrogenase may be prepared and used in the form of cells expressing the enzymes, as crude extracts, or as isolated or purified preparations. The formate dehydrogenase may be prepared as lyophilizates, in powder form (e.g., acetone powders), or prepared as enzyme solutions. In some embodiments, the formate dehydrogenases can be in the form of substantially pure preparations.
[0158] In some embodiments, the formate dehydrogenase polypeptides can be attached to a solid substrate. The substrate can be a solid phase, surface, and/or membrane. A solid support can be composed of organic polymers such as polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy, and polyacrylamide, as well as co-polymers and grafts thereof. A solid support can also be inorganic, such as glass, silica, controlled pore glass (CPG), reverse phase silica or metal, such as gold or platinum. The configuration of the substrate can be in the form of beads, spheres, particles, granules, a gel, a membrane or a surface. Surfaces can be planar, substantially planar, or non-planar. Solid supports can be porous or non-porous and can have swelling or non-swelling characteristics. A solid support can be configured in the form of a well, depression, or other container, vessel, feature, or location. A plurality of support can be configured on an array at various locations, addressable for robotic delivery of reagents, or by detection methods and/or instruments.
[0159] In some embodiments of the method, at least about 90% of the substrate is reduced to the product in at least about 90% of the enantiomeric excess in less than 24 hours, when the method is conducted with at least about 200g/L of the substrate and less than about 1g/L of the formate dehydrogenase polypeptide, wherein the formate dehydrogenase polypeptide used in the method comprises an amino acid sequences selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, and 200.
[0160] As is known by those of skill in the art, formate dehydrogenase catalysed reduction reaction typically requires a cofactor. Reduction reactions catalysed by the engineered formate dehydrogenase described herein also typically require a cofactor, although many embodiments of the engineered formate dehydrogenase require far less co-factor than reactions catalysed with the artificial formate dehydrogenase enzyme. As used herein, the term “cofactor” refers to a non-protein compound that operates in combination with a formate dehydrogenase enzyme. Cofactors suitable for use with the engineered formate dehydrogenase enzymes described herein include, but are not limited to, NADP+ (nicotinamide adenine dinucleotide phosphate), NADPH (the reduced form of NAD+), NAD+ (nicotinamide adenine dinucleotide) and NADH (the reduced form of NAD+). Generally, the reduced form of the co-factor is added to the reaction mixture. The reduced NAD(P)H form is regenerated from the oxidised NAD(P)+ form using an engineered formate dehydrogenase as a co-factor regeneration system.
[0161] The term “cofactor regeneration system” refers to a set of reactants that participate in a reaction that reduces the oxidized form of the cofactor (e.g., NADP+ to NADPH). Cofactors oxidized by the formate dehydrogenased-catalysed reduction of the keto substrate are regenerated in reduced form by the cofactor regeneration system. Co-factors regeneration systems comprise a stoichiometric reductant that is a source of reducing hydrogen equivalents and is capable of reducing the oxidized form of the cofactor. The cofactor regeneration system may further comprise a catalyst, for example an enzyme catalysts, that catalyses the reduction of the oxidized form of the co-factor by the reductant. Co-factor regeneration systems to regenerate NADH or NADPH from NAD+ or NADP+, respectively, are known in the art and is used in the methods described herein.
[0162] The formate dehydrogenase-catalysed reduction reaction described herein are generally, carried out in a solvent. Suitable solvents including water, organic solvents (e.g., isopropanol, DMSO, Acetone, DMF, ethyl acetate, butyl acetate, 1-octanol, heptane, octane, methyl t-butyl ether (MTBE), toluene, and the like), ionic liquids (1-ethyl 4-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium hexaflurophosphate, and the like). In some embodiments aqueous solvents, including water and aqueous co-solvent systems, are used.
[0163] Exemplary, aqueous co-solvent systems have water and one or more organic solvent. In general, an organic solvent component of an aqueous co-solvent system is selected such that it does not completely inactivate the formate dehydrogenase enzyme. Appropriate co-solvent systems can be readily identified by measuring the enzymatic activity of the specified engineered formate dehydrogenase enzyme with a defined substrate of interests in the candidate solvent system, utilizing an enzyme activity assay, such as those described herein.
[0164] The organic solvent components of an aqueous co-solvent system may be miscible with the aqueous component, providing a single liquid phase, or may be partly miscible or immiscible with the aqueous component, providing two liquid phases. Generally, when an aqueous co-solvent system is employed, it is selected to be biphasic, with water dispersed in an organic solvent, or vice versa. Generally, when an aqueous co-solvent system is utilized, it is desirable to select an organic solvent that can be readily separated from the aqueous phase. In general, the ratio of water to organic solvent in the co-solvent system is typically in the range of from about 90:10 to about 10:90 (v/v) organic solvent to water, and between 80:20 and 20:80 (v/v) organic solvent to water. The co-solvent system may be pre-formed prior to addition to the reaction mixture, or it may be formed in-situ in the reaction vessel.
[0165] The aqueous solvent (water or aqueous co-solvent system) may be pH-buffered or unbuffered. Generally, the reduction can be carried out at a pH of about 10 or below, usually in the range of from about 5 to about 10. In some embodiments, the reduction is carried out at a pH of about 9 or below, usually in the range of from about 5 to about 9. In some embodiments, the reduction is carried out at a pH of about 8 or below, often in the range of from about 5 to about 8 and usually in the range of from about 6 to about 8. The reduction may also be carried out at a pH of about 7.8 or below, or 7.5 or below. Alternatively, the reduction may be carried out a neutral pH, i.e., about 7.
[0166] During the course of the reduction reactions, the pH of the reaction mixture may change. The pH of the reaction mixture may be maintained at a desired pH or within a desired pH range by the addition of an acid or a base during the course of the reaction. Alternatively, the pH may be controlled by using an aqueous solvent that comprises a buffer. Suitable buffers to maintain desired pH ranges are known in the art and include, for example, phosphate buffer, triethanolamine buffer, and the like. Combination of buffering and acid or base addition may also be used.
EXAMPLES
Example 1
[0167] Expression and Purification of Formate Dehydrogenase (FDH)
[0168] The pET28a(+) vector was used to transform CaCl2 competent Escherichia coli BL21 (DE3) cells. Cultures were grown in agitation at 37 °C. A 100-ml over-night pre-culture of transformed cells were inoculated in 2 L of LB medium, 50 µg/ml kanamycin. Once the culture had reached OD600 = 1, protein expression was induced by 0.1 mM isopropyl ß-d-1-thiogalactopyranoside (IPTG) and cells were further grown at 18 °C for 20 h. They were harvested by centrifugation at 6000g for 10 min. The pellet was washed twice in the washing buffer (20 mM phosphate buffer, 0.5 M NaCl pH 7.4) and resuspended in 30 ml of the same buffer. Cells were lysed using a French press (Constant Systems Cell Disruptor OneShot; Constant Systems, Kennesaw, GA, USA) set at 1.35 KPa; debris were removed by centrifugation at 20,000g for 20 min and the supernatant was loaded onto a 10-ml Ni-NTA column (GE Healthcare, Munich, Germany), pre-equilibrated with the washing buffer. Elution was performed in the elution buffer (20 mM phosphate buffer, 0.5 M NaCl, 0.250 M imidazole pH 7.4) and 1-ml fractions were harvested. Fractions with Abs280 higher than 0.4 were combined and loaded on a pre-equilibrated gel filtration Superdex 200 column (GE Healthcare, Munich, Germany). The protein expressions were checked using SDS-PAGE (Fig. 7) and protein concentrations were determined using Bradford assay (Table 13).
Example 2
[0169] Generations of Mutations
[0170] All mutations were introduced by using the QuikChange® site-directed mutagenesis kit (Stratagene, Agilent Technologies Company, La Jolla, CA, USA) according to the manufacturer’s instructions. A pET-28a(+)-arFDH vector generated was used as template. DNA in the first rounds of mutagenic PCR. In this vector, the arfdh gene is cloned between NcoI and XhoI restriction sites. Mutations were combined by consecutive rounds of site-directed mutagenesis. All mutations were confirmed by sequencing.
Example 3
[0171] Screening of the mutant library:
[0172] Colonies were randomly picked into the primary 96-deep well plates containing LB Medium (300uL) with ampicillin (50µg/mL) per well. After incubated at 37°C , 800rpm overnight, the preculture (20µL) was transferred from the primary plate to the secondary 96-deep well plate containing LB medium (430µL) with ampicillin (50µg/mL) and was further incubated at 37°C, 800 rpm for 2 to 3 h, then the isopropyl-ß-D-thiogalactopyranoside (IPTG) was added to the culture plates with the final concentration of 0.2 mM to induce protein expression at 16°C for 24 h. Next, the cells were harvested by centrifugation at 2500 x g, 4°C for 15 min and then were resuspended in 200 µL lysis buffer (lysozyme, 750 mg/L, DNase,10 mg/L, potassium phosphate, 100 mM, pH 7.5), the suspension was further incubated at 30°C, 800 rpm for 2 h. A clear cell-free extract was obtained by centrifugation at 2500 x g, 4°C for 10 min. An aliquot (100 µL) of the supernatant was used to evaluate the mutant’s reactivity with NAD(P)+ by mixing with 100 µL of screening buffer (sodium formate, 0.2 M; NAD(P)+, 2 mM; potassium phosphate, 100 mM, pH 7.5). The concentration of the NAD(P)+ in the screening buffer and the addition amount of cell-free extract was stepwise decreased to 0.5 mM and 20 µL with the increased activity of the engineered FDH variants, respectively. The NAD(P)+ reduction was monitored at 340 nm, 30°C for 5 min. Positive hits with higher 340 nm absorbance change were sequenced, cultured by shake flask fermentation, and subjected to Ni-affinity chromatography for further characterization. The protein expressions of variant were checked using SDS-PAGE (Fig. 7) and protein concentrations were determined using Bradford assay (Table 13).
Example 4
[0173] Activity Assay of arFDH for substrate Phenylpyruvic acid
[0174] arFDH was assayed in 0.1 M phosphate buffer (pH 7.0) containing 300 mM phenylpyruvic acid, 0.1mM NAD(P)+ sodium formate as a co-substrate (1.5 eq. of substrate), at 37°C. By altering the quantities of one substrate at a fixed and saturating concentration of the second, kinetic constants were determined from duplicate or triple measurements of starting rates. The thermostability of FDH was examined by incubation of 0.2 mg/ml protein solution in 0.1 M phosphate buffer (pH 7.0) containing 0.01 M EDTA at 60.5°C.
Example 5
[0175] Activity Assay of arFDH for substrate 2-oxo-4-phenylbutyric acid
[0176] arFDH was assayed in 0.1 M phosphate buffer (pH 7.0) containing 300 mM 2-oxo-4-phenylbutyric acid, 0.1mM NAD(P)+ sodium formate as a co-substrate (1.5 eq. of substrate), at 37°C. By altering the quantities of one substrate at a fixed and saturating concentration of the second, kinetic constants were determined from duplicate or triple measurements of starting rates. The thermostability of FDH was examined by incubation of 0.2 mg/ml protein solution in 0.1 M phosphate buffer (pH 7.0) containing 0.01 M EDTA at 60.5°C.
Example 6
[0177] Activity Assay of arFDH for substrate 4-hydroxyphenylpyruvic acid
[0178] arFDH was assayed in 0.1 M phosphate buffer (pH 7.0) containing 300 mM 4-hydroxyphenylpyruvic acid, 0.1mM NAD(P)+ sodium formate as a co-substrate (1.5 eq. of substrate), at 37°C. By altering the quantities of one substrate at a fixed and saturating concentration of the second, kinetic constants were determined from duplicate or triple measurements of starting rates. The thermostability of FDH was examined by incubation of 0.2 mg/ml protein solution in 0.1 M phosphate buffer (pH 7.0) containing 0.01 M EDTA at 60.5°C.
Example 7
[0179] Activity Assay of arFDH for substrate pyruvate
[0180] arFDH was assayed in 0.1 M phosphate buffer (pH 7.0) containing 300 mM pyruvate, 0.1mM NAD(P)+ sodium formate as a co-substrate (1.5 eq. of substrate), at 37°C. By altering the quantities of one substrate at a fixed and saturating concentration of the second, kinetic constants were determined from duplicate or triple measurements of starting rates. The thermostability of FDH was examined by incubation of 0.2 mg/ml protein solution in 0.1 M phosphate buffer (pH 7.0) containing 0.01 M EDTA at 60.5°C.
Example 8
[0181] Activity Assay of arFDH in the presence of NADP+ and NAD+
[0182] The engineered formate dehydrogenase (arFDH) activity was measured in a 1.4-mL quartz cuvette containing 1 mL of the assay mixture at 30°C by monitoring the change of absorbance at 340 nm within 1 min on a UV-spectrometer. The reaction mixture (1 mL) contained potassium phosphate buffer (100mM, pH 7.5), sodium formate (500mM), NADP+/NAD+ (0.5mM), and appropriately diluted purified formate dehydrogenase variants. One unit of enzyme activity was defined as the amount of FDH required for the formation of 1µM NAD(P)H in 1 min. The concentration of sodium formate for Km determination were varied from 10mM to 800mM in a potassium phosphate buffer (100 mM, pH 7.5) containing 1mM NAD(P)+. Similarly, the concentrations of NAD(P)+ for the measurement of Km were varied from 0.1 mM to 10 mM in potassium phosphate buffer (100 mM, pH 7.5) containing 500 mM sodium formate. The reaction mixture was incubated at 30°C, then monitored the increase of the absorbance at 340 nm.
Example 9
[0183] Biocatalytic cofactor recycling using engineered Formate Dehydrogenase for NAD(P)H Regeneration in combination with Ketoreductase (KRED)
[0184] The biocatalytic cofactor recycling were carried out in 2-mL Eppendorf tubes containing 1 mL reaction mixture and agitated at 30°C for 24 hr. The reaction mixture contained 100 mM acetophenone, 5% (v/v) ethanol, 100 Units of KRED, 2.4 units of engineered formate dehydrogenase, potassium phosphate buffer (100mM, pH 6.0) and 150mM sodium formate. After 24 hours of incubation, the reaction mixtures were extracted with 1mL EtOAc, the organic layer was dried over anhydrous Na2SO4 analysed by chiral GC.
Example 10
[0185] Biocatalytic cofactor recycling using engineered Formate Dehydrogenase for NAD(P)H Regeneration in combination with Imine reductase (IRED)
[0186] The biocatalytic cofactor recycling were carried out in 2-mL Eppendorf tubes containing 1 mL reaction mixture and agitated at 30°C for 24 hr. The reaction mixture contained 100mM of piperideine, 0.1 mM NAD(P)+, 15% (v/v) DMSO, 2U IRED, 1.8 U engineered formate dehydrogenase, potassium phosphate buffer (100mM, pH 6.0) and 150mM sodium formate. After 24 hours of incubation, the reaction mixtures were extracted with 1mL EtOAc, the organic layer was dried over anhydrous Na2SO4 analysed by chiral GC.
Example 11
[0187] Biocatalytic cofactor recycling using engineered Formate Dehydrogenase for NAD(P)H Regeneration in combination with Baeyer-Villiger Monoxygenase (BVMO)
[0188] The biocatalytic cofactor recycling were carried out in 2-mL Eppendorf tubes containing 1 mL reaction mixture and agitated at 30°C for 24 hr. The reaction mixture contained 50mM cyclohexanone, 0.1 mM NAD(P)+, 2% (v/v) Ethanol, 1.3 U BVMO enzyme, 1.3 U engineered formate dehydrogenase, potassium phosphate buffer (100 mM, pH 7.0), 75mM sodium formate. After 24 hours of incubation, the reaction mixtures were extracted with 1mL EtOAc, the organic layer was dried over anhydrous Na2SO4 analysed by chiral GC.
Example 12
[0189] Biocatalytic cofactor recycling using engineered Formate Dehydrogenase for NAD(P)H Regeneration in combination with Leucine Dehydrogenase (LeuDH)
[0190] The biocatalytic reaction was carried out at 10mL scale in 50 mL flask employing LeuDH enzyme. Th reaction mixture contains 500 mM NH4Cl-NH3.H2O buffer (pH 9.5), 100 mM trimethylpyruvic acid (TMP), 200 mM sodium formate and 0.1 g of LeuDH enzyme and engineered formate dehydrogenase enzyme. The reaction mixture was incubated at 30°C and 200 rpm for 35h, and 100µL samples were periodically removed for high-performance liquid chromatography (HPLC) analysis.
Example 13
[0191] Biocatalytic cofactor recycling using engineered Formate Dehydrogenase for NAD(P)H Regeneration in combination with Amine Dehydrogenase (AmDH)
[0192] The biocatalytic reaction was carried out by adding 20µL of ketone (Cyclohexanone) to 580 µL ethyl acetate. The methylamine-HCl and cyclohexanone stock solutions were made up in water and DMSO respectively. DMSO concentrations was kept to a constant final concentration of 1% in the total reaction mixtures. The reactions were incubated at 25°C with shaking at 150 r.p.m. Reaction conditions where ammonia was the amine donor contained the following in a 3 mL total reaction volume: 10mM cyclohexanone, 12 mM ammonium formate, 3 U/mL engineered formate dehydrogenase, 0.5 mM NAD(P)H with 1mg/mL of AmDH, made up to 3 mL total volume with 2M ammonium formate buffer pH 8.0. Aliquots of 200 µL were taken every 1 h between t=0-8h and then t=24h, with t=0 time points being taken directly after the addition of enzyme. Aliquots were quenched with 20 µL of 10 M NaOH and then extracted with 600 µL ethyl acetate after which the organic layer was dried using MgSO4 and then analysed using GC.
Example 14
[0193] Biocatalytic cofactor recycling using engineered Formate Dehydrogenase for NAD(P)H Regeneration in combination with Ene-Reductase (ERED)
[0194] The biocatalytic reaction was carried out using ene-reductase and engineered formate dehydrogenase present in a conical tube together with 700µL of Tris-HCl buffer (50mM, pH 7.5) and 2-cyclohexene-1-one stock solution (100mM, 150µL). The reaction was started by adding 150µL of NAD(P)H stock solution (6.0 mM) and carried out at 40°C in a water bath with shaking. After centrifugation, 100µL samples of the supernatant were removed from the mixture and added in 900 µL of Tris-HCl buffer (50 mM, pH 7.5) to perform the activity assay. The activity was measured by high-performance liquid chromatograph (HPLC) with 100µL samples were periodically removed for analysis.
Example 15
[0195] Biocatalytic cofactor recycling using engineered Formate Dehydrogenase for NAD(P)H Regeneration in combination with Alcohol Dehydrogenase (ADH)
[0196] The biocatalytic cofactor recycling were carried out in 2-mL Eppendorf tubes containing 1 mL reaction mixture and agitated at 35°C for 12 hr. The reaction mixture contained 100 mM acetophenone, 5% (v/v) ethanol, 50 Units of ADH, 2.0 units of engineered formate dehydrogenase, potassium phosphate buffer (100mM, pH 7.0) and 150mM sodium formate. After 24 hours of incubation, the reaction mixtures were extracted with 1mL EtOAc, the organic layer was dried over anhydrous Na2SO4 analysed by chiral GC.
Example 16
[0197] Biocatalytic cofactor recycling using engineered Formate Dehydrogenase for NAD(P)H Regeneration in combination with Glucose Dehydrogenase (GDH)
[0198] The biocatalytic cofactor recycling were carried out in 2-mL Eppendorf tubes containing 1 mL reaction mixture and agitated at 30°C for 24 hr. The reaction mixture contained 100 mM acetophenone, 5% (v/v) ethanol, 50 Units of GDH, 2.5 units of engineered formate dehydrogenase, potassium phosphate buffer (100mM, pH 7.0) and 150mM sodium formate. After 24 hours of incubation, the reaction mixtures were extracted with 1mL EtOAc, the organic layer was dried over anhydrous Na2SO4 analysed by chiral GC.
Example 17
[0199] Immobilization of Engineered Formate dehydrogenase by CLEA method
[0200] The engineered formate dehydrogenase (50 U) was mixed in Tris-HCl buffer (50mM, pH 7.5, 1.5mL) and then BSA (20 mg) was added in the mixture under stirring. The ammonium sulphate stock solution (5.3 M) was prepared by dissolving solid ammonium sulphate in Tris-HCl buffer (50mM, pH 7.5), and then the stock solution was added in above enzyme solution drop by drop to reach final ammonium sulphate concentration of 4.0 M. After keeping the mixture for 60 min at 4 °C for complete precipitation of enzymes, glutaraldehyde stock solution (4.7 M) or oxidized dextran stock solution (0.4mM) was added to a final concentration of 15% (v/v). The mixture was kept at 4°C for 3h with shaking. The immobilized FDH were collected by centrifugation at 10,614 x g for 5 min and washed three times with Tris-HCl buffer (50 mM, pH 7.5). The resulting FDH-CLEAs were stored at 4°C for further use.
Example 18
[0201] Co-Immobilization of Engineered Formate dehydrogenase and KRED by CLEA method
[0202] The engineered formate dehydrogenase (50 U) and KRED (11 U) were mixed in Tris-HCl buffer (50mM, pH 7.5, 1.5mL) and then BSA (20 mg) was added in the mixture under stirring. The ammonium sulphate stock solution (5.3 M) was prepared by dissolving solid ammonium sulphate in Tris-HCl buffer (50mM, pH 7.5), and then the stock solution was added in above enzyme solution drop by drop to reach final ammonium sulphate concentration of 4.0 M. After keeping the mixture for 60 min at 4 °C for complete precipitation of enzymes, glutaraldehyde stock solution (4.7 M) or oxidized dextran stock solution (0.4mM) was added to a final concentration of 15% (v/v). The mixture was kept at 4°C for 3h with shaking. The co-immobilized FDH and KRED were collected by centrifugation at 10,614 x g for 5 min and washed three times with Tris-HCl buffer (50 mM, pH 7.5). The resulting FDH-KRED-CLEAs were stored at 4°C for further use.
Example 19
[0203] Immobilization of Engineered Formate dehydrogenase by Biomimetic immobilization (BI) method
[0204] The FDH (50 U) in a sodium phosphate buffer (50mM, pH 7.0, 1.8 mL) was mixed with 628 µL of a hydrolyzed tetramethyl orthosilicate (TMOS) solution. TMOS was hydrolyzed with hydrochloric acid (HCl, 1 mM) to a final concentration of 1.0 M. The mixture was agitated for 3 h at 4 °C. The immobilized FDH was collected by centrifuging at 6,793 x g for 5 min and washed three times with a sodium phosphate buffer (50 mM, pH 7.0). The resulting FDH were used in the subsequent experiments. Control experiments, wherein silica was prepared without adding the enzymes, were then performed.
Example 20
[0205] Co-Immobilization of Engineered Formate dehydrogenase and KRED by Biomimetic immobilization (BI) method
[0206] The KRED (14 U) and FDH (50 U) in a sodium phosphate buffer (50mM, pH 7.0, 1.8 mL) was mixed with 628 µL of a hydrolyzed tetramethyl orthosilicate (TMOS) solution. TMOS was hydrolyzed with hydrochloric acid (HCl, 1 mM) to a final concentration of 1.0 M. The mixture was agitated for 3 h at 4 °C. The co-immobilized KRED and FDHwere collected by centrifuging at 6,793 x g for 5 min and washed three times with a sodium phosphate buffer (50 mM, pH 7.0). The resulting KRED-FDH were used in the subsequent experiments. Control experiments, wherein silica was prepared without adding the enzymes, were then performed.
Example 21
[0207] Immobilization of Engineered Formate dehydrogenase by Calcium Alginate
[0208] The immobilization of engineered formate dehydrogenase was prepared by dissolving 9 g of sodium alginate in 300 ml of growth medium, and stirring until complete dissolution of the sodium alginate was achieved. The resulting solution contained 3% alginate by weight. To prevent premature gel formation, the phosphate concentration in the medium was adjusted to less than 100µM. Approximately 250 g of wet cells were thoroughly suspended in the alginate solution, and the mixture was allowed to stand to facilitate the escape of air bubbles. The yeast-alginate mixture was then dripped from a height of 20 cm into 1000 ml of crosslinking solution. (The crosslinking solution was prepared by adding an additional 0.05M of CaCl2 to the growth media. The calcium crosslinking solution was agitated using a magnetic stirrer. Gel formation occurred at room temperature upon direct contact between the sodium alginate drops and the calcium solution. Relatively small alginate beads were preferred to minimize mass transfer resistance. A diameter of 0.5-2 mm was readily achieved using a syringe and needle. The beads were fully hardened within 1-2 hours. The concentration of CaCl2 was approximately one-fourth of the strength used for enzyme immobilization, and the beads were subsequently washed with fresh calcium crosslinking solution and stored in 4°C for further use.
Example 22
[0209] Immobilization of Engineered Formate dehydrogenase by Chitosan beads
[0210] For hydrogel chitosan beads (HGBs) preparation, 20 g of chitosan was dissolved in 1 L of a mixed acid solution containing 2% (w/v) of acetic acid, 1% (w/v) of lactic acid, and 1% (w/v) of citric acid. The resulting chitosan solution was introduced dropwise into 0.8 N NaOH by a peristaltic pump. The resultant HGBs were washed with deionized water until pH became neutral. dried chitosan beads (DBs) were prepared by drying the HGBs obtained above at 60 °C for 24 h. For core–shell chitosan beads (CSB) preparation, DBs were soaked in a 0.5% (w/v) acetic acid solution for 60 s and then neutralized by the addition of an equal molar amount of NaOH. To attain high efficiency of engineered formate dehydrogenase biocatalyst, the immobilization conditions, such as pH and glutaraldehyde and engineered formate dehydrogenase concentrations, were optimized. HGBs and CSBs were activated with 0.05–8.0% (w/v) glutaraldehyde in a buffer with a pH range of 5.0–8.0 (acetate buffer pH 5.0–6.0; phosphate buffer pH 7.0–8.0) at 4 °C for 24 h. After that, the activated chitosan beads were incubated with an engineered formate dehydrogenase solution (25–800 U per g of beads; or 0.97–30.3 mg per g of beads) at 4 °C for 24 h with mild agitation. The immobilized engineered formate dehydrogenase enzymes were washed with 1 M NaCl to remove an electrostatically adsorbed enzyme and then were washed 3 times with 50 mM acetate buffer pH 5.5. The resultant immobilized engineered formate dehydrogenase enzyme was kept at 4 °C until further use. The immobilized activity and activity yield served as parameters to optimize immobilization conditions. The immobilized activity was calculated as immobilized activity (U) per gram of beads and the activity yield (%) was expressed as activity on beads (U)/[initial enzyme (U) - unbound enzyme (U)] × 100.
Example 23
[0211] Immobilization of Engineered Formate dehydrogenase by Polymeric matrix
[0212] Dilbeads (10g) were washed with methanol and treated with a solution of Polyethyleneimine (PEI) (mol. wt. 70000, 100 g/L water; 100 mL) overnight. The supernatant was decanted, and the polymer was washed with distilled water till the pH of washings reached neutrality. The polymer was dried at 60°C in oven. This polymer was further used for engineered formate dehydrogenase immobilization. The PEI modified polymer (10g) was mixed with engineered formate dehydrogenase enzyme solution (100 mL), pH of the medium was adjusted to 7.0 and shaken overnight at room temperature at 150 rpm on an orbital shaker. The supernatant was collected, and the polymer was washed with deionized water (3x 100 mL). The engineered formate dehydrogenase enzyme bound polymer was further crosslinked with dextran aldehyde. The dextran aldehyde was prepared by dissolving dextran (1.7 g) in deionized water (50 mL). Sodium periodate (3.5 g) was added while stirring. After 3h, the oxidised dextran was dialyzed in cellulose acetate membrane tube (mol. wt. cutoff 10,000) against distilled water at 4°C for 24 hr. The dialyzed solution (100 mL). The dextran aldehyde solution (10 mL) was diluted with distilled water to 100 mL and the polymer beads bearing engineered formate dehydrogenase were added. The contents were shaken at room temperature overnight at 150 rpm. The polymer was then washed with deionized water (3 x 100 mL), air dried and stored in 4 °C for further use.
Example 24
[0213] Immobilization of Engineered Formate dehydrogenase by mesoporous silica
[0214] Aqueous solutions of the Mesoporous silica (MPS) particles were prepared by dispersing 5 mg of dry mesoporous silica particles in 1ml phosphate-citrate buffer, using vortexing for 10 min at 10 rpm followed by sonication (at a power of 70 W) for 20 min in order to dissolve any particle aggregates, and a final step of vortexing for 5min. Engineered formate dehydrogenase-particle samples were prepared by mixing 20µl of engineered formate dehydrogenase solution (20mg/ml in phosphate-citrate buffer) with 200 µl of MPS solutions (5mg/ml) diluted to a final volume of 500 µl with phosphate-citrate buffer. Each sample contained a total amount of 400 µg enzyme/mg MPS. Reference samples of free Engineered formate dehydrogenase were prepared in the same way but replacing the 200 µl MPS solution with buffer. In the latter case the final protein concentration 0.8mg/ml corresponds to a volume fraction of about 0.1%, whereas the accumulation in the particle pores leads to much higher local volume fractions of 20-60%. The samples were incubated at 25ºC for 48 h during gentle stirring, and then centrifuged for 6 min. The pelleted protein-particles complexes were re-suspended and washed three times with 500 µl of phosphate citrate buffer by repeated centrifugation and resuspension. The purified MPS particles with the immobilized engineered formate dehydrogenase were finally re-suspended by adding 100 µl of buffer and vortexing for a few minutes until homogenous samples were obtained for the spectroscopic measurements and further conduction of the reaction assay.
Example 25
[0215] Immobilization of Engineered Formate dehydrogenase by Zeolite
[0216] We prepared the engineered formate dehydrogenase preparation ZSM-5 zeolites catalysts as follows. First, 100 mg of engineered formate dehydrogenase preparation was dissolved in 10 ml of acetate buffer solution (pH 5, 20 mM), and then 0.5 g of ZSM-5 zeolite was added. The mixed dispersion was continuously shaken for 12 h to ensure adequate adsorption of engineered formate dehydrogenase preparation onto the surface of the ZSM-5 support. The final engineered formate dehydrogenase preparation ZSM-5 catalyst was obtained after crosslinking the engineered formate dehydrogenase preparation on the ZSM-5 surface by immersion in a glutaraldehyde solution (0.5%) for 4 h at 25°C, with stirring at 120 r min-1. The residual un-crosslinked engineered formate dehydrogenase preparation was removed by continuous washing until no protein was detected in the washing solution. The amount of engineered formate dehydrogenase preparation immobilized on the zeolite was calculated from the difference between the initial amount of engineered formate dehydrogenase preparation and the amount of un-crosslinked engineered formate dehydrogenase preparation.
Example 26
[0217] Activity Assay for Immobilized Formate Dehydrogenase
[0218] In order to measure engineered FDH recycling activity, FDH-CLEAs or FDH-BI was mixed with 700 µL of Tris-HCl buffer (50 mM, pH 7.5) and sodium formate stock solution (440 mM, 150 µL). The reaction was started by adding 150 µL of NAD(P)+ stock solution (6.0 mM) and carried out at 40°C in a water bath with shaking. After centrifugation,100 µL samples of the supernatant were removed from the mixture and added in 900 µL of Tris-HCl buffer (50 mM, pH 7.5) to perform the activity assay. The activity was determined by measuring the increased NAD(P)H concentration at 340 nm. The absorption values were limited to a range from 0.2 to 0.8. One unit of FDH activity (U) was defined as the amount of enzyme required to generate 1µmol of NAD(P)H per minute. The immobilization efficiency (%) and activity recovery (%) of the immobilized formate dehydrogenase was calculated with the following equations.
[0219] Immobilization efficiency (%) = (Total FDH used for immobilization-total FDH content in supernatant/Total protein content used for immobilization) X 100%
[0220] Activity recovery (%) = (Total activity of immobilized FDH/Total activity of free FDH) X 100%
Example 27
[0221] Thermal inactivation study of formate dehydrogenase
[0222] The thermal stability of the enzymes was studied in a sodium-phosphate buffer (0.1 M, pH 7.0). Several Eppendorf tubes (0.5 ml) with 100µl formate dehydrogenase enzyme solution (0.25 mg/ml) were prepared for each experiment. The tubes were incubated in a water bath at different temperatures (20–55°C, ±0.1°?). At fixed time intervals, a tube was transferred from the bath to cold water (4°C) for 5 min. Then, the solution was centrifuged for 3 min at 12 000 rpm using an Eppendorf 5415D centrifuge. The residual FDH activity was measured as described previously.
Example 28
[0223] pH inactivation study of formate dehydrogenase
[0224] The pH stability of the enzymes was studied in a sodium-phosphate buffer (0.1 M) with varying pH range from 3 to 9. Several Eppendorf tubes (0.5 ml) with 100µl formate dehydrogenase enzyme solution (0.25 mg/ml) were prepared for each experiment. The tubes were incubated in a water bath at temperatures of 30°C. At fixed time intervals, a tube was transferred from the bath to cold water (4°C) for 5 min. Then, the solution was centrifuged for 3 min at 12 000 rpm using an Eppendorf 5415D centrifuge. The residual FDH activity was measured as described previously.
Example 29
[0225] Comparative Activity Assay of arFDH and Commercial FDH in the presence of NAD(P)+
[0226] The engineered formate dehydrogenase (arFDH) and commercial FDH activity was measured in a 1.4-mL quartz cuvette containing 1 mL of the assay mixture at 20-22°C by monitoring the change of absorbance at 340 nm within 1 min on a UV-spectrometer. The reaction mixture (1 mL) contained potassium phosphate buffer (100mM, pH 7.0), sodium formate (162mM), NAD(P)+ (1.62mM), and appropriately diluted purified formate dehydrogenase variants and commercial FDH of 5µL of a 2 mg/mL enzyme solution to 0.5mL of the phosphate buffer. One unit of enzyme activity was defined as the amount of FDH required for the formation of 1µM NAD(P)H in 1 min. The reaction mixture was incubated at 20-22°C, then monitored the increase of the absorbance at 340 nm.
Example 30
[0227] Cell Transformation Procedure for arFDH expression in BL21
[0228] The transformation protocol involves adding 40 µl of BL21 DE3 competent cells to a 1.5 ml tube, followed by the addition of 1 µl of plasmid DNA from the plasmid tube. The mixture is then incubated on ice for 30 minutes, after which the tube is placed in a water bath at 42°C for 60 seconds and subsequently returned to ice for 5 minutes. Following this, 850 µl of plain LB broth is added to the tube, and the mixture is incubated at 37°C in a shaker incubator for 1 hour. The cells are then pelleted at 3500 RPM for 5 minutes, and the supernatant is decanted, leaving 50 µl of media for resuspension. The cells are then resuspended in the remaining LB broth and plated onto LB agar plates containing kanamycin (50 µg/ml). For master plate streaking, single isolated colonies are picked from the transformation plate and streaked onto a labelled master plate. In the expression protocol, transformed colonies are picked from the master plate and inoculated into 5 ml of LB+Kan media, where they are grown overnight at 37°C. A 5 ml overnight culture is then inoculated into 100 ml of LB medium with 50 µg/ml kanamycin. Once the culture reaches an OD600 of 0.6, protein expression is induced by adding 0.1 mM IPTG, and the cells are grown at 25°C for 16 hours. Afterward, the cells are harvested by centrifugation at 4700 RPM for 10 minutes. They are then resuspended in 1 ml of PBS buffer at pH 7.5, containing 1 mg/ml lysozyme, and incubated on ice for 1 hour. The volume is made up to 5 ml with PBS, and lysis is carried out by sonication. The crude lysates are analyzed using SDS-PAGE.
[0229] Examples for Substrates Activity with the engineered formate dehydrogenase (FDH):
SEQ ID NO Conversion Enantiomeric excess
1 + +
3 + +
7 ++ ++
10 + +
18 + +
36 ++ ++
58 ++ +
80 ++ ++
87 ++ +
91 +++ +
94 +++ +
100 +++ ++
153 +++ ++
198 +++ +
200 +++ ++

[0230] In Table 3: Several engineered formate dehydrogenases that had been tested for substrate phenylpyruvic acid to convert into (R)-phenyllactic acid and converted greater 10% of conversion of the substrate to chiral alcohol when evaluated under the conditions described in the Example 4. In the conversion column, single plus “+” indicates conversion of >10%, two pluses “++” indicates conversion of >50%, three pluses “+++” indicates conversion of >95%. In the Enantiomeric excess column, “+” indicates <90% e.e. (R-enantiomer), two pluses “++” indicates >99% e.e. (R-enantiomer).
SEQ ID NO Conversion Enantiomeric excess
1 + +
4 ++ +
6 + ++
13 ++ +
21 ++ +
24 + +
38 ++ ++
42 +++ +
68 +++ ++
110 +++ +
122 +++ +
126 ++++ ++
142 ++++ +
198 ++++ +
200 ++++ ++

[0231] In Table 4: Several engineered formate dehydrogenases that had been tested for substrate 2-oxo-4-phenylbutyric acid to convert into (R)-2-Hydroxy-4-phenylbutyric acid and converted greater 10% of conversion of the substrate to chiral alcohol when evaluated under the conditions described in the Example 5. In the conversion column, single plus “+” indicates conversion of >10%, two pluses “++” indicates conversion of >50%, three pluses “+++” indicates conversion of >95%, four pluses “++++” indicates conversion of >99.9%. In the Enantiomeric excess column, “+” indicates <90% e.e. (R-enantiomer), two pluses “++” indicates >99% e.e. (R-enantiomer).

SEQ ID NO Conversion Enantiomeric excess
1 + +
4 ++ +
5 +++ +
6 + +
8 +++ ++
20 ++ +
24 + ++
34 +++ +
38 ++ +
41 + ++
86 +++ ++
91 +++ +
127 +++ +
198 ++++ ++
199 ++++ +
200 ++++ ++
[0232] In Table 5: Several engineered formate dehydrogenases that had been tested for substrate 4-hydroxyphenylpyruvic acid to convert into (2S)-2-hydroxy-3-(4-hydroxyphenyl)propanoic acid and converted greater 10% of conversion of the substrate to chiral alcohol when evaluated under the conditions described in the Example 6. In the conversion column, single plus “+” indicates conversion of >10%, two pluses “++” indicates conversion of >50%, three pluses “+++” indicates conversion of >95%, four pluses “++++” indicates conversion of >99.9%.. In the Enantiomeric excess column, “+” indicates <90% e.e. (S-enantiomer), two pluses “++” indicates >99% e.e. (S-enantiomer).
SEQ ID NO Conversion Enantiomeric excess
1 + +
7 ++ +
8 +++ +
10 + ++
13 ++ ++
18 + +
22 +++ +
33 + ++
36 ++ ++
70 +++ +
108 +++ +
154 ++ +
182 ++++ ++
198 ++++ +
200 ++++ ++
[0233] In Table 6: Several engineered formate dehydrogenases that had been tested for substrate pyruvate to convert into (S)-lactate and converted greater 10% of conversion of the substrate to chiral alcohol when evaluated under the conditions described in the Example 7. In the conversion column, single plus “+” indicates conversion of >10%, two pluses “++” indicates conversion of >50%, three pluses “+++” indicates conversion of >95%, four pluses “++++” indicates conversion of >99.9%. In the Enantiomeric excess column, “+” indicates <90% e.e. (S-enantiomer), two pluses “++” indicates >99% e.e. (S-enantiomer).
SEQ ID NO Conversion
1 +
3 +
6 +
15 ++
10 +
18 +
24 +
33 +
41 +
59 ++
61 ++
80 ++
87 ++
90 ++
92 ++
94 ++
97 ++++
99 ++
100 +++
105 +++
120 +++
144 +++
176 +++
177 ++++
180 +++
184 +++
186 ++++
198 ++++
199 ++++
200 ++++
[0234] In Table 7: Several engineered formate dehydrogenases that had been tested for substrate sodium formate to carbon dioxide, and recycling efficiency for conversion of NADP+ to NADPH and NAD+ to NADH. The cofactor recycling ability for NAD(P)+ to NAD(P)H with formate as a co-substrate was greater than 10% when evaluated under the conditions described in the Example 8. In the conversion column, single plus “+” indicates conversion of >10%, two pluses “++” indicates conversion of >50%, three pluses “+++” indicates conversion of >95%, four pluses “++++” indicates conversion of >99.9%.
Temperature (°C) SEQ ID NO Stability
15 1, 6,10,12, 26,32,42,54,60,78,84, 100,106,121,129,131,149,156, 160,172,182,198, 199,200 +
20 1, 6,10,12, 26,32,42,54,60,78,84, 100,106,121,129,131,149,156, 160,172,182,198, 199,200 ++
25 1, 6,10,12, 26,32,42,54,60,78,84, 100,106,121,129,131,149,156, 160,172,182,198, 199,200 ++
30 1, 6,10,12, 26,32,42,54,60,78,84, 100,106,121,129,131,149,156, 160,172,182,198, 199,200 ++
35 1, 6,10,12, 26,32,42,54,60,78,84, 100,106,121,129,131,149,156, 160,172,182,198, 199,200 ++
40 1, 6,10,12, 26,32,42,54,60,78,84, 100,106,121,129,131,149,156, 160,172,182,198, 199,200 ++
45 1, 6,10,12, 26,32,42,54,60,78,84, 100,106,121,129,131,149,156, 160,172,182,198, 199,200 ++
50 1, 6,10,12, 26,32,42,54,60,78,84, 100,106,121,129,131,149,156, 160,172,182,198, 199,200 ++
55 1, 6,10,12, 26,32,42,54,60,78,84, 100,106,121,129,131,149,156, 160,172,182,198, 199,200 ++
60 1, 6,10,12, 26,32,42,54,60,78,84, 100,106,121,129,131,149,156, 160,172,182,198, 199,200 +
[0235] In Table 8: Several engineered formate dehydrogenases that had been tested for substrate sodium formate to carbon dioxide, and recycling efficiency for conversion of NAD(P)+ to NAD(P)H. The cofactor recycling ability for NAD(P)+ to NAD(P)H with formate as a co-substrate was greater than 10% when evaluated under the conditions described in the Example 27. In the stability column, a single plus “+” indicates that the polypeptide exhibits measurable activity after heat treatment of 2 hr at temperature range from 15-60°C, two pluses “++” indicates that the polypeptide has greater than 200% improvement in activity as compared to SEQ ID NO: 1, when comparing activity for both proteins after heat treatment of 2 hours at 15-60°C.
pH SEQ ID NO Stability
4.0 1, 6,10,12, 26,32,42,54,60,78,84, 100,106,121,129,131,149,156, 160,172,182,198, 199,200 +
4.5 1, 6,10,12, 26,32,42,54,60,78,84, 100,106,121,129,131,149,156, 160,172,182,198, 199,200 +
5.0 1, 6,10,12, 26,32,42,54,60,78,84, 100,106,121,129,131,149,156, 160,172,182,198, 199,200 +
5.5 1, 6,10,12, 26,32,42,54,60,78,84, 100,106,121,129,131,149,156, 160,172,182,198, 199,200 ++
6.0 1, 6,10,12, 26,32,42,54,60,78,84, 100,106,121,129,131,149,156, 160,172,182,198, 199,200 ++
6.5 1, 6,10,12, 26,32,42,54,60,78,84, 100,106,121,129,131,149,156, 160,172,182,198, 199,200 ++
7.0 1, 6,10,12, 26,32,42,54,60,78,84, 100,106,121,129,131,149,156, 160,172,182,198, 199,200 ++
7.5 1, 6,10,12, 26,32,42,54,60,78,84, 100,106,121,129,131,149,156, 160,172,182,198, 199,200 ++
8.0 1, 6,10,12, 26,32,42,54,60,78,84, 100,106,121,129,131,149,156, 160,172,182,198, 199,200 ++
8.5 1, 6,10,12, 26,32,42,54,60,78,84, 100,106,121,129,131,149,156, 160,172,182,198, 199,200 +
9.0 1, 6,10,12, 26,32,42,54,60,78,84, 100,106,121,129,131,149,156, 160,172,182,198, 199,200 +
[0236] In Table 9: Several engineered formate dehydrogenases that had been tested for substrate sodium formate to carbon dioxide, and recycling efficiency for conversion of NAD(P)+ to NAD(P)H. The cofactor recycling ability for NAD(P)+ to NAD(P)H with formate as a co-substrate was greater than 10% when evaluated under the conditions described in the Example 28. In the stability column, a single plus “+” indicates that the polypeptide exhibits measurable activity after pH treatment of 2 hr at pH range from 4.0-9.0, two pluses “++” indicates that the polypeptide has greater than 200% improvement in activity as compared to SEQ ID NO: 1, when comparing activity for both proteins after pH treatment of 2 hr at pH range from 4.0-9.0.
SEQ ID No. No of Mutations relative to SEQ ID No: 1 Activity
(HCOONa) Activity
(Pyruvate) Stability

1 0 +++ + +
2 5 +++ ++ +
3 5 ++ + ++
4 5 ++ ++ ++
5 5 ++ +++ +
6 6 + + ++
7 6 ++ ++ ++
8 6 +++ +++ ++
9 6 ++ ++ +
10 7 ++ + ++
11 7 ++ ++ +
12 7 ++ ++ +
13 7 ++ ++ +
14 7 ++ +++ +
15 7 +++ +++ ++
16 7 ++ +++ ++
17 7 +++ +++ +
18 8 + + ++
19 8 ++++ ++++ ++
20 8 ++ ++ ++
21 8 ++ ++ ++
22 9 +++ +++ ++
23 9 ++++ ++++ ++
24 9 + + ++
25 9 +++ +++ ++
26 10 +++ +++ ++
27 10 +++ ++++ ++
28 10 ++++ ++++ ++
29 10 +++ +++ ++
30 10 +++ +++ ++
31 10 ++++ ++++ ++
32 10 ++++ +++ +
33 10 + + +
34 11 +++ +++ +
35 11 +++ +++ ++
36 11 ++ ++ +
37 11 ++ ++ ++
38 12 ++ ++ ++
39 12 ++ ++ ++
40 12 +++ +++ +
41 12 + + ++
42 13 +++ +++ +
43 13 ++ ++++ ++
44 13 +++ +++ ++
45 13 ++++ ++++ ++
46 13 ++++ ++++ ++
47 13 +++ +++ +
48 13 +++ +++ ++
49 13 ++++ ++++ ++
50 14 ++ ++++ ++
51 14 ++++ ++++ ++
52 14 +++ ++++ ++
53 14 ++++ ++++ ++
54 15 ++ ++++ ++
55 15 ++++ ++++ ++
56 15 ++ ++ +
57 15 +++ +++ +
58 16 ++ ++ +
59 16 ++ ++ ++
60 16 +++ +++ ++
61 16 ++ ++ ++
62 16 +++ +++ ++
63 16 ++ ++++ ++
64 16 +++ +++ +
65 16 ++++ ++++ ++
66 17 +++ ++++ ++
67 17 +++ +++ ++
68 17 +++ +++ +
69 17 +++ +++ +
70 18 +++ +++ +
71 18 +++ +++ +
72 18 ++++ ++++ ++
73 18 ++++ ++++ ++
74 18 +++ +++ +
75 18 ++ ++++ ++
76 18 ++ ++ ++
77 18 ++ ++ ++
78 19 +++ +++ ++
79 19 ++ ++ +
80 19 ++ ++ +
81 19 +++ +++ ++
82 20 ++++ ++++ ++
83 20 +++ +++ ++
84 20 +++ ++++ ++
85 20 ++ ++++ ++
86 20 +++ +++ +
87 20 ++ ++ +
88 20 ++ ++ ++
89 20 ++ ++ ++
90 21 ++ ++ ++
91 21 +++ +++ ++
92 21 ++ ++ +
93 21 ++ ++ +
94 21 +++ +++ +
95 21 ++ ++ ++
96 21 ++ ++ ++
97 21 ++++ ++++ ++
98 21 ++ ++++ ++
99 21 ++ ++ ++
100 23 +++ +++ +
101 23 ++ ++ ++
102 23 ++ ++ +
103 23 ++ ++ +
104 23 +++ ++++ ++
105 23 +++ +++ ++
106 23 ++++ ++++ ++
107 23 ++++ ++++ ++
108 23 +++ +++ +
109 23 ++++ ++++ ++
110 23 +++ +++ ++
111 23 ++ ++ +
112 23 +++ +++ ++
113 23 +++ ++++ ++
114 23 +++ +++ +
115 23 ++++ ++++ ++
116 24 +++ +++ +
117 24 ++ ++ +
118 24 ++ ++ +
119 24 +++ +++ ++
120 24 +++ +++ +
121 24 ++ ++ ++
122 24 +++ +++ ++
123 24 ++++ ++++ ++
124 24 ++++ ++++ ++
125 24 ++ ++ +
126 24 ++++ ++++ ++
127 24 +++ +++ +
128 24 ++ ++ ++
129 24 ++++ ++++ ++
130 24 + ++ +
131 25 + ++ ++
132 25 +++ +++ ++
133 25 ++++ ++++ ++
134 25 +++ +++ +
135 25 ++ ++ +
136 25 ++++ ++++ ++
137 25 ++ ++ ++
138 25 ++++ ++++ ++
139 25 ++++ ++++ ++
140 25 +++ ++++ ++
141 25 ++ ++ +
142 25 ++++ ++++ ++
143 25 ++ ++ +
144 25 ++ +++ +
145 25 +++ +++ +
146 25 ++++ ++++ ++
147 25 ++++ ++++ ++
148 25 ++ ++++ ++
149 25 ++++ ++++ ++
150 26 +++ +++ +
151 26 ++ ++++ ++
152 26 ++++ ++++ ++
153 26 ++ ++++ ++
154 26 ++ ++ ++
155 26 ++ ++ +
156 26 +++ +++ ++
157 26 ++ ++ ++
158 26 ++ ++ ++
159 26 ++++ ++++ ++
160 26 +++ +++ ++
161 26 ++ ++++ ++
162 26 +++ +++ +
163 26 +++ +++ +
164 26 +++ +++ ++
165 26 +++ +++ +
166 26 ++++ ++++ ++
167 27 ++++ ++++ ++
168 27 +++ +++ +
169 27 +++ ++++ ++
170 27 +++ +++ ++
171 27 +++ +++ +
172 27 ++++ ++++ ++
173 27 ++++ ++++ ++
174 27 +++ +++ ++
175 27 +++ ++++ ++
176 27 +++ +++ ++
177 27 ++++ ++++ ++
178 27 ++++ ++++ ++
179 27 ++ ++ +
180 28 +++ +++ ++
181 28 +++ +++ +
182 28 ++++ ++++ ++
183 28 +++ +++ ++
184 28 +++ +++ ++
185 28 ++++ ++++ ++
186 28 ++++ ++++ ++
187 28 +++ +++ ++
188 28 +++ +++ +
189 29 +++ +++ +
190 29 +++ +++ ++
191 29 +++ +++ ++
192 29 +++ ++ +
193 29 ++ ++ +
194 29 ++ ++ ++
195 29 +++ +++ +
196 30 +++ +++ ++
197 30 ++ ++ ++
198 31 +++ ++++ ++
199 32 +++ +++ ++
200 31 ++++ ++++ ++
[0237] In Table 10, above, in the activity column, single plus “+” indicates an activity improvement of 10-20% of the activity of SEQ ID NO: 1, two pluses “++” indicates an activity improvement of 60-80% of SEQ ID NO: 1, three pluses “+++” indicates an activity improvement of 100-120% of SEQ ID NO: 1 and four pluses “++++” indicates an activity improvement of 150-200% of SEQ ID NO: 1. In the stability column, a single plus “+” indicates that the polypeptide exhibits measurable activity after heat treatment of 2 hr at 55°C, two pluses “++” indicates that the polypeptide has greater than 200% improvement in activity as compared to SEQ ID NO: 1, when comparing activity for both proteins after heat treatment of 2 hours at 55°C.
Time (in minutes) Absorbance (OD) at 340nm for SEQ ID NO: 211 Absorbance (OD) at 340nm for SEQ ID NO: 212 Absorbance (OD) at 340nm for SEQ ID NO: 213 Absorbance (OD) at 340nm for SEQ ID NO: 214 Absorbance (OD) at 340nm for SEQ ID 1
0 0.00 0 0 0.00 0.00
5 0.103 0.205 0.991 0.269 1.399
10 0.118 0.224 0.821 0.408 2.003
15 0.093 0.343 0.624 0.502 2.538
20 0.258 0.335 0.754 0.639 2.58
25 0.136 0.359 0.794 0.706 2.714
30 0.153 0.363 0.995 0.796 2.807
[0238] In Table 11: The activity of formate dehydrogenases (SEQ ID NO: 211, 212, 213 and 214) and artificial FDH (SEQ ID NO: 1) had been tested substrate sodium formate to carbon dioxide, and recycling efficiency for conversion of NAD(P)+ to NAD(P)H. The cofactor recycling ability for NAD(P)+ to NAD(P)H with formate as a co-substrate was greater in SEQ ID NO: 1when evaluated under the conditions described in the Example 29.
S. NO Organisms Accession ID
1 Mycolicibacterium vaccae Q93GV1
2 Candida dubliniensis 8J3O
3 Rhodococcus jostii A0A1H4ISR4
4 Pseudomonas sp. 101 P33160
5 Saccharomyces cerevisiae Q08911
6 Ancylobacter aquaticus D3G885
7 Candida boidinii O13437
8 Candida methylica CAA57036.1
9 Burkholderia stabilis ACF35003.1
10 Lentilactobacillus buchneri AEB72361.1
11 Granulicella mallensis AEU36496.1
12 Burkholderia dolosa A0A892IHP6
13 Bacillus sp. F1 CBL95273
14 Moraxella sp.C-1 O08375
15 Arabidopsis thaliana AF208029
16 Komagataella phaffii XP_002493171.1
17 Ogataea polymorpha XP_018212858.1
18 Ascoidea rubescens XP_020048692.1
19 Lithohypha guttulata XP_064753633.1
20 Hypoxylon fragiforme XP_049116698.1
21 Debaryomyces fabryi XP_015464814.1
22 Truncatella angustata XP_045959367.1
23 Botrytis fragariae XP_037190069.1
24 Sclerotinia sclerotiorum XP_001590273.1
25 Cryphonectria parasitica XP_040781956.1
26 Penicillium brevicompactum XP_056810746.1
27 Capronia coronata XP_007726405.1
28 Cladophialophora psammophila XP_007749595.1
29 Paecilomyces variotii XP_028486516.1
30 Filobasidium floriforme XP_046040391.1
31 Cladophialophora bantiana XP_016613795.1
32 Aureobasidium melanogenum XP_040882049.1
33 Phaeoacremonium minimum XP_007918553.1
34 Exophiala xenobiotica XP_013312301.1
35 Fonsecaea multimorphosa XP_016630859.1
36 Fonsecaea erecta XP_018694713.1
37 Didymosphaeria variabile XP_056068191.1
38 Capronia epimyces XP_007732874.1
39 Gaeumannomyces tritici XP_009223986.1
40 Aspergillus ruber XP_040640940.1
41 Aspergillus chevalieri XP_043137557.1
42 Scedosporium apiospermum XP_016646331.1
43 Colletotrichum truncatum XP_070062469.1
44 Rhinocladiella mackenziei XP_013276995.1
45 Fonsecaea pedrosoi XP_013279965.1
46 Cordyceps fumosorosea XP_018708014.1
47 Beauveria bassiana XP_008596053.1
48 Exophiala oligosperma XP_016258587.1
49 Aspergillus pseudoviridinutans XP_043160652.1
50 Lachnellula hyalina XP_031001124.1
51 Ascochyta rabiei XP_038796251.1
52 Neohortaea acidophila XP_033585297.1
53 Penicillium taxi XP_057066262.1
54 Xylaria bambusicola XP_047834941.1
55 Talaromyces stipitatus XP_002488147.1
56 Aspergillus lentulus XP_033414702.1
57 Exophiala aquamarina XP_013264072.1
58 Pyricularia oryzae XP_003719810.1
59 Aspergillus nidulans XP_050467117.1
60 Lasiodiplodia theobromae XP_035370354.1
61 Talaromyces atroroseus XP_020116886.1
62 Cladophialophora carrionii XP_008728045.1
63 Cladophialophora immunda XP_016247723.1
64 Aspergillus viridinutans XP_043129675.1
65 Exophiala viscosa XP_062669056.1
66 Apiospora aurea XP_066707134.1
67 Cadophora gregata XP_058360223.1
68 Aspergillus niger XP_025454923.1
69 Saxophila tyrrhenica XP_064661258.1
70 Aspergillus candidus XP_024668481.1
71 Coccidioides posadasii str. Silveira XP_003071148.1
72 Aspergillus fischeri XP_001257699.1
73 Apiospora marii XP_066678337.1
74 Aspergillus ibericus XP_025569209.1
75 Aspergillus eucalypticola XP_025390402.1
76 Alternaria arborescens XP_028507415.1
77 Aspergillus udagawae XP_043145784.1
78 Aspergillus campestris XP_024695464.1
79 Aspergillus piperis XP_025515752.1
80 Microdochium trichocladiopsis XP_046013782.1
81 Coccidioides immitis XP_001248879.1
82 Colletotrichum fructicola Nara gc5 XP_031891161.1
83 Exophiala mesophila XP_016223083.1
84 Alternaria burnsii XP_038789413.1
85 Blastomyces dermatitidis XP_045281214.1
86 Colletotrichum aenigma XP_037180751.1
87 Aspergillus japonicus XP_025526163.1
88 Cladophialophora yegresii XP_007755117.1
89 Aspergillus terreus XP_001214863.1
90 Aspergillus uvarum XP_025492586.1
91 Alternaria alternata XP_018382854.1
92 Colletotrichum siamense XP_036497302.1
93 Sparassis crispa XP_027619450.1
94 Scheffersomyces xylosifermentans XP_064777102.1
95 Rhodofomes roseus XP_047781246.1
96 Vanrija pseudolonga XP_062625161.1
97 Cryptococcus wingfieldii XP_019035043.1
98 Fonsecaea monophora XP_022514256.1
99 Cryptococcus gattii XP_003194677.1
100 Fusarium graminearum XP_011324753.1
[0239] Table 12: The curated 100 formate dehydrogenase database used in for deriving the artificial formate dehydrogenase (SEQ ID NO: 1), for improved cofactor recycling of NAD(P)+/NAD(P)H.
S. No Variant ID Protein Concentration (mg protein/ ml of lysate)
01 SEQ ID NO: 1 29.003
02 SEQ ID NO: 12 29.98
03 SEQ ID NO: 30 31.36
04 SEQ ID NO: 198 31.62
05 SEQ ID NO: 199 31.95
06 SEQ ID NO: 200 29.52

[0240] Table 13: The determined protein concentration of recombinant engineered formate dehydrogenase variants from the Bradford assay.
ADVANTAGES
[0241] Formate dehydrogenases are commonly utilized to recycle NAD(P)+ to NADPH cofactors by employing formate and its salts as a substrate, resulting in the production of carbon dioxide. This disclosure presents an engineered biocatalyst in the form of a formate dehydrogenase polypeptide that promotes reduction of bulky 2-oxo-acids into bulky 2-hydroxy-acids. Additionally, the same engineered formate dehydrogenase is capable of recycling NAD(P)+ to NAD(P)H using formate as a co-substrate which was reduced during the process. Eliminating the use of a secondary enzyme like glucose dehydrogenase or Ketoreductases for cofactor recycling. Thus, simplifying the process ketone reduction using a single enzyme system, and by reducing the number of operational steps. Additionally, the engineered formate dehydrogenase has improved thermal (20°-55°C), and at least three-fold increase in the cofactor NAD(P)+ recycling ability as compared to any reported formate dehydrogenases.
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[0270] Kumar, P., Sigamani, G. & Roopa. L., (2023). METHOD FOR ENGINEERING PROTEINS (Indian Patent Application No. 202341064088). Indian Patent Office. ,CLAIMS:We Claim:
1. A recombinant engineered formate dehydrogenase polypeptide comprising of an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 and that includes the feature of residues corresponding to X24 is Ala, X79 is Leu, X81 is Thr, X147 is Ser, X314 is Glu, X336 is Ser; X386 is Asp; and X389 is Arg;

2. The recombinant engineered formate dehydrogenase polypeptide of claim 1, which comprises an amino acid sequence having one or more of the following features:
X5 is Leu or Ala or Ile;
X13 is Val or Glu or Thr;
X16 is Tyr or Met or Gln;
X19 is Thr or Lys or Ala;
X21 is Cys or Pro or Ile;
X27 is Lys or Thr or Gln;
X28 is Ile, Val or Pro;
X29 is Asp or Glu or Lys;
X30 is His or Ala; X33 is Ser or Asp;
X36 is Ile or Ala or Ser;
X37 is Leu, Ala or Val;
X38 is Cys; X49 is Gln or Thr or Ala;
X62 is Glu or Pro or Arg;
X67 is Asn or Gln or Leu;
X72 is Lys or Ile or Ala;
X84 is Phe;
X93 is Lys or Ile;
X94 is Val, Ala or Leu;
X106 is Pro or Ala;
X131 is Glu or Asp;
X132 is Ser or Thr;
X134 is Lys or Met or Asn;
X138 is Val or Met or Leu;
X139 is Arg or Ile;
X149 is Asp or Asn or His;
X161 is Ser, Thr or Ala;
X167 is Leu or Val or Thr;
X171 is Glu or Gln or Asn;
X173 is Ala or Ile;
X174 is Arg;
X186 is His or Gly or Gln;
X190 is Leu, Val or Ala;
X194 is Asp or Gln or Asn;
X212 is Ala, Gly or Val;
X216 is Val or Ala;
X217 is Arg or Lys;
X219 is Arg or Lys;
X228 is Glu or Asp or Leu;
X229 is Ser or Ala or Asp;
X232 is Lys or Arg or Leu;
X235 is Asn or His or Asp;
X236 is Lys or Val or Ala;
X239 is Glu or Asp;
X240 is Asp or Pro or Thr;
X270 is Ser or Met or Leu;
X302 is Arg or Gln or His;
X318 is Lys or Ala or Gln;
X320 is Glu or Asp;
X347 is Cys;
X357 is Glu or Leu;
X370 is Asp or Glu or Ser;
X375 is Arg or Val;
X385 is Glu or Asn or Lys;
X388 is Lys or Arg or Gly;
X389 is Arg; X395 is Leu or Glu or Ser;
X396 is Lys or Arg or His;
X397 is Phe;
X398 is Lys or Arg or Glu;
Wherein optionally the amino acid sequence has one or more residue differences at other amino acid residues as compared to the SEQ ID NO: 1.

3. The recombinant engineered formate dehydrogenase polypeptide of claim 2, where in the amino acid sequence includes at least one or more of the following features:
X98 is Gly;
X99 is Gly;
X100 is Ala;
X121 is Gly;
X122 is Ala or Ser;
X123 is Val,
X124 is Ala or Ser;
X126 is Glu, Ala or Gly;
X127 is Val, Ala or Leu;
X140 is Asn or Gln;
X142 is Gln or Asn;
X283 is Ser, Asn, Ala, Gly or Val;
X284 is Gly, Val, Ala or Leu;
X310 is Ala or Gly;
X335 is Ala or Gly;
X337 is Ser, Asn, Ala or Gly;
X345 is His, Ala, Gly, Phe or Thr;
X382 is Ile, Phe or Gln;
Wherein optionally the amino acid sequence has one or more residue differences at other amino acid residues as compared to the SEQ ID NO: 1.

4. The recombinant engineered formate dehydrogenase polypeptide of claim 3, wherein the amino acid sequence includes at least one or more of the following features:
X223 is Asn;
X224 is Arg;
X380 is Gln;
Wherein optionally the amino acid sequence has one or more residue differences at other amino acid residues as compared to the SEQ ID NO: 1.

5. The recombinant engineered formate dehydrogenase polypeptide of claim 1, wherein the amino acid sequence corresponds to the SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200.

6. The method of preparing compound of structural formula (I) with at least 90% enantiomeric excess from compound of structural formula (II) using any of the recombinant engineered formate dehydrogenase polypeptides of claims 1 to 5, in the presence of formate or its corresponding salts as co-substrate and NAD(P)+/NAD(P)H cofactor as a hydride donor in a suitable organic solvent under suitable reaction conditions.

7. The recombinant engineered formate dehydrogenase polypeptide of any of the claims 1 to 5, wherein the recombinant engineered formate dehydrogenase polypeptide exhibits at least 5.2-fold increase in cofactor NAD+ to NADH recycling rate within 5 minutes of the reaction time compared to the formate dehydrogenases corresponding to SEQ ID NOs: 211, 212, 213, or 214.

8. The recombinant engineered formate dehydrogenase polypeptide of any of the claims 1 to 5, wherein the amino acid sequences correspond to the SEQ ID NOs: 198, 199 or 200 does the recycling of cofactors NADP+ to NADPH and NAD+ to NADH in a suitable organic solvent under suitable reaction conditions using formate or its corresponding salts.

9. The recombinant engineered formate dehydrogenase polypeptide of any of the claims 1 to 5 used in the method of claims 6 to 8, wherein,
a) at least 95% of the substrate is converted to the product in less than 24 hours in a reaction mixture comprising 5 g/L to 100 g/L of substrate and 0.5 g/L to 2 g/L of the engineered formate dehydrogenase polypeptide.
b) the reaction is carried out under suitable reaction conditions comprising a phosphate buffer of 100 mM at pH 4.0 to 9.0, a temperature of 20-55°C.
c) the reaction mixture contains a co-substrate selected from formate or its corresponding salts—sodium formate (HCOONa), ammonium formate (NH4HCOO), or formic acid (HCOOH)- at concentrations ranging from 2 g/L to 10 g/L, along with a cofactor NAD(P)H or NAD(P)+ loaded at 0.5 g/L to 5 g/L;
d) the reaction mixture contains a suitable organic solvent selected from isopropanol, DMSO, acetone, ethyl acetate, butyl acetate, 1-octanol, or DMF;

10. The recombinant engineered formate dehydrogenase polypeptide of claim 1 is used in the method of any one of claims 6 to 9, wherein the engineered formate dehydrogenase polynucleotide having the sequence of SEQ ID NO: 201 is expressed in an E. coli host using the pET28b(+) vector under the control of T7 promotor, in a 20 mM phosphate buffer at pH 7.5, with an IPTG concentration of 0.05 mM and an induction temperature of 25°C, and wherein the said recombinant engineered formate dehydrogenase polypeptide is used in the form of whole cells, or an extract or lysate of such cells.