DESC:DESCRIPTION
FIELD OF THE INVENTION
[0001] This invention relates to the fields of Biology, Life Science, Computational Biology, Biocatalysis, and Biochemistry
BACKGROUND OF THE INVENTION
[0002] This invention specifically aims to engineer threonine deaminase to perform full transamination reactions, potentially utilizing external amine donors. The engineered enzyme would ideally convert a-keto acids into non-natural amino acids, leveraging the enzyme's existing mechanism. Unlike standard threonine deaminases that typically release PLP during the a, ß-elimination reaction, the engineered enzyme would retain the cofactor in a reactive state, enabling continuous synthesis of valuable amino acids and derivatives. The proposed engineering would involve strategic mutations at the active site to facilitate the attachment and exchange of amino groups, mimicking the dual functionality seen in aminotransferases.
[0003] This innovation could expand the enzyme's utility in pharmaceutical and industrial applications, making it a versatile tool for synthesizing a wide range of bioactive compounds and fine chemicals.
[0004] Threonine Deaminase (ThrD, EC 4.3.1.19) is a pyridoxal-5'-phosphate (PLP) based enzyme encoded by ilvA gene, which in various microorganisms plays a crucial role in amino acid metabolism for the synthesis of isoleucine and valine from L-threonine. It catalyses the a, ß-elimination of L-threonine or L-serine to produce 2-ketobutyrate or pyruvate, respectively with ammonia as the by-product. These enzymes belong to the type II-fold of PLP-dependent enzymes and share a similar reaction to transaminases where the PLP is covalently attached to the catalytic lysine through a Schiff base.
[0005] The mechanism of action of Threonine Deaminase (ThrD) begins when the amino group (-NH2) of L-threonine reacts with the PLP cofactor, forming a Schiff base (imine) intermediate (Fig.1). This step involves the transfer of the amino group from L-threonine or L-serine to PLP. The Schiff base intermediate then undergoes an a,ß-elimination reaction, wherein the hydroxyl group at the ß-carbon is dehydrated leading to the formation of a 2-amino-butenoic acid intermediate and water. The intermediate is then attacked by the nucleophilic water. This results in the release of ammonia (NH3) from threonine/serine and the formation of 2-ketobutyric acid or pyruvic acid, and ammonia is released into the active site of the enzyme (Favrot et. al., 2018). Biochemically, threonine deaminases are essential for producing various pharmacological intermediates, including isoleucine, 2-aminobutyric acid, and 2-hydroxybutyric acid. These enzymes have a unique deamination process that supports a variety of synthetic a-keto acids, unlike other L-amino acid deaminases. They are renowned for their adaptability, importance in biological processes, and production of valuable chemical compounds.
[0006] A significant number of studies have been conducted to investigate ThrD enzymes from different microorganisms, including Escherichia coli (E. coli), Arabidopsis thaliana, Chryseobacterium takakiae, and Bacillus subtilis (B. subtilis). These focused studies aim to uncover the specific traits, regulatory mechanisms, and structural variances exhibited by ThrD enzymes among various species for a deeper understanding of the diverse functions and adaptations of ThrD enzymes across different biological systems. An engineered threonine deaminase from Corynebacterium glutamicum, having six mutations (V111A/V119N/K123S/V137I/K260S/R261T), improves the catalytic efficiency (kcat/Km) by 90.6 fold, with an efficient production of both natural and unnatural a-keto acids, including 2-oxobutyric acid (83.4 g/L, 99% conversion), phenylpyruvic acid (72.5 g/L, 95% conversion), 2-oxovaleric acid (21.1 g/L, 91% conversion), and 2-oxo-4-phenylbutyric acid (29.9 g/L, 84% conversion) (Song et. al., 2020). Threonine deaminases are also involved in the defluorination of 4-fluoro-L-threonine to 2-oxobutanoate, with the release of PLP (Wu et. al., 2020). A threonine deaminase from Escherichia coli with a double mutation of F352A/R362F showed both higher activity and much stronger resistance to Ile inhibition compared to those of wild enzymes. Overexpression of this mutant in E. coli JW3591 significantly increased the production of 2-ketobutyrate and Ile (Chen et. al., 2012). A variant of threonine deaminase (H480Y) designed by conventional mutagenesis, in a diploid sake yeast of S. cerevisiae, was markedly insensitive to feedback inhibition by Ile but was not upregulated by valine, leading to intracellular accumulation of Ile and extracellular overproduction of 2-methyl-1-butanol, a fusel alcohol derived from Ile, in yeast cells. (Isogai et. al., 2022)
[0007] Threonine deaminases also produce natural and synthetic a-keto acids, which are important building blocks for fine chemical synthesis. Some a-keto acids used as starting points for the synthesis of many fine chemicals include:
[0008] Use of 2-keto-butyric acid as a precursor for the synthesis of 2-hydroxybutyrate, which is in turn an intermediate in the synthesis of peroxisome proliferator-activated receptor a (PPARa) antagonist (R)-K-13675. (Yamazaki et al., 2008). Use of 2-keto-butyric acid as a precursor for the synthesis of S-amino-butyric acid and S-amino-butyramide, which are intermediates in the synthesis of the anti-epileptic drugs levetiracetam and brivaracetam (Gayke et al., 2022), and the anti-tuberculosis drug ethambutol (Yendapally and Lee et. al., 2008). 2-keto acids can also be used in the synthesis of non-natural amino acids. Non-natural amino acids are distinct from natural amino acids as they are not found in natural polypeptide chains and are derived as secondary metabolites in various organisms or synthesized by chemical means. Many non-natural amino acids are structural analogues of natural amino acids and therefore find applications as amino acid derivatives or analogues in the synthesis of APIs such as (2R,3S)-N-benzoyl-3-phenylisoserine for the synthesis of paclitaxel, a drug used to treat cancer (Blaskovich, 2016). Non-natural amino acids also find use in the synthesis of anti-microbial peptides, such as 2-aminooctanoic acid for its application in lactoferricin B peptide derivatives (Almahboub et al. 2018). L-4-fluorophenylalanine is reported as an antimicrobial agent by interfering with the formation of L-phenylalanine acylated tRNA during protein translation (Sharpe, M., et. al., 2004). Glufosinate ([(3S)-3-amino-3-carboxypropyl] (methyl)phosphinate) is a key herbicide to manage glyphosate-resistant weeds mainly because it is a broad-spectrum herbicide, and transgenic glufosinate-resistant crops are available. Glufosinate is a rapid herbicide that was first discovered as the only natural herbicide targeting glutamine synthetase (Takano et. al., 2020).
[0009] Various biocatalytic routes for the synthesis of non-natural amino acids from a-keto acids are reported using engineered amino acid dehydrogenases (AADH) and ?-transaminases (?-TA) (Narancic, T. et. al., 2019). Threonine deaminases, enzymes bound with pyridoxal-phosphate cofactors, undergo a similar reaction cycle to other PLP/PMP-bound enzymes such as aminotransferases, amino acid racemases, and amino acid synthases. While threonine deaminases do not show full aminotransferase activity (Leoncini, R., et. al., 1998), reasonable engineering to the enzyme's active site could modify threonine deaminase for aminotransferase activity, specifically for the synthesis of non-natural amino acids.
[0010] The engineered ThrD could facilitate the synthesis of important intermediates and compounds in pharmaceutical applications. For instance, 2-keto-butyric acid, a product of ThrD activity, can be used as a precursor for synthesizing compounds like the anti-epileptic drugs levetiracetam and brivaracetam, as well as anti-tuberculosis drug ethambutol. To achieve this, the engineered enzyme would undergo targeted mutations at the active site to enable the transfer of an amine group from an external donor to the a-keto acid substrate, thus mimicking the full cycle of transamination reactions observed in aminotransferases. This innovative approach would not only enhance the enzyme's utility in deamination but also empower it with transaminase-like activity, significantly broadening its application in biotechnological and pharmaceutical fields.
OBJECTIVES OF THE INVENTION
[0011] The primary goal of this invention is to develop an engineered artificial threonine deaminase capable of transforming bulky a-keto-acids into corresponding S-amino acids, or bulky a-keto-esters into their respective S-amino-acid esters, with L-threonine serving as the amine donor. Specifically, the enzyme is designed to convert specific bulky a-ketoacids, such as (3-carboxy-3-oxopropyl)(methyl)phosphinate (2a) or 4-fluorophenyl-pyruvic acid (2b), into their respective S-amino acids, phosphinothricin (4a) or L-4-fluorophenylalanine (4b). Additionally, it can convert bulky a-keto-esters like (4-methoxy-3,4-dioxobutyl)(methyl)phosphinate (5a) or methyl-3-(4-fluorophenyl)-2-oxopropanoate (5b) into their corresponding S-amino acid esters, such as [(3S)-3-amino-4-methoxy-4-oxobutyl]-(methyl) phosphinate (6a) or methyl (2S)-2-amino-3-(4-fluorophenyl) propanoate (6b).
[0012] This invention also seeks to enhance the utilization of the amine donor L-threonine (1), facilitating its conversion to 2-oxo-butyric acid (3) as depicted in Figure 2 A, B. Furthermore, it aims to improve the aminotransferase activity of the pyridoxal phosphate (PLP)-bound engineered artificial threonine deaminase. Moreover, the polynucleotide encoding this engineered enzyme is operably linked to one or more promoter sequences to boost the production of recombinant engineered artificial threonine deaminase in a recombinant host cell using an expression vector and expressed in such a cell.
SUMMARY
[0013] Threonine Deaminase (ThrD, EC 4.3.1.19) is a pyridoxal-5'-phosphate (PLP) based enzyme encoded by ‘ilvA’ gene that plays a crucial role in amino acid metabolism. It catalyses the a, ß-elimination of L-threonine or L-serine to produce 2-ketobutyrate or pyruvate, respectively with ammonia as the by-product. These enzymes belong to the type II-fold of PLP-dependent enzymes and share a similar reaction to transaminases where the PLP is covalently attached to the catalytic lysine through a Schiff base. Threonine deaminases are robust enzymes that are reported to produce natural and synthetic a-keto acids, important building blocks in the fine chemical synthesis. They are also capable of tolerating higher substrate loads, in such cases where the enzyme added to the reaction system is 0.25% w/w with respect to the substrate added into the reaction medium, i.e., the enzyme can tolerate up to 400 folds greater substrate loading. Since, threonine deaminases are enzyme that are bound with pyridoxal-phosphate cofactors, they undergo a similar reaction cycle to other PLP bound enzymes such as aminotransferases and ?-transaminases. Threonine deaminases are reported to show “half-transamination” activity in biological systems when the apoenzyme is coupled with pyruvate, to replenish the biological activity of the threonine deaminase and maintain a metabolic balance of PLP and pyridoxamine-5'-phosphate (PMP) in the cellular system.
[0014] Threonine deaminases have not been observed to exhibit full amino-transferase activity, which can be attributed to the distinct mechanisms employed by the active sites of threonine deaminases compared to transaminases. Transaminases are efficient in facilitating the complete transamination cycle, beginning with the entry of external amines into the active site. The initial phase of the transaminase reaction involves transferring an amine from a donor (such as L-alanine or isopropylamine) to pyridoxal phosphate (PLP), resulting in the formation of pyridoxamine phosphate (PMP) within the active site. The subsequent phase sees the PMP transferring the amine to an incoming ketone (the substrate), which leads to the reformation of the PLP state. Conversely, the action of threonine deaminase involves a distinct a,ß-elimination reaction of L-threonine or L-serine substrates, where the PLP cofactor forms a Schiff base intermediate directly with the catalytic lysine, bypassing the PMP intermediate stage.
[0015] However, it's noted that threonine deaminases can engage in the latter part of the transamination process when PMP is present in the active site, despite their primary function not involving the full transamination cycle.
[0016] Our Primary objective was to take advantage of the robustness and versatility of threonine deaminases to develop an artificial threonine deaminase that shows aminotransferase activity against bulky a-keto-acids. In this invention, we present an engineered artificial polypeptide that was developed to show amino-transferase activity. Specifically, we present an engineered threonine deaminase developed for the conversion of any one of the bulky a-keto-acids such as (3-carboxy-3-oxopropyl)(methyl)phosphinate (2a), or 4-fluorophenyl-pyruvic acid (2b) or bulky a-keto-esters such as (4-methoxy-3,4-dioxobutyl)(methyl)phosphinate (5a), or methyl-3-(4-fluorophenyl)-2-oxopropanoate (5b), in the presence of L-threonine (1) as the amine donor, and either PLP or PMP cofactor with to yield bulky S-amino acids, phosphinothricin (4a), or L-4-fluorophenylalanine (4b) or their esters , [(3S)-3-amino-4-methoxy-4-oxobutyl]-(methyl) phosphinate (6a), or methyl (2S)-2-amino-3-(4-fluorophenyl) propanoate (6b), respectively with the release of the byproduct 2-oxo-butyric acid (3) using a reaction system that comprises of an engineered artificial threonine deaminase. The derived a-bulky amino acids find uses in the agrochemical and pharmaceutical industry. Glufosinate ([(3S)-3-amino-3-carboxypropyl] (methyl) phosphinate) (4a) is a key herbicide to manage glyphosate-resistant weeds mainly because it is a broad-spectrum herbicide. L-4-fluorophenylalanine (4b) is reported as an antimicrobial agent by interfering with the formation of L-phenylalanine-acylated tRNA during protein translation. The byproduct 2-oxo-butyric acid (3) is used as a precursor for the synthesis of S-amino-butyric acid and S-amino-butyramide, which are intermediates in the synthesis of the anti-epileptic drugs levetiracetam, brivaracetam and the anti-tuberculosis drug ethambutol. The engineered artificial threonine deaminase (SEQ ID NO: 1) can take the bulky side chains of the a-keto-acids or the a-keto-esters and using either PLP or PMP in the active site convert these substrates into their respective S-amino acids or S-amino-acid esters (amino transferase activity), whilst maintaining the binding mode required for threonine deaminase activity. To design the artificial threonine deaminase of the present invention, a computational based method was used, that leverages sequence and structural information from PLP based enzymes to design and generate artificial threonine deaminase sequences with motifs imbibed from other S-selective PLP based enzymes. The artificial threonine deaminase as described in SEQ ID NO: 1, was then subjected to multiple rounds of engineering design and iterative characterisation to derive high active variants with both a,ß-elimination and transamination activity. The engineered artificial Threonine deaminase of SEQ ID NO: 1 and the corresponding variants were expressed in E. coli BL21 (DE3) using the plasmid pET28a(+). The expressed enzymes were quantified using molecular biology techniques such as SDS PAGE and Bradford’s assay. The engineered artificial threonine deaminase showed =90% conversion for bulky a-keto acids and a-keto esters to bulky S-amino acids and S-amino esters, respectively, and at least two-fold improvement in the catalytic efficiency of threonine deaminase activity at an L-threonine substrate concentration of 560mM with an enzyme concentration as low as 0.06 g/L when compared to previously reported wild-type threonine deaminases.
DESCRIPTION OF INVENTION
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1: Proposed reaction mechanism of threonine deaminase for native threonine deamination activity to yield 2-oxo-butyric acid. The reaction proceeds with the formation of Schiff’s intermediate with the L-threonine substrate (1), followed by the a,ß-elimination reaction (2) resulting in the dehydration of hydroxyl and release of 2-amino-2-butenoic acid intermediate (3). The 2-amino-2-butenoic acid intermediate undergoes nucleophilic attack by the released water to form 2-oxo-butyric acid and ammonia.
[0018] FIG. 2: Scheme of reactions catalysed by the engineered threonine deaminase. (A) Conversion of L-threonine (1) to 2-oxo-butyric acid (3) and any one of the bulky a-ketoacids (2), namely (3-carboxy-3-oxopropyl)(methyl)phosphinate (2a), or 4-fluorophenyl-pyruvic acid (2b), to their respective bulky S-amino acids (4) namely, phosphinothricin (4a), or L-4-fluorophenylalanine (4b) in the presence of either PLP or PMP. (B) Conversion of L-threonine (1) to 2-oxo-butyric acid (3) and any one of the bulky a-keto-esters (5), namely (4-methoxy-3,4-dioxobutyl)(methyl)phosphinate (5a), or methyl-3-(4-fluorophenyl)-2-oxopropanoate (5b), to their respective bulky S-amino acids esters (6) namely, [(3S)-3-amino-4-methoxy-4-oxobutyl]-(methyl) phosphinate (6a), or methyl (2S)-2-amino-3-(4-fluorophenyl)propanoate (6b) in the presence of either PLP or PMP.
[0019] FIG. 3: Proposed reaction mechanism for first half of transamination activity of the engineered artificial threonine deaminase. “?” indicates a residue side chain incorporated in the active site that can donate a proton to alter the reaction kinetics towards the transaminase reaction. In the beginning, the reaction proceeds in a manner similar to the native threonine deaminase reaction of a,ß-elimination (1). The presence of a proton donor near the catalytic lysine is proposed to not lead to the formation of the geminal diamine intermediates that results in the formation of the the schiff’s base with the catalytic lysine and PLP (4). The planar quinoid intermediate with the L-threonine substrate eventual opens up to nucleophilic attack from the water as a result of the a,ß-elimination reaction of the previous steps leading to the formation of PMP and 2-oxo-butyric acid (5).
[0020] FIG. 4: Schematic presentation of the workflow for the development of artificial threonine deaminase of the present invention for the Conversion of L-threonine (1) to 2-oxo-butyric acid (3) and any one of the bulky a-ketoacids and esters (2a, 2b, 5a or 5b), to their respective bulky S-amino acids or S-amino esters (4a, 4b, 6a, 6b) in the presence of either PLP or PMP. KTA: Kcat Torsion angle Analysis method; LSFG: Local Spherical Feature Grid method.
[0021] FIG. 5: Generation of Artificial threonine deaminase sequences from base templates T1 (WP_050116487.1), T2 (WP_000785596.1), T3 (WP_156265197.1) and T4 (WP_202668756.1). (A) The database of PLP-dependent enzymes. (B) Identification of high energy regions in base templates T1-T4 to derive Localized Spherical Feature Grids (LSFGs) of high energy regions. (C) Using Kcat Torsion angle Analysis (KTA) to filter S-specific PLP dependent enzymes to build an internal database of LSFGs in the regions around proton donors and stable regions of the S-specific PLP based enzymes. (D) LSFG matching to replace high energy regions in the base templates with the proton donors and stable regions of the S-selective PLP dependent enzymes of the internal database thereby generating artificial threonine deaminase sequences. (E) Computational validation of artificial threonine deaminase sequences as described in STEPS (7)-(10) in Figure 4, to derive the Artificial threonine deaminase described in SEQ ID NO. 1
[0022] FIG. 6: (A) Three-dimensional structure of engineered artificial Threonine deaminase where Pyridoxal 5'-phosphate (PLP) is represented as spheres. The encircled portion represents the active site. The images (B), (C) and (D) represents the modelled binding conformation of substrates L-Threonine, 4-(hydroxymethylphosphinyl)-2-oxobutyric acid and 4-fluoro-oxo-benzenepropanoic acid, respectively and their interaction profile. Conventional hydrogen bonding interactions, halogen interactions and, alkyl interactions are shown in dashed lines.
[0023] FIG. 7. Expression vector designed to house the artificial threonine deaminase for heterologous expression in E. coli.
[0024] FIG. 8. Assessment of recombinant engineered threonine deaminase expression using SDS-PAGE. The bands 1-8 correspond to recombinant engineered threonine deaminase and its variants as given in SEQ ID NO. 1, 5, 6, 10, 12, 13, and 15, respectively.
DETAIL DESCRIPTION OF THE INVENTION
Terminologies Explained / Abbreviations
[0025] All technical and scientific terms used herein, unless otherwise defined, are generally intended to have the same meaning as would be expected of a person of ordinary skill in the field to which this invention relates. The following terms are meant to mean the following things as they are used here.
[0026] “Encoding,” “encode” herein refers to transforming coding polynucleotide sequence into polypeptide sequence or protein or enzyme to perform the reactions.
[0027] “Polypeptide” and “protein” or “amino acid sequence” refer to any polymer containing at least two amino acids covalently joined by an amide bond regardless of length or post-translational modification.
[0028] “Threonine Deaminase or Thr-deaminase or TD or ThrD” used interchangeably herein, to refer to a polypeptide capable of converting a-amino acid to a-keto acids, specifically L-threonine to 2-ketobutyric acid. In some embodiments, the polypeptide is also capable of converting different a-amino acids such as L-Ser, and pyruvic acid in the presence of PLP cofactor.
[0029] “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).
[0030] "Derived from" designates the original polypeptide and/or the gene that codes for that polypeptide, which forms the basis of the engineering or procedures.
[0031] “Naturally occurring” and “wild type” are interchangeable in this context to denote polypeptides or polynucleotides that arise naturally and have not undergone deliberate human modification.
[0032] "Substrate" describes a chemical compound that an enzyme is intended to change into another material or chemical compound known as a product.
[0033] “Cofactor” refers to a substance that is necessary or beneficial for the activity of an enzyme. In the context of Threonine deaminase, the cofactor is generally a pyridoxal-5'-phosphate.
[0034] “PLP cofactor” or pyridoxal-5'-phosphate and “PMP cofactor” or pyridoxamine 5'-phosphate refers to cofactors that bind to threonine deaminase.
[0035] “Thermostable protein” refers to a polypeptide that maintains similar activity (more than e.g., 50% to 70%) after exposure to high or low temperature (10 – 20°C or 30 - 60°C) for a period of time (e.g., 0.5 – 24 hrs) compared to the untreated polypeptide.
[0036] “Conversion” refers to the enzymatic conversion of a substrate into a product, and the biocatalytic conversion of that substrate into a product.
[0037] “Percentage conversion” refers to the conversion of substrate into a product within a given period and reaction medium. The percentage conversion can be explained by the conversion of substrate into a product.
[0038] “Reaction medium” refers to a solution comprising a mixture of two or more components (e.g., enzyme, substrate, cofactor, cofactor recycling enzyme, appropriate buffer. in which reaction takes place.
[0039] “pH stable protein” refers to a polypeptide or an amino acid sequence which is stable or maintains similar activity after exposure to high or low temperatures (10 – 20°C or 30 - 60°C) for a period of time (e.g., 0.5 – 24 hrs) compared to the untreated polypeptide.
[0040] "Acidic amino acid or residue" refers to hydrophilic amino acid residues, which are acidic and possess a side chain that can donate a proton (H+). Because they have a negative charge at neutral pH, aspartate (Asp, D) and glutamate (Glu, E) are the two common acidic amino acids.
[0041] "Basic amino acid or residue" describes residues with side chains that are positively charged at physiological pH and have a nitrogen atom that can accept a proton (H+). Lysine (Lys, L), arginine (Arg, R), and histidine (His, H) are the three common basic amino acids.
[0042] "Polar amino acid or residue" refers to those that have an affinity for water molecules on their side chains, which allows them to establish hydrogen bonds. These amino acids have a polarity that enables them to interact successfully in aquatic environments despite not being totally hydrophobic or fully charged. Serine (Ser, S), threonine (Thr, T), tyrosine (Tyr, Y), asparagine (Asn, N), and glutamine (Gln, Q) are a few examples of polar amino acids.
[0043] "Non-polar amino acid or residue" refers to residues that, when present in a protein, are distinguished by their unfavourable interactions with water due to their hydrophobic side chains. Within the protein structure, these amino acids have a tendency to group together, creating a hydrophobic core that is protected from the aqueous environment. Glycine (Gly, G), alanine (Ala, A), valine (Val, V), leucine (Leu, L), isoleucine (Ile, I), proline (Pro, P), tryptophan (Trp, W), phenylalanine (Phe, F), methionine (Met, M), and cysteine (Cys, C) are a few examples of non-polar amino acids.
[0044] "Hydrophilic amino acid or residue" refers to these amino acids that are present on the surface of proteins, where they have the ability to interact with water. These comprise amino acids with charged side chains like aspartate (Asp, D) and glutamate (Glu, E), as well as polar side chains like serine (Ser, S), threonine (Thr, T), and tyrosine (Tyr, Y) that may form hydrogen bonds with water. Hydrophilic amino acids are necessary for proteins to function.
[0045] "Hydrophobic" amino acids refer to amino acids with side chains that are non-polar and tend to avoid water. These include alanine (Ala, A), valine (Val, V), leucine (Leu, L), isoleucine (Ile, I), proline (Pro, P), phenylalanine (Phe, F), and cysteine (Cys, C).
[0046] "Aromatic amino acid or residue" refers to amino acids that include an aromatic ring in their structure. Among the 20 standard amino acids, phenylalanine (Phe, F), tryptophan (Trp, W), and tyrosine (Tyr, W) are classified as aromatic due to their side chains containing a benzene ring.
[0047] "Aliphatic amino acid or residue" refers to the aliphatic side chain functional group that is present in amino acids. These amino acids are hydrophobic and non-polar, which means they usually reside in the hydrophobic core of proteins and have a tendency to avoid water. The length of the carbon atoms in the hydrocarbon chain causes these amino acids to become more hydrophobic. Aliphatic amino acids include, for example, proline (Pro, P), valine (Val, V), leucine (Leu, L), isoleucine (Ile, I), and alanine (Ala, A).
[0048] "Amino acid difference or residue difference" refers to a polypeptide chain that differs from a reference sequence. An amino acid in one place might move to another, for instance, causing a change in the polypeptide chain.
[0049] “Charged residues” refers to amino acids that hold charges in them such as positively charged (Lys, K and Arg, R) or negatively charged (Glu, E and Asp, D) amino acids.
[0050] “Salt bridges” refer to the interactions between positively charged residue and negatively charged residue. The positively charged residues such as Lys or Arg interact with negatively charged residues such as Asp or Glu. These interactions stabilize the quaternary structure of the enzymes.
[0051] “Salt bridge variants” refers to designing engineered variants based on the improving the salt-bridge interactions between certain residues across the enzyme structure.
[0052] “Disulfide variants” refers to designing engineered variants based on formation of di-sulfide bridges to improve enzyme stability.
[0053] “Denovo design” refers to designing engineered variants based on an ab-initio method that aims at improving the stability and local interactions of the residue side chains in regions across the enzyme structure.
[0054] “Artificial threonine deaminase (Ar-TDA)”, “engineered artificial threonine deaminase”, “engineered threonine deaminase”, “recombinant engineered threonine deaminase”, “artificial engineered threonine deaminase”, “engineered artificial polypeptide” are used interchangeably herein, to denote the mutated polypeptide sequence of threonine deaminase as claimed in this invention.
[0055] “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.
[0056] “Recombinant engineered formate dehydrogenase”, herein refers to denote the engineered threonine deaminase derived using the method mentioned in the embodiment. The engineered threonine deaminase 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.
[0057] “Phylogenetic based substitutions” herein to denote to introduce a canonical substitution at a given hotspots to improve catalytic efficiency for the substrates.
[0058] “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.
[0059] 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.
[0060] “PLP-dependent enzymes” and “PLP-bound enzymes” are used herein to refer to the class of enzymes that utilize pyridoxal 5'-phosphate (PLP) as a coenzyme at their active site.
[0061] “PLP-dependent enzyme database” refers herein to a curated collection of enzymes that utilize pyridoxal 5'-phosphate (PLP) as a coenzyme at their active site.
[0062] “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.
[0063] “Induced fit method” refers to a computational modeling approach in which the binding of a substrate induces a conformational change in the active site of an enzyme, thereby optimizing the interaction and enhancing specificity and catalytic efficiency.
[0064] “Binding energy” refers to the computed energy associated with the interaction between a substrate and an enzyme, accounting for both the direct molecular interactions and the conformational changes induced in the enzyme's active site.
[0065] “Threonine deaminase activity” refers to the a,ß-elimination reaction catalysed by threonine deaminase enzymes. Specifically, the conversion of L-threonine or L-serine to 2-oxo-butyric acid or pyruvic acid, respectively with the release of ammonia, in the presence of PLP cofactor.
[0066] “Amino-transferase activity” refers to the reversible amine transfer reaction catalysed by various PLP-dependent enzymes such as aminotransferase and ?-transaminases. For the purposes of this embodiment, aminotransferase activity refers to the conversion of a-keto compounds into their corresponding a-amine compounds in the presence of the enzyme and PMP cofactor.
[0067] “Cosubstrate” refers to the sacrificial substrate that is consumed in an enzymatic biocatalysis reaction during the conversion of the substrate to product, thereby forming a biproduct during the reaction process. For the purposes of this embodiment, L-threonine acts as the co-substrate during the aminotransferase activity of the recombinant engineered threonine deaminase of the present invention, thereby acting as the amine donor for transamination and in turn getting converted into 2-oxo-butyric acid or 2-oxo-butyrate.
[0068] The primary objective of the present invention was to utilize the robustness of threonine deaminase enzymes for the synthesis of non-natural amino-acids compounds of industrial and pharmaceutical relevance. Specifically, the present invention seeks to develop an amino-transferase activity in a threonine deaminase polypeptide. A reaction mechanism was proposed to alter the a, ß elimination reaction in a way that yields an active PMP in the active site to be used in the aminotransferase reaction for the conversion of bulky a-ketoacids and a-keto-esters to their respective amines (Fig. 3). The present invention provides an engineered artificial threonine deaminase showing amino-transferase and threonine deaminase activity in a reaction mixture that comprises of L-threonine (1), bulky ketoacids and either PLP or PMP to derive a product of 2-oxo-butyrate (3) and bulky amino acids. Specifically, the engineered artificial threonine deaminase converts bulky a-ketoacids to their respective bulky amino acids with a side reaction of converting L-threonine to 2-oxo-butyrate.
[0069] In some embodiments, the engineered artificial threonine deaminase takes the bulky a-ketoacids chosen from (3-carboxy-3-oxopropyl)(methyl)phosphinate (2a), or 4-fluorophenyl-pyruvic acid (2b) as substrate to get L-phosphinothricin (4a), or L-4-fluorophenylalanine (4b) as product, respectively in the presence of either PLP or PMP. In some embodiments, bulky-a-keto-esters can replace bulky ketoacids to derive bulky amino acid esters. In such cases, the engineered threonine deaminase can take any one of the bulky keto-esters, namely (4-methoxy-3,4-dioxobutyl)(methyl)phosphinate (5a), or methyl-3-(4-fluorophenyl)-2-oxopropanoate (5b) as substrate, and convert them to their respective bulky amino acids esters namely, [(3S)-3-amino-4-methoxy-4-oxobutyl]-(methyl) phosphinate (6a), or methyl (2S)-2-amino-3-(4-fluorophenyl)propanoate (6b) in the presence of either PLP or PMP.
[0070] In some embodiments, the present disclosure also provides threonine deaminase polynucleotides encoding the engineered artificial threonine deaminase polypeptides given herein as SEQ ID NO 201. The polynucleotide can include promoters and other regulatory elements useful for expression of the encoded engineered threonine deaminase and utilizes codons optimized for specific desired expression systems.
[0071] The artificial engineered threonine deaminase enzyme of the present invention was developed using a combination of computational and molecular biology techniques as described in the scheme in Fig.4. The sequences of PLP bound enzymes (STEP 1, Table 8 and Fig. 5A) such as Acetyltransferases, aldolases, Alliinases, Aminomutases, aminotransferases, coenzyme ligases, cyclases, cytidylyltransferases, deaminases, decarboxylases, dehydrases, dehydratases, desulfurases, dicarboxylases, epimerases, hydrolases, hydroxymethyltransferases, isomerases, kinases, kynureninases, ligases, lyases, oxidases, Oxidoreductases, palmitoyltransferases, phosphatases, phospholyases, phosphorylases, racemases, signalling proteins, sulfhydrolases, sulfhydrylases, synthases, deaminases, transaminases, and transferases were processed using an in silico method (STEP 2). The threonine deaminase sequences were segregated from the list of PLP bound enzymes (STEP 3) and a binding energy value was determined for the substrate L-threonine (L-Thr) by an induced fit method.
[0072] Top 4 sequences, database ID: WP_050116487.1, WP_000785596.1, WP_156265197.1 and WP_202668756.1, were chosen, based on L-Thr binding energy values, as base templates to build an artificial threonine deaminase (STEP 4). The high energy regions across these enzymes were determined and stored as using the Localized Spherical Feature Grids (LSFG) (Kumar, P et. al., 2023) (Fig. 5B). The remainder of the PLP based enzymes were categorized and S-selective enzymes were filtered using the Kcat Torsional angle Analysis (KTA) method (STEP 5) (Kumar, P. et al., 2021) and using the LSFG method, the localized regions around proton donors and low-energy stable regions were characterized and compiled in an internal database (Fig. 5C).
[0073] The objective of this in silico method was to characterise the active site of PLP bound enzymes, to introduce an amino-acid side chain capable of donating a proton to the catalytic lysine. As per the proposed reaction mechanism, reaction in the threonine deaminase forms Schiff’s base with PLP. A proton donor is able to divert the reaction to yield and active PMP after the a,ß elimination reaction to be used as the catalyst for the transamination reaction for the conversion of any one of the bulky S-ketoacids and esters (2a, 2b, 5a, or 5b), to their respective bulky S-amino acids or amino esters (4a, 4b, 6a, or 6b). Artificial threonine deaminase sequences were generated using the method of localized spherical feature grid matching (Kumar, P. et al., 2023), wherein, a proton donor or donors were introduced into the active site near the catalytic lysine of the base template sequences, followed by introduction of motifs from regions of stability, derived from an internal database of low energy regions exclusive to S-selective PLP bound enzymes (STEP 6, Fig. 5D). This resulted in the generation of artificial threonine deaminases with motifs derived from S-selective PLP based enzymes (SEQ ID NO: 1, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211). A computational proton donor validation was performed, wherein, using a molecular dynamics simulation method, feasibility of proton donation from the proton donor to the catalytic lysine was assessed (STEP 7). Post this, a computational quantum chemical-based reaction characterisation step was conducted (STEP 8) to determine the feasibility of the newly generated sequences to catalyse the a,ß-elimination reaction (STEP 9) and transamination reactions (STEP 10), based on the proposed reaction mechanism described above. STEPs (6)-(10) were repeated iteratively until the artificial engineered threonine deaminase showed both transamination activity and threonine deaminase activity in the presence of L-Thr and either bulky a-keto acids or bulky a-keto esters. Residues such as D24, D71, K101, K108, D142, K299, K334, R371, K431, K458, and E459 contributed to formation of salt-bridge interactions aiding in improving the stability of the artificial threonine deaminase enzymes and were consistently observed in the top ranked artificial threonine sequences. The engineered artificial threonine deaminase derived from the above method was described by the polypeptide given in SEQ ID NO. 1 (STEP 11, Fig. 5E) and the residues that contributed to its stability were deemed as features. The artificial engineered threonine deaminase was subjected to rational-based engineering (STEP 12), which involved the steps of improving the binding affinity of the bulky a-keto acids and bulky a-keto esters in the active site of the artificial engineered threonine deaminase. The artificial engineered threonine deaminase was studied, and mutations were developed from hotspots that were chosen based on the interactions of the side chains with the substrate molecules bound in the active site (STEP 13). To enhance the activity of the threonine deaminase towards the three substrates namely (3-carboxy-3-oxopropyl)(methyl)phosphinate (2a) and 4-fluorophenyl-pyruvic acid (2b), and L threonine, binding mode studies were conducted for these molecules by modelling them into the active site of the engineered artificial threonine deaminase (Fig. 6A) site in the presence of the PLP-lysine Shiff’s base intermediate (Fig 6B, C and D, respectively).
[0074] The substitutions for hotspots of the artificial engineered threonine deaminase that are in the region of the protein that is 8Å away from the catalytic Shiff’s base centre were derived from the various approaches such as electrostatic modulation and a partial de novo approach that increases the stability of the enzyme by introducing salt-bridges and hydrophobic clusters at the periphery regions of the enzyme (STEP 14). The mutations derived for improving substrate interactions and for improving the stability of the artificial threonine deaminase, were combined to generate variants that showed a synergistic effect in the improvement of catalytic efficiency (STEP 15). The generated variants were characterized using computational proton donor validation as described in STEP 7 (STEP 16) and biochemical characterization for the required activity (STEPs 17, 18, 19). Finally, variants that showed both transamination activity and threonine deaminase activity in the presence of L-Thr and either bulky a-keto acids or bulky a-keto esters, were classified as top variants. These variants were used as the basis for generation of more variants by combining them with other variants that show the required activity (STEP 20)
[0075] In some embodiments, the engineered threonine deaminase polypeptides comprise an amino acid sequence that has one or more residue differences as compared to SEQ ID NO: 1. The residue differences can be non-conservative substitutions, conservative substitutions, or a combination of non-conservative and conservative substitutions.
[0076] The present invention details engineered artificial threonine deaminase polypeptides that show 90% sequence identity to SEQ ID NO. 1 and contains the features of X24D, X71D or K, X101K, X108K or C, X142D or C, X299K, X334K, X371R, X431K, X458K, and X459E. In some embodiments, the engineered artificial threonine deaminase polypeptides comprise amino acid sequences that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% identical to SEQ ID NO:1.
[0077] In some embodiments, the engineered artificial threonine deaminase polypeptides also contain one or more of the following residue differences when compared to the artificial sequence of SEQ ID No. 1: The residue corresponding to X20 is V or I; The residue corresponding to X28 is A or V; The residue corresponding to X70 is M or L; The residue corresponding to X96 is F or L; The residue corresponding to X98 is S or A; The residue corresponding to X153 is W or F; The residue corresponding to X161 is M or A; The residue corresponding to X171 is L or M; The residue corresponding to X183 is V or I; The residue corresponding to X193 is A or V; The residue corresponding to X211 is A or G; The residue corresponding to X221 is K or R; The residue corresponding to X251 is T or P; The residue corresponding to X284 is V or I; The residue corresponding to X313 is I or V;
[0078] In some embodiments, the engineered artificial threonine deaminase polypeptides can have an amino acid sequence that differs by one or more residues from the SEQ ID NO:1 at the following residue positions corresponding to X3, X19, X40, X42, X45, X47, X55, X69, X75, X86, X87, X88, X89, X90, X92, X111, X114, X115, X123, X124, X130, X133, X135, X136, X140, X143, X146, X148, X156, X164, X235, X240, X244, X249, X273, X294, X301, X302, X306, X348, X354, X357, X360, X361, X375, X386, X389, X390, X393, X397, X398, X400, X401, X402, X403, X405, X429, X432, X442, X445, X449, X452, X457, X472, X473, X478, X482, X483, X484, X487, X490, X491, X493, X494, X497, X504, and X509.
[0079] Furthermore, in some embodiments, the engineered artificial threonine deaminase polypeptides contain additionally the following residue differences when compared to SEQ ID No. 1: The residue corresponding to X3 is D or V; The residue corresponding to X19 is C or A; The residue corresponding to X40 is L or I; The residue corresponding to X42 is E, S or L; The residue corresponding to X45 is D or A; The residue corresponding to X47 is V or T; The residue corresponding to X55 is Q or R; The residue corresponding to X69 is F or M; The residue corresponding to X75 is E or A; The residue corresponding to X87 is K or A; The residue corresponding to X92 is Y or Q; The residue corresponding to X111 is T, V or E; The residue corresponding to X114 is P or A; The residue corresponding to X115 is P or D; The residue corresponding to X123 is S or G or E; The residue corresponding to X124 is L or F; The residue corresponding to X130 is A or L; The residue corresponding to X133 is E, A or F; The residue corresponding to X135 is L, F, D, V or K; The residue corresponding to X136 is E, D or M; The residue corresponding to X140 is A, G or E; The residue corresponding to X143 is L or I; The residue corresponding to X146 is A or S; The residue corresponding to X148 is Q or L; The residue corresponding to X164 is C or A; The residue corresponding to X235 is I or V; The residue corresponding to X249 is E or D; The residue corresponding to X273 is C or A; The residue corresponding to X294 is C or A; The residue corresponding to X301 is A or Q; The residue corresponding to X302 is L or Q; The residue corresponding to X306 is R or Q; The residue corresponding to X348 is E or Q; The residue corresponding to X354 is K or R; The residue corresponding to X357 is Q or E; The residue corresponding to X360 is H or G; The residue corresponding to X361 is V or G; The residue corresponding to X375 is K or E; The residue corresponding to X386 is S, T or I; The residue corresponding to X389 is L or H; The residue corresponding to X390 is E or V; The residue corresponding to X393 is K or E; The residue corresponding to X397 is Q or S; The residue corresponding to X398 is M or E; The residue corresponding to X400 is N or R; The residue corresponding to X401 is D or A; The residue corresponding to X402 is G or K; The residue corresponding to X403 is G or D; The residue corresponding to X405 is S or Q; The residue corresponding to X429 is H or K; The residue corresponding to X432 is Q or R; The residue corresponding to X442 is N or E; The residue corresponding to X445 is M or G; The residue corresponding to X449 is R or K; The residue corresponding to X452 is N or H; The residue corresponding to X457 is Y or H; The residue corresponding to X472 is Y or F; The residue corresponding to X473 is A or G; The residue corresponding to X478 is A or G; The residue corresponding to X482 is G or S; The residue corresponding to X483 is D or A; The residue corresponding to X484 is H or S; The residue corresponding to X487 is D or Q; The residue corresponding to X490 is T or Q; The residue corresponding to X491 is R or H; The residue corresponding to X493 is N or A; The residue corresponding to X494 is E or A; The residue corresponding to X497 is W or Y; The residue corresponding to X504 is N or D; The residue corresponding to X509 is R or K;
[0080] Furthermore, in some embodiments, the engineered artificial threonine deaminase polypeptides contain additionally the following residue differences when compared to SEQ ID No. 1: The residue corresponding to X89 is E, K, S, T or N; The residue corresponding to X240 is W, Y, or E; The residue corresponding to X88 is D, T or G; The residue corresponding to 90 is L or H; The residue corresponding to X156 is P or S; The residue corresponding to X86 is S or Y; The residue corresponding to X244 is V, W, Y or Q
[0081] In some embodiments, the engineered artificial threonine deaminase sequence given 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, and 200 can have an amino acid difference by one or more substitutions in combination with one or multiple that are at least about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% identical to SEQ ID NO:1 (Table 1).
[0082] The engineered variants significantly improved the activity and stability of the engineered artificial threonine deaminase enzymes for biocatalytic applications.
[0083] As demonstrated in Table 1, variants such as SEQ ID NO: 2, which comprises five substitutions (I40L, A75E, Q301A, E357Q, G478A), exhibit a 2-fold increase (++) in threonine deaminase activity while maintaining the same transaminase activity (+) as SEQ ID NO: 1. Other variants with additional substitutions exhibit varying degrees of activity enhancement. The variants of artificial threonine deaminase were developed to be effective at lower enzyme concentrations, i.e., the enzyme required for successful conversion of the bulky a-keto acids or esters to their respective a-amino acids or esters is a concentration of 0.25% w/w of L-threonine added in the reaction mixture. In some embodiments the engineered threonine deaminase is designed through the incorporation of one or more substitutions to work under the conditions of 45-60 °C. The engineered threonine deaminase enzyme with one or more substitutions incorporated into the sequence not only facilitated the =90% conversion of bulky a-keto acids or esters into their respective a-amino-acid or a-amino-acid esters but also at least 70% or up to 90% increase in the catalytic efficiency of converting at least 560mM of L-threonine to 2-oxo-butyrate within 60 minutes using an enzyme concentration that is as low as a hundredth of the concentration of L-threonine added into the reaction mixture.
[0084] In some embodiments of the process herein, the engineered threonine deaminase can be present in the forms of whole cells, including whole cells transformed with polynucleotide constructs. In some embodiments, the engineered threonine deaminase can be present in the form of cell extracts and/or lysate thereof and may be employed in a variety of different forms, including solid (e.g., lyophilized, spray-dried) or semisolid (e.g., a crude paste). In some embodiments, the engineered threonine deaminase is isolated and can be in a substantially purified form.
[0085] In some embodiments, the engineered threonine deaminase polypeptides of the present disclosure are capable of converting L-threonine (1) to 2-oxo-butyric acid (3) and any one of the bulky ketoacids (2), (3-carboxy-3-oxopropyl)(methyl)phosphinate (2a), or 4-fluorophenyl-pyruvic acid (2b) to their respective bulky amino acids (4) namely, phosphinothricin (4a), or L-4-fluorophenylalanine (4b) or any one of the bulky keto-esters (5), namely (4-methoxy-3,4-dioxobutyl)(methyl)phosphinate (5a), or methyl-3-(4-fluorophenyl)-2-oxopropanoate (5b), to their respective bulky amino acids esters (6) namely, [(3S)-3-amino-4-methoxy-4-oxobutyl]-(methyl) phosphinate (6a), or methyl (2S)-2-amino-3-(4-fluorophenyl)propanoate (6b) in the presence of either PLP or PMP and comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more identical to SEQ ID NO: 1.
[0086] In some embodiments, the improved enzymatic activity of the engineered artificial threonine deaminase polypeptides is also associated with other improvements in enzyme properties. In some embodiments, the improvement in enzyme property is concerning thermal stability, such as at 30°C, 40°C, 45°C, 50°C, 55°C, and 60°C or higher.
[0087] The engineered artificial threonine deaminase reaction can typically be performed at a pH of 10.0 or lower, more typically in the range of 6.5 to 10.0. In certain variations, the procedure is executed at a pH of around 8.0 or lower, often within the range of approximately 6.5 to 8.0. The process of generating amino acids with engineered artificial threonine deaminase can, in certain implementations, be carried out in a pH range of approximately 9.0 to 11.0, more specifically at 10.0. In certain variations, the procedure could be executed at a neutral pH, or roughly 7.0.
[0088] In some embodiments, the threonine deaminase that has been artificially generated or engineered, is produced using well-established protein synthesis techniques. Specifically, the process of creating an engineered artificial Threonine deaminase gene involves first preparing the host organism E. coli BL21 (DE3) and then inserting it using the plasmid pET28a(+). In this plasmid, the artificial threonine deaminase gene is organized between the restriction sites of NcoI and EcoRI, controlled by the T7 promoter (Fig. 7). Next comes the process of cultivating the host, which involves causing the expression of the engineered threonine deaminase gene within the host after the logarithmic growth phase. This is achieved by culturing the host at a temperature that is below what is ideal for host cell growth and survival. There are no restrictions on the use of other conventional promoters as an inducible promoter in the process of creating the artificial threonine deaminase. For example, when transcription is employed with the expression host E. coli, an inducible promoter that is triggered by isopropyl -D-1-thiogalactopyranoside (IPTG) can be used. Examples of this kind of promoter include T7, Lac, Tac, Trp, and Trc. The cultured host can be used as whole cell catalysts for the biocatalysis described in the present invention. Furthermore, the host cell was lysed by means of lysis techniques such as sonication, lysis buffer etc. to produce a crude lysate to be used in the biocatalysis described in the present invention. Additionally, the amount of protein produced was quantified by molecular biology techniques such as SDS page analysis (Fig. 8) and Bradford’s assay (Table 2).
[0089] Table 1: List of sequences and their corresponding residue differences (substitutions) with respect to SEQ ID No. 1 generated during the engineering process of the artificial threonine deaminase.

SEQ ID No. Residue difference from SEQ ID No. 1 No. of Residue difference from SEQ ID No: 1 Expression Threonine deaminase Activity Transaminase
Activity
SEQ ID NO: 1 NA 0 ++ + +
SEQ ID NO: 2 X3D, X92Y, X96L, X183I, X402G 5 ++ ++ +
SEQ ID NO: 3 X87K, X130A, X284I, X313V, X398M 5 ++ + +
SEQ ID NO: 4 X3D, X135L, X171M, X211G, X306R, X360H 6 ++ ++ +
SEQ ID NO: 5 X28V, X114P, X130A, X135K, X146A, X183I, X445M, X452N 8 +++ ++ ++
SEQ ID NO: 6 X92Y, X96L, X143L, X146A, X153F, X164C, X302L, X361V, X457Y 9 +++ ++ ++
SEQ ID NO: 7 X3D, X19C, X114P, X115P, X124L, X251P, X313V, X398M, X494E, X509R 10 +++ ++ ++
SEQ ID NO: 8 X3D, X19C, X92Y, X130A, X136M, X146A, X171M, X193V, X400N, X405S, X442N 11 ++ ++ ++
SEQ ID NO: 9 X3D, X28V, X87K, X130A, X133F, X135L, X164C, X171M, X273C, X301A, X400N, X457Y 12 ++ + +
SEQ ID NO: 10 X3D, X87K, X114P, X115P, X123E, X135K, X164C, X193V, X284I, X398M, X402G, X452N, X457Y 13 +++ + +
SEQ ID NO: 11 X20I, X92Y, X114P, X130A, X136M, X143L, X235I, X251P, X273C, X301A, X302L, X491R, X497W 13 ++ ++ +
SEQ ID NO: 12 X47V, X87K, X114P, X124L, X130A, X135K, X136E, X143L, X164C, X221R, X251P, X306R, X386I, X497W, X504N 15 +++ ++ +
SEQ ID NO: 13 X42L, X55Q, X92Y, X114P, X123E, X136M, X164C, X193V, X221R, X235I, X249E, X302L, X361V, X398M, X497W, X509R 16 +++ ++ +
SEQ ID NO: 14 X42L, X55Q, X87K, X92Y, X114P, X123E, X124L, X143L, X164C, X249E, X251P, X301A, X313V, X361V, X390E, X445M, X497W 17 ++ ++ +
SEQ ID NO: 15 X19C, X42E, X47V, X55Q, X75E, X87K, X98A, X130A, X133E, X146A, X164C, X249E, X284I, X302L, X386S, X398M, X442N, X509R 18 +++ +++ +
SEQ ID NO: 16 X19C, X42L, X47V, X69F, X75E, X92Y, X123E, X124L, X143L, X146A, X171M, X235I, X273C, X284I, X405S, X429H, X445M, X478A, X490T, X504N 20 ++ ++ +
SEQ ID NO: 17 X3D, X28V, X42E, X75E, X87K, X92Y, X114P, X123S, X124L, X133F, X135K, X136M, X146A, X153F, X249E, X348E, X398M, X457Y, X491R, X494E, X509R 21 ++ ++ +
SEQ ID NO: 18 X3D, X20I, X42E, X75E, X87K, X92Y, X96L, X114P, X115P, X123S, X124L, X130A, X135L, X143L, X146A, X164C, X249E, X294C, X302L, X390E, X442N, X473A, X478A, X509R 24 +++ ++ +++
SEQ ID NO: 19 X3D, X19C, X42L, X55Q, X69F, X70L, X87K, X115P, X123S, X124L, X133E, X136M, X143L, X146A, X153F, X164C, X249E, X273C, X294C, X301A, X302L, X360H, X390E, X445M, X473A 25 +++ ++ ++
SEQ ID NO: 20 X3D, X19C, X20I, X42L, X47V, X55Q, X69F, X92Y, X115P, X130A, X133E, X135L, X136E, X143L, X146A, X164C, X171M, X273C, X294C, X302L, X306R, X348E, X390E, X402G, X405S, X429H 26 ++ + ++
SEQ ID NO: 21 X3D, X19C, X20I, X47V, X69F, X75E, X92Y, X114P, X123E, X124L, X130A, X133E, X135K, X136M, X143L, X146A, X161A, X164C, X249E, X273C, X301A, X306R, X357Q, X361V, X386I, X390E, X445M, X452N, X490T 29 ++ ++ ++
SEQ ID NO: 22 X3D, X19C, X42L, X55Q, X69F, X87K, X115P, X123E, X124L, X130A, X133E, X135L, X136M, X143L, X146A, X164C, X183I, X221R, X235I, X249E, X273C, X301A, X302L, X348E, X357Q, X386S, X398M, X402G, X405S, X497W 30 +++ ++ ++
SEQ ID NO: 23 X3D, X42L, X47V, X55Q, X69F, X75E, X87K, X92Y, X114P, X115P, X133E, X135K, X136M, X143L, X146A, X164C, X171M, X211G, X235I, X249E, X273C, X302L, X348E, X357Q, X398M, X400N, X402G, X429H, X449R, X494E 30 ++ ++ +
SEQ ID NO: 24 X19C, X42L, X47V, X55Q, X69F, X75E, X87K, X92Y, X98A, X114P, X115P, X123E, X130A, X133E, X135L, X136M, X143L, X146A, X164C, X235I, X273C, X313V, X348E, X386S, X429H, X442N, X445M, X452N, X457Y, X473A, X504N 31 +++ ++ ++
SEQ ID NO: 25 X3D, X19C, X20I, X42L, X47V, X55Q, X75E, X87K, X92Y, X115P, X123E, X124L, X130A, X133E, X135K, X136M, X143L, X146A, X164C, X235I, X249E, X273C, X284I, X294C, X306R, X348E, X386S, X402G, X429H, X478A, X484H, X509R 32 +++ ++ +
SEQ ID NO: 26 X3D, X19C, X42L, X47V, X55Q, X69F, X75E, X87K, X114P, X115P, X123S, X130A, X133F, X135K, X136M, X143L, X146A, X164C, X183I, X221R, X235I, X249E, X273C, X301A, X306R, X348E, X357Q, X361V, X390E, X457Y, X473A, X497W, X504N 33 ++ ++ +++
SEQ ID NO: 27 X3D, X19C, X20I, X42L, X47V, X55Q, X69F, X75E, X87K, X92Y, X114P, X115P, X123S, X124L, X130A, X133E, X135K, X143L, X146A, X164C, X171M, X249E, X273C, X302L, X306R, X360H, X402G, X405S, X429H, X484H, X491R, X494E, X504N 33 +++ +++ +++
SEQ ID NO: 28 X3D, X19C, X20I, X42L, X47V, X55Q, X69F, X75E, X87K, X92Y, X114P, X115P, X123S, X124L, X130A, X133E, X135K, X136M, X143L, X146A, X164C, X235I, X249E, X273C, X284I, X301A, X302L, X357Q, X361V, X386I, X405S, X442N, X473A, X484H, X509R 35 ++ ++ ++
SEQ ID NO: 29 X3D, X19C, X42L, X47V, X55Q, X69F, X75E, X87K, X92Y, X114P, X115P, X123E, X124L, X130A, X133E, X135K, X136M, X143L, X146A, X164C, X235I, X249E, X273C, X284I, X313V, X357Q, X361V, X398M, X445M, X452N, X473A, X484H, X490T, X494E, X504N, X509R 36 ++ ++ +
SEQ ID NO: 30 X3D, X19C, X42L, X47V, X55Q, X69F, X75E, X87K, X92Y, X114P, X115P, X123E, X124L, X130A, X133F, X135K, X136E, X143L, X146A, X164C, X183I, X235I, X249E, X273C, X294C, X313V, X357Q, X360H, X386I, X398M, X402G, X442N, X452N, X484H, X494E, X504N 36 ++ ++ +++
SEQ ID NO: 31 X3D, X19C, X42L, X47V, X55Q, X69F, X75E, X87K, X92Y, X114P, X115P, X123S, X124L, X130A, X133F, X135K, X136E, X143L, X146A, X164C, X221R, X235I, X249E, X273C, X302L, X313V, X361V, X402G, X405S, X429H, X445M, X473A, X478A, X484H, X490T, X494E 36 ++ +++ +++
SEQ ID NO: 32 X3D, X19C, X20I, X42L, X47V, X55Q, X69F, X75E, X87K, X92Y, X114P, X115P, X123S, X124L, X130A, X133F, X135L, X136E, X143L, X146A, X153F, X164C, X235I, X249E, X273C, X294C, X302L, X361V, X386S, X398M, X405S, X429H, X442N, X490T, X491R, X497W, X504N 37 +++ +++ +++
SEQ ID NO: 33 X3D, X19C, X42E, X47V, X55Q, X69F, X70L, X75E, X87K, X92Y, X114P, X115P, X123S, X124L, X130A, X133F, X135L, X136M, X143L, X146A, X164C, X211G, X235I, X249E, X273C, X301A, X306R, X361V, X386S, X390E, X400N, X405S, X457Y, X473A, X484H, X494E, X504N 37 +++ +++ +++
SEQ ID NO: 34 X115P, X183I, X193V, X235I, X306R 5 + + -
SEQ ID NO: 35 X55Q, X171M, X249E, X251P, X473A 5 + + -
SEQ ID NO: 36 X42L, X98A, X130A, X221R, X442N 5 + - +
SEQ ID NO: 37 X19C, X47V, X221R, X284I, X484H 5 + - +
SEQ ID NO: 38 X19C, X96L, X130A, X171M, X301A, X361V 6 + + -
SEQ ID NO: 39 X28V, X123E, X124L, X161A, X306R, X490T 6 + + -
SEQ ID NO: 40 X19C, X20I, X193V, X273C, X348E, X509R 6 + + -
SEQ ID NO: 41 X19C, X20I, X96L, X124L, X235I, X405S, X452N 7 + + -
SEQ ID NO: 42 X20I, X55Q, X135K, X221R, X235I, X302L, X457Y 7 + - +
SEQ ID NO: 43 X114P, X143L, X164C, X183I, X221R, X390E, X490T 7 + - +
SEQ ID NO: 44 X133E, X136E, X146A, X153F, X211G, X361V, X386I 7 + - +
SEQ ID NO: 45 X114P, X115P, X124L, X211G, X313V, X386S, X494E 7 + + -
SEQ ID NO: 46 X20I, X28V, X123E, X133F, X273C, X386S, X402G 7 + + -
SEQ ID NO: 47 X75E, X87K, X143L, X171M, X221R, X386S, X491R 7 + + -
SEQ ID NO: 48 X42E, X96L, X114P, X130A, X133F, X183I, X386S, X504N 8 + + -
SEQ ID NO: 49 X96L, X115P, X143L, X171M, X249E, X273C, X400N, X491R 8 + + -
SEQ ID NO: 50 X19C, X70L, X87K, X92Y, X114P, X193V, X301A, X478A 8 + + -
SEQ ID NO: 51 X75E, X92Y, X96L, X135K, X193V, X235I, X360H, X457Y, X473A 9 + + -
SEQ ID NO: 52 X115P, X123S, X124L, X135L, X161A, X211G, X294C, X398M, X429H 9 + - +
SEQ ID NO: 53 X55Q, X130A, X133E, X193V, X235I, X251P, X301A, X429H, X457Y 9 + - +
SEQ ID NO: 54 X47V, X55Q, X98A, X164C, X273C, X313V, X360H, X390E, X402G 9 + - +
SEQ ID NO: 55 X3D, X19C, X42E, X133F, X146A, X153F, X221R, X398M, X442N, X478A 10 + + -
SEQ ID NO: 56 X20I, X87K, X124L, X130A, X143L, X161A, X235I, X386S, X490T, X509R 10 + + -
SEQ ID NO: 57 X55Q, X70L, X92Y, X96L, X115P, X133F, X164C, X386I, X445M, X494E 10 + + -
SEQ ID NO: 58 X3D, X19C, X69F, X133F, X143L, X211G, X313V, X348E, X473A, X494E 10 + - +
SEQ ID NO: 59 X3D, X42L, X87K, X96L, X123E, X124L, X211G, X294C, X452N, X504N 10 + - +
SEQ ID NO: 60 X47V, X69F, X96L, X124L, X143L, X146A, X284I, X302L, X449R, X509R 10 + + -
SEQ ID NO: 61 X69F, X87K, X123S, X124L, X135K, X143L, X161A, X183I, X294C, X306R, X405S 11 + + -
SEQ ID NO: 62 X3D, X19C, X69F, X130A, X133E, X146A, X183I, X211G, X390E, X402G, X457Y 11 + + -
SEQ ID NO: 63 X20I, X47V, X123S, X133F, X135L, X143L, X164C, X171M, X402G, X405S, X452N 11 + + -
SEQ ID NO: 64 X42L, X70L, X98A, X115P, X123E, X124L, X130A, X133F, X164C, X294C, X302L, X509R 12 + - +
SEQ ID NO: 65 X42E, X69F, X87K, X124L, X146A, X164C, X221R, X235I, X313V, X348E, X457Y, X494E 12 + - +
SEQ ID NO: 66 X98A, X114P, X123E, X130A, X133F, X135L, X143L, X249E, X313V, X361V, X390E, X429H 12 + + -
SEQ ID NO: 67 X19C, X42L, X98A, X114P, X123S, X135K, X136E, X146A, X211G, X348E, X360H, X442N, X497W 13 + + -
SEQ ID NO: 68 X70L, X123S, X124L, X133E, X143L, X153F, X164C, X235I, X249E, X294C, X348E, X357Q, X504N 13 + + -
SEQ ID NO: 69 X3D, X55Q, X69F, X96L, X114P, X115P, X143L, X193V, X273C, X398M, X429H, X452N, X473A 13 + + -
SEQ ID NO: 70 X3D, X19C, X20I, X92Y, X115P, X123E, X133F, X143L, X313V, X348E, X386S, X457Y, X490T 13 + + -
SEQ ID NO: 71 X3D, X55Q, X69F, X75E, X96L, X114P, X123E, X193V, X235I, X301A, X457Y, X494E, X497W 13 + + -
SEQ ID NO: 72 X3D, X19C, X42L, X47V, X98A, X115P, X164C, X249E, X313V, X390E, X445M, X478A, X509R 13 + + -
SEQ ID NO: 73 X42E, X75E, X87K, X143L, X164C, X171M, X221R, X235I, X249E, X429H, X452N, X490T, X497W 13 + + -
SEQ ID NO: 74 X28V, X47V, X92Y, X123E, X135K, X136M, X146A, X171M, X235I, X249E, X357Q, X390E, X400N, X491R 14 + + -
SEQ ID NO: 75 X19C, X28V, X42L, X75E, X87K, X114P, X136E, X146A, X161A, X164C, X360H, X400N, X490T, X494E 14 + + -
SEQ ID NO: 76 X42E, X47V, X69F, X92Y, X114P, X135L, X143L, X161A, X171M, X235I, X402G, X442N, X490T, X497W 14 + + -
SEQ ID NO: 77 X47V, X55Q, X69F, X87K, X123S, X130A, X146A, X164C, X193V, X211G, X273C, X405S, X457Y, X494E, X497W 15 + - +
SEQ ID NO: 78 X3D, X19C, X28V, X42L, X47V, X70L, X87K, X92Y, X124L, X133E, X235I, X294C, X457Y, X494E, X504N 15 + - +
SEQ ID NO: 79 X19C, X47V, X55Q, X75E, X87K, X135L, X136M, X161A, X211G, X249E, X273C, X357Q, X490T, X491R, X494E 15 + - +
SEQ ID NO: 80 X28V, X42E, X75E, X92Y, X124L, X130A, X135K, X136M, X164C, X171M, X273C, X294C, X301A, X302L, X473A, X494E 16 + + -
SEQ ID NO: 81 X19C, X42L, X70L, X87K, X92Y, X123S, X124L, X133E, X153F, X235I, X249E, X302L, X348E, X402G, X445M, X484H 16 + + -
SEQ ID NO: 82 X42E, X75E, X87K, X96L, X114P, X130A, X136E, X235I, X249E, X273C, X313V, X348E, X390E, X442N, X445M, X449R 16 + + -
SEQ ID NO: 83 X3D, X42L, X55Q, X70L, X75E, X92Y, X96L, X123S, X133E, X146A, X249E, X306R, X348E, X390E, X400N, X449R 16 + - +
SEQ ID NO: 84 X55Q, X87K, X92Y, X114P, X115P, X130A, X133F, X164C, X171M, X183I, X249E, X348E, X361V, X405S, X473A, X497W 16 + - +
SEQ ID NO: 85 X3D, X19C, X20I, X87K, X130A, X135L, X136E, X146A, X164C, X273C, X284I, X348E, X400N, X402G, X429H, X452N 16 + + -
SEQ ID NO: 86 X20I, X42L, X69F, X75E, X98A, X114P, X123E, X124L, X135K, X136E, X249E, X348E, X386I, X400N, X491R, X497W 16 + + -
SEQ ID NO: 87 X3D, X19C, X47V, X55Q, X92Y, X96L, X115P, X124L, X146A, X164C, X249E, X251P, X301A, X306R, X398M, X445M, X497W 17 + + -
SEQ ID NO: 88 X19C, X47V, X69F, X70L, X114P, X123S, X130A, X133E, X135L, X235I, X273C, X284I, X294C, X302L, X360H, X497W, X509R 17 + + -
SEQ ID NO: 89 X3D, X28V, X55Q, X69F, X92Y, X123E, X133E, X135K, X136E, X164C, X221R, X249E, X301A, X402G, X445M, X473A, X497W 17 + - +
SEQ ID NO: 90 X3D, X19C, X42L, X47V, X55Q, X75E, X92Y, X130A, X133E, X153F, X161A, X235I, X273C, X301A, X357Q, X386S, X445M, X491R 18 + - +
SEQ ID NO: 91 X3D, X55Q, X70L, X87K, X114P, X123E, X133F, X136M, X146A, X164C, X235I, X249E, X251P, X294C, X390E, X405S, X445M, X484H 18 + + -
SEQ ID NO: 92 X3D, X19C, X42E, X55Q, X114P, X124L, X130A, X135L, X136M, X153F, X235I, X249E, X251P, X301A, X306R, X452N, X484H, X504N 18 + + -
SEQ ID NO: 93 X19C, X42E, X55Q, X92Y, X98A, X114P, X123E, X133F, X146A, X164C, X171M, X249E, X273C, X360H, X386S, X400N, X429H, X457Y 18 + + -
SEQ ID NO: 94 X3D, X19C, X42E, X47V, X87K, X114P, X115P, X123E, X124L, X135K, X146A, X161A, X284I, X301A, X490T, X491R, X497W, X509R 18 + + -
SEQ ID NO: 95 X42L, X47V, X55Q, X69F, X70L, X75E, X130A, X133F, X135K, X143L, X146A, X153F, X249E, X302L, X306R, X400N, X449R, X478A 18 + + -
SEQ ID NO: 96 X47V, X75E, X87K, X114P, X115P, X124L, X133E, X135L, X136M, X143L, X164C, X211G, X284I, X402G, X405S, X457Y, X490T, X494E 18 + + -
SEQ ID NO: 97 X3D, X42L, X69F, X75E, X92Y, X114P, X124L, X143L, X146A, X153F, X183I, X235I, X273C, X302L, X361V, X386S, X402G, X473A, X478A 19 + + -
SEQ ID NO: 98 X3D, X47V, X69F, X75E, X87K, X114P, X115P, X123S, X130A, X143L, X193V, X235I, X284I, X390E, X400N, X405S, X442N, X457Y, X491R 19 + + -
SEQ ID NO: 99 X3D, X42L, X55Q, X69F, X87K, X115P, X124L, X130A, X143L, X146A, X153F, X164C, X251P, X302L, X357Q, X402G, X429H, X494E, X504N 19 + + -
SEQ ID NO: 100 X20I, X42E, X55Q, X75E, X92Y, X96L, X115P, X123E, X135K, X136M, X143L, X146A, X164C, X235I, X302L, X306R, X360H, X449R, X473A, X504N 20 + + -
SEQ ID NO: 101 X3D, X20I, X42E, X47V, X69F, X87K, X92Y, X114P, X123E, X124L, X133E, X136E, X164C, X313V, X360H, X402G, X429H, X442N, X494E, X497W 20 + + -
SEQ ID NO: 102 X19C, X28V, X42E, X69F, X92Y, X98A, X115P, X123E, X124L, X130A, X133F, X135K, X143L, X146A, X348E, X360H, X361V, X398M, X402G, X405S 20 + - +
SEQ ID NO: 103 X19C, X42L, X55Q, X69F, X92Y, X98A, X114P, X130A, X136M, X143L, X164C, X211G, X235I, X273C, X348E, X361V, X398M, X445M, X490T, X491R 20 + - +
SEQ ID NO: 104 X28V, X42E, X47V, X55Q, X69F, X123E, X124L, X130A, X143L, X146A, X164C, X221R, X235I, X249E, X301A, X400N, X445M, X457Y, X484H, X504N 20 + - +
SEQ ID NO: 105 X3D, X19C, X47V, X55Q, X70L, X87K, X92Y, X114P, X124L, X136M, X146A, X164C, X235I, X284I, X357Q, X360H, X390E, X452N, X457Y, X509R 20 + + -
SEQ ID NO: 106 X3D, X55Q, X69F, X75E, X87K, X92Y, X115P, X135K, X136M, X146A, X153F, X161A, X164C, X273C, X360H, X361V, X390E, X442N, X452N, X504N 20 + + -
SEQ ID NO: 107 X3D, X19C, X42L, X47V, X55Q, X123E, X124L, X130A, X133E, X136E, X143L, X164C, X211G, X273C, X284I, X301A, X302L, X442N, X449R, X478A, X497W 21 + + -
SEQ ID NO: 108 X19C, X42E, X55Q, X70L, X87K, X114P, X115P, X124L, X136M, X143L, X146A, X164C, X171M, X235I, X273C, X306R, X361V, X400N, X449R, X491R, X494E 21 + - +
SEQ ID NO: 109 X3D, X19C, X28V, X55Q, X115P, X123S, X124L, X130A, X135K, X136E, X143L, X146A, X161A, X235I, X273C, X357Q, X390E, X473A, X490T, X504N, X509R 21 + - +
SEQ ID NO: 110 X3D, X42E, X55Q, X69F, X75E, X87K, X92Y, X96L, X115P, X123E, X130A, X135L, X136E, X235I, X249E, X301A, X302L, X313V, X348E, X398M, X473A, X478A 22 + + -
SEQ ID NO: 111 X3D, X20I, X42L, X55Q, X75E, X87K, X92Y, X114P, X123E, X133F, X136E, X143L, X146A, X164C, X235I, X302L, X306R, X313V, X357Q, X442N, X491R, X494E 22 + - +
SEQ ID NO: 112 X3D, X19C, X42L, X69F, X75E, X87K, X92Y, X115P, X124L, X133F, X136M, X143L, X146A, X183I, X249E, X313V, X348E, X357Q, X361V, X400N, X402G, X497W 22 + - +
SEQ ID NO: 113 X3D, X19C, X42E, X47V, X55Q, X69F, X70L, X87K, X96L, X114P, X123E, X130A, X135L, X143L, X164C, X235I, X360H, X386I, X442N, X449R, X491R, X494E 22 + + -
SEQ ID NO: 114 X3D, X19C, X42L, X75E, X92Y, X98A, X115P, X123S, X124L, X130A, X135K, X136E, X143L, X146A, X193V, X235I, X294C, X306R, X386S, X442N, X491R, X494E 22 + - +
SEQ ID NO: 115 X3D, X19C, X47V, X75E, X87K, X92Y, X114P, X123E, X124L, X133E, X143L, X164C, X183I, X221R, X235I, X249E, X301A, X348E, X429H, X452N, X490T, X509R 22 + - +
SEQ ID NO: 116 X3D, X19C, X28V, X55Q, X69F, X75E, X87K, X115P, X124L, X135L, X146A, X164C, X235I, X249E, X251P, X273C, X294C, X357Q, X445M, X490T, X504N, X509R 22 + + -
SEQ ID NO: 117 X3D, X19C, X42L, X47V, X55Q, X69F, X92Y, X123S, X124L, X130A, X133F, X136E, X143L, X183I, X249E, X251P, X302L, X306R, X357Q, X361V, X400N, X405S, X509R 23 + + -
SEQ ID NO: 118 X42E, X47V, X87K, X92Y, X114P, X115P, X124L, X130A, X133F, X135K, X136E, X193V, X235I, X249E, X273C, X284I, X302L, X360H, X361V, X398M, X429H, X478A, X494E 23 + - +
SEQ ID NO: 119 X3D, X19C, X55Q, X69F, X75E, X87K, X92Y, X114P, X115P, X130A, X135L, X143L, X161A, X164C, X273C, X284I, X361V, X386I, X402G, X457Y, X478A, X491R, X504N 23 + + -
SEQ ID NO: 120 X3D, X19C, X47V, X55Q, X75E, X87K, X114P, X115P, X123E, X124L, X135L, X136E, X146A, X171M, X235I, X249E, X284I, X386S, X398M, X402G, X405S, X429H, X478A, X490T 24 + - +
SEQ ID NO: 121 X19C, X20I, X42E, X47V, X87K, X92Y, X114P, X115P, X124L, X133F, X135K, X136E, X143L, X146A, X164C, X221R, X249E, X357Q, X400N, X402G, X478A, X484H, X494E, X509R 24 + + -
SEQ ID NO: 122 X3D, X19C, X47V, X55Q, X87K, X92Y, X115P, X123S, X124L, X135K, X136M, X143L, X153F, X164C, X249E, X273C, X313V, X348E, X357Q, X360H, X400N, X445M, X449R, X494E 24 + + -
SEQ ID NO: 123 X19C, X42E, X47V, X69F, X70L, X75E, X87K, X92Y, X114P, X115P, X123S, X124L, X130A, X136M, X164C, X183I, X235I, X306R, X348E, X357Q, X400N, X405S, X449R, X457Y 24 + - +
SEQ ID NO: 124 X3D, X47V, X55Q, X69F, X70L, X87K, X92Y, X114P, X123E, X124L, X130A, X133E, X135K, X136E, X164C, X221R, X235I, X302L, X360H, X442N, X457Y, X484H, X504N, X509R 24 + - +
SEQ ID NO: 125 X19C, X55Q, X70L, X75E, X92Y, X114P, X115P, X124L, X130A, X133F, X136E, X143L, X146A, X164C, X235I, X249E, X301A, X313V, X452N, X473A, X484H, X491R, X494E, X504N 24 + - +
SEQ ID NO: 126 X3D, X19C, X47V, X55Q, X69F, X87K, X114P, X115P, X123S, X124L, X133F, X135K, X143L, X193V, X235I, X273C, X284I, X302L, X386S, X429H, X473A, X478A, X490T, X504N 24 + + -
SEQ ID NO: 127 X3D, X19C, X20I, X42L, X55Q, X92Y, X114P, X115P, X123S, X124L, X130A, X135K, X136E, X143L, X146A, X235I, X251P, X273C, X348E, X386S, X449R, X452N, X484H, X494E, X497W 25 + - +
SEQ ID NO: 128 X3D, X19C, X42E, X47V, X69F, X75E, X87K, X92Y, X114P, X123S, X124L, X133F, X135L, X146A, X153F, X164C, X183I, X235I, X301A, X398M, X452N, X478A, X491R, X497W, X509R 25 + - +
SEQ ID NO: 129 X3D, X42E, X47V, X55Q, X75E, X87K, X92Y, X96L, X114P, X115P, X130A, X133F, X146A, X153F, X164C, X235I, X249E, X273C, X306R, X405S, X445M, X449R, X473A, X484H, X491R 25 + - +
SEQ ID NO: 130 X3D, X19C, X42E, X47V, X69F, X87K, X92Y, X114P, X124L, X130A, X133F, X135K, X146A, X161A, X164C, X211G, X249E, X273C, X348E, X361V, X400N, X402G, X473A, X478A, X491R, X504N 26 + - +
SEQ ID NO: 131 X3D, X19C, X20I, X42L, X55Q, X69F, X75E, X87K, X115P, X123E, X124L, X130A, X133F, X135K, X136M, X143L, X164C, X251P, X294C, X301A, X306R, X357Q, X429H, X442N, X473A, X484H 26 + + -
SEQ ID NO: 132 X3D, X19C, X42L, X47V, X55Q, X87K, X92Y, X96L, X115P, X123S, X124L, X133F, X135L, X143L, X164C, X235I, X249E, X284I, X301A, X302L, X386I, X405S, X442N, X484H, X497W, X504N 26 + + -
SEQ ID NO: 133 X3D, X19C, X42E, X47V, X69F, X75E, X115P, X123E, X130A, X133E, X143L, X146A, X164C, X171M, X235I, X249E, X273C, X284I, X301A, X348E, X386I, X390E, X398M, X405S, X452N, X497W 26 + - +
SEQ ID NO: 134 X19C, X42L, X47V, X69F, X75E, X87K, X96L, X114P, X115P, X123S, X130A, X135K, X136E, X143L, X164C, X235I, X249E, X251P, X306R, X357Q, X361V, X402G, X405S, X452N, X473A, X490T 26 + - +
SEQ ID NO: 135 X19C, X47V, X55Q, X69F, X75E, X92Y, X98A, X114P, X115P, X123S, X124L, X130A, X133F, X143L, X146A, X153F, X164C, X235I, X294C, X357Q, X390E, X405S, X449R, X457Y, X478A, X484H 26 + + -
SEQ ID NO: 136 X3D, X19C, X42L, X47V, X55Q, X69F, X75E, X92Y, X98A, X114P, X124L, X133F, X136M, X143L, X146A, X211G, X249E, X273C, X306R, X390E, X400N, X429H, X442N, X449R, X473A, X478A 26 + + -
SEQ ID NO: 137 X3D, X19C, X42E, X47V, X55Q, X69F, X70L, X92Y, X114P, X115P, X123S, X124L, X130A, X136M, X143L, X146A, X164C, X221R, X249E, X348E, X357Q, X390E, X405S, X442N, X478A, X494E, X497W 27 + + -
SEQ ID NO: 138 X42L, X47V, X55Q, X70L, X75E, X87K, X92Y, X114P, X115P, X123S, X124L, X130A, X135K, X136M, X143L, X161A, X164C, X235I, X273C, X306R, X398M, X400N, X442N, X452N, X457Y, X490T, X504N 27 + + -
SEQ ID NO: 139 X19C, X28V, X42E, X47V, X55Q, X69F, X75E, X92Y, X114P, X115P, X123E, X124L, X130A, X136E, X146A, X161A, X164C, X235I, X249E, X294C, X402G, X452N, X457Y, X473A, X478A, X484H, X491R 27 + - +
SEQ ID NO: 140 X19C, X42L, X47V, X69F, X70L, X75E, X87K, X92Y, X114P, X115P, X123S, X130A, X133E, X135L, X143L, X146A, X164C, X249E, X273C, X284I, X357Q, X360H, X386I, X390E, X398M, X452N, X490T, X491R 28 + - +
SEQ ID NO: 141 X3D, X19C, X47V, X55Q, X75E, X87K, X92Y, X114P, X115P, X123E, X124L, X130A, X133E, X143L, X146A, X164C, X211G, X249E, X273C, X313V, X386I, X402G, X449R, X452N, X457Y, X484H, X490T, X504N 28 + + -
SEQ ID NO: 142 X3D, X19C, X42E, X47V, X55Q, X69F, X75E, X87K, X92Y, X124L, X130A, X133F, X135L, X136E, X153F, X164C, X235I, X249E, X273C, X313V, X357Q, X361V, X429H, X449R, X478A, X484H, X494E, X497W 28 + + -
SEQ ID NO: 143 X3D, X19C, X20I, X42L, X47V, X55Q, X69F, X87K, X92Y, X114P, X123E, X124L, X133E, X135K, X136M, X143L, X146A, X221R, X235I, X249E, X294C, X302L, X360H, X390E, X398M, X449R, X494E, X509R 28 + + -
SEQ ID NO: 144 X3D, X19C, X47V, X69F, X75E, X87K, X92Y, X115P, X123S, X124L, X130A, X133E, X136E, X143L, X146A, X164C, X235I, X249E, X251P, X284I, X294C, X357Q, X361V, X405S, X445M, X449R, X491R, X509R 28 + + -
SEQ ID NO: 145 X19C, X42L, X47V, X55Q, X69F, X75E, X92Y, X114P, X115P, X123E, X124L, X130A, X133F, X135L, X136E, X143L, X211G, X221R, X249E, X273C, X348E, X405S, X445M, X452N, X457Y, X478A, X494E, X509R 28 + + -
SEQ ID NO: 146 X3D, X19C, X28V, X42L, X69F, X75E, X87K, X92Y, X114P, X115P, X123S, X124L, X130A, X135K, X136M, X143L, X146A, X164C, X249E, X251P, X302L, X348E, X357Q, X405S, X457Y, X484H, X490T, X509R 28 + + -
SEQ ID NO: 147 X19C, X42L, X47V, X55Q, X69F, X87K, X114P, X115P, X123S, X124L, X133E, X135L, X136M, X143L, X146A, X164C, X193V, X211G, X235I, X249E, X306R, X361V, X442N, X452N, X457Y, X473A, X478A, X484H, X490T 29 + + -
SEQ ID NO: 148 X3D, X19C, X28V, X42L, X47V, X55Q, X69F, X70L, X75E, X87K, X92Y, X114P, X124L, X133E, X136E, X143L, X164C, X235I, X249E, X273C, X302L, X398M, X402G, X429H, X452N, X473A, X484H, X494E, X509R 29 + + -
SEQ ID NO: 149 X3D, X19C, X20I, X42E, X69F, X87K, X114P, X115P, X124L, X130A, X133E, X135K, X136M, X143L, X146A, X164C, X235I, X249E, X251P, X273C, X360H, X386I, X390E, X442N, X445M, X449R, X478A, X484H, X509R 29 + + -
SEQ ID NO: 150 X3D, X19C, X28V, X42L, X47V, X55Q, X69F, X75E, X87K, X92Y, X114P, X123E, X124L, X130A, X133F, X143L, X146A, X164C, X235I, X273C, X313V, X357Q, X390E, X429H, X445M, X457Y, X478A, X484H, X491R, X504N 30 + + -
SEQ ID NO: 151 X3D, X42L, X47V, X55Q, X69F, X75E, X87K, X92Y, X114P, X115P, X123E, X124L, X135L, X136M, X143L, X164C, X183I, X211G, X235I, X249E, X273C, X302L, X348E, X390E, X398M, X400N, X442N, X491R, X504N, X509R 30 + + -
SEQ ID NO: 152 X19C, X47V, X55Q, X69F, X70L, X75E, X87K, X92Y, X115P, X123E, X130A, X133E, X135K, X136M, X143L, X146A, X164C, X235I, X249E, X273C, X284I, X348E, X360H, X361V, X400N, X405S, X449R, X478A, X494E, X509R 30 + - +
SEQ ID NO: 153 X3D, X19C, X42L, X55Q, X75E, X87K, X92Y, X114P, X123S, X124L, X130A, X133E, X135K, X136E, X143L, X153F, X164C, X193V, X235I, X249E, X273C, X294C, X301A, X361V, X398M, X402G, X473A, X491R, X504N, X509R 30 + - +
SEQ ID NO: 154 X3D, X19C, X20I, X42L, X47V, X55Q, X69F, X75E, X87K, X92Y, X115P, X123E, X124L, X133F, X136M, X143L, X146A, X164C, X183I, X249E, X273C, X294C, X357Q, X360H, X429H, X449R, X478A, X484H, X491R, X509R 30 + - +
SEQ ID NO: 155 X3D, X19C, X47V, X55Q, X69F, X87K, X92Y, X114P, X115P, X130A, X133E, X135L, X136E, X143L, X146A, X164C, X193V, X235I, X249E, X273C, X302L, X306R, X313V, X357Q, X400N, X445M, X449R, X457Y, X504N, X509R 30 + + -
SEQ ID NO: 156 X3D, X19C, X47V, X55Q, X69F, X75E, X87K, X92Y, X114P, X115P, X130A, X133E, X135K, X136E, X143L, X164C, X183I, X193V, X235I, X249E, X273C, X301A, X302L, X357Q, X360H, X361V, X386I, X429H, X452N, X504N 30 + + -
SEQ ID NO: 157 X3D, X19C, X20I, X42E, X47V, X55Q, X69F, X87K, X92Y, X114P, X115P, X123S, X124L, X130A, X135K, X136E, X143L, X153F, X164C, X235I, X249E, X273C, X301A, X306R, X357Q, X361V, X400N, X402G, X445M, X478A, X490T 31 + + -
SEQ ID NO: 158 X3D, X19C, X42E, X55Q, X69F, X75E, X87K, X92Y, X114P, X115P, X123S, X124L, X130A, X133E, X135L, X136E, X146A, X171M, X183I, X235I, X249E, X273C, X306R, X360H, X398M, X429H, X442N, X445M, X473A, X484H, X494E 31 + - +
SEQ ID NO: 159 X3D, X19C, X42L, X47V, X55Q, X69F, X75E, X87K, X98A, X114P, X115P, X123S, X124L, X133F, X135K, X136M, X143L, X146A, X164C, X221R, X235I, X273C, X294C, X361V, X390E, X402G, X405S, X429H, X442N, X473A, X478A 31 + - +
SEQ ID NO: 160 X3D, X19C, X20I, X42E, X47V, X55Q, X69F, X87K, X92Y, X114P, X115P, X123E, X124L, X130A, X133F, X135K, X136M, X143L, X146A, X235I, X249E, X273C, X294C, X302L, X313V, X361V, X400N, X405S, X442N, X452N, X478A, X504N 32 + + -
SEQ ID NO: 161 X3D, X19C, X20I, X47V, X55Q, X75E, X87K, X92Y, X114P, X115P, X123S, X124L, X130A, X133E, X135L, X136M, X143L, X146A, X164C, X235I, X249E, X273C, X301A, X313V, X357Q, X360H, X398M, X442N, X449R, X473A, X494E, X504N 32 + + -
SEQ ID NO: 162 X3D, X19C, X42L, X47V, X55Q, X69F, X75E, X87K, X92Y, X96L, X114P, X115P, X123E, X124L, X130A, X136M, X143L, X146A, X161A, X164C, X235I, X249E, X273C, X294C, X348E, X360H, X361V, X400N, X402G, X405S, X494E, X504N 32 + + -
SEQ ID NO: 163 X3D, X19C, X42L, X47V, X55Q, X69F, X75E, X87K, X92Y, X96L, X114P, X115P, X123S, X124L, X130A, X135L, X136M, X143L, X164C, X211G, X235I, X249E, X273C, X301A, X306R, X361V, X398M, X405S, X442N, X449R, X452N, X491R 32 + + -
SEQ ID NO: 164 X3D, X19C, X42L, X47V, X55Q, X69F, X70L, X75E, X87K, X114P, X115P, X123E, X124L, X130A, X133E, X135L, X136M, X143L, X146A, X235I, X249E, X273C, X284I, X306R, X360H, X390E, X398M, X402G, X429H, X457Y, X473A, X491R 32 + - +
SEQ ID NO: 165 X3D, X19C, X42E, X47V, X55Q, X69F, X87K, X92Y, X114P, X115P, X123S, X124L, X133E, X135K, X136E, X143L, X146A, X161A, X164C, X235I, X249E, X273C, X284I, X306R, X357Q, X361V, X386I, X405S, X445M, X449R, X452N, X484H 32 + - +
SEQ ID NO: 166 X3D, X19C, X20I, X42L, X47V, X55Q, X69F, X75E, X87K, X92Y, X114P, X123S, X124L, X130A, X133F, X135L, X143L, X146A, X164C, X193V, X235I, X249E, X273C, X294C, X301A, X386I, X449R, X452N, X457Y, X478A, X490T, X509R 32 + + -
SEQ ID NO: 167 X3D, X47V, X55Q, X69F, X75E, X87K, X92Y, X114P, X115P, X123E, X124L, X130A, X133F, X135L, X136M, X143L, X146A, X164C, X193V, X235I, X249E, X251P, X273C, X306R, X361V, X400N, X405S, X442N, X445M, X473A, X484H, X494E, X497W 33 + + -
SEQ ID NO: 168 X3D, X19C, X42L, X55Q, X69F, X75E, X87K, X92Y, X96L, X114P, X115P, X123S, X124L, X130A, X133E, X135K, X143L, X146A, X164C, X171M, X235I, X249E, X273C, X357Q, X360H, X361V, X386S, X400N, X445M, X449R, X491R, X494E, X509R 33 + + -
SEQ ID NO: 169 X3D, X19C, X42E, X47V, X55Q, X69F, X75E, X87K, X92Y, X96L, X114P, X115P, X124L, X130A, X133E, X136M, X143L, X146A, X161A, X164C, X235I, X249E, X273C, X301A, X348E, X361V, X398M, X402G, X405S, X452N, X457Y, X484H, X504N 33 + + -
SEQ ID NO: 170 X3D, X19C, X42E, X47V, X55Q, X69F, X70L, X75E, X87K, X92Y, X114P, X115P, X123S, X124L, X130A, X133E, X135K, X136E, X143L, X146A, X161A, X164C, X235I, X273C, X301A, X306R, X398M, X400N, X442N, X449R, X484H, X490T, X494E, X497W 34 + + -
SEQ ID NO: 171 X3D, X19C, X42L, X47V, X55Q, X69F, X75E, X87K, X92Y, X114P, X115P, X123E, X124L, X133F, X135L, X136E, X143L, X146A, X164C, X211G, X221R, X235I, X249E, X273C, X294C, X301A, X306R, X386I, X400N, X449R, X452N, X457Y, X473A, X490T 34 + + -
SEQ ID NO: 172 X3D, X19C, X42L, X47V, X55Q, X69F, X70L, X75E, X87K, X92Y, X114P, X115P, X123E, X124L, X130A, X133F, X135L, X136M, X143L, X146A, X164C, X211G, X249E, X273C, X294C, X357Q, X360H, X390E, X398M, X402G, X442N, X457Y, X473A, X504N 34 + + -
SEQ ID NO: 173 X3D, X19C, X42L, X47V, X55Q, X69F, X75E, X87K, X92Y, X114P, X123E, X124L, X130A, X133E, X135L, X136M, X143L, X146A, X164C, X235I, X249E, X251P, X273C, X284I, X294C, X306R, X361V, X386S, X400N, X402G, X445M, X449R, X452N, X478A 34 + + -
SEQ ID NO: 174 X3D, X19C, X42E, X47V, X55Q, X69F, X70L, X75E, X87K, X92Y, X114P, X115P, X123E, X124L, X130A, X133E, X135L, X136M, X143L, X153F, X164C, X235I, X249E, X273C, X306R, X348E, X361V, X405S, X429H, X442N, X449R, X452N, X473A, X484H 34 + + -
SEQ ID NO: 175 X3D, X19C, X42E, X47V, X55Q, X69F, X70L, X87K, X92Y, X114P, X115P, X123S, X124L, X130A, X133E, X135L, X136M, X143L, X146A, X164C, X235I, X249E, X273C, X284I, X348E, X390E, X398M, X429H, X442N, X445M, X473A, X484H, X490T, X504N 34 + + -
SEQ ID NO: 176 X3D, X19C, X42E, X47V, X69F, X75E, X87K, X92Y, X96L, X114P, X115P, X123S, X124L, X130A, X133E, X135L, X136E, X143L, X146A, X164C, X211G, X235I, X249E, X273C, X294C, X301A, X357Q, X390E, X400N, X402G, X405S, X442N, X445M, X449R 34 + + -
SEQ ID NO: 177 X3D, X19C, X42L, X47V, X55Q, X69F, X75E, X87K, X92Y, X98A, X114P, X115P, X123S, X124L, X130A, X133F, X135K, X136M, X143L, X146A, X164C, X235I, X249E, X273C, X284I, X294C, X301A, X357Q, X360H, X361V, X390E, X402G, X457Y, X473A, X504N 35 + - +
SEQ ID NO: 178 X3D, X19C, X20I, X42L, X47V, X55Q, X69F, X75E, X87K, X92Y, X114P, X115P, X123S, X124L, X130A, X133E, X135L, X136M, X143L, X146A, X164C, X221R, X235I, X249E, X273C, X301A, X302L, X348E, X386S, X390E, X400N, X405S, X429H, X491R, X494E 35 + - +
SEQ ID NO: 179 X3D, X19C, X42E, X47V, X55Q, X69F, X75E, X87K, X92Y, X98A, X114P, X115P, X123S, X124L, X130A, X133E, X135L, X136M, X143L, X146A, X164C, X183I, X235I, X249E, X273C, X294C, X302L, X357Q, X360H, X386S, X405S, X449R, X452N, X478A, X484H, X509R 36 + - +
SEQ ID NO: 180 X3D, X19C, X42L, X47V, X55Q, X69F, X75E, X87K, X92Y, X98A, X114P, X115P, X123S, X124L, X130A, X133F, X135L, X136E, X143L, X146A, X164C, X235I, X249E, X251P, X273C, X301A, X348E, X398M, X405S, X449R, X452N, X457Y, X478A, X484H, X494E, X504N 36 + + -
SEQ ID NO: 181 X3D, X19C, X42L, X47V, X55Q, X69F, X75E, X87K, X92Y, X96L, X114P, X115P, X123E, X124L, X130A, X133E, X135K, X136M, X143L, X146A, X164C, X211G, X235I, X249E, X273C, X306R, X348E, X357Q, X361V, X398M, X400N, X442N, X445M, X452N, X484H, X509R 36 + + -
SEQ ID NO: 182 X3D, X19C, X42L, X47V, X55Q, X69F, X75E, X87K, X92Y, X114P, X115P, X123E, X124L, X130A, X133E, X135K, X136M, X143L, X146A, X164C, X193V, X235I, X249E, X251P, X273C, X306R, X361V, X398M, X405S, X442N, X449R, X452N, X457Y, X484H, X490T, X494E 36 + + -
SEQ ID NO: 183 X3D, X19C, X42E, X47V, X55Q, X69F, X75E, X87K, X92Y, X114P, X115P, X123E, X124L, X130A, X133F, X135L, X136M, X143L, X146A, X161A, X164C, X171M, X235I, X249E, X273C, X301A, X348E, X398M, X402G, X405S, X429H, X449R, X457Y, X490T, X497W, X509R 36 + - +
SEQ ID NO: 184 X3D, X19C, X42E, X47V, X55Q, X69F, X75E, X87K, X92Y, X114P, X115P, X123S, X124L, X130A, X133F, X135K, X136E, X143L, X146A, X164C, X171M, X221R, X235I, X249E, X273C, X294C, X302L, X348E, X357Q, X402G, X405S, X449R, X452N, X478A, X491R, X497W 36 + - +
SEQ ID NO: 185 X3D, X19C, X42E, X47V, X55Q, X69F, X75E, X87K, X92Y, X114P, X115P, X123S, X124L, X130A, X133F, X135K, X136M, X143L, X146A, X164C, X183I, X235I, X249E, X251P, X273C, X301A, X348E, X398M, X400N, X429H, X445M, X449R, X452N, X490T, X491R, X509R 36 + + -
SEQ ID NO: 186 X3D, X19C, X42L, X47V, X55Q, X69F, X70L, X75E, X87K, X92Y, X114P, X115P, X123E, X124L, X130A, X133F, X135L, X136M, X143L, X146A, X161A, X164C, X235I, X249E, X273C, X301A, X360H, X386I, X429H, X442N, X457Y, X473A, X478A, X484H, X491R, X504N 36 + + -
SEQ ID NO: 187 X3D, X19C, X42L, X47V, X55Q, X69F, X70L, X75E, X87K, X92Y, X98A, X114P, X115P, X123E, X124L, X130A, X133E, X135L, X136M, X143L, X146A, X164C, X235I, X249E, X273C, X294C, X390E, X400N, X402G, X405S, X429H, X442N, X457Y, X484H, X497W, X509R 36 + - +
SEQ ID NO: 188 X3D, X19C, X20I, X42L, X47V, X55Q, X69F, X75E, X87K, X92Y, X114P, X115P, X123S, X124L, X130A, X133F, X135K, X136M, X143L, X146A, X164C, X183I, X235I, X249E, X273C, X294C, X302L, X306R, X398M, X442N, X445M, X452N, X457Y, X484H, X494E, X504N 36 + + -
SEQ ID NO: 189 X3D, X19C, X42L, X47V, X55Q, X69F, X70L, X75E, X87K, X92Y, X114P, X115P, X123S, X124L, X130A, X133E, X135L, X136M, X143L, X146A, X164C, X193V, X235I, X249E, X273C, X301A, X302L, X360H, X390E, X398M, X400N, X405S, X473A, X478A, X484H, X497W 36 + - +
SEQ ID NO: 190 X3D, X19C, X42E, X47V, X55Q, X69F, X75E, X87K, X92Y, X114P, X115P, X123S, X124L, X130A, X133E, X135K, X136E, X143L, X146A, X164C, X183I, X235I, X249E, X273C, X302L, X306R, X313V, X357Q, X360H, X398M, X400N, X429H, X442N, X449R, X452N, X497W 36 + - +
SEQ ID NO: 191 X3D, X19C, X42E, X47V, X55Q, X69F, X70L, X75E, X87K, X92Y, X114P, X115P, X123S, X124L, X130A, X133F, X135K, X136E, X143L, X146A, X164C, X235I, X249E, X251P, X273C, X306R, X348E, X360H, X361V, X402G, X405S, X429H, X449R, X491R, X497W, X504N 36 + - +
SEQ ID NO: 192 X3D, X19C, X42L, X47V, X55Q, X69F, X70L, X75E, X87K, X92Y, X114P, X115P, X123S, X124L, X130A, X133F, X135K, X136E, X143L, X146A, X164C, X193V, X235I, X249E, X273C, X306R, X348E, X361V, X398M, X405S, X442N, X445M, X478A, X490T, X494E, X497W 36 + + -
SEQ ID NO: 193 X3D, X19C, X42E, X47V, X55Q, X69F, X75E, X87K, X92Y, X114P, X115P, X123S, X124L, X130A, X133F, X135K, X136E, X143L, X146A, X164C, X193V, X235I, X249E, X273C, X301A, X313V, X348E, X357Q, X361V, X390E, X402G, X445M, X449R, X457Y, X473A, X478A, X484H 37 + - +
SEQ ID NO: 194 X3D, X19C, X42E, X47V, X55Q, X69F, X75E, X87K, X92Y, X114P, X115P, X123S, X124L, X130A, X133F, X135K, X136E, X143L, X146A, X164C, X171M, X235I, X249E, X273C, X284I, X306R, X348E, X360H, X390E, X398M, X402G, X429H, X445M, X449R, X452N, X497W, X504N 37 + + -
SEQ ID NO: 195 X3D, X19C, X42L, X47V, X55Q, X69F, X75E, X87K, X92Y, X96L, X114P, X115P, X123E, X124L, X130A, X133F, X135K, X136M, X143L, X146A, X153F, X164C, X235I, X249E, X273C, X301A, X302L, X306R, X348E, X386I, X398M, X405S, X449R, X457Y, X478A, X490T, X509R 37 + + -
SEQ ID NO: 196 X3D, X19C, X42L, X47V, X55Q, X69F, X75E, X87K, X92Y, X114P, X115P, X123E, X124L, X130A, X133F, X135K, X136M, X143L, X146A, X164C, X221R, X235I, X249E, X273C, X306R, X313V, X357Q, X361V, X390E, X402G, X429H, X452N, X473A, X478A, X490T, X504N, X509R 37 + - +
SEQ ID NO: 197 X3D, X19C, X42L, X47V, X55Q, X69F, X75E, X87K, X92Y, X98A, X114P, X115P, X123S, X124L, X130A, X133F, X135K, X136E, X143L, X146A, X164C, X193V, X235I, X249E, X273C, X294C, X301A, X357Q, X400N, X402G, X405S, X445M, X452N, X457Y, X491R, X494E, X497W 37 + - +
SEQ ID NO: 198 X3D, X19C, X42L, X47V, X55Q, X69F, X75E, X87K, X92Y, X98A, X114P, X115P, X123E, X124L, X130A, X133E, X135L, X136M, X143L, X146A, X161A, X164C, X235I, X249E, X273C, X294C, X301A, X348E, X386S, X390E, X402G, X429H, X442N, X449R, X452N, X457Y, X494E 37 + - +
SEQ ID NO: 199 X3D, X19C, X20I, X42L, X47V, X55Q, X69F, X75E, X87K, X92Y, X98A, X114P, X115P, X123E, X124L, X130A, X133F, X135L, X136M, X143L, X146A, X164C, X235I, X249E, X273C, X301A, X357Q, X390E, X398M, X442N, X445M, X452N, X457Y, X473A, X491R, X494E, X504N 37 + + -
SEQ ID NO: 200 X3D, X19C, X20I, X28V, X42L, X47V, X55Q, X69F, X75E, X87K, X92Y, X114P, X115P, X123E, X124L, X130A, X133F, X135K, X136M, X143L, X146A, X164C, X235I, X249E, X273C, X301A, X357Q, X360H, X386S, X400N, X473A, X478A, X490T, X491R, X494E, X504N, X509R 37 + + -

[0090] Threonine deaminase (TD) Activity column: "+" = 1-fold increase in TD activity; "++" = 2-fold increase in TD activity; "+++" = 3-fold increase in TD activity; "-" = No TD activity; Transaminase (TA) Activity column: "+" = 1-fold increase in TA activity; "++" = 2-fold increase in TA activity; "+++" = 3-fold increase in TA activity; "-" = No TA activity; Expression column: "+" = Low expression; "++" = Moderate expression; "+++" = High expression; NA – Not applicable
Examples:
Example 1
[0091] Molecular cloning of threonine deaminase
[0092] The threonine deaminase gene was cloned into the pET28a(+) vector between the NcoI and EcoRI sites. Mutations were introduced via site-directed mutagenesis. For transformation, 40 µL of E. coli BL21 (DE3) competent cells were transferred into a 1.5 mL tube, followed by the addition of 1 µL of plasmid DNA. The mixture was incubated on ice for 30 minutes, then subjected to heat shock at 42°C for 60 seconds, and immediately placed on ice for 5 minutes. Subsequently, 850 µL of plain LB broth was added to the tube, and the culture was incubated at 37°C in a shaking incubator for 1 hour. The cells were then pelleted by centrifugation at 3,500 RPM for 5 minutes, and the supernatant was decanted, leaving behind 50 µL of media for resuspension. The cells were resuspended in the remaining LB broth and plated onto LB agar plates containing kanamycin (50 µg/mL).
[0093] Master Plate Streaking
[0094] Single isolated colonies from the transformation plate were selected and streaked onto a labeled master plate to maintain stock cultures.
Example 2
[0095] Expression of artificial engineered threonine deaminase enzymes
[0096] pET28a(+) plasmid vector housing threonine deaminase gene was transformed into chemically competent E. coli BL21 (DE3) cells. The transformed cells were grown overnight with 200 rpm agitation at 37 °C in 5ml Luria-Bertani (LB) medium plus kanamycin (50 µg/ml) in a 50-ml flask. These cultures were then diluted into 100 ml of LB medium plus kanamycin (50 µg/ml) and grown at 37 °C in a 1,000-ml flask with agitation 200 rpm to an OD600 of 0.6. Then, the cultures were induced with 0.1 mM IPTG and incubated for a further 16 h at 25 °C. The cells were then recovered by centrifugation at 4700 rpm for 10 min, washed in 50 mM Tris–HCl buffer (pH 7.5) and similarly pelleted. The cell pellets were frozen for use in the biotransformation. The cell pellets were suspended in lysis buffer (100 mM PBS, pH 7.5), and lysed using sonication. Crude lysates were analyzed using SDS-PAGE (Fig. 8) and the protein concentration was determined using Bradford’s assay method.
[0097] Table 2: The determined protein concentration of recombinant engineered threonine deaminase variants from the Bradford assay
S. No Variant ID Protein Concentration (mg protein/ ml of lysate)
01 SEQ ID NO: 1 25.3
02 SEQ ID NO: 5 31.36
03 SEQ ID NO: 6 31.23
04 SEQ ID NO: 10 31.62
05 SEQ ID NO: 12 31.95
06 SEQ ID NO: 13 29.52
07 SEQ ID NO: 15 29.59
Example 3
[0098] Threonine deaminase activity assay
[0099] Reaction buffer is prepared with 100mM Potassium phosphate (1.628gm Potassium Phosphate Dibasic and 88.78 mg Potassium Phosphate Monobasic in sterile 80 mL MiliQ water then adjust the pH of the buffer to 8.0 with 4N NaOH and makeup the volume to 100mL). L-Threonine substrate solution (0.119gm L-threonine in 2mL MilliQ) and PLP solution (0.0247gm PLP in 2mL of MilliQ) were prepared and placed on ice. 900µl of the reaction buffer was added to 25 to 30 mg of enzyme powder from the vial that contained 300mg of enzyme powder, mixed and then 80µl of substrate from the substrate stock and 20 µl of PLP from the PLP solution was added to the reaction mixture. The reaction mixture was incubated incubated on a thermomixer for 15 min at 28 °C and 180 rpm. The reaction was terminated by adding 1% semicarbazide and 0.9% sodium acetate. The reaction mixture was centrifuged at 10,000 rpm for 15 min. The supernatant was collected and filtered through 0.45µm filters into HPLC vials.
Example 4
[0100] Threonine deaminase activity assay at elevated temperature.
[0101] The reaction mixture contained 400 mM threonine, 100 mM Tris-HCl (pH 7.5), and 20 mg lysed extract. The reactions were carried out at 37 °C for 10 minutes and then terminated by adding 2ml 10% HCl. The activity was measured by the amount of L-threonine diminished.
Example 5
[0102] Enzyme assay with bulky substrates for half-transaminating activity
[0103] The reaction mixture contained 10mM bulky a-keto acid ((3-carboxy-3-oxopropyl)(methyl)phosphinate), 20 mg lyophilized extract of engineered threonine deaminase enzyme and 1mM PMP in 100 mM Tris-HCl (pH 8.5). The reactions were carried out at 37°C for 60 minutes and then terminated by adding 2ml 10% HCl. The activity was measured based on the amount of L-phosphinothricin formed.
Example 6
[0104] Enzyme assay with bulky substrates for half-transaminating activity
[0105] The reaction mixture contained 10mM bulky a-keto acid (4-fluorophenyl-pyruvic acid), 20 mg lyophilized extract of engineered threonine deaminase enzyme and 1mM PMP in 100 mM Tris-HCl (pH 8.5). The reactions were carried out at 37 °C for 60 minutes and then terminated by adding 2ml 10% HCl. The activity was measured based on the amount of 4-fluorophenyalanine formed.
Example 7
[0106] Enzyme assay with bulky substrates for half-transaminating activity
[0107] The reaction mixture contained 10mM bulky a-keto ester, 4-methoxy-3,4-dioxobutyl)(methyl)phosphinate, 20 mg lyophilized extract of engineered threonine deaminase enzyme and 1mM PMP in 100 mM Tris-HCl (pH 8.5). The reactions were carried out at 37 °C for 60 minutes and then terminated by adding 2ml 10% HCl. The activity was measured based on the amount of [(3S)-3-amino-4-methoxy-4-oxobutyl]-(methyl) phosphinate formed.
Example 8
[0108] Enzyme assay with bulky substrates for half-transaminating activity
[0109] The reaction mixture contained 10mM bulky a-keto ester, methy-3-(4-fluorophenyl)-2-oxopropanoate , 20 mg lyophilized extract of engineered threonine deaminase enzyme and 1mM PMP in 100 mM Tris-HCl (pH 8.5). The reactions were carried out at 37 °C for 60 minutes and then terminated by adding 2ml 10% HCl. The activity was measured based on the amount of methyl (2S)-2-amino-3-(4-fluorophenyl)propanoate formed.
Example 9
[0110] Coupled synthesis of 2-oxo-butyric acid and L-phosphinothricin (full transamination reactions)
[0111] The reaction mixture contained 10mM bulky a-keto acid ((3-carboxy-3-oxopropyl)(methyl)phosphinate), 10mM L-threonine, 20 mg lyophilized extract of engineered threonine deaminase enzyme and 1mM PMP/PLP in 100 mM Tris-HCl (pH 8.5). The reactions were carried out at 37 °C for 60 minutes and then terminated by adding 2ml 10% HCl. The activity was measured based on the amount of L-phosphinothricin formed. Table 3 provides the SEQ ID NOs. for the engineered artificial threonine deaminase, along with the number of amino acid mutations from SEQ ID NO. 1 and activity for the synthesis of 2-oxo-butyric acid and L-phosphinothricin.
[0112] Table 3: Activity of variants for coupled synthesis of 2-oxo-butyric acid and L-phosphinothricin
SEQ ID No. No. of Residue difference from SEQ ID No: 1 Expression Threonine deaminase Activity Transaminase Activity
1 0 ++ + +
2 5 ++ ++ +
6 9 +++ ++ ++
10 13 +++ + +
14 17 ++ ++ +
18 24 +++ ++ +++
22 30 +++ ++ ++
26 33 ++ ++ +++
30 36 ++ ++ +++

[0113] Threonine deaminase (TD) Activity column: "+" = 1-fold increase in TD activity; "++" = 2-fold increase in TD activity; "+++" = 3-fold increase in TD activity; "-" = No TD activity; Transaminase (TA) Activity column: "+" = 1-fold increase in TA activity; "++" = 2-fold increase in TA activity; "+++" = 3-fold increase in TA activity; "-" = No TA activity; Expression column: "+" = Low expression; "++" = Moderate expression; "+++" = High expression.
Example 10
[0114] Coupled synthesis of 2-oxo-butyric acid and 4-flurophenyalanine (full transamination reactions)
[0115] The reaction mixture contained 10mM bulky a-keto acid (4-fluorophenyl-pyruvic acid), 10mM L-threonine, 20 mg lyophilized extract of engineered threonine deaminase enzyme and 1mM PMP/PLP in 100 mM Tris-HCl (pH 8.5). The reactions were carried out at 37 °C for 60 minutes and then terminated by adding 2ml 10% HCl. The activity was measured based on the amount of L-4-fluorophenylalanine formed. Table 4 provides the SEQ ID NOs. for the engineered artificial threonine deaminase variants, along with the number of amino acid mutations from SEQ ID NO. 1 and activity for the synthesis of 2-oxo-butyric acid and 4-fluorophenyalanine.
[0116] Table 4: Activity of variants for coupled synthesis of 2-oxo-butyric acid and 4-fluoro-phenyalanine
SEQ ID No. No. of Residue difference from SEQ ID No: 1 Expression Threonine deaminase Activity Transaminase Activity
1 0 ++ + +
4 6 ++ ++ +
8 11 ++ ++ ++
12 15 +++ ++ +
16 20 ++ ++ +
20 26 ++ + ++
24 31 +++ ++ ++
28 35 ++ ++ ++
32 37 +++ +++ +++

[0117] Threonine deaminase (TD) Activity column: "+" = 1-fold increase in TD activity; "++" = 2-fold increase in TD activity; "+++" = 3-fold increase in TD activity; "-" = No TD activity; Transaminase (TA) Activity column: "+" = 1-fold increase in TA activity; "++" = 2-fold increase in TA activity; "+++" = 3-fold increase in TA activity; "-" = No TA activity; Expression column: "+" = Low expression; "++" = Moderate expression; "+++" = High expression.
Example 11
[0118] Coupled synthesis of 2-oxo-butyric acid and [(3S)-3-amino-4-methoxy-4-oxobutyl]-(methyl) phosphinate (full transamination reactions)
[0119] The reaction mixture contained 10mM bulky a-keto acid (4-methoxy-3,4-dioxobutyl)(methyl)phosphinate, 10mM L-threonine, 20 mg lyophilized extract of engineered threonine deaminase enzyme and 1mM PMP/PLP in 100 mM Tris-HCl (pH 8.5). The reactions were carried out at 37 °C for 60 minutes and then terminated by adding 2ml 10% HCl. The activity was measured based on the amount of [(3S)-3-amino-4-methoxy-4-oxobutyl]-(methyl) phosphinate formed. Table 5 provides the SEQ ID NOs. for the engineered artificial threonine deaminase variants, along with the number of amino acid mutations from SEQ ID NO. 1 and activity for the synthesis of 2-oxo-butyric acid and [(3S)-3-amino-4-methoxy-4-oxobutyl]-(methyl) phosphinate.
[0120] Table 5: Activity of variants for coupled synthesis of 2-oxo-butyric acid and [(3S)-3-amino-4-methoxy-4-oxobutyl]-(methyl) phosphinate
SEQ ID No. No. of Residue difference from SEQ ID No: 1 Expression Threonine deaminase Activity Transaminase Activity
1 0 ++ + +
3 5 ++ + +
7 10 +++ ++ ++
11 13 ++ ++ +
15 18 +++ +++ +
19 25 +++ ++ ++
23 30 ++ ++ +
27 33 +++ +++ +++
31 36 ++ +++ +++

[0121] Threonine deaminase (TD) Activity column: "+" = 1-fold increase in TD activity; "++" = 2-fold increase in TD activity; "+++" = 3-fold increase in TD activity; "-" = No TD activity; Transaminase (TA) Activity column: "+" = 1-fold increase in TA activity; "++" = 2-fold increase in TA activity; "+++" = 3-fold increase in TA activity; "-" = No TA activity; Expression column: "+" = Low expression; "++" = Moderate expression; "+++" = High expression.
Example 12
[0122] Coupled synthesis of 2-oxo-butyric acid and methyl (2S)-2-amino-3-(4-fluorophenyl)propanoate (full transamination reactions)
[0123] The reaction mixture contained 10mM bulky a-keto acid methy-3-(4-fluorophenyl)-2-oxopropanoate, 10mM L-threonine, 20 mg lyophilized extract of engineered threonine deaminase enzyme and 1mM PMP/PLP in 100 mM Tris-HCl (pH 8.5). The reactions were carried out at 37 °C for 60 minutes and then terminated by adding 2ml 10% HCl. The activity was measured based on the amount of methyl (2S)-2-amino-3-(4-fluorophenyl)propanoate formed. Table 6 provides the SEQ ID NOs. for the engineered artificial threonine deaminase variants, along with the number of amino acid mutations from SEQ ID NO. 1 and activity for the synthesis of 2-oxo-butyric acid and methyl (2S)-2-amino-3-(4-fluorophenyl)propanoate.
[0124] Table 6: Activity of variants for coupled synthesis of 2-oxo-butyric acid and methyl (2S)-2-amino-3-(4-fluorophenyl)propanoate
SEQ ID No. No. of Residue difference from SEQ ID No: 1 Expression Threonine deaminase Activity Transaminase Activity
1 0 ++ + +
5 8 +++ ++ ++
9 12 ++ + +
13 16 +++ ++ +
17 21 ++ ++ +
21 29 ++ ++ ++
25 32 +++ ++ +
29 36 ++ ++ +
33 37 +++ +++ +++

[0125] Threonine deaminase (TD) Activity column: "+" = 1-fold increase in TD activity; "++" = 2-fold increase in TD activity; "+++" = 3-fold increase in TD activity; "-" = No TD activity; Transaminase (TA) Activity column: "+" = 1-fold increase in TA activity; "++" = 2-fold increase in TA activity; "+++" = 3-fold increase in TA activity; "-" = No TA activity; Expression column: "+" = Low expression; "++" = Moderate expression; "+++" = High expression.
Example 13
[0126] Engineered artificial threonine deaminase tested for the synthesis of 2-oxo-butyric acid from L-threonine
[0127] The reaction was performed by incubating 500 g of L-threonine and 500 mg of enzyme and 1mM PMP/PLP in 7.5 L of 0.1 M phosphate buffer (pH 8.0) for 1 hour at 30°C and then terminated by adding 2ml 10% HCl. The activity was measured based on the amount of 2-oxo-butyric acid formed. Table 7 provides the SEQ ID NOs. for the engineered artificial threonine deaminase variants and activity for the synthesis of 2-oxo-butyric acid.
[0128] Table 7: Activity of variants for synthesis of 2-oxo-butyric acid from L-threonine
SEQ ID No. Expression Threonine deaminase Activity
212 ++ +
213 +++ +
214 ++ +
215 ++ +
16 ++ ++
23 ++ ++
31 ++ +++
33 +++ +++
[0129] Threonine deaminase (TD) Activity column: "+" = 1-fold increase in TD activity; "++" = 2-fold increase in TD activity; "+++" = 3-fold increase in TD activity; "-" = No TD activity; Expression column: "+" = Low expression; "++" = Moderate expression; "+++" = High expression.
[0130] Table 8: Representative sequences (their corresponding UniProt IDs and source organisms) from the database used for deriving the artificial threonine deaminase polypeptide SEQ ID No. 1.

Sl. NO Organisms Accession ID
1 Paracoccus denitrificans A1B8Z1
2 Mycobacterium tuberculosis Q79FW0
3 Arabidopsis thaliana B0F481
4 Escherichia coli P00509
5 Thermotoga maritima Q9X0D0
6 Salmonella typhimurium Q8ZNF3
7 Thermus thermophilus Q5SHH5
8 Nostoc sp. Q8YY48
9 Alkalihalobacillus alcalophilus Q9RME2
10 Mycobacterium tuberculosis P9WQ77
11 Mycobacterium tuberculosis P9WQ73
12 Staphylococcus aureus Q2FV61
13 Thermotoga maritima Q9X2A5
14 Arabidopsis thaliana Q93ZN9
15 Ruegeria sp. Q1GD43
16 Saccharolobus solfataricus Q97VM5
17 Pseudomonas aeruginosa Q9I700
18 Thermoproteus uzoniensis F2L0W0
19 Geoglobus acetivorans A0A0A7GJ30
20 Pyrococcus horikoshii O57809
21 Cyberlindnera saturnus Q7M523
22 Thermus thermophilus Q5SLL4
23 Pseudomonas sp Q00740
24 Pseudomonas sp Q53IZ1
25 Thermus thermophilus Q5SKW8
26 Thermus thermophilus Q5SKW7
27 Escherichia coli P00861
28 Vibrio vulnificus Q7MK24
29 Levilactobacillus brevis D6PXK5
30 Burkholderia cenocepacia B4ECY9
31 Plasmodium falciparum Q8I566
32 Arabidopsis thaliana Q94C74
33 Acetoanaerobium sticklandii E3PRJ5
34 Acetoanaerobium sticklandii E3PRJ4
35 Clostridium subterminale Q9XBQ8
36 Acetoanaerobium sticklandii E3PY95
37 Acetoanaerobium sticklandii E3PY96
38 Arabidopsis thaliana P42799
39 Stenotrophomonas maltophilia B2FT35
40 Oryctolagus cuniculus P00489
41 Hordeum vulgare subsp. vulgare F2E0G2
42 Rattus norvegicus Q76EQ0
43 Mycobacterium tuberculosis P9WQA9
44 Bacillus anthracis A0A6L7HC45
45 Achromobacter obae Q7M181
46 Escherichia coli P0A6B4
47 Staphylococcus aureus P63479
48 Aeromonas hydrophila subsp. hydrophila A0KLG5
49 Bacillus subtilis P94494
50 Rhizobium freirei PRF 81 N6UXY4
51 Aeropyrum pernix Q9YBL2
52 Escherichia coli P16703
53 Thermus thermophilus Q5SLE6
54 Microcystis aeruginosa A8YBS4
55 Entamoeba histolytica O15570
56 Anopheles gambiae Q7PRG3
57 Corynebacterium glutamicum Q8NTR2
58 Arthrobacter sp. KNK168 F7J696
59 Chromobacterium violaceum Q7NWG4
60 Escherichia coli P77581
61 Aspergillus fumigatus Q4WH08
62 Mycobacterium tuberculosis P9WQ81
63 Serratia sp. A0A0J9X1Q5
64 Aedes aegypti Q0IG34
65 Treponema denticola Q73MU2
66 Curtobacterium pusillum A0A1S4NYF0
67 Citrobacter WP_032945726.1
68 [Enterobacter] lignolyticus WP_062739950.1
69 Kluyvera ascorbata WP_035896403.1
70 Phytobacter massiliensis WP_044185615.1
71 Klebsiella WP_014227448.1
72 Trabulsiella odontotermitis WP_152958650.1
73 Silvania confinis WP_271266116.1
74 Salmonella WP_001518457.1
75 Lelliottia nimipressuralis WP_100780792.1
76 Trabulsiella guamensis WP_156964210.1
77 Yokenella regensburgei WP_087943222.1
78 Raoultella WP_100686190.1
79 Yersinia thracia WP_050116487.1
80 Escherichia coli WP_000785596.1
81 Leclercia pneumoniae WP_207292615.1
82 Lelliottia aquatilis WP_095284423.1
83 Phytobacter WP_039077000.1
84 Raoultella WP_015585418.1
85 Scandinavium WP_258964547.1
86 Leclercia WP_156265197.1
87 Kosakonia WP_090398681.1
88 Scandinavium goeteborgense WP_174081415.1
89 Leclercia adecarboxylata WP_098946308.1
90 Superficieibacter electus WP_103677621.1
91 Lelliottia amnigena WP_202668756.1
92 Shimwellia pseudoproteus WP_199016216.1
93 Thermococcus profundus Q9V2W5
94 Aquifex aeolicus O66442
95 Sulfurisphaera tokodaii F9VN77
96 Salmonella typhimurium P40732
97 Toxoplasma gondii S8EY38
98 Rhizobium meliloti Q02635
99 Chromohalobacter salexigens Q9ZEU7
100 Citrobacter freundii P31013
ADVANTAGES/SIGNIFICANCE OF THE INVENTION
[0131] The invention provides an artificial engineered threonine deaminase enzyme that shows amino-transferase activity against bulky a-keto-acid molecules. In particular, the present invention provides a one-pot solution taking any of the bulky a-keto acid (3-carboxy-3-oxopropyl)(methyl)phosphinate (2a), or 4-fluorophenyl-pyruvic acid (2b), and L-threonine (1) as substrates for the coupled synthesis of pharmaceutically relevant intermediate compounds, specifically any of the following chosen from, phosphinothricin (4a), or L-4-fluorophenylalanine (4b) and 2-oxo-butyric acid. In some embodiments, the a-keto-acids can be replaced with their esters to derive corresponding a-amino acid esters. This is advantageous over the regular methods of biosynthesis of these compounds as generally they would be synthesized in different reaction systems using a different catalyst. Furthermore, the engineered artificial threonine deaminase offers a viable alternative to conventional amino-transferase reactions using transaminases, because of the robust, versatile nature of the threonine deaminase enzymes and their characteristic high substrate tolerance. The engineered threonine deaminase provided in the present invention also shows stability and improvement in catalytic efficiency over the wild-type enzyme under the conditions of temperature range from 30-60 °C, pH range of 6.5-10.0 and at increased substrate loading of up to 560 mM of L-threonine.
OTHER PUBLICATIONS
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We Claim:
1. A recombinant engineered threonine deaminase polypeptide comprising an amino acids sequence that is at least 90% identical to SEQ ID NO :1 and that includes the feature of residues corresponding to X24D, X71D or K, X101K, X108K or C, X142D or C, X299K, X334K, X371R, X431K, X458K, and X459E.

2. The recombinant engineered threonine deaminase polypeptide of claim 1 which comprises an amino acid sequence having one or more of the following features:
X20 is V or I;
X28 is A or V;
X70 is M or L;
X96 is F or L;
X98 is S or A;
X153 is W or F;
X161 is M or A;
X171 is L or M;
X183 is V or I;
X193 is A or V;
X211 is A or G;
X221 is K or R;
X251 is T or P;
X284 is V or I;
X313 is I or V.

3. The recombinant engineered threonine deaminase polypeptide of claim 2 which comprises an amino acid sequence having one or more of the following features:
X69 is F or M;
X75 is E or A;
X87 is K or A;
X92 is Y or Q;
X111 is T, V or E;
X114 is P or A;
X115 is P or D;
X123 is S or G or E;
X124 is L or F;
X130 is A or L;
X133 is E, A or F;
X135 is L, F, D, V or K;
X136 is E, D or M;
X140 is A, G or E;
X143 is L or I;
X146 is A or S;
X148 is Q or L;
X164 is C or A;
X235 is I or V;
X249 is E or D;
X273 is C or A;
X294 is C or A;
X301 is A or Q;
X302 is L or Q;
X306 is R or Q;
X348 is E or Q;
X354 is K or R;
X357 is Q or E;
X360 is H or G;
X361 is V or G;
X375 is K or E;
X386 is S, T or I;
X389 is L or H;
X390 is E or V;
X393 is K or E;
X397 is Q or S;
X398 is M or E;
X400 is N or R;
X401 is D or A;
X402 is G or K;
X403 is G or D;
X405 is S or Q;
X429 is H or K;
X432 is Q or R;
X442 is N or E;
X445 is M or G;
X449 is R or K;
X452 is N or H;
X457 is Y or H;
X472 is Y or F;
X473 is A or G;
X478 is A or G;
X482 is G or S;
X483 is D or A;
X484 is H or S;
X487 is D or Q;
X490 is T or Q;
X491 is R or H;
X493 is N or A;
X494 is E or A;
X497 is W or Y;
X504 is N or D;
X509 is R or K..

4. The recombinant engineered threonine deaminase polypeptide of claim 3 which comprises an amino acid sequence having one or more of the following features:
X86 is S or Y;
X88 is D, T or G;
X89 is E, K, S, T or N;
X90 is L or H;
X156 is P or S;
X240 is W, Y, or E;
X244 is V, W, Y or Q.

5. The recombinant engineered threonine deaminase polypeptide of claim 1-4 is given 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, and 200 can have an amino acid difference by one or more substitutions in combination with one or multiple that are at least about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% identical to SEQ ID NO:1.

6. A biocatalytic process for producing bulky S-amino acids or S-amino acid esters and 2-oxo-butyric acid, comprising contacting a bulky a-keto acid or a-keto ester with the recombinant engineered threonine deaminase polypeptides of claims 1-5 and co-substrate L-threonine in the presence of a PLP or PMP cofactor; and under suitable reaction conditions wherein:
a. the bulky a-keto acids are selected from the group consisting of 3-carboxy-3-oxopropyl (methyl) phosphinate, or 4-fluorophenyl-pyruvic acid, to produce S-amino acids such as L-phosphinothricin, or L-4-fluorophenylalanine, respectively.
b. the bulky a-keto esters are selected from the group consisting of 4-methoxy-3,4-dioxobutyl (methyl) phosphinate, or methyl-3-(4-fluorophenyl)-2-oxopropanoate to produce S-amino acid esters such as [(3S)-3-amino-4-methoxy-4-oxobutyl] (methyl) phosphinate, or methyl (2S)-2-amino-3-(4-fluorophenyl) propanoate, respectively.

7. The method of claim 6, wherein the said suitable reaction conditions comprise: a temperature range of 28°C to 60°C, pH range of 6.5 to 10.0, a-keto acid or a-keto ester concentration range of 5mM to 20 mM, a L-threonine concentration of 10 mM to 560 mM, a PLP/PMP concentration of 0.5 mM to 1 mM, a recombinant engineered threonine deaminase concentration of 0.05 g/L to 30 g/L and a buffer selected from the group consisting of Potassium phosphate buffer (100mM) or Tris-HCl buffer (100mM).

8. The recombinant engineered threonine deaminase polypeptides of claims 1-5 is used in the method of claim 7 in the form of whole cells, or an extract or lysate of such cells, or in purified form, wherein the recombinant engineered threonine deaminase polypeptide is encoded by a polynucleotide having the sequence of SEQ ID NO: 201, expressed in an E. coli host using the pET28a(+) vector under the control of T7 promotor, in a 20 mM phosphate buffer at pH 7.5, with an IPTG concentration of 0.1 mM and an induction temperature of 25 °C.

9. The recombinant engineered threonine deaminase polypeptides of claims 1-5, as compared to the wild-type enzyme described in SEQ ID NO: 212, 213, 214, or 215, exhibits at least a 70% to 90% increase in catalytic efficiency under suitable reaction conditions comprising a temperature of 30°C and a pH of 8.0, when tested at a substrate concentration of 500 grams of L-threonine, a PLP concentration of 1 mM and an enzyme concentration as low as 0.5 grams in a reaction volume of 7.5 L, with at least 99.9% conversion in at least 60 minutes of the reaction time.