Engineered lactate dehydrogenases and uses thereof

Abstract

The present disclosure provides polypeptides (e.g., LDH polypeptides), polynucleotides and expression vectors and host cells comprising the same, methods of producing polypeptides (e.g., LDH polypeptides), methods of catalyzing the NAD(P)H-dependent reduction 2-oxoglutarate to 2-hydroxyglutarate and methods of catalyzing the oxidation of NAD(P)H to NAD(P)+.

Description

ENGINEERED LACTATE DEHYDROGENASES AND USES THEREOFFIELD

[0001] The present disclosure relates generally to engineered NAD(P)H-dependent lactate dehydrogenases useful in the reduction of a-ketoglutarate (i.e. , 2-oxoglutarate), as well as related polynucleotides, expression vectors, host cells, methods of production, methods of catalyzing the NAD(P)H-dependent reduction of a-ketoglutarate (or 2-oxoglutarate) to 2-hydroxy glutarate, and methods of catalyzing the oxidation of NAD(P)H to NAD(P)+.BACKGROUND

[0002] Enzymes are protein molecules that serve to accelerate the chemical reactions of living cells (often by several orders of magnitude). Without enzy mes, most biochemical reactions would be too slow to carry out life processes. Enzymes display great specificity’ and are not permanently modified by their participation in reactions. Because they are not changed during the reactions, enzymes may be cost effectively used as catalysts for a desired chemical transformation.

[0003] Lactate dehydrogenase (LDH or LD) enzymes natively catalyze the reduction of pyruvate to either L- or D-lactate using nicotinamide cofactors NADH or NADPH as reducing agents. Some wild-ty pe lactate dehydrogenases may display higher selectivity for NADH over NADPH. Lactate dehydrogenases have been used in biocatalytic cascade reactions to regenerate the oxidized form of nicotinamide cofactors in order to drive thermodynamic equilibria. For example, in reactions involving oxidation of primary or secondary alcohols, alcohol dehydrogenase enzymes will use a nicotinamide cofactor, NAD+ or NADP+ as an electron acceptor, generating reduced NADH or NADPH as a coproduct. In order to use the nicotinamide cofactors in a catalytic fashion (i.e., less than 1 equivalent relative to substrate), the oxidized state of the cofactor must be regenerated using a sacrificial oxidant. Lactate dehydrogenases have been used to recycle oxidized NAD(P)+ by catalyzing the NAD(P)H-dependent reduction of pyruvate.SUMMARY

[0004] The present disclosure provides, inter alia, polypeptides (e.g., LDH polypeptides), polynucleotides encoding the same, expression vectors, host cells, methods of producing polypeptides, and methods of catalyzing the NAD(P)H-dependent reduction of 2-oxoglutarate to 2-hydroxy glutarate.

[0005] The present disclosure provides lactate dehydrogenases useful in the NAD(P)H- dependent reduction of a-ketoglutarate (i.e., 2-oxoglutarate) and in the oxidation of NAD(P)H to NAD(P)+. The lactate dehydrogenases of the present disclosure may also be useful for catalyzing reactions in the process of generating macrocyclic peptides.

[0006] In some embodiments, the lactate dehydrogenases and lactase dehydogenase-catalyzed methods of NAD(P)H-dependent reduction of 2-oxoglutarate to 2-hydroxy glutarate provided herein are useful in the production of macrocyclic peptides, e.g., enlicitide. The structure of enlicitide is shown below as Formula 1. In some embodiments, A is decanoate.Formula 1

[0007] In one aspect, provided herein is an engineered polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 4, wherein the polypeptide comprises an amino acid substitution at one or more amino acid positions selected from 12, 88, 98, 99, 166, 199, 205, 229, and 308, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 4.

[0008] Also provided herein is an engineered polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2, wherein the polypeptide comprises an amino acid substitution at one or more amino acid positions selected from 12, 88, 98, 99, 166, 199, 205, 229, and 308, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2.

[0009] Also provided herein is an engineered polypeptide comprising an amino acid sequence having at least 98% sequence identity to any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

[0010] In some embodiments of any of the preceding, the polypeptide is capable of catalyzing the NAD(P)H-dependent reduction of pyruvate to L- or D-lactate and / or the NAD(P)H-dependent reduction of a-ketoglutarate to 2-hydroxy glutarate. In some embodiments of any of the preceding, the polypeptide is capable of catalyzing the NAD(P)H-dependent reduction of pyruvate to L- or D-lactate. In some embodiments of any of the preceding, the polypeptide is capable of catalyzing the NAD(P)H-dependent reduction of a-ketoglutarate to 2- hydroxy glutarate. In some embodiments of any of the preceding, the polypeptide is capable of catalyzing the NAD(P)H-dependent reduction of pyruvate to L- or D-lactate and the NAD(P)H- dependent reduction of a-ketoglutarate to 2-hydroxy glutarate.

[0011] Also provided herein is a polynucleotide encoding at least one polypeptide of the disclosure, wherein the polynucleotide does not comprise SEQ ID NO: 1 or SEQ ID NO: 3.

[0012] Also provided herein is a polynucleotide comprising at least 80% sequence identity to any one of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, or 31, wherein the polynucleotide does not comprise SEQ ID NO: 1 or SEQ ID NO: 3.

[0013] Also provided herein is an expression vector comprising at least one of the polynucleotides disclosed herein.

[0014] Also provided herein is a host cell comprising at least one of the polynucleotides disclosed herein or at least one of the expression vectors disclosed herein.

[0015] Also provided herein is a method of producing a polypeptide, the method comprising culturing any one of the host cells disclosed herein under conditions such that the polypeptide encoded by the polynucleotide is produced.

[0016] Also provided herein is a method of catalyzing the NAD(P)H-dependent reduction of 2- oxoglutarate to 2-hydroxyglutarate, the method comprising contacting the 2-oxoglutarate with at least one polypeptide of the disclosure.

[0017] The summary of the technology described above is non-limiting and other features and advantages of the technology will be apparent from the following detailed description, and from the claims.DETAILED DESCRIPTION

[0018] The present disclosure relates to engineered lactate dehydrogenase polypeptides. As is described in the Examples, the present disclosure provides engineered LDH polypeptides derived from wild-type Streptococcus thermophilus LDH, which exhibit improved enzy me properties relative to wild-type Streptococcus thermophilus LDH, and which were discovered through iterative rounds of directed evolution as described herein. For example, the novel engineered LDH polypeptides of the disclosure may exhibit one or more of the following properties relative to a reference polypeptide (e.g., a wild-ty pe Streptococcus thermophilus LDH): increased activity726079 in catalyzing the NAD(P)H-dependent conversion of 2-oxoglutarate to 2-hydroxy glutarate, increased activity in catalyzing the oxidation of NAD(P)H to NAD(P)+, and / or increased stability'. The Examples describe the discover}' of LDH polypeptide variants with enhanced activity in the NADH-dependent reduction of 2-oxoglutarate with >1 OO-fold of activity improvement relative to wild-type Streptococcus thermophilus LDH.

[0019] The engineered lactate dehydrogenases of the disclosure are able to selectively utilize 2- oxoglutarate as an electron acceptor over pyruvate. The engineered lactate dehydrogenases of the disclosure and may be useful for catalyzing reactions in the process of generating macrocyclic peptides.

[0020] The present disclosure also relates to polynucleotides and expression vectors encoding the LDH polypeptides of the present disclosure, host cells comprising the polynucleotides or expression vectors, methods of producing the LDH polypeptides, and methods of catalyzing the NAD(P)H-dependent reduction of 2-oxoglutarate to 2-hydroxy glutarate and methods of catalyzing the oxidation of NAD(P)H to NAD(P)+.Definitions

[0021] Listed below are definitions of various terms used herein. These definitions apply to the terms as they are used throughout this specification and claims, unless otherwise limited in specific instances, either individually or as part of a larger group.

[0022] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and peptide chemistry are those well-known and commonly employed in the art.

[0023] As used herein, the articles “a” and “an” refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting and is used interchangeably with the term “including, but not limited to.”

[0024] As used herein, the term “about” in quantitative terms refers to plus or minus 10% of the value it modifies (rounded up to the nearest whole number if the value is not sub-dividable, such as a number of molecules or nucleotides).

[0025] Except where otherwise indicated, all numbers expressing quantities used in the disclosure are to be understood as being modified in all instances by the term “about,” whether or not the term “about” is present in front of the number. Accordingly, unless indicated to the26079 contrary, the numerical parameters set forth in the following specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very’ least, and not to be considered as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding conventions.

[0026] As used herein, the terms “at least one” item or “one or more” item each include a single item selected from a list as well as combinations of two or more items selected from a list (e g., a list of mutations). Similarly, the terms “at least two” items and “two or more” items each include combinations of two items selected from a list as well as combinations of three or more items selected from a list.

[0027] All ranges disclosed herein are inclusive of the recited endpoint and independently combinable. For example, if a range is from about 1 to about 50, it is deemed to include, for example, 1, 50, 7, 34, 46.1, 23.7, or any other value or range within the range. The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value and include values approximating these ranges and / or values, except where otherwise indicated.

[0028] As used herein, the term “comprising” may include the embodiments “consisting of’ and “consisting essentially of.” The terms “comprise(s),” “include(s),” “having,” “has,” “may,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that require the presence of the named ingredients / steps and permit the presence of other ingredients / steps. However, such description should be construed as also describing compositions or processes as “consisting of and “consisting essentially of the enumerated components, which allows the presence of only the named components or compounds, along with any acceptable carriers or fluids, and excludes other components or compounds.

[0029] “Derived from” as used herein in the context of enzy mes, identifies the originating enzyme, and / or the gene encoding such enzyme, upon which the enzyme was based. For example, the LDH polypeptide variants of SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, and 32 were obtained by artificially evolving over multiple generations the gene encoding the wild-type Streptococcus thermophilus LDH enzyme of SEQ ID NO: 2 or the wild-type Streptococcus thermophilus LDH enz me with an added C-terminal His tag (SEQ ID NO: 4). Thus, the evolved LDH variant enzymes are “derived from” the Streptococcus thermophilus LDH enzyme of SEQ ID NO: 2 or SEQ ID NO: 4.

[0030] As used herein, "reference sequence’7refers to a defined sequence used as a basis for a sequence comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotide or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, or the full length of the nucleic acid or polypeptide. Since two polynucleotides or polypeptides may each (1) comprise a sequence (i.e., a portion of the complete sequence) that is similar between the two sequences, and (2) may further comprise a sequence that is divergent between the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptide are typically performed by comparing sequences of the two polynucleotides over a ■‘comparison window” to identify and compare local regions of sequence similarity.

[0031] As used herein, “polynucleotide” and “nucleic acid’ are used interchangeably to refer to two or more nucleotides that are covalently linked together. The polynucleotide may be wholly comprised of ribonucleotides (i.e., RNA), wholly comprised of 2' deoxyribonucleotides (i.e.. DNA), or comprised of mixtures of ribo- and 2' deoxyribonucleotides. The polynucleotide may include modified nucleotides, including nucleotides which have modifications at the sugar, nucleobase, and / or phosphodiester backbone (intemucleoside linkage), and nucleoside analogues. For example, while the nucleosides will typically be linked together via standard phosphodiester linkages, the polynucleotides may include one or more non-standard linkages. Moreover, while a polynucleotide may be typically composed of the naturally occurring encoding nucleobases (i.e., adenine, guanine, uracil, thymine, and cytosine), it may also include one or more modified and / or synthetic nucleobases, such as, for example, inosine, xanthine, hypoxanthine, etc. In some embodiments, such modified or synthetic nucleobases are nucleobases encoding amino acid sequences. The polynucleotide may be single-stranded or double-stranded, or the polynucleotide may include both single-stranded regions and double-stranded regions.

[0032] The abbreviations used for the genetically encoding nucleosides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless specifically delineated, the abbreviated nucleosides may be either ribonucleosides or 2'- deoxyribonucleosides. The nucleosides may be specified as being either ribonucleosides or 2'- deoxyribonucleosides on an individual basis or on an aggregate basis. When nucleic acid sequences are presented as a string of one-letter abbreviations, the sequences are presented in the 5' to 3' direction in accordance with common convention, and the phosphates are not indicated.

[0033] As used herein, the terms “protein,” “polypeptide,” and “peptide” are used interchangeably herein to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation,phosphorylation, lipidation, myristoylation, ubiquitination, and the like). Included within this definition are D- and L-amino acids, and mixtures of D- and L-amino acids, as well as polymers comprising D- and L-amino acids, and mixtures of D- and L-amino acids. Proteins, polypeptides, and peptides may include a tag (e.g.. an epitope tag), such as a histidine tag.

[0034] As used herein, the terms '“amino acid’7or “residue” as used in context of the polypeptides disclosed herein refers to the specific monomer at a sequence position. Amino acids are referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by International Union of Pure and Applied Chemistry (IUPAC) - International Union of Biochemistry (IUB) Biochemical Nomenclature Commission.Nucleotides, likewise, may be referred to by their commonly accepted single letter codes.

[0035] The abbreviations used for the genetically encoded amino acids are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartate (Asp or D), cysteine (Cys or C), glutamate (Glu or E), glutamine (Gin or Q), histidine (His or H), isoleucine (He or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Vai or V).

[0036] “Hydrophilic amino acid” or “hydrophilic residue” refers to an amino acid or residue having a side chain exhibiting a hydrophobicity of less than zero according to the normalized consensus hydrophobicity scale of Eisenberg et al., 1984, J. Mol. Biol. 179: 125-142. Genetically encoded hydrophilic amino acids include L-Thr (T), L-Ser (S), L-His (H), L-Glu (E), L-Asn (N), L-Gln (Q), L-Asp (D), L-Lys (K), and L-Arg (R).

[0037] “Acidic amino acid” or “acidic residue” refers to a hydrophilic amino acid or residue having a side chain exhibiting a pKa value of less than about 6 when the amino acid is included in a peptide or polypeptide. Acidic amino acids typically have negatively charged side chains at physiological pH due to loss of a hydrogen ion. Genetically encoded acidic amino acids include L-Glu (E) and L-Asp (D).

[0038] “Basic amino acid” or “basic residue” refers to a hydrophilic amino acid or residue having a side chain exhibiting a pKa value of greater than about 6 when the amino acid is included in a peptide or polypeptide. Basic amino acids typically have positively charged side chains at physiological pH due to association with hydronium ion. Genetically encoded basic amino acids include L-Arg (R) and L-Lys (K).

[0039] “Polar amino acid” or “polar residue” refers to a hydrophilic amino acid or residue having a side chain that is uncharged at physiological pH, but which has at least one bond in which the pair of electrons shared in common by two atoms is held more closely by one of theatoms. Genetically encoded polar amino acids include L-Asn (N), L-Gln (Q). L-Ser (S), and L- Thr (T).

[0040] “Hydrophobic amino acid” or “hydrophobic residue” refers to an amino acid or residue having a side chain exhibiting a hydrophobicity of greater than zero according to the normalized consensus hydrophobicity scale of Eisenberg et al., 1984. J. Mol. Biol. 179: 125-142. Genetically encoded hydrophobic amino acids include L-Pro (P), L-Ile (I), L-Phe (F), L-Val (V), L-Leu (L), L-Trp (W), L-Met (M), L-Ala (A), and L-Tyr (Y).

[0041] “Aromatic amino acid” or “aromatic residue” refers to a hydrophilic or hydrophobic amino acid or residue having a side chain that includes at least one aromatic or heteroaromatic ring. Genetically encoded aromatic amino acids include L-Phe (F), L-Tyr (Y), L-His (H), and L- Trp (W). L-His (H) histidine is also classified herein as a hydrophilic residue or as a constrained residue.

[0042] As used herein, “constrained amino acid” or “constrained residue” refers to an amino acid or residue that has a constrained geometry. Herein, constrained residues include L-Pro (P) and L-His (H). Histidine has a constrained geometry because it has a relatively small imidazole ring. Proline has a constrained geometry' because it also has a five-membered ring.

[0043] “Non-polar amino acid” or “non-polar residue” refers to a hydrophobic amino acid or residue that has a side chain that is uncharged at physiological pH and that has bonds in which the pair of electrons shared in common by two atoms is generally held equally by each of the two atoms (i.e., the side chain is not polar). Genetically encoded non-polar amino acids include L-Gly (G), L-Leu (L), L-Val (V), L-Ile (I), L-Met (M), and L-Ala (A).

[0044] As used herein, “aliphatic amino acid” or “aliphatic residue” refers to a hydrophobic amino acid or residue having an aliphatic hydrocarbon side chain. Genetically encoded aliphatic amino acids include L-Ala (A), L-Val (V), L-Leu (L), and L-Ile (I).

[0045] The ability of L-Cys (C) (and other amino acids with SH-containing side chains) to exist in a peptide in either the reduced free SH or oxidized disulfide-bridged form affects whether L- Cys (C) contributes net hydrophobic or hydrophilic character to a peptide. While L-Cys (C) exhibits a hydrophobicity of 0.29 according to the normalized consensus scale of Eisenberg (Eisenberg et al., 1984, supra), it is to be understood that for purposes of the present disclosure, L-Cys (C) is categorized into its own unique group. It is noted that L-Cys (C) is unusual in that it can form disulfide bridges with other L-Cys (C) amino acids or other sulfanyl- or sulfhydryl- containing amino acids. The “cysteine-like residues” include cysteine and other amino acids that contain sulfhydryl moieties that are available for formation of disulfide bridges.

[0046] As used herein, "small amino acid’7or "small residue” refers to an amino acid or residue having a side chain that is composed of a total of three or fewer carbon and / or heteroatoms (excluding the a carbon and hydrogens). The small amino acids or residues may be further categorized as aliphatic, non-polar, polar or acidic small amino acids or residues, in accordance with the above definitions. Genetically encoded small amino acids include L-Ala (A), L-Val (V), L-Cys (C), L-Asn (N), L-Ser (S), L-Thr (T), and L-Asp (D).

[0047] “Hydroxyl-containing amino acid” or "hydroxyl-containing residue” refers to an amino acid containing a hydroxyl (-OH) moiety. Genetically encoded hydroxyl-containing amino acids include L-Ser (S), L-Thr (T), and L-Tyr (Y).

[0048] As used herein, '‘conservative amino acid substitution” refers to a substitution of a residue with a different residue having a similar side chain, and thus typically involves substitution of an amino acid in the polypeptide with an amino acid within the same or similar defined class of amino acids. By way of example and not limitation, in some embodiments, an amino acid with an aliphatic side chain is substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with an hydroxyl side chain is substituted with another amino acid with an hydroxyl side chain (e.g., serine and threonine); an amino acid having an aromatic side chain is substituted with another amino acid having an aromatic side chain (e.g.. phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain is substituted with another amino acid with a basic side chain (e.g., lysine and arginine); an amino acid with an acidic side chain is substituted with another amino acid with an acidic side chain (e.g., aspartic acid and glutamic acid); and / or a hy drophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively.

[0049] As used herein, “non-conservative substitution” refers to substitution of an ammo acid in the polypeptide with an amino acid with significantly differing side chain properties. Nonconservative substitutions may use amino acids betw een, rather than within, the defined groups and affect (a) the structure of the peptide backbone in the area of the substitution (e.g., proline for glycine) (b) the charge or hydrophobicity, or (c) the bulk of the side chain. By way of example and not limitation, an exemplary non-conservative substitution can be an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.

[0050] “Mutation” refers to any change in a polypeptide or polynucleotide sequence, and encompasses any number (i.e., one or more) of substitutions, deletions, insertions, and / or rearrangements present in a sequence compared to a reference sequence.

[0051] As used herein, "deletion" refers to modification to a polypeptide by removal of one or more amino acids relative to a reference polypeptide (e.g., enzy me). Deletions can comprise removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids making up the reference polypeptide while retaining activity (e.g., enzymatic activity) and / or retaining the improved properties of an evolved polypeptide. Deletions can be directed to the internal portions and / or terminal portions of the polypeptide. In various embodiments, the deletion can comprise a continuous segment or can be discontinuous. Deletions are typically indicated by in amino acid sequences.

[0052] As used herein, “insertion” refers to modification to a polypeptide by addition of one or more amino acids relative to a reference polypeptide (e.g., enzyme). Insertions can be in the internal portions of the polypeptide, or to the carboxy or amino terminus. Insertions as used herein include fusion proteins. The insertion can be a contiguous segment of amino acids or separated by one or more of the amino acids in the naturally occurring polypeptide.

[0053] As used herein with respect to amino acid sequences, a “substitution” refers to a difference in the amino acid residue at a position of a polypeptide sequence relative to the amino acid residue at a corresponding position in a reference sequence. In some instances, the present disclosure provides specific amino acid differences denoted by the conventional notation “AnB,” where A is the single letter identifier of the residue in the reference sequence, n is the number of the residue position in the reference sequence, and B is the single letter identifier of the residue substitution in the sequence of the engineered polypeptide.

[0054] The term “amino acid substitution set” or “substitution set” refers to a group of amino acid substitutions in a polypeptide sequence, as compared to a reference sequence. For example, a substitution set may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions. When used in reference to a polypeptide comprising a substitution set (e.g., a polypeptide comprising “a substitution set at positions 148. 157, and 246”), the polypeptide would comprise (and the substitution set would include) substitutions at each of the positions that followed (e.g., in the foregoing example at each of positions 148, 157, and 246).

[0055] “Corresponding to,” “reference to” or “relative to” when used in the context of the numbering of a given amino acid or polynucleotide sequence refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given polymer is designated with respect to the reference sequence rather than by the actual26079 numerical position of the residue within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, although the gaps are present, the numbering of the residue in the given amino acid or polynucleotide sequence is made with respect to the reference sequence to which it has been aligned.

[0056] A '‘functional fragment’’ and '‘biologically active fragment” are used interchangeably herein to refer to a polypeptide that has an amino-terminal and / or carboxy -terminal deletion(s) and / or internal deletion(s), but where the remaining amino acid sequence is identical to the corresponding positions in the reference sequence and that retains substantially all of the activity of the full-length polypeptide.

[0057] As used herein, “isolated polypeptide” refers to a composition in which the polypeptide is substantially separated from other contaminants that naturally accompany it (e.g., protein, lipids, and polynucleotides). The term embraces polypeptides that have been removed or purified from their naturally occurring environment or expression system (e.g., within a host cell or via in vitro synthesis). The recombinant polypeptides may be present within a cell, present in the cellular medium, or prepared in various forms, such as lysates or isolated preparations. As such, in some embodiments, the recombinant poly peptides can be an isolated polypeptide.

[0058] As used herein, “substantially pure polypeptide” or “purified protein” refers to a composition in which the polypeptide species is the predominant species present (i.e., on a molar or weight basis it is more abundant than any other individual macromolecular species in the composition), and is generally a substantially purified composition when the object species comprises at least about 50 percent of the macromolecular species present by mole or % weight. However, in some embodiments, an enzyme comprising composition comprises enzymes that are less than 50% pure (e.g., about 10%, about 20%, about 30%, or about 40% pure). Generally, a substantially pure enzyme or polypeptide composition comprises about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecular species by mole or % weight present in the composition. In some embodiments, the object species is purified to essential homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species. In some embodiments, the isolated recombinant polypeptides are substantially pure polypeptide compositions.26079

[0059] “Improved enzyme property” refers to any property of an enzyme that exhibits an improvement as compared to a reference enzyme. For the enzymes described herein, the comparison is generally made to a wild-type enzy me (e.g., SEQ ID NO: 2 or SEQ ID NO: 4), although in some embodiments, the reference enzyme can be another improved enzyme. Enzyme properties for which improvement may be desirable include, but are not limited to. enzymatic activity (which may be expressed in terms of percent conversion of the substrate), thermal stability, soluble expression, pH activity' profile, cofactor requirements, refractoriness to inhibitors (e.g., product inhibition), stereospecificity, and stereoselectivity' (including enantioselectivity).

[0060] “Increased enzymatic activity ” refers to an improved property of the enzymes, which can be represented by an increase in specific activity (e.g., amount, selectivity, or quantity of product produced, time of activity, and / or amount of product produced per time unit per weight of enzyme) or an increase in percent conversion of the substrate to the product (e.g., percent conversion of starting amount of substrate to product in a specified time period using a specified amount of enzy me) as compared to a reference enzy me. Exemplary methods to determine enzyme activity7are provided in the Examples. Any property7relating to enzyme activity may be affected, including the classical enzy me properties of K™, Nmax, or kcor, changes of which can lead to increased enzymatic activity. Improvements in enzyme activity' can be at least about 1.1 times the enzymatic activity of the corresponding wild-type enzyme, at least about 2 times, at least about 5 times, at least about 10 times, at least about 20 times, at least about 25 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 150 times, at least about 200 times, at least about 500 times, at least about 1000 times, at least about 3000 times, at least about 5000 times, at least about 7000 times or more enzymatic activity than the reference enzyme, e.g., a naturally occurring enzyme or another enzyme from which the polypeptides were derived. The term “fold” is also used in relation to the enzy matic activity', where “fold” is used interchangeably with “times” in this respect. For example, the terms “2-fold” and “2 times” are used interchangeably. In some examples, the enzyme exhibits improved enzymatic activity in the range of 100 to 3000 times, 3000 to 7000 times, or more than 7000 times greater than that of the parent enzyme. It is understood by the skilled artisan that the activity of any enzyme is diffusion limited such that the catalytic turnover rate cannot exceed the diffusion rate of the substrate, including any required cofactors. The theoretical maximum of the diffusion limit, or kcat / K™. is generally about I08to 109( T's-1). Hence, any improvements in the enzyme activity will have an upper limit related to the diffusion rate of the substrates acted on by the enzyme. Enzyme activity can be measured by any suitable approach, e.g., an enzyme activity assay or by any of the26079 traditional methods for assaying chemical reactions, including but not limited to high- performance liquid chromatography (HPLC), HPLC-mass spectrometry (MS), ultra-performance liquid chromatography (UPLC), UPLC-MS, thin-layer chromatography (TLC), and nuclear magnetic resonance (NMR). Comparisons of enzy me activities may be made using a defined preparation of enzyme, a defined assay under a set condition, and one or more defined substrates, as further described in detail herein. Generally, when lysates are compared, the numbers of cells and the amount of protein assayed are determined as well as use of identical expression systems and identical host cells to minimize variations in amount of enzyme produced by the host cells and present in the lysates.

[0061] As used herein, a '‘vector’ is a DNA construct for introducing a DNA sequence into a cell. In some embodiments, the vector is an expression vector that is operably linked to a suitable control sequence capable of effecting the expression of the polypeptide encoded by the polynucleotide (e.g., DNA) sequence in a suitable host. In some embodiments, an “expression vector” has a promoter sequence operably linked to the polynucleotide (e.g., DNA) sequence (e.g., transgene) to drive expression in a host cell, and in some embodiments, also comprises a transcription terminator sequence.

[0062] As used herein with respect to polypeptides, the terms “expression” and “production” and “produce” includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from a cell.

[0063] As used herein, an amino acid or nucleotide sequence (e g., a promoter sequence, signal peptide, terminator sequence, and the like) is “heterologous” to another sequence with which it is operably linked if the two sequences are not associated in nature. For example, a “heterologous polynucleotide” is any polynucleotide that is introduced into a host cell by laboratory' techniques, and the term includes polynucleotides that are removed from a host cell, subjected to laboratory manipulation, and then reintroduced into a host cell.

[0064] As used herein, the terms “host cell” and “host strain” refer to suitable hosts for an expression vector comprising a polynucleotide (e.g., DNA) provided herein (e.g., a polynucleotide encoding an LDH polypeptide disclosed herein). In some embodiments, the host cells are prokaryotic or eukaryotic cells that have been transformed or transfected with vectors constructed using recombinant DNA techniques as known in the art.

[0065] The term “analogue” means a polypeptide having more than 70% sequence identity but less than 100% sequence identity (e.g., more than 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,2607983%. 84%. 85%. 86%. 87%. 88%. 89% 90%. 91%, 92%, 93%, 94%, 95%. 96%. 97%. 98%. or 99% but less than 100% sequence identity) with a reference polypeptide. In some embodiments, “analogues” means polypeptides that contain one or more non-naturally occurring amino acid residues including, but not limited, to homoarginine, ornithine and non aline, as well as naturally occurring amino acids. In some embodiments, analogues also include one or more D-amino acid residues and non-peptide linkages between two or more amino acid residues.

[0066] “Coding sequence” refers to that portion of a polynucleotide (e.g., a gene) that encodes an amino acid sequence of a polypeptide.

[0067] “Naturally occurring” or “wild-type” generally refers to a form found in nature. As used herein, a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a source in nature and that has not been intentionally modified by human manipulation, with the sole exception that wild-ty pe polypeptide or polynucleotide sequences as identified herein may include a tag, such as a histidine (His) tag. For example, the Streptococcus thermophilus LDH sequence of SEQ ID NO: 4, which includes a C-terminal His tag (and thus is not found in nature), may be referred to as a wild-type Streptococcus thermophilus LDF herein. Herein, “wild-type” polypeptide or polynucleotide sequences may be denoted “WT.”

[0068] “Operably linked” is defined herein as a configuration in which a control sequence is appropriately placed at a position relative to a polynucleotide sequence (i.e., in a functional relationship) such that the control sequence directs the expression of the polynucleotide and / or a polypeptide encoded by the polynucleotide.

[0069] A “promoter sequence” is a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide. The control sequence may comprise an appropriate promoter sequence. The promoter sequence contains transcriptional control sequences, which mediate the expression of the polynucleotide. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

[0070] The terms “engineered,” “recombinant,” “variant,” and “non-naturally occurring,” when used with reference to, e g., a polynucleotide, polypeptide, or cell, 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. Non-limiting examples include, among others, 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. In the present26079 disclosure, reference to a polypeptide having an amino acid sequence that is not a wild-type amino acid sequence as used herein, for example, will be understood to refer to an engineered polypeptide.

[0071] A “selectable marker'’ is a gene, the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs. and the like. Exemplary selectable markers are described herein.

[0072] “Percentage of sequence identity,” “percent identity,” and “percent identical” are used herein to refer to comparisons between polynucleotide sequences or polypeptide sequences, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Determination of optimal alignment and percent sequence identity can be performed using the BLAST and BLAST 2.0 algorithms (see e.g., Altschul et al., 1990, J. Mol. Biol. 215: 403-410; and Altschul et al., 1977, Nucleic Acids Res. 3389-3402). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website.

[0073] Briefly, the BLAST analyses involve first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as, the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence26079 is reached. The BLAST algorithm parameters W. T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M = 5, N = -4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff. 1989, PROC. NATL. ACAD. SCI. USA 89: 10915).

[0074] Numerous other algorithms are available that function similarly to BLAST in providing percent identity for two sequences. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homolog)’ algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection (see generally, Cunent Protocols in Molecular Biolog)’, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)). Additionally, determination of sequence alignment and percent sequence identity' can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison WI), using default parameters provided.

[0075] “Conversion” refers to the enzymatic transformation of a substrate to the corresponding product. “Percent conversion” refers to the percent of the substrate that is converted to the product within a period of time under specified conditions. Thus, for example, the “enzymatic activity” or “activity” of a polypeptide can be expressed as “percent conversion” of the substrate to the product.

[0076] “Thermostable” refers to a polypeptide that maintains similar activity (more than 60% to 80%, for example) after exposure to elevated temperatures (e.g., 40 °C to 80 °C) for a period of time (e.g., 0.5 h to 24 h) compared to the untreated enzyme.

[0077] As used herein, the terms “biocatalysis,” “biocatalytic.” “biotransformation.” and “biosynthesis” refer to the use of enzymes to perform chemical reactions on organic compounds.

[0078] The term “effective amount” means an amount sufficient to produce the desired result. One of general skill in the art may determine what the effective amount by using routine experimentation.

[0079] The terms “isolated” and “punfied” are used to refer to a molecule (e.g., an isolated nucleic acid or polypeptide) or other component that is removed from at least one other26079 component with which it is naturally associated. The term “purified” does not require absolute purity, rather it is intended as a relative definition.

[0080] The terms “lactate dehydrogenase,” “LDH,” “LDH enzy mes,” and “LDH polypeptides” are used interchangeably herein to refer to a polypeptide having a capability of catalyzing an NAD(P)H-dependent reaction by converting NAD(P)H to NAD(P)+. For example. LDH enzymes disclosed herein are capable of catalyzing the NAD(P)H-dependent reduction of pyruvate to L- or D-lactate and / or the NAD(P)H-dependent reduction of a-ketoglutarate (i.e., 2- oxoglutarate) to 2-hydroxyglutarate. “LDH polypeptide” as used herein includes naturally occurring (wild-type) LDH polypeptides as well as non-naturally occurring engineered polypeptides generated by human manipulation. In an embodiment, an LDH polypeptide as disclosed herein may have an amino acid sequence selected from any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, and 32.

[0081] The term “NAD(P)H” as used herein refers to cofactor reduced nicotinamide adenine dinucleotide (NADH) and / or cofactor reduced nicotinamide adenine dinucleotide phosphate (NADPH). The term “NAD(P)+” refers to cofactor(s) NADP+ and / or NAD+. As used herein, the term “cofactor” refers to a non-protein compound that operates in combination with a lactate dehydrogenase. Cofactors suitable for use with the engineered lactate dehydrogenases described herein include, but are not limited to, NADP+ (nicotinamide adenine dinucleotide phosphate), NADPH (the reduced form of NADP+), NAD+ (nicotinamide adenine dinucleotide) and NADH (the reduced form of NAD+). Generally, the reduced form of the cofactor is added to the reaction mixture. The reduced NAD(P)H form can be optionally regenerated from the oxidized NAD(P)+ form using a cofactor regeneration system. The term “cofactor regeneration system” refers to a set of reactants that participate in a reaction that reduces the oxidized form of the cofactor (e.g., NAD(P)+ to NAD(P)H). Cofactor regeneration systems to regenerate NADH or NADPH from NAD+ or NADP+, respectively, are known in the art and may be used in the methods described herein. For example, NAD(P)+ may be recycled back to NAD(P)H by an alcohol dehydrogenase (ADH) enzyme. The LDH polypeptides of the disclosure may also be used in a cofactor regeneration system. For example, it may be advantageous to regenerate the oxidized form of NAD(P)+. The LDH polypeptides may be used in a method of catalyzing the oxidation of NAD(P)H to NAD(P)+. For example, NADH can be recycled back to NAD+ by an LDH polypeptide of the disclosure using a-ketoglutarate as the stoichiometric oxidant (i.e., electron acceptor).26079LDH Polypeptides

[0082] This disclosure provides polypeptides (e.g., LDH polypeptides) capable of catalyzing the NAD(P)H-dependent reduction of pyruvate to L- or D-lactate and / or the NAD(P)H- dependent reduction of a-ketoglutarate (i. e. , 2-oxoglutarate) to 2-hydroxyglutarate. In some embodiments, the LDH polypeptides described herein preferentially utilize a-ketoglutarate (i.e., 2-oxoglutarate) as an electron acceptor rather than pyruvate. In some embodiments, the LDH polypeptides described herein exhibit preferential selectivity for NADH over NADPH.

[0083] In some embodiments, the LDH polypeptides described herein are capable of catalyzing the NAD(P)H-dependent reduction of 2-oxoglutarate to 2-hydroxyglutarate with selectivity for NADH over NADPH as shown below:

[0084] In some embodiments, the LDH polypeptides of the disclosure are capable of catalyzing reactions in the process of generating macrocyclic peptides.

[0085] In some embodiments, the LDH polypeptides of the disclosure are capable of catalyzing the oxidation of NAD(P)H to NAD(P)+. For example, NADH can be recycled back to NAD+ by an LDH polypeptide of the disclosure using a-ketoglutarate as the electron acceptor. In some embodiments, the LDH polypeptides of the disclosure are capable of regenerating oxidized NAD(P)+ in the context of an alcohol dehydrogenase reaction.

[0086] In certain embodiments, a polypeptide (e.g., an LDH polypeptide) described herein has an amino acid sequence comprising one or more amino acid differences as compared to a reference amino acid sequence of a wild-type polypeptide that results in an improved enzyme property.

[0087] In some embodiments, the LDH polypeptides described herein are a product of directed evolution from a wild-type LDH sequence, which was itself identified by screening a panel of wild-type LDH polypeptides((i.e., a wild-type LDH polypeptide as set forth in SEQ ID NO: 4. which includes a C-terminal His tag or a wild-type LDH polypeptide as set forth in SEQ ID NO: 2, which lacks a C-terminal His tag).26079

[0088] Enzyme properties for which improvements relative to a reference polypeptide are desirable include, but are not limited to, enzymatic activity, thermal stability, pH activity profile, cofactor requirements, cofactor selectivity, refractoriness to inhibitors (e.g., product inhibition), stereospecificity, stereoselectivity, and solvent stability. The improvements can relate to a single enzyme property, such as enzymatic activity, or a combination of different enzyme properties, such as enzymatic activity and stereoselectivity.

[0089] In some embodiments, the polypeptide (e.g., the LDH polypeptide) of the disclosure may demonstrate one or more improvements relative to a reference polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. including, but not limited to, increases in enzymatic activity, thermal stability, and / or solvent stability. In some embodiments, the polypeptide (e.g., the LDH polypeptide) of the disclosure may demonstrate at least two improvements relative the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4, including, but not limited to, increases in enzymatic activity, thermal stability, and / or solvent stability.

[0090] For example, in some embodiments, the polypeptide has one or more of the following properties relative to a reference polypeptide (e.g., a reference polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4): a) increased activity in converting 2- oxoglutarate to 2-hydroxyglutarate; b) increased activity in catalyzing the oxidation of NAD(P)H to NAD(P)+; and / or c) increased stability. In some embodiments, the polypeptide has the following properties relative to a reference polypeptide (e.g., a reference polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4): a) increased activity' in converting 2-oxoglutarate to 2-hydroxyglutarate, and b) increased activity' in catalyzing the oxidation of NAD(P)H to NAD(P)+. In some embodiments, the polypeptide has the following properties relative to a reference polypeptide (e.g., a reference polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4): a) increased activity' in converting 2-oxoglutarate to 2-hydroxyglutarate, and b) increased stability'. In some embodiments, the polypeptide has the following properties relative to a reference polypeptide (e.g., a reference polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4): a) increased activity in converting 2-oxoglutarate to 2-hydroxyglutarate, b) increased activity in catalyzing the oxidation of NAD(P)H to NAD(P)+; and c) increased stability. In some embodiments, the reference polypeptide comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the reference polypeptide comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the reference polypeptide comprises the ammo acid sequence of SEQ ID NO: 4.26079

[0091] In some embodiments, the polypeptide has the following properties relative to a reference polypeptide that is another engineered polypeptide of the disclosure: a) increased activity in converting 2-oxoglutarate to 2-hydroxyglutarate; b) increased activity7in catalyzing the oxidation of NAD(P)H to NAD(P)+; c) increased selectivity for NADH over NADPH; and / or d) increased stability. In some embodiments, the reference polypeptide comprises the amino acid sequence of SEQ ID NO: any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 20, 22, 24, 26, 28, or 30.

[0092] In some embodiments, the LDH polypeptides of the disclosure may demonstrate improvements in the rate of enzymatic activity, i.e., the rate of converting the substrate to the product. In some embodiments, the LDH polypeptides are capable of converting the substrate to the product at a rate that is at least about 1.1-fold, about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30- fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 150-fold, about 200-fold, about 300-fold, about 400-fold, about 500-fold, about 600-fold, about 700-fold, about 800-fold, about 900-fold, about 1000- fold, or more than about 1000-fold the rate exhibited by the polypeptides of SEQ ID NO: 2 or SEQ ID NO: 4.

[0093] In some embodiments, the LDH polypeptides of the disclosure may demonstrate increased activity in converting 2-oxoglutarate to 2-hydroxyglutarate, wherein the polypeptide has at least about 1. 1-fold, about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 150- fold, about 200-fold, about 300-fold, about 400-fold, about 500-fold, about 600-fold, about 700- fold, about 800-fold, about 900-fold, about 1000- fold, or more than about 1000-fold increased activity relative to a reference polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the LDH polypeptides of the disclosure may demonstrate increased activity7in converting 2-oxoglutarate to 2-hydroxyglutarate, wherein the polypeptide has at least 1. 1-fold, about 5-fold, about 10-fold, about 100-fold, about 500-fold, about 1000-fold, about 2000-fold, about 3000-fold, about 4000-fold, or about 5000-fold increased activity relative to a reference polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

[0094] In some embodiments, the LDH polypeptides of the disclosure may demonstrate increased activity in catalyzing the oxidation of NAD(P)H to NAD(P)+, wherein the polypeptide has at least about 1.1 -fold, about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 150-26079 fold, about 200-fold, about 300-fold, about 400-fold, about 500-fold, about 600-fold, about 700- fold, about 800-fold, about 900-fold, about 1000- fold, or more than about 1000-fold increased activity relative to a reference polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the LDH polypeptides of the disclosure may demonstrate increased activity in catalyzing the oxidation of NAD(P)H to NAD(P)+. wherein the polypeptide has at least I . I -fold, about 5-fold, about 10-fold, about 100-fold, about 500-fold, about 1000-fold, about 2000-fold, about 3000-fold, about 4000-fold, or about 5000-fold increased activity' relative to a reference polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

[0095] In some embodiments, the LDH polypeptides of the disclosure may demonstrate increased stability, wherein the polypeptide has at least about 1.1-fold, about 1.1-fold, about 1.5- fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 150-fold, about 200-fold, about 300-fold, about 400-fold, about 500-fold, about 600-fold, about 700-fold, about 800-fold, about 900-fold, about 1000- fold, or more than about 1000-fold increase in stability' relative to a reference polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the LDH polypeptides of the disclosure may demonstrate increased stability, wherein the polypeptide has at least 1.1 -fold, about 5-fold, about 10-fold, about 100-fold, or about 500-fold increase in stability' relative to a reference polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

[0096] In some embodiments, a polypeptide (e.g., LDH polypeptide) of the disclosure is a polypeptide that comprises an amino acid sequence having at least 80% but less than 1 0% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% but less than 100%) sequence identity to SEQ ID NO: 2. In some embodiments, a polypeptide (e.g., LDH polypeptide) of the disclosure is a polypeptide that comprises an amino acid sequence having at least 80% but less than 100% (e.g.. 80%. 81%. 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% but less than 100%) sequence identity to SEQ ID NO: 4.

[0097] The difference(s) between the variant(s) and SEQ ID NO: 2 or SEQ ID NO: 4 can be amino acid insertions, deletions, substitutions, or any combinations of such changes. In some embodiments, the amino acid sequence difference(s) are substitutions. In some embodiments, the amino acid sequence difference(s) can comprise non-conservative, conservative, or a combination of non-conservative and conservative amino acid substitutions. In some26079 embodiments, the ammo acid sequence difference(s) is / are conservative ammo acid substitution(s). In other embodiments, the amino acid sequence difference(s) is / are nonconservative amino acid substitution(s). In some embodiments, the amino acid sequence differences are a combination of non-conservative and conservative amino acid substitutions.

[0098] In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% but less than 100% sequence identity to SEQ ID NO: 2. In other words, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity' to SEQ ID NO: 2, wherein the polypeptide does not comprise SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., an LDH poly peptide) comprising an amino acid sequence having at least 80% but less than 100% sequence identity to SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 85% but less than 100% sequence identity to SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 90% but less than 100% sequence identity’ to SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 91% but less than 100% sequence identity to SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 92% but less than 100% sequence identity to SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 93% but less than 100% sequence identity to SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 94% but less than 100% sequence identity to SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 95% but less than 100% sequence identity to SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 96% but less than 100% sequence identity' to SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 97% but less than 100% sequence identity to SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 98% but less than 100% sequence identity to SEQ ID26079NO: 2. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 99% but less than 100% sequence identity to SEQ ID NO: 2.

[0099] In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2, wherein the polypeptide comprises at least one amino acid substitution at one or more positions selected from 12, 88, 98, 99, 1 6, 199, 205, 229, and 308, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2.

[0100] In some embodiments, the polypeptide comprises 2, 3, 4, 5, 6, 7, 8, or 9 substitutions at two or more amino acid positions selected from 12, 88, 98, 99, 166, 199, 205, 229, and 308, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2.

[0101] In some embodiments, the polypeptide comprises: a) an amino acid substitution at amino acid position 88; b) an amino acid substitution set at amino acid positions 88, 98, 99, and 229; c) an amino acid substitution set at amino acid positions 88, 229, and 308; d) an amino acid substitution set at amino acid positions 88 and 308; e) an amino acid substitution set at amino acid positions 88, 98, and 308; or f) an amino acid substitution set at amino acid positions 12, 88, 98, 166, 199, 205, 229, and 308, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the polypeptide comprises an amino acid substitution at amino acid position 88, w herein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the polypeptide comprises an ammo acid substitution set at amino acid positions 88, 98, 99, and 229, w herein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the polypeptide comprises an amino acid substitution set at amino acid positions 88, 229, and 308, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the polypeptide comprises an amino acid substitution set at amino acid positions 88 and 308, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the polypeptide comprises an amino acid substitution set at amino acid positions 88, 98, and 308, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the polypeptide comprises an amino acid substitution set at amino acid positions 12, 88, 98, 166, 199, 205, 229, and 308, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2.26079

[0102] In some embodiments, the polypeptide comprises one or more amino acid substitutions set forth in Table 1.

[0103] In some embodiments, the polypeptide comprises one of the following amino acid substitutions or amino acid substitution sets: a) Q88L; b) Q88L, V98T, G99D, and I229K; c) Q88L, I229R, and E308V; d) Q88L and E308I; e) Q88L, I229K, and E308V; f) Q88L. V98I, and E308V; or g) L12V, Q88L, V98I, L166M, L199C, D205L, I229R, and E308V, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the polypeptide comprises a Q88L substitution, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the polypeptide comprises Q88L, V98T, G99D, and I229K substitutions, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the polypeptide comprises Q88L, I229R, and E308V substitutions, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the polypeptide comprises Q88L and E308I substitutions, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the polypeptide comprises Q88L, I229K, and E308V substitutions, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the polypeptide comprises Q88L. V98I, and E308V substitutions, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the polypeptide comprises L12V, Q88L, V98I, L166M, L199C, D205L, I229R, and E308V substitutions, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2.

[0104] In some embodiments, the ammo acid sequence comprises any one of SEQ ID NO: 20, 22, 24, 26, 28, 30, or 32. In some embodiments, the amino acid sequence consists of any one of SEQ ID NO: 20, 22, 24, 26, 28, 30, or 32.

[0105] In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 20, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 20, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 20, wherein the polypeptide does not comprise the sequence of26079SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising the amino acid sequence of SEQ ID NO: 20. Also provided herein is a polypeptide (e.g., an LDH polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 20. Also provided herein is a polypeptide (e.g., an LDH polypeptide) consisting of the amino acid sequence of SEQ ID NO: 20.

[0106] In some embodiments, provided herein is a polypeptide (e g., an LDH polypeptide) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 22, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 22, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 99% sequence identity’ to SEQ ID NO: 22, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e g., an LDH polypeptide) comprising the amino acid sequence of SEQ ID NO: 22. Also provided herein is a polypeptide (e.g., an LDH polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 22. Also provided herein is a polypeptide (e.g.. an LDH polypeptide) consisting of the amino acid sequence of SEQ ID NO: 22.

[0107] In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 24, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 24, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 99% sequence identity' to SEQ ID NO: 24, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising the amino acid sequence of SEQ ID NO: 24. Also provided herein is a polypeptide (e.g., an LDH polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 24. Also provided herein is a polypeptide (e.g., an LDH polypeptide) consisting of the amino acid sequence of SEQ ID NO: 24.

[0108] In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 26 wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g.. an LDH polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 26, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 99% sequence identity’ to SEQ ID NO: 26, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising the amino acid sequence of SEQ ID NO: 26. Also provided herein is a polypeptide (e.g., an LDH polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 26. Also provided herein is a polypeptide (e.g., an LDH polypeptide) consisting of the amino acid sequence of SEQ ID NO: 26.

[0109] In some embodiments, provided herein is a polypeptide (e g., an LDH polypeptide) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity' to SEQ ID NO: 28, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 28, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 99% sequence identity’ to SEQ ID NO: 28, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising the amino acid sequence of SEQ ID NO: 28. Also provided herein is a polypeptide (e.g., an LDH polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 28. Also provided herein is a polypeptide (e.g.. an LDH polypeptide) consisting of the amino acid sequence of SEQ ID NO: 28.

[0110] In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. or higher sequence identity’ to SEQ ID NO: 30, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 98% sequence identity’ to SEQ ID NO: 30, wherein the polypeptide does not comprise thesequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 30, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e g., an LDEI polypeptide) comprising the amino acid sequence of SEQ ID NO: 30. Also provided herein is a polypeptide (e g., an LDH polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 30. Also provided herein is a polypeptide (e.g., an LDH polypeptide) consisting of the amino acid sequence of SEQ ID NO: 30.[OHl] In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 32, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 32, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 99% sequence identity' to SEQ ID NO: 32, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising the amino acid sequence of SEQ ID NO: 32. Also provided herein is a polypeptide (e.g., an LDH polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 32. Also provided herein is a polypeptide (e.g., an LDH polypeptide) consisting of the amino acid sequence of SEQ ID NO: 32.

[0112] In some embodiments, the polypeptide further comprises a tag (e.g., an epitope tag). Any suitable tag may be used, e.g., a His tag, a FLAG tag, a fluorescent protein tag (e.g., green fluorescent protein (GFP), yellow fluorescent protein (YFP), or red fluorescent protein (RFP)), a hemagglutinin (HA) tag, an ALFA-tag, a V5-tag, a Myc-tag, a SPOT-tag, a T7-tag, or an NE-tag. In some embodiments, the epitope tag is a His tag. In some embodiments, the His tag comprises the amino acid sequence of HHHHHH (SEQ ID NO: 33). In some embodiments, the polypeptide comprises an epitope tag at the C-terminus. In some embodiments, the polypeptide comprises a His tag at the C-terminus. In some embodiments, the polypeptide comprises a His tag comprising of SEQ ID NO: 33 at the C-terminus. In some embodiments, the polypeptide consisting of a His tag comprising of SEQ ID NO: 33 at the C-terminus. In some embodiments, the His tag may be attached to the polypeptide via a linker positioned between the His tag and the polypeptide and connecting the two. In some embodiments, the linker is an amino acid linker. In some26079 embodiments the amino acid linker comprises 1, 2, 3, 4, 5, or 6 amino acids. In some embodiments the amino acid linker comprises two amino acids. In some embodiments the amino acid linker comprises leucine and glutamate. In some embodiments the His tag and the linker comprise the amino acid sequence of SEQ ID NO: 34.

[0113] For example, an LDH polypeptide sequence provided in the disclosure may already comprise a tag, a tag may be added to a polypeptide sequence that does not comprise a tag, or the tag on a polypeptide sequence already comprising a tag may be removed or replaced with a different tag. For example, the polypeptide sequence of any one of even numbered SEQ ID NO: 4-18 may be modified to no longer include a C-terminal His tag having the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 34. For example, the polypeptide sequence of any one of even numbered SEQ ID NO: 20-32 may be modified to add a tag, including, but not limited to, a C- terminal His tag having the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 34.

[0114] In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% but less than 100% sequence identity to SEQ ID NO: 4. In other words, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%. 85%. 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. or 99% sequence identity' to SEQ ID NO: 4, wherein the polypeptide does not comprise SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 80% but less than 100% sequence identity' to SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 85% but less than 100% sequence identity to SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 90% but less than 100% sequence identity to SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g.. an LDH polypeptide) comprising an amino acid sequence having at least 91% but less than 100% sequence identity to SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 92% but less than 100% sequence identity to SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 93% but less than 100% sequence identity to SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 94% but less than 100% sequence identity to SEQ ID NO: 4. In some embodiments,26079 provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 95% but less than 100% sequence identity to SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 96% but less than 100% sequence identity to SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 97% but less than 100% sequence identity to SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 98% but less than 100% sequence identity to SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 99% but less than 100% sequence identity to SEQ ID NO: 4.

[0115] In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. or 99% sequence identity to SEQ ID NO: 4, wherein the polypeptide comprises at least one amino acid substitution at one or more amino acid positions selected from 12, 88, 98, 99, 166, 199, 205, 229, and 308, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 4.

[0116] In some embodiments, the polypeptide comprises 2, 3. 4, 5, 6. 7, 8, or 9 amino acid substitutions at two or more amino acid positions selected from 12, 88, 98, 99, 166, 199, 205, 229, and 308, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 4.

[0117] In some embodiments, the polypeptide comprises: a) an amino acid substitution at amino acid position 88; b) an amino acid substitution set at amino acid positions 88, 98, 99, and 229; c) an amino acid substitution set at amino acid positions 88, 229, and 308; d) an amino acid substitution set at amino acid positions 88 and 308; e) an amino acid substitution set at amino acid positions 88, 98, and 308; or f) an amino acid substitution set at amino acid positions 12, 88, 98. 166, 199. 205, 229, and 308. wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 4. In some embodiments, the polypeptide comprises an amino acid substitution at amino acid position 88, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 4. In some embodiments, the polypeptide comprises an amino acid substitution set at amino acid positions 88, 98, 99, and 229, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 4. In some embodiments, the polypeptide comprises an amino acid substitution set at amino acid positions 88, 229, and 308, wherein the amino acid positions of the polypeptide are numbered26079 with reference to SEQ ID NO: 4. In some embodiments, the polypeptide comprises an amino acid substitution set at amino acid positions 88 and 308, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 4. In some embodiments, the polypeptide comprises an amino acid substitution set at amino acid positions 88, 98, and 308, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 4. In some embodiments, the polypeptide comprises an amino acid substitution set at amino acid positions 12, 88, 98, 166, 199, 205, 229, and 308, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 4.

[0118] In some embodiments, the polypeptide comprises one of the following amino acid substitutions or amino acid substitution sets: a) Q88L; b) Q88L, V98T, G99D, and I229K; c) Q88L, I229R, and E308V; d) Q88L and E308I; e) Q88L, I229K, and E308V; f) Q88L, V98I, and E308V; or g) L12V, Q88L, V98I, L166M, L199C, D205L, I229R, and E308V, wherein the amino acid positions of the polypeptide are numbered w ith reference to SEQ ID NO: 4. In some embodiments, the polypeptide comprises a Q88L substitution, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 4. In some embodiments, the polypeptide comprises Q88L, V98T, G99D, and I229K substitutions, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 4. In some embodiments, the polypeptide comprises Q88L. I229R. and E308V substitutions, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 4. In some embodiments, the polypeptide comprises Q88L and E308I substitutions, w erein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 4. In some embodiments, the polypeptide comprises Q88L. I229K, and E308V substitutions, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 4. In some embodiments, the polypeptide comprises Q88L, V98I, and E308V substitutions, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 4. In some embodiments, the polypeptide comprises L12V, Q88L, V98I, L166M, L199C, D205L, I229R, and E308V substitutions, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 4.

[0119] In some embodiments, the amino acid sequence comprises any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, or 18. In some embodiments, the amino acid sequence consists of any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, or 18.

[0120] In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 6, wherein the polypeptide does not26079 comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 6, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 99% sequence identity’ to SEQ ID NO: 6, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising the amino acid sequence of SEQ ID NO: 6. Also provided herein is a polypeptide (e.g., an LDH polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 6. Also provided herein is a polypeptide (e.g., an LDH polypeptide) consisting of the amino acid sequence of SEQ ID NO: 6.

[0121] In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 8, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 8, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 99% sequence identity' to SEQ ID NO: 8, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising the amino acid sequence of SEQ ID NO: 8. Also provided herein is a polypeptide (e.g., an LDH polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 8. Also provided herein is a polypeptide (e.g., an LDH polypeptide) consisting of the amino acid sequence of SEQ ID NO: 8.

[0122] In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 10, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 10, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 99% sequence identity' to SEQ ID NO: 10, wherein the polypeptide does not comprise the sequence of26079SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising the amino acid sequence of SEQ ID NO: 10. Also provided herein is a polypeptide (e.g., an LDH polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 10. Also provided herein is a polypeptide (e.g., an LDH polypeptide) consisting of the amino acid sequence of SEQ ID NO: 10.

[0123] In some embodiments, provided herein is a polypeptide (e g., an LDH polypeptide) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 12, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 12, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 99% sequence identity’ to SEQ ID NO: 12, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e g., an LDH polypeptide) comprising the amino acid sequence of SEQ ID NO: 12. Also provided herein is a polypeptide (e.g., an LDH polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 12. Also provided herein is a polypeptide (e.g.. an LDH polypeptide) consisting of the amino acid sequence of SEQ ID NO: 12.

[0124] In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 14, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 14, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 99% sequence identity' to SEQ ID NO: 14, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising the amino acid sequence of SEQ ID NO: 14. Also provided herein is a polypeptide (e.g., an LDH polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 14. Also provided herein is a polypeptide (e.g., an LDH polypeptide) consisting of the amino acid sequence of SEQ ID NO: 14.26079

[0125] In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 16, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g.. an LDH polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 16, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 99% sequence identity’ to SEQ ID NO: 16, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising the amino acid sequence of SEQ ID NO: 16. Also provided herein is a polypeptide (e.g., an LDH polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 16. Also provided herein is a polypeptide (e.g., an LDH polypeptide) consisting of the amino acid sequence of SEQ ID NO: 16.

[0126] In some embodiments, provided herein is a polypeptide (e g., an LDH polypeptide) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity' to SEQ ID NO: 18, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 18, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising an amino acid sequence having at least 99% sequence identity’ to SEQ ID NO: 18, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., an LDH polypeptide) comprising the amino acid sequence of SEQ ID NO: 18. Also provided herein is a polypeptide (e.g., an LDH polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 18. Also provided herein is a polypeptide (e.g.. an LDH polypeptide) consisting of the amino acid sequence of SEQ ID NO: 18.

[0127] In some embodiments, provided herein is a polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%. 94%, 95%. 96%. 97%. 98%. 99%. or 100%. sequence identity to any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%,2607994%, 95%, 96%, 97%, 98%, or 99%, sequence identity to any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NO: 6, 8. 10. 12. 14. 16, 18, 20, 22, 24, 26, 28, 30, or 32, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide comprising an amino acid sequence having at least 91% sequence identity to any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide comprising an amino acid sequence having at least 92% sequence identity to any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32, wherein the poly peptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide comprising an amino acid sequence having at least 93% sequence identity to any one of SEQ ID NO: 6. 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide comprising an amino acid sequence having at least 94% sequence identity to any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide comprising an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide comprising an amino acid sequence having at least 96% sequence identity’ to any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide comprising an amino acid sequence having at least 97% sequence identity to any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22. 24. 26. 28, 30, or 32, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide comprising an amino acid sequence having at least 98% sequence identity to any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide comprising an amino acid sequence having at least 99% sequence identity to any one of SEQ ID NO: 6, 8,2607910, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

[0128] In some embodiments, a polypeptide (e.g., an LDH polypeptide) of the disclosure comprises any of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32. In some embodiments, a polypeptide (e.g., LDH polypeptide) of the disclosure consists of any of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32.

[0129] In some embodiments, the polypeptide is isolated.

[0130] In some embodiments, the polypeptide is a lactate dehydrogenase.

[0131] In some embodiments of any of the preceding, the polypeptide is capable of catalyzing the NAD(P)H-dependent reduction of pyruvate to L- or D-lactate and / or the NAD(P)H- dependent reduction of a-ketoglutarate to 2-hydroxy glutarate. In some embodiments of any of the preceding, the polypeptide is capable of catalyzing the NAD(P)H-dependent reduction of pyruvate to L- or D-lactate. In some embodiments of any of the preceding, the polypeptide is capable of catalyzing the NAD(P)H-dependent reduction of a-ketoglutarate to 2- hydroxy glutarate. In some embodiments of any of the preceding, the polypeptide is capable of catalyzing the NAD(P)H-dependent reduction of pyruvate to L- or D-lactate and the NAD(P)H- dependent reduction of a-ketoglutarate to 2-hydroxy glutarate.

[0132] Also provided herein are functional fragments of any of the polypeptides disclosed herein. Also provided herein are analogues of any of the polypeptides disclosed herein.

[0133] In addition to the positions of residue differences specified above, any of the engineered LDH polypeptides disclosed herein can further comprise other residue differences relative to SEQ ID NO: 2 or SEQ ID NO: 4 at other residue positions than those of amino acid differences disclosed above and in Table 1, e.g., residue positions other than 12, 88, 98, 99, 166, 199, 205, 229, and 308. Residue differences at these other residue positions can provide for additional variations in the amino acid sequence without adversely affecting the ability of the polypeptide to catalyze the desired reaction. Accordingly, in some embodiments, in addition to the amino acid residue differences present in any one of the engineered LDH polypeptides selected from SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32, the sequence can further comprise 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, or 1-30, 1-35, 1-40, 1-45, or 1-50 residue differences at other amino acid residue positions as compared to the SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the number of amino acid residue differences as compared to the reference sequence can be at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45 or 50 residue positions. In some embodiments, the number of amino acid residue differences as compared to the26079 reference sequence can be at 1, 2, 3. 4, 5, 6, 7. 8, 9, 10, 11, 12, 13, 14, 15, 16, 18. 20. 21. 22. 23. 24, or 25 residue positions. The residue difference at these other positions can be conservative changes or non-conservative changes. In some embodiments, the residue differences can comprise conservative substitutions and non-conservative substitutions as compared to SEQ ID NO: 2 or SEQ ID NO: 4.

[0134] In some embodiments, the engineered LDH polypeptide can comprise a deletion at one or more amino acid positions as compared to any one of the engineered LDH polypeptides described herein, such as the exemplary engineered polypeptides of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32. Thus, for each embodiment of the engineered LDH polypeptides of the disclosure, the amino acid sequence can comprise deletions of one or more amino acids, 2 or more amino acids, 3 or more amino acids, 4 or more amino acids, 5 or more amino acids, 6 or more amino acids, 8 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, up to 15% of the total number of amino acids, up to 20% of the total number of amino acids, or up to 30% of the total number of amino acids of the LDH polypeptides, where the associated functional activity and / or improved properties of the engineered LDH polypeptides described herein is maintained. In some embodiments, the deletions can comprise 1-2, 1-3. 1-4, 1-5, 1-6, 1-7, 1-8, 1- 9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, or 1-50 amino acid residues. In some embodiments, the number of deletions can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50 ammo acid residues. In some embodiments, the deletions can comprise deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 amino acid residues.

[0135] In some embodiments, the engineered LDH polypeptide herein can have an amino acid sequence comprising an insertion as compared to any one of the engineered LDH polypeptides described herein, such as the exemplary engineered polypeptides of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32. The insertions can comprise one or more amino acids, 2 or more amino acids, 3 or more amino acids, 4 or more amino acids, 5 or more amino acids, 6 or more amino acids, 8 or more amino acids, 10 or more amino acids, 15 or more amino acids, 20 or more amino acids, 30 or more amino acids, 40 or more amino acids, or 50 or more amino acids, where the associated functional activity and / or improved properties of the engineered LDH polypeptide described herein is maintained. The insertions can be to amino or carboxy terminus, or internal portions of the LDH polypeptide.

[0136] In some embodiments, the engineered LDH polypeptide herein can have an amino acid sequence comprising a sequence selected from SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,2607926, 28, 30, or 32, and optionally one or several (e.g., up to 3, 4, 5. or up to 10) amino acid residue deletions, insertions and / or substitutions. In some embodiments, the amino acid sequence has optionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1- 30, 1-35, 1-40, 1-45, or 1-50 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the number of amino acid sequence has optionally 1. 2, 3, 4. 5, 6, 7, 8. 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the amino acid sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the substitutions can be conservative or non-conservative substitutions.

[0137] Also provided herein are compositions comprising any of the polypeptides disclosed herein. For example, the composition may include an effective amount of the polypeptide for catalyzing the NAD(P)H-dependent reduction of pyruvate to either L- or D-lactate. In some embodiments, the composition may include an effective amount of the polypeptide for catalyzing the NAD(P)H-dependent reduction of 2-oxoglutarate to 2-hydroxy glutarate. The composition may include one or more carriers or diluents.

[0138] In some embodiments, the polypeptides of the disclosure can be in the form of fusion polypeptides in which the engineered polypeptides are fused to other polypeptides, such as, by way of example and not limitation, antibody tags (e.g., myc epitope), purification sequences (e.g., His tags for binding to metals), and cell localization signals (e.g., secretion signals). Thus, the engineered polypeptides described herein can be used with or without fusions to other polypeptides.

[0139] It is to be understood that the polypeptides described herein are not restricted to the genetically encoded amino acids. In addition to the genetically encoded amino acids, the polypeptides described herein may be comprised, either in whole or in part, of naturally- occurring and / or synthetic non-encoded amino acids.

[0140] In some embodiments, the engineered polypeptides can be provided on a solid support, such as a membrane, resin, solid carrier, or other solid phase material. A solid support can be composed of organic polymers such as polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy, and polyacrylamide, as well as co-polymers and grafts thereof. A solid support can also be inorganic, such as glass, silica, controlled pore glass (CPG), reverse phase silica or metal, such as gold or platinum. The configuration of a solid support can be in the form of beads, spheres, particles, granules, a gel, a membrane or a surface. Surfaces can be planar, substantially planar, or non-planar. Solid supports can be porous or non-porous, and26079 can have swelling or non-swelling characteristics. A solid support can be configured in the form of a well, depression, or other container, vessel, feature, or location.

[0141] In some embodiments, the engineered LDH polypeptides of the present disclosure can be immobilized on a solid support such that they retain their improved activity, stereoselectivity, and / or other improved properties relative to the reference engineered polypeptide. In some embodiments, the immobilized polypeptides can be retained after a desired reaction (e.g., by retaining beads on which polypeptide is immobilized) and then reused or recycled in subsequent reactions. Such immobilized enzyme processes allow for further efficiency and cost reduction. Accordingly, it is further contemplated that any of the methods of using the LDH polypeptides of the present disclosure can be carried out using the same LDH polypeptides bound or immobilized on a solid support.

[0142] In some embodiments, the polypeptides described herein can be provided in the form of kits. The enzymes in the kits may be present individually or as a plurality of enzy mes. The kits can further include reagents for carrying out the enzymatic reactions, substrates for assessing the activity of enzy mes, as well as reagents for detecting the products. The kits can also include reagent dispensers and instructions for use of the kits.

[0143] In some embodiments, the kits of the present disclosure include arrays comprising a plurality of different LDH polypeptides at different addressable position, wherein the different polypeptides are different variants of a reference sequence each having at least one different improved enzyme property. In some embodiments, a plurality7of polypeptides immobilized on solid supports can be configured on an array at various locations, addressable for robotic delivery7of reagents, or by detection methods and / or instruments. The array can be used to test a variety of substrate compounds for conversion by the polypeptides.Polynucleotides Encoding LDH Polypeptides

[0144] In another aspect, the present disclosure provides polynucleotides encoding the polypeptides (e.g., LDH polypeptides) disclosed herein. The polynucleotides may be operatively linked to one or more heterologous regulatory sequences that control gene expression to create a recombinant polynucleotide capable of expressing the polypeptide. Expression vectors containing a heterologous polynucleotide encoding the polypeptide (e.g., LDH polypeptide) can be introduced into appropriate host cells to express the corresponding polypeptide.

[0145] Because of the knowledge of the codons corresponding to the various amino acids, availability7of a protein sequence provides a description of all the polynucleotides capable of encoding the subject. The degeneracy of the genetic code, where the same amino acids are26079 encoded by alternative or synonymous codons allows an extremely large number of nucleic acids to be made, all of which encode the polypeptides (e.g., LDH polypeptides) disclosed herein. Thus, having identified a particular amino acid sequence, those skilled in the art could make any number of different nucleic acids by simply modifying the sequence of one or more codons in a way that does not change the amino acid sequence of the protein. In this regard, the present disclosure specifically contemplates each and every possible variation of polynucleotides that could be made by selecting combinations based on the possible codon choices, and all such variations are to be considered specifically disclosed for any polypeptide disclosed herein.

[0146] In various embodiments, the codons are preferably selected to be suitable for the host cell in which the polypeptide is being produced. For example, preferred codons used in bacteria are used to express the gene in bacteria; preferred codons used in yeast are used for expression in yeast; and preferred codons used in mammals are used for expression in mammalian cells. By way of example, the polynucleotide of SEQ ID NO: 3 provided herein has been codon optimized for expression in E. coli.

[0147] In certain embodiments, all codons need not be replaced to optimize the codon usage of the polypeptide (e.g., LDH polypeptide) since the natural sequence will comprise preferred codons and because use of preferred codons may not be required for all amino acid residues. Consequently, codon optimized polynucleotides encoding the polypeptides (e.g.. LDH polypeptides) may contain preferred codons at about 40%, 50%, 60%, 70%, 80%, or greater than 90% of codon positions of the full-length coding region.

[0148] Provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%. 88%, 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. or 99%) sequence identity to the polynucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, or 31, wherein the polynucleotide does not comprise SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%. 97%. 98%. or 99%) sequence identity to the polynucleotide sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, or 31, wherein the polynucleotide does not comprise SEQ ID NO: 1 or SEQ ID NO: 3. In some examples, the polynucleotide sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, or 17 is modified to no longer code for a C-terminal His tag having the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 34.

[0149] In some embodiments, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of any26079 one of SEQ ID NO: 5. 7. 9, 11, 13, 15, 17. 19. 21. 23. 25. 27. 29, or 31. wherein the polynucleotide does not comprise SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, provided herein is a polynucleotide comprising at least 80% sequence identity to the polynucleotide sequence of any one of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, or 31, wherein the polynucleotide does not comprise SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, provided herein is a polynucleotide comprising at least 90% sequence identity to the polynucleotide sequence of any one of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, or 31, wherein the polynucleotide does not comprise SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, provided herein is a polynucleotide comprising at least 91% sequence identity to the polynucleotide sequence of any one of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, or 31, wherein the polynucleotide does not comprise SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, provided herein is a polynucleotide comprising at least 92% sequence identity to the polynucleotide sequence of any one of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, or 31, wherein the polynucleotide does not comprise SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, provided herein is a polynucleotide comprising at least 93% sequence identity to the polynucleotide sequence of any one of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, or 31, wherein the polynucleotide does not comprise SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, provided herein is a polynucleotide comprising at least 94% sequence identity to the polynucleotide sequence of any one of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, or 31, wherein the polynucleotide does not comprise SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, provided herein is a polynucleotide comprising at least 95% sequence identity to the polynucleotide sequence of any one of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, or 31, wherein the polynucleotide does not comprise SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, provided herein is a polynucleotide comprising at least 96% sequence identity to the polynucleotide sequence of any one of SEQ ID NO: 5, 7. 9, 11, 13, 15, 17. 19. 21, 23, 25, 27, 29, or 31. wherein the polynucleotide does not comprise SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, provided herein is a polynucleotide comprising at least 97% sequence identity to the polynucleotide sequence of any one of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 1 , 29, or 31, wherein the polynucleotide does not comprise SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, provided herein is a polynucleotide comprising at least 98% sequence identity to the polynucleotide sequence of any one of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, or 31, wherein the polynucleotide does not comprise SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, provided herein is a polynucleotide comprising at least 99% sequence26079 identity to the polynucleotide sequence of any one of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21. 23, 25, 27, 29, or 31, wherein the polynucleotide does not comprise SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, provided herein is a polynucleotide comprising any one of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17. 19, 21, 23, 25, 27, 29, or 31. In some embodiments, provided herein is a polynucleotide consisting of any one of SEQ ID NO: 5, 7. 9, 11, 13, 15, 17, 19. 21. 23. 25. 27, 29, or 31. In some embodiments, provided herein is a polynucleotide comprising any one of SEQ ID NO: 5, 7, 9, 11, 13, 15, or 17. In some embodiments, provided herein is a polynucleotide consisting any one of SEQ ID NO: 5, 7, 9, 11, 13, 15, or 17. In some embodiments, provided herein is a polynucleotide comprising any one of SEQ ID NO: 19, 21, 23, 25, 27. 29. or 31. In some embodiments, provided herein is a polynucleotide consisting any one of SEQ ID NO: 19, 21, 23, 25, 27, 29, or 31.

[0150] In some embodiments, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%. 93%, 94%. 95%, 96%, 97%, 98%, or 99%) sequence identity to the polynucleotide sequence of SEQ ID NO: 3.

[0151] In some embodiments, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%. 97%. 98%. 99%. or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 5, wherein the polynucleotide does not comprise SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 5. In some embodiments, the polynucleotide consists of SEQ ID NO: 5.

[0152] In some embodiments, provided herein is a polynucleotide comprising at least 80% (e.g.. 80%. 81%. 82%. 83%. 84%. 85%. 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 7, wherein the polynucleotide does not comprise SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 7. In some embodiments, the polynucleotide consists of SEQ ID NO: 7.

[0153] In some embodiments, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 9, wherein the polynucleotide does not comprise SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 9. In some embodiments, the polynucleotide consists of SEQ ID NO: 9.26079

[0154] In some embodiments, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 11. wherein the polynucleotide does not comprise SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 11. In some embodiments, the polynucleotide consists of SEQ ID NO: 11.

[0155] In some embodiments, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%. 99%. or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 13, wherein the polynucleotide does not comprise SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 13. In some embodiments, the polynucleotide consists of SEQ ID NO: 13.

[0156] In some embodiments, provided herein is a polynucleotide comprising at least 80% (e.g.. 80%. 81%. 82%. 83%. 84%. 85%. 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 15, wherein the polynucleotide does not comprise SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 15. In some embodiments, the polynucleotide consists of SEQ ID NO: 15.

[0157] In some embodiments, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 17. wherein the polynucleotide does not comprise SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 17. In some embodiments, the polynucleotide consists of SEQ ID NO: 17.

[0158] In some embodiments, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%. 97%. 98%. 99%. or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 19, wherein the polynucleotide does not comprise SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 19. In some embodiments, the polynucleotide consists of SEQ ID NO: 19.

[0159] In some embodiments, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 21, wherein the poly nucleotide does not comprise SEQ ID NO: 1 or SEQ ID NO: 3. In26079 some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 21. In some embodiments, the polynucleotide consists of SEQ ID NO: 21.

[0160] In some embodiments, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%. 97%. 98%. 99%. or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 23, wherein the polynucleotide does not comprise SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 23. In some embodiments, the polynucleotide consists of SEQ ID NO: 23.

[0161] In some embodiments, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 25, wherein the poly nucleotide does not comprise SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 25. In some embodiments, the polynucleotide consists of SEQ ID NO: 25.

[0162] In some embodiments, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 27. wherein the polynucleotide does not comprise SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 27. In some embodiments, the polynucleotide consists of SEQ ID NO: 27.

[0163] In some embodiments, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%. 92%. 93%. 94%. 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 29, wherein the polynucleotide does not comprise SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 29. In some embodiments, the polynucleotide consists of SEQ ID NO: 29.

[0164] In some embodiments, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 31, wherein the polynucleotide does not comprise SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 31. In some embodiments, the polynucleotide consists of SEQ ID NO: 31.

[0165] In various embodiments, an isolated polynucleotide encoding polypeptide (e.g., an LDH polypeptide) may be manipulated in a variety7of ways to provide for expression of the26079 polypeptide. Manipulation of the isolated polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. Techniques for modifying polynucleotides and nucleic acid sequences utilizing recombinant DNA methods are well known in the art. For example, guidance is provided in Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3rdEd., Cold Spring Harbor Laboratory Press; and Current Protocols in Molecular Biology, Ausubel. F. ed., Greene Pub. Associates, 1998, updates to 2006.

[0166] In some embodiments, an isolated polynucleotide encoding any of the polypeptides herein is manipulated in a variety of ways to facilitate expression of the polypeptide. In some embodiments, the polynucleotides encoding the polypeptides comprise expression vectors where one or more control sequences is present to regulate the expression of the polynucleotides and / or polypeptides. Manipulation of the isolated polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector utilized. Techniques for modify ing polynucleotides and nucleic acid sequences utilizing recombinant DNA methods are well known in the art.

[0167] In some embodiments, the control sequences include among others, promoters, leader sequences, polyadenylation sequences, propeptide sequences, signal peptide sequences, and transcription terminators. In some embodiments, the control sequence is a promoter. In some embodiments, the promoter is a heterologous promoter.

[0168] In some embodiments, the control sequence is a suitable promoter sequence. In some embodiments, suitable promoters are selected based on the host cell selection. For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the present disclosure, include, but are not limited to, promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokary otic beta-lactamase gene (See e.g., Villa-Kamaroff et al., Proc. Natl Acad. Sci. USA 75: 3727-3731, 1978), as well as the tac promoter (See e.g., DeBoer et al.. Proc. Natl Acad. Sci. USA 80: 21-25, 1983). Exemplary promoters for filamentous fungal host cells, include, but are not limited to, promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alphaamylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (See e.g., WO2607996 / 00787), as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase), and mutant, truncated, and hybrid promoters thereof. Exemplary yeast cell promoters can be from the genes can be from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GALI), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP), and Saccharomyces cerevisiae 3-phosphogly cerate kinase. Other useful promoters for yeast host cells are know n in the art {see e.g., Romanos et al., Yeast 8:423-488, 1992).

[0169] In some embodiments, the control sequence is a suitable transcription terminator sequence (i.e., a sequence recognized by a host cell to terminate transcription). In some embodiments, the terminator sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the enzy me polypeptide. Any suitable terminator that is functional in the host cell of choice finds use in the present disclosure. Exemplary’ transcription terminators for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus idulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum try psin-like protease. Exemplary’ terminators for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are known in the art {See e.g., Romanos et al., supra).

[0170] In some embodiments, the control sequence is a suitable leader sequence (i.e., a nontranslated region of an mRNA that is important for translation by the host cell). In some embodiments, the leader sequence is operably linked to the 5' terminus of the polynucleotide sequence encoding the polypeptide. Any suitable leader sequence that is functional in the host cell of choice find use in the present disclosure. Exemplary’ leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, and Aspergillus nidulans triose phosphate isomerase. Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3- phosphogly cerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0171] In some embodiments, the control sequence is a polyadenylation sequence (i.e.. a sequence operably linked to the 3' terminus of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA). Any suitable polyadenylation sequence that is functional in the host cell of choice may26079 be used in the present disclosure. Exemplar}' polyadenylation sequences for filamentous fungal host cells include, but are not limited to, the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase. Useful poly adenylation sequences for yeast host cells are known (See e.g., Guo and Sherman, Mol. Cell. Biol., 15:5983-5990, 1995).

[0172] In some embodiments, the control sequence is a signal peptide (i.e., a coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell’s secretory pathway). In some embodiments, the 5' end of the coding sequence of the nucleic acid sequence inherently contains a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide. Alternatively, in some embodiments, the 5' end of the coding sequence contains a signal peptide coding region that is foreign to the coding sequence. Any suitable signal peptide coding region that directs the expressed polypeptide into the secretory pathway of a host cell of choice finds use for expression of the engineered polypeptide(s). Effective signal peptide coding regions for bacterial host cells are the signal peptide coding regions include, but are not limited to, those obtained from the genes for Bacillus NC1B 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis betalactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are known in the art (See e.g., Simonen and Palva, Microbiol. Rev., 57: 109-137, 1993). In some embodiments, effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to. the signal peptide coding regions obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase. Useful signal peptides for yeast host cells include, but are not limited to, those from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase.

[0173] In some embodiments, regulator}’ sequences are also utilized. These sequences facilitate the regulation of the expression of the polypeptide relative to the grow th of the host cell.Examples of regulator}' systems are those that cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. In prokaryotic host cells, suitable regulatory sequences include, but are not limited to, the lac, tac, and trp operator systems. In yeast host cells, suitable regulatory systems include, but are not limited to, the ADH2 system or GALI system. In filamentous fungi, suitable regulator}'26079 sequences include, but are not limited to, the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter.

[0174] In another aspect, the present disclosure provides a recombinant expression vector comprising a polynucleotide encoding a polypeptide (e.g., an LDH polypeptide), and one or more expression regulating regions such as a promoter, a terminator, a replication origin, a leader sequence, a signal peptide, or a regulatory sequence, depending on the type of host into which it is to be introduced. In some embodiments, one or more nucleic acid and control sequences as described herein are joined together to produce recombinant expression vectors that include one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the enzyme polypeptide at such sites. Alternatively, in some embodiments, a nucleic acid sequence of the present disclosure is expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In some embodiments involving the creation of the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

[0175] The recombinant expression vector may be any suitable vector (e.g., a plasmid or virus), that can be subjected to recombinant DNA procedures and bring about the expression of the enzyme polynucleotide sequence. The choice of the vector typically depends on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

[0176] In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, such as a plasmid, an extra-chromosomal element, a minichromosome, or an artificial chromosome). The vector may contain any means for assuring self-replication. In some embodiments, the vector, when introduced into the host cell, is integrated into the genome of the host cell and replicates together with the chromosome(s) into which it has been integrated. Furthermore, in some embodiments, a single vector or plasmid, or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, and / or a transposon is utilized.

[0177] In some embodiments, the expression vector contains one or more selectable markers, which permit easy selection of transformed cells. Examples of bacterial selectable markers include, but are not limited to, the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers, which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to,26079ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase; e.g., from . nidulans or A. orzyae), argB (ornithine carbamoyltransferases), bar (phosphinothricin acetyltransferase; e.g., from S. hygroscopicus), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase; e.g.. from A. nidulans or A. orzyae), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof.

[0178] In another aspect, the present disclosure provides a host cell comprising at least one polynucleotide encoding at least one polypeptide of the present disclosure, the polynucleotide(s) being operatively linked to one or more control sequences for expression of the at least one polypeptide in the host cell. In some embodiments, the at least one polynucleotide is part of an expression vector. Host cells suitable for use in expressing the polypeptides encoded by the polynucleotide(s) or expression vector(s) of the present disclosure are well known in the art and described herein.

[0179] In another aspect, the present disclosure provides a method of producing a polypeptide, the method comprising culturing a host cell comprising at least one polynucleotide encoding at least one polypeptide of the present disclosure under conditions such that the polypeptide encoded by the polynucleotide is produced.

[0180] In some embodiments, the expression vectors of the present disclosure contain one or more element(s) that permits integration of the vector into the host cell’s genome or autonomous replication of the vector in the cell independent of the genome. In some embodiments involving integration into the host cell genome, the vectors rely on the nucleic acid sequence encoding the polypeptide or any other element of the vector for integration of the vector into the genome by homologous or nonhomologous recombination.

[0181] In some embodiments, the expression vectors contain additional nucleic acid sequences for directing integration by homologous recombination into the genome of the host cell. The additional nucleic acid sequences enable the vector to be integrated into the host cell genome at a precise location(s) in the chromosome(s) of the host cell. To increase the likelihood of integration at a precise location, the integrational elements preferably contain a sufficient number of nucleotides, such as 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000 base pairs, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may include any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding nucleic acid26079 sequences. In other examples, the vector may be integrated into the genome of the host cell by non-homologous recombination.

[0182] For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. Examples of bacterial origins of replication are P15A ori or the origins of replication of plasmids pBR322, pET30a(+). pUC19, pACYC177 (which contains the P15A ori), or pACYC184 (which contains the P15A ori) permitting replication in E. coli, and pUBl 10, pE194, or pTA1060 permitting replication in Bacillus. Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication. ARS1, ARS4, the combination of ARS 1 and CEN3, and the combination of ARS4 and CEN6. The origin of replication may be one having a mutation which makes its functioning temperature-sensitive in the host cell (See e.g., Ehrlich, Proc. Natl. Acad. Sci. USA 75:1433, 1978).

[0183] In some embodiments, more than one copy of a polynucleotide sequence of the present disclosure is inserted into the host cell to increase production of the gene product. An increase in the copy number of the nucleic acid sequence can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the nucleic acid sequence where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the nucleic acid sequence, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

[0184] Many of the expression vectors for use in the present disclosure are commercially available. Suitable commercial expression vectors include, but are not limited to, NOVAGEN® pET E. coli T7 expression vectors, such as pET30a(+) vector (Millipore Sigma) and the p3xFLAG® expression vectors (Sigma-Aldrich Chemicals). Other suitable expression vectors include, but are not limited to, pBluescriptll SK(-) and pBK-CMV (Stratagene), and plasmids derived from pBR322 (Gibco BRL), pUC (Gibco BRL), pREP4, pCEP4 (Invitrogen) or pPoly (See e.g., Lathe et al., Gene 57: 193-201, 1987).

[0185] Thus, in some embodiments, a vector comprising a sequence encoding at least one variant polypeptide (e.g., an LDH polypeptide) is transformed into a host cell in order to allow propagation of the vector and expression of the variant polypeptide(s). In some embodiments, the transformed host cell described above is cultured in a suitable nutrient medium under conditions permitting the expression of the variant polypeptides(s). Any suitable medium useful for culturing the host cells finds use in the present disclosure, including, but not limited to minimal or complex media containing appropriate supplements. In some embodiments, host cells are grow n in HTP media. Suitable media are available from various commercial suppliers or may be26079 prepared according to published recipes (e.g., in catalogues of the American Type Culture Collection).Host Cells for Expression of LDH Polypeptides

[0186] The present disclosure also provides a host cell comprising a polynucleotide encoding a polypeptide (e.g., an LDH polypeptide) disclosed herein, or an expression vector comprising a polynucleotide encoding a polypeptide (e.g., an LDH polypeptide) disclosed herein. In some embodiments, the polynucleotide is operatively linked to one or more control sequences for expression of the polypeptide in the host cell. In some embodiments, the control sequence is a promoter. In some embodiments, the promoter is a heterologous promoter.

[0187] Host cells for use in expressing the polypeptides described herein are well known in the art and include, but are not limited to, prokaryotic cells (e.g., bacterial cells (e.g., E. coli, B. subtilis, B. licheniformis , B. megaterium, B. stearothermophilus , B. amyloliquefaciens .Lactobacillus ke ir. Lactobacillus brevis, Lactobacillus minor, Streptomyces and Salmonella typhimurium cells)) or eukaryotic cells (e.g., fungal cells (e.g., yeast cells (e.g., Saccharomyces cerevisiae or Pichia pas lor is) or mammalian cells). Appropriate culture mediums and growth conditions for the above-described host cells are well known in the art.

[0188] Polynucleotides for expression of the polypeptides (e.g.. LDH polypeptides) may be introduced into cells by various methods known in the art. Techniques include among others, electroporation, biolistic particle bombardment, liposome mediated transfection, calcium chloride transfection, and protoplast fusion. Various methods for introducing polynucleotides into cells will be apparent to the skilled artisan.

[0189] In some embodiments, the host cell is a filamentous fungal host cell. For example, in some embodiments, the filamentous fungal host cells are of any suitable genus and species, including, but not limited to Achlya, Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Cephalosporium, Chrysosporium, Cochliobolus, Corynascus, Cryphonectria, Cryptococcus, Coprinus, Coriolus, Diplodia, Endothis, Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea, Myceliophthora, Mucor, Neurospora, Penicillium, Podospora, Phlebia, Piromyces, Pyricularia, Rhizomucor, Rhizopus, Schizophyllum, Scytalidium, Sporotrichum, Talaromyces, Thermoascus, Thielavia, Trametes, Tolypocladium, Trichoderma, Verticillium, and / or Volvariella. and / or teleomorphs, or anamorphs, and synonyms, basionyms, or taxonomic equivalents thereof.

[0190] In some embodiments, the host cell is a yeast cell, including but not limited to cells of Candida, Hansenula, Saccharomyces , Schizosaccharomyces, Pichia, Kluyveromyces, or26079Yarrowia species. In some embodiments of the present disclosure, the yeast cell is Hansenula polymorpha, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces diastaticus , Saccharomyces norbensis, Saccharomyces kluyveri, Schizosaccharomyces pombe. Pichia pastoris, Pichia fmlandica, Pichia trehalophila, Pichia kodamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia quercuum. Pichia pijperi, Pichia stipitis, Pichia methanolica. Pichia angusta, Kluyveromyces lactis, Candida albicans, or Yarrowia lipolytica.

[0191] In some embodiments, the host cell is a prokaryotic cell. Suitable prokaryotic cells include, but are not limited to, Gram-positive. Gram-negative and Gram-variable bacterial cells. Any suitable bacterial organism finds use in the present disclosure, including but not limited to Agrobacterium, Alicyclobacillus , Anabaena, Anacystis, Acinetobacter, Acidothermus, Arthrobacter, Azobacter, Bacillus, Bifidobacterium, Brevibacterium, Butyrivibrio, Buchnera, Campestris, Camplyobacter, Clostridium, Corynebacterium, Chromatium, Coprococcus, Escherichia, Enterococcus, Enterobacter , Erwinia, Fusobacterium, Faecalibacterium, Francisella, Flavobacterium. Geobacillus, Haemophilus, Helicobacter, Klebsiella, Lactobacillus, Lactococcus, Ilyobacter, Micrococcus, Microbacterium, Mesorhizobium, Methylobacterium, Methylobacterium, Mycobacterium, Neisseria, Pantoea, Pseudomonas, Prochlorococcus, Rhodobacter, Rhodopseudomonas, Rhodopseudomonas, Roseburia, Rhodospirillum. Rhodococcus, Scenedesmus, Streptomyces, Streptococcus, Synecoccus, Saccharomonospora, Staphylococcus, Serratia, Salmonella, Shigella, Thermoanaerobacterium, Tropheryma, Tularensis, Temecula, Thermosynechococcus, Thermococcus, Ureaplasma, Xanthomonas, Xylella, Yersinia and Zymomonas. In some embodiments, the host cell is a species of Agrobacterium, Acinetobacter, Azobacter, Bacillus, Bifidobacterium, Buchnera. Geobacillus, Campylobacter, Clostridium, Corynebacterium, Escherichia, Enterococcus, Erwinia, Flavobacterium, Lactobacillus, Lactococcus, Pantoea, Pseudomonas, Staphylococcus, Salmonella, Streptococcus, Streptomyces, or Zymomonas. In some embodiments, the bacterial host strain is non-pathogenic to humans. In some embodiments the bacterial host strain is an industrial strain. Numerous bacterial industrial strains are known and suitable in the present disclosure. In some embodiments of the present disclosure, the bacterial host cell is an Agrobacterium species (e.g., A. radiobacter, A. rhizogenes, and A. rubi). In some embodiments of the present disclosure, the bacterial host cell is an Arthrobacter species (e.g., A. aurescens, A. citreus, A. globiformis, A. hydrocarboglutamicus, A. mysorens, A. nicotianae, A. parafflneus, A. protophonniae, A. roseoparqffinus, A. sulfur eus, or A. ureafaciens) . In some embodiments of the present disclosure, the bacterial host cell is a Bacillus species (e.g., B. thuringensis , B. anthracis,26079B. megaterium, B. subtilis, B. lentus, B. circulans, B. pumilus, B. lautus, B.coagulans, B. brevis, B. firmus, B. alkaophius, B. licheniformis , B. clausii, B. stearother mophilus, B. halodurans, or B. amyloliquefaciens). In some embodiments, the host cell is an industrial Bacillus strain including but not limited to B. subtilis, B. pumilus, B. licheniformis, B. megaterium, B. clausii, B. stearothermophilus, or B. amyloliquefaciens. In some embodiments, the Bacillus host cells are B. subtilis. B. licheniformis, B. megaterium. B. stearothermophilus. and / or B. amyloliquefaciens. In some embodiments, the bacterial host cell is aClostridium species (e.g., C. acetobutylicum, C. tetani E88, C. lituseburense, C. saccharobutylicum, C. perfringens, and C. beijerinckii). In some embodiments, the bacterial host cell is a Corynebacterium species (e.g., C. glutamicum and C. acetoacidophilum). In some embodiments the bacterial host cell is an Escherichia species (e.g., E. coli). In some embodiments, the host cell is Escherichia coli W3110. In some embodiments the host is Escherichia coli BL21 or BL21(DE3). In some embodiments, the bacterial host cell is an Erwinia species (e.g., E. uredovora, E. carotovora, E. ananas, E. herbicola, E. punctata, and E. terreus). In some embodiments, the bacterial host cell is aPantoea species (e.g., P. citrea or P. agglomerans). In some embodiments the bacterial host cell is a Pseudomonas species (e.g., P. putida, P. aeruginosa, P. mevalonii, and P. sp. D-01 10). In some embodiments, the bacterial host cell is a Streptococcus species (e.g., S. equisimiles, S. pyogenes, and S. uberis). In some embodiments, the bacterial host cell is a Streptomyces species (e.g.. S. ambofaciens, S. achromogenes, S. avermitilis, S. coelicolor, S. aureofaciens. S. aureus, S. fungicidicus, S. griseus, or S. lividans). In some embodiments, the bacterial host cell is a Zymomonas species (e.g., Z. mobilis, or Z. lipolytica).

[0192] Many prokary otic and eukaryotic strains that find use in the present disclosure are readily available to the public from a number of culture collections such as American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

[0193] In some embodiments, host cells are genetically modified to have characteristics that improve protein secretion, protein stability and / or other properties desirable for expression and / or secretion of a protein. Genetic modification can be achieved by genetic engineering techniques and / or classical microbiological techniques (e.g., chemical or UV mutagenesis and subsequent selection). Indeed, in some embodiments, combinations of recombinant modification and classical selection techniques are used to produce the host cells. Using recombinant technology, nucleic acid molecules can be introduced, deleted, inhibited or modified, in a manner that results in increased yields of LDH variant(s) within the host cell and / or in the culture medium. In one26079 genetic engineering approach, homologous recombination is used to induce targeted gene modifications by specifically targeting a gene in vivo to suppress expression of the encoded protein. In alternative approaches, siRNA, antisense and / or ribozyme technology find use in inhibiting gene expression. A variety' of methods are known in the art for reducing expression of protein in cells, including, but not limited to deletion of all or part of the gene encoding the protein and site-specific mutagenesis to disrupt expression or activity of the gene product. (See e.g., Chaveroche et al., Nucl. Acids Res., 28:22 e97, 2000; Cho et al., Molec. Plant Microbe Interact., 19:7-15, 2006; Maruyama and Kitamoto, Biotechnol. Lett., 30: 1811-1817, 2008; Takahashi et al., Mol. Gen. Genom, 272: 344-352, 2004; and You et al., Arch.Microbiol. ,191 :615-622, 2009, all of which are incorporated by reference herein). Random mutagenesis, followed by screening for desired mutations may also be used (See e.g., Combier et al., FEMS Microbiol. Lett., 220: 141-8, 2003; and Firon et al., Eukary. Cell 2:247-55, 2003, both of which are incorporated by reference).

[0194] Introduction of a vector or polynucleotide construct into a host cell can be accomplished using any suitable method known in the art, including but not limited to calcium phosphate transfection, diethylaminoethyl (DEAE)-dextran mediated transfection, polyethylene glycol (PEG)-mediated transformation, electroporation, or other common techniques known in the art.

[0195] In some embodiments, the present disclosure provides a method of producing a polypeptide, the method comprising culturing a host cell comprising at least one polynucleotide encoding at least one polypeptide of the present disclosure under conditions such that the polypeptide encoded by the polynucleotide is produced. In some embodiments, the present disclosure provides a method of producing a polypeptide, the method comprising culturing a host cell comprising at least one polynucleotide encoding at least one polypeptide of the present disclosure under conditions such that the polypeptide encoded by the polynucleotide is produced, further comprising the step of recovering the polypeptide. In some embodiments, the present disclosure provides a method of producing a polypeptide, the method comprising culturing a host cell comprising at least one polynucleotide encoding at least one polypeptide of the present disclosure under conditions such that the polypeptide encoded by the polynucleotide is produced, further comprising the step of recovering the polypeptide, and further comprising the step of purifying the polypeptide.

[0196] In some embodiments, the engineered host cells (i.e., recombinant host cells) of the present disclosure are cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying the polynucleotide(s). Culture conditions, such as temperature, pH and the like, may be those previously used w ith the host cell26079 selected for expression, and are well-known to those skilled in the art. As noted, many standard references and texts are available for the culture and production of many cells, including cells of bacterial, plant, animal (especially mammalian) and archaebacterial origin.

[0197] In some embodiments, cells expressing an LDH polypeptide of the present disclosure are grown under batch or continuous fermentations conditions. Classical batch fermentation is a closed system, wherein the compositions of the medium are set at the beginning of the fermentation and is not subject to artificial alternations during the fermentation. A variation of the batch system is a fed-batch fermentation that also finds use in the present disclosure. In this variation, the substrate is added in increments as the fermentation progresses. Fed-batch systems are useful when catabolite repression is likely to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the medium. Batch and fed-batch fermentations are common and well known in the art. Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth. Continuous fermentation systems strive to maintain steady state growth conditions. Methods for modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology.

[0198] More than one copy of a polynucleotide sequence of the present disclosure may be inserted into the host cell to increase production of the gene product. An increase in the copynumber of the nucleic acid sequence can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the nucleic acid sequence where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the nucleic acid sequence, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

[0199] In some embodiments, cell-free transcription and translation systems find use in producing the polypeptide(s) (e.g., LDH polypeptide(s)). Several systems are commercially available, and the methods are well-known to those skilled in the art.Methods of Evolving LDHs

[0200] Evolution (e.g., directed evolution) may be used to identify polypeptides (e.g., LDH polypeptides) of the present disclosure. For example, in some embodiments, to make an LDH polypeptide of the present disclosure, an LDH polypeptide may be obtained (or derived) from26079 any suitable bacterial species, e.g.. Streptococcus thermophilus. In some embodiments, the parent polynucleotide sequence is codon optimized to enhance expression of the LDH polypeptide in a specified host cell (e.g., E. coli). For example, as is described in Example 1, a parental polynucleotide sequence, designated as SEQ ID NO: 1, which encodes SEQ ID NO: 2, was codon optimized for expression in E. coli and the codon-optimized polynucleotide (SEQ ID NO: 3) was cloned into an expression vector containing a T7 promoter upstream of a Lac operator sequence, a multiple-cloning site, a T7 terminator sequence, an open-reading frame encoding LacI, a pBR322 origin of replication and a KanR selection marker. Clones expressing the active LDH in E. coli may be identified and the genes sequenced to confirm their identity.

[0201] The polypeptides (e.g., LDH polypeptides) of the disclosure may be obtained by subjecting a polynucleotide encoding a parent sequence to mutagenesis and / or directed evolution methods. An exemplary7directed evolution technique is mutagenesis and / or DNA shuffling as described in Stemmer, 1994. Proc. Natl. Acad. Sci. USA 91: 10747-10751; WO 95 / 22625; WO 97 / 20078; WO 97 / 35966; WO 98 / 27230; WO 00 / 42651; WO 01 / 75767 and U.S. Pat. No. 6,537,746. Other directed evolution procedures that can be used include, among others, staggered extension process (StEP), in vitro recombination (Zhao et al., 1998, Nat. Biotechnol. 16:258- 261), mutagenic polymerase chain reaction (PCR) (Caldwell et al., 1994, PCR Methods Appl. 3:S136-S140), and cassette mutagenesis (Black et al., 1996. Proc. Natl. Acad. Sci. USA 93:3525- 3529).

[0202] The clones obtained following mutagenesis treatment may be screened for LDH polypeptides having a desired improved enzy me property. Measuring enzyme activity7from the expression libraries can be performed using standard chemistry analytical techniques for measuring substrates and products such as UPLC-MS, as well as the standard biochemistry technique of monitoring the rate of decrease (via a decrease in absorbance or fluorescence) of NADH or NADPH concentration, as it is converted into NAD+ or NADP+. In this reaction, the NADH or NADPH is consumed (oxidized) by the LDH as the LDH reduces pyruvate to L- or D- lactate and / or as the LDH reduces 2-oxoglutarate to 2-hydroxyglutarate. The rate of decrease of NADH or NADPH concentration, as measured by the decrease in absorbance or fluorescence, per unit time indicates the relative (enzy matic) activity of the LDH polypeptide in a fixed amount of the lysate (or a lyophilized powder made therefrom). For example, an LDH activity assay as described in Example 4 may be used.

[0203] Where the improved enzy me property desired is thermal stability, enzyme activity may be measured after subjecting the enzyme preparations to a defined temperature and measuring the amount of enzy me activity remaining after heat treatments. Any suitable approach may be used,26079 e.g., differential scanning colorimetry (DSC) a biochemical assay, or spectroscopy. Clones containing a polynucleotide encoding an LDH polypeptide may then be isolated, sequenced to identify the nucleotide sequence changes (if any), and used to express the enzy me in a host cell.

[0204] Where the sequence of the polypeptide is known, the polynucleotides encoding the enzyme can be prepared, e.g., by standard solid-phase methods, according to known synthetic methods. In some embodiments, fragments of up to about 100 bases can be individually synthesized, then joined (e.g., by enzy matic or chemical litigation methods, or polymerase mediated methods) to form any desired continuous sequence. For example, polynucleotides and oligonucleotides of the disclosure can be prepared by chemical synthesis using, e.g., the classical phosphoramidite method described by Beaucage et al., 1981, Tet. Lett. 22: 1859-69, or the method described by Matthes et al., 1984, EMBO J. 3:801-05, e.g., as it is typically practiced in automated synthetic methods. According to the phosphoramidite method, oligonucleotides are synthesized, e.g.. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors. In addition, essentially any nucleic acid can be obtained from any of a variety of commercial sources, such as Integrated DNA Technologies, Coralville, IA, The Midland Certified Reagent Company, Midland, Tex., GENEART® Gene Synthesis (Thermo Fisher Scientific), and many others.

[0205] Polypeptides (e.g., LDH polypeptides) expressed in a host cell can be recovered from the cells and or the culture medium using any one or more of the well-known techniques for protein purification, including, among others, lysozyme treatment, sonication, filtration, saltingout, ultra-centrifugation, and chromatography. Suitable solutions for lysing and the high efficiency extraction of proteins from bacteria, such as E. coli, are commercially available under the trade name CELLYTIC B® from Sigma- Aldrich.

[0206] Chromatographic techniques for isolation of the polypeptide (e g., LDH polypeptide) include, among others, reverse phase chromatography high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, and affinity chromatography. Conditions for purifying a particular enzyme will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, and the like, and will be apparent to those having skill in the art.

[0207] In some embodiments, affinity techniques may be used to isolate the improved polypeptides (e.g., LDH polypeptides). For affinity chromatography purification, the protein sequence can be tagged with a recognition sequence to enable purification. For example, tags include cellulose-binding domains, poly His-tags, di-His chelates, FLAG-tags and many others26079 that will be apparent to those having skill in the art. Antibodies can also be used as affinity purification reagents. Any antibody that specifically binds the LDH polypeptide may be used.Methods of Using LDH Polypeptides

[0208] Also provided herein are methods of using a polypeptide (e.g.. an LDH polypeptide) of the disclosure. In some examples, a polypeptide (e g., an LDH polypeptide) of the disclosure is used in a method of catalyzing the NAD(P)H-dependent reduction of pyruvate to L- or D-lactate and / or the NAD(P)H-dependent reduction of a-ketoglutarate (i.e., 2-oxoglutarate) to 2- hydroxy glutarate. In some embodiments, a polypeptide (e.g., a LDH polypeptide) of the disclosure is used in a method of catalyzing the NAD(P)H-dependent reduction of a- ketoglutarate (i.e., 2-oxoglutarate) to 2 -hydroxy glutarate. In some embodiments, the LDH polypeptides described herein exhibit preferential selectivity' for NADH over NADPH.

[0209] The method of catalyzing the NAD(P)H-dependent reduction of a-ketoglutarate (i.e., 2- oxoglutarate) to 2-hydroxyglutarate may also be described as a method of catalyzing the oxidation of NAD(P)H to NAD(P)+ in the presence of 2-oxoglutarate. For example, NAD(P)H can be recycled back to NAD(P)+ by an LDH polypeptide of the disclosure, for example using a- ketoglutarate as the stoichiometric oxidant (i.e., electron acceptor). In some embodiments, the LDH polypeptides of the disclosure are capable of regenerating oxidized NAD(P)+ in the context of an alcohol dehydrogenase reaction.

[0210] For example, provided herein is a method of catalyzing the NAD(P)H-dependent reduction of a-ketoglutarate (i.e., 2-oxoglutarate) to 2-hydroxyglutarate, the method comprising contacting the 2-oxoglutarate with any one of the polypeptides (e.g., LDH polypeptides) disclosed herein. In some embodiments, provided herein is a method of catalyzing the NAD(P)H- dependent reduction of a-ketoglutarate (i.e., 2-oxoglutarate) to 2-hydroxyglutarate, the method comprising incubating the 2-oxoglutarate in the presence of any one of the polypeptides (e.g., LDH polypeptides) disclosed herein.

[0211] In some embodiments, the method of catalyzing the NAD(P)H-dependent reduction of a-ketoglutarate (i.e., 2-oxoglutarate) to 2-hydroxyglutarate, comprises contacting the 2- oxoglutarate with a polypeptide (e.g., an LDH polypeptide) as disclosed herein under reaction conditions suitable for converting the a-ketoglutarate (i.e., 2-oxoglutarate) to 2-hydroxyglutarate. In some embodiments, the method of catalyzing the NAD(P)H-dependent reduction of a- ketoglutarate (i.e., 2-oxoglutarate) to 2-hydroxyglutarate, comprises incubating the 2- oxoglutarate in the presence of a polypeptide (e.g., an LDH polypeptide) as disclosed herein26079 under reaction conditions suitable for converting the a-ketoglutarate (i . e.. 2-oxoglutarate) to 2- hydroxy glutarate.

[0212] In some embodiments, the method of catalyzing the oxidation of NAD(P)H to NAD(P)+, comprises contacting the NAD(P)H with a polypeptide (e.g., an LDH polypeptide) as disclosed herein under reaction conditions suitable for converting the NAD(P)H to NAD(P)+. In some embodiments, the method of catalyzing the oxidation of NAD(P)H to NAD(P)+, comprises incubating the NAD(P)H in the presence of a polypeptide (e.g., an LDH polypeptide) as disclosed herein under reaction conditions suitable for converting the NAD(P)H to NAD(P)+. In some embodiments, a-ketoglutarate (i.e., 2-oxoglutarate) is used as the stoichiometric oxidant (i. e. , electron acceptor) in the method of catalyzing the oxidation of NAD(P)H to NAD(P)+.

[0213] Any polypeptide (e.g., LDH polypeptide) or combination of polypeptides disclosed herein may be used in the methods for catalyzing the NAD(P)H-dependent reduction of a- ketoglutarate (i.e., 2-oxoglutarate) to 2 -hydroxy glutarate or the method of catalyzing the oxidation of NAD(P)H to NAD(P)+. As noted herein, in some embodiments, the polypeptide (e.g., LDH polypeptide) comprises an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a reference sequence comprising the sequence of SEQ ID NO: 2 or 4, wherein the polypeptide does not comprise SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, these polypeptides can have one or more modifications to the amino acid sequence of SEQ ID NO: 2 or 4. The modifications can include substitutions, deletions, and insertions. The substitutions can be non-conservative substitutions, conservative substitutions, or a combination of nonconservative and conservative substitutions.

[0214] For example, in some embodiments, the polypeptide (e.g., LDH polypeptide) comprises an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the polypeptide comprises an amino acid sequence having at least about 98% sequence identity to any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the polypeptide comprises any of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26. 28. 30. or 32. In some embodiments, the polypeptide consists of any of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32.26079

[0215] In some embodiments, provided herein is a method of catalyzing the NAD(P)H- dependent reduction of a-ketoglutarate (i.e., 2-oxoglutarate) to 2 -hydroxy glutarate, the method comprising contacting the 2-oxoglutarate with any one of the polypeptides (e.g., LDH polypeptides) disclosed herein in the presence of NAD(P)H, wherein at least about 60%, 65%, 70%. 75%. 80%. 85%. 90%. 95%. 96%. 97%. 98%. 99%. or more of the 2-oxoglutarate is converted to 2 -hydroxy glutarate. In some embodiments, provided herein is a method of catalyzing the NAD(P)H-dependent reduction of a-ketoglutarate (i.e., 2-oxoglutarate) to 2- hydroxyglutarate, wherein at least about 90% (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%. 99%. or higher) of the 2-oxoglutarate is converted to 2-hydroxyglutarate.

[0216] As is known by those of skill in the art, LDH catalyzed reduction reactions typically require a cofactor. Reduction reactions catalyzed by the LDH enzymes described herein also typically require a cofactor, though the cofactor selectivity may be changed or improved in some embodiments relative to a wild-type polypeptide. For example, cofactors suitable for use with the LDH polypeptides described herein include, but are not limited to, NADP+ (nicotinamide adenine dinucleotide phosphate), NADPH (the reduced form of NADP+), NAD+ (nicotinamide adenine dinucleotide) and NADH (the reduced form of NAD+).

[0217] In some embodiments, provided herein is a method of catalyzing the oxidation of NAD(P)H to NAD(P)+, the method comprising contacting the NAD(P)H with any one of the polypeptides (e.g., LDH polypeptides) disclosed herein, wherein at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the NAD(P)H is converted to NAD(P)+. In some embodiments, provided herein is a method of catalyzing the oxidation of NAD(P)H to NAD(P)+, wherein at least about 90% (e.g., at least about 90%. 91%. 92%. 93%. 94%, 95%, 96%, 97%, 98%, 99%, or higher) of the NAD(P)H is converted to NAD(P)+. In some embodiments, provided herein is a method of catalyzing the oxidation of NAD(P)H to NAD(P)+, the method comprising contacting the NAD(P)H with any one of the polypeptides (e.g., LDH polypeptides) disclosed herein in the presence of a-ketoglutarate (i.e., 2-oxoglutarate).

[0218] The LDH-catalyzed reactions described herein are generally carried out in a solvent. Suitable solvents include water, organic solvents (e.g., ethyl acetate, butyl acetate, 1-octanol, heptane, octane, methyl t-buty 1 ether (MTBE), dimethylacetamide (DMAc), toluene, and the like), and ionic liquids (e.g., 1-ethyl 4-methylimidazolium tetrafluoroborate, l-butyl-3- methylimidazolium tetrafluoroborate, l-butyl-3-methylimidazolium hexafluorophosphate, and the like). In some embodiments, aqueous solvents, including water and aqueous co-solvent systems, are used.

[0219] Exemplary aqueous co-solvent systems have water and one or more organic solvent. In general, an organic solvent component of an aqueous co-solvent system is selected such that it does not completely inactivate the LDH enzyme. Appropriate co-solvent systems can be readily identified by measuring the enzymatic activity of the specified engineered LDH enzyme with a defined substrate of interest in the candidate solvent system, utilizing an enzyme activity assay, such as those described herein.

[0220] The aqueous solvent (water or aqueous co-solvent system) may be pH-buffered or unbuffered. In some embodiments, the method of catalyzing the NAD(P)H-dependent reduction of 2-oxoglutarate to 2-hydroxyglutarate or the method of catalyzing the oxidation of NAD(P)H to NAD(P)+ is carried out at a pH of about 10 or below, e.g., in a range of from about 5 to about 10. In some embodiments, the methods of the disclosure are carried out at a pH of about 9 or below, e.g., in a range of from about 5 to about 9. In some embodiments, the methods of the disclosure are carried out at a pH of about 8.5 or below, e.g., in a range of from about 5 to about 8.5, in a range of from about 6 to about 8.5, in a range of from about 7 to about 8.5, or in a range of from about 7.5 to about 8.5. The methods may also be carried out at a pH of about 8 or below, 7.8 or below, or 7.5 or below. Alternatively, the methods may be carried out a neutral pH, i.e., about 7.

[0221] During the course of the reactions, the pH of the reaction mixture may change. The pH of the reaction mixture may be maintained at a desired pH or within a desired pH range by the addition of an acid or a base during the course of the reaction. Alternatively, the pH may be controlled by using an aqueous solvent that comprises a buffer. Suitable buffers to maintain desired pH ranges are known in the art and include, for example, phosphate buffer, triethanolamine buffer, and the like. Combinations of buffering and acid or base addition may also be used.

[0222] In some embodiments, the method of catalyzing the NAD(P)H-dependent reduction of 2-oxoglutarate to 2-hydroxyglutarate or the method of catalyzing the oxidation of NAD(P)H to NAD(P)+ is performed at a pH of less than about 11, e.g., less than about 11, about 10.5. about 10. about 9.5, about 9, about 8.5, about 8, about 7.5, about 7. In some embodiments, the methods of the disclosure are performed at a pH of from about 7 to about 11, e.g., 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.8, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5. 10.6, 10.7, 10.8, 10.9, or 11.0. In some embodiments, the methods of the disclosure are performed at a pH of from 7 to 7.5, from 7 to 8, from 7 to 9, from 7.5 to 8.5, from 7.5 to 9, from 7.5 to 8, from 8 to 9, from 8.5 to 9, from 9 to 11, from 9 to 10.5, from 9 to 10, from 9 to 9.5, from 9.5 to 11, from 9.5 to 10.5, from 9.5 to 10, from 10 to 11, or from 10 to 10.5.

[0223] In some embodiments, the method of catalyzing the NAD(P)H-dependent reduction of 2-oxoglutarate to 2-hydroxy glutarate or the method of catalyzing the oxidation of NAD(P)H to NAD(P)+ is performed at a temperature of from about 15 °C to about 45 °C, e.g., about 15 °C, 16 °C, 17 °C, 18 °C, 19 °C, 20 °C, 21 °C. 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C. 35 °C, 36 °C, 37 °C. 38 °C, 39 °C, 40 °C. 41 °C, 42 °C, 43 °C, 44 °C, or 45 °C. In some embodiments, the methods of the disclosure are performed at a temperature of from about 20 °C to about 40 °C, e.g., about 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, about 31 °C, 33 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, or 40 °C.

[0224] In some embodiments, the method of catalyzing the NAD(P)H-dependent reduction of 2-oxoglutarate to 2-hydroxyglutarate comprises contacting or incubating from about 0.01 to about 10 g / L, about 0.01 to about 8 g / L, about 0.01 to about 5 g / L, 0.05 to about 10 g / L, 0.05 to about 8 g / L, 0.05 to about 5 g / L. about 0.1 to about 10 g / L, about 0.1 to about 8 g / L, about 0.1 to about 5 g / L, about 0.5 to about 10 g / L, about 0.5 to about 8 g / L, about 0.5 to about 5 g / L, about 1 to about 10 g / L, about 1 to about 8 g / L, about 1 to about 7 g / L, about 1 to about 6 g / L, about 1 to about 5 g / L, or about 2 to about 6 g / L of 2-oxoglutarate with any one of the polypeptides (e.g., LDH polypeptides) disclosed herein or any combination of the polypeptides disclosed herein in the presence of NAD(P)H. In some embodiments, the method of catalyzing the reduction of 2- oxoglutarate to 2-hydroxyglutarate comprises contacting or incubating from about 10 g / L, about 9 g / L, about 8 g / L, about 7 g / L, about 6.5 g / L, about 6 g / L, about 5.5 g / L, about 5 g / L, about 4 g / L, about 4.5 g / L, about 3 g / L, about 2.5 g / L, about 2 g / L, about 1 g / L, about 0.5 g / L, about 0.25 g / L, about 0. 1 g / L, about 0.05 g / L, about 0.025 g / L, or about 0.01 g / L of 2-oxoglutarate with any one of the polypeptides (e.g., LDH polypeptides) disclosed herein or any combination of the polypeptides disclosed herein in the presence of NAD(P)H.

[0225] In some embodiments, the method of catalyzing the oxidation of NAD(P)H to NAD(P)+ in the presence of 2-oxoglutarate is carried out under an NAD(P)H concentration of about 0.01 to about 3 g / L, about 0.03 to about 3 g / L, about 0.05 to about 2 g / L. about 0.05 to about 1 g / L, about 0. 1 to about 2 g / L, about 0. 1 to about 1 g / L, about 0.2 to about 1 g / L, about 0.2 to about 0.8 g / L, or about 0.25 to about 0.8 g / L. In some embodiments, the method is carried out under an NAD(P)H concentration of about 3 g / L, about 2.5 g / L, about 2 g / L, about 1.5 g / L, about 1 g / L, about 0.8 g / L, about 0.7 g / L. about 0.6 g / L, about 0.5 g / L, about 0.4 g / L, about 0.3 g / L, about 0.2 g / L, about 0. 1 g / L, about 0.05 g / L, about 0.03 g / L, or about 0.01 g / L.

[0226] In some embodiments, the method of catalyzing the NAD(P)H-dependent reduction of 2-oxoglutarate to 2-hydroxyglutarate or the method of catalyzing the oxidation of NAD(P)H is26079 performed in the presence of from about 0.01 g / L to about 1 g / L, 0.01 to about 0.8 g / L, about 0.01 to about 0.5 g / L, about 0.02 to about 0.3 g / L, about 0.02 to about 0.2 g / L, about 0.02 to about 0.2 g / L, about 0.02 to about 0.05 g / L, about 0.02 to about 0.03 g / L of any one of the polypeptides (e.g., LDH polypeptides) disclosed herein or any combination of the polypeptides disclosed herein. In some embodiments, the method is carried out in the presence of about 1 g / L, about 0.8 g / L, about 0.5 g / L, about 0.3 g / L, about 0.2 g / L, about 0.1 g / L, about 0.05 g / L, about 0.03 g / L, about 0.025 g / L, about 0.02 g / L or about 0.01 g / L of any one of the polypeptides (e.g., LDH polypeptides) disclosed herein or any combination of the poly peptides disclosed herein. In some embodiments, the method is carried out in the presence of from about 0.01% w / w to about 15% w / w (e.g., about 0.01% w / w, 0.02% w / w, 0.03% w / w, 0.04% w / w, 0.05% w / w, 0.06% w / w, 0.07% w / w, 0.08% w / w, 0.09% w / w, 0.1% w / w, 0.15% w / w, 0.2% w / w, 0.25% w / w, 0.3% w / w, 0.35% w / w, 0.4% w / w, 0.45% w / w, 0.5% w / w, 0.6% w / w, 0.7% w / w, 0.8% w / w, 0.9% w / w, 1% w / w, 2% w / w, 3% w / 2, 4% w / w, 5% w / w, 6% w / w. 7% w / w, 8% w / w, 9% w / w, 10% w / w, 11% w / w, 12% w / w, 13% w / w, 14% w / w or 15% w / w) of any one of the polypeptides (e.g.. LDH polypeptides) disclosed herein or any combination of the polypeptides disclosed herein.

[0227] Any of the methods disclosed herein may further comprise fdtering the product, e.g., 2- hydroxyglutarate. Any suitable approach may be used for filtration. In some embodiments, the pH is adjusted to from about 6 to about 8 (e.g., about 6.0, 6.1. 6.2, 6.3, 6.4, 6.5. 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0).

[0228] In some embodiments, the method of catalyzing the NAD(P)H-dependent reduction of 2-oxoglutarate to 2-hydroxy glutarate or the method of catalyzing the oxidation of NAD(P)H comprises contacting or incubating the 2-oxoglutarate with the polypeptide (e.g., LDH polypeptide) in the presence of NAD(P)H for any suitable period of time, e.g., about 1 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h,12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, 25 h, 26 h, 27 h, 28 h,29 h, 30 h, 31 h, 32 h, 33 h, 34 h, 35 h, 36 h, 37 h. 38 h, 39 h, 40 h, 41 h, 42 h, 43 h, 44 h, 45 h,46 h, 47 h, 48 h, 36 h, 60 h, or longer. In some embodiments, the period of time is about 1 min to about 1 h, about 1 min to about 30 min, about 1 min to about 10 min, about 5 min to about 30 min, about 10 min to about 30 min, about 30 min to about 1 h, about 1 h to about 36 h, about 1 h to about 24 h, about 1 h to 23 h, about 1 h to about 22 h, about 1 h to about 21 h, about 1 h to about 20 h. about 1 h to about 19 h, about 1 h to about 18 h. about 1 h to about 17 h, about 1 h to about 16 h, about 1 h to about 15 h, about 1 h to about 14 h, about 1 h to about 13 h, about 1 h to about 12 h, about 1 h to about 11 h, about 1 h to about 10 h, about 1 h to about 9 h, about 1 h to about 8 h, about 1 h to about 7 h, about 1 h to about 6 h, about 1 h to about 5 h, about 1 h to about260794 h, about 1 h to about 3 h, about 1 h to about 2 h, about 5 h to about 36 h, about 5 h to about 24 h, about 5 h to 23 h, about 5 h to about 22 h, about 5 h to about 21 h, about 5 h to about 20 h, about 5 h to about 19 h, about 5 h to about 18 h, about 5 h to about 17 h, about 5 h to about 16 h, about 5 h to about 15 h, about 5 h to about 14 h, about 5 h to about 13 h, about 5 h to about 12 h. about 5 h to about 11 h, about 5 h to about 10 h. about 5 h to about 9 h. about 5 h to about 8 h, about 5 h to about 7 h, about 5 h to about 6 h, about 10 h to about 36 h, about 10 h to about 24 h, about 10 h to 23 h, about 10 h to about 22 h, about 10 h to about 21 h, about 10 h to about 20 h, about 10 h to about 19 h. about 10 h to about 18 h, about 10 h to about 17 h, about 10 h to about 16 h, about 10 h to about 15 h. about 10 h to about 14 h, about 10 h to about 13 h, about 10 h to about 12 h, about 10 h to about 11 h, about 12 h to about 36 h, about 12 h to about 24 h, about 12 h to 23 h, about 12 h to about 22 h, about 12 h to about 21 h, about 12 h to about 20 h, about 12 h to about 19 h, about 12 h to about 18 h, about 12 h to about 17 h, about 12 h to about 16 h, about 12 h to about 15 h, about 12 h to about 14 h. about 12 h to about 13 h, about 18 h to about 36 h, about 18 h to about 24 h. about 18 h to 23 h. about 18 h to about 22 h, about 18 h to about 21 h, about 18 h to about 20 h, or about 18 h to about 19 h.EXAMPLES

[0229] The following examples are meant to be illustrative and should not be construed as further limiting. The contents of the figures and all references, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference.Example 1 : Gene synthesis

[0230] The DNA sequence encoding NAD(P)H-dependent wild-type lactate dehydrogenase polypeptide from Streptococcus thermophilus (SEQ ID NO: 1, GenBank ID: WP_011227325. 1 (SEQ ID NO: 2)) was identified in silico by BLAST searching using several confirmed lactate dehydrogenase enzymes as query' sequences. SEQ ID NO: 1 was then codon optimized for A coli expression and chemically synthesized to generate reference sequence polynucleotide SEQ ID NO: 3. A C-terminal hexahistidine tag (SEQ ID NO: 33) with a linker (SEQ ID NO: 34) was included as part of SEQ ID NO: 3. The sequence for the LDH polypeptide having the wild-type amino acid sequence with the C-terminal hexahistidine tag (SEQ ID NO: 33) is provided as SEQ ID NO: 4. The gene (SEQ ID NO: 3) was cloned into pET30a(+) vector containing a T7 promoter upstream of a Lac operator sequence, a multiple-cloning site, a T7 terminator sequence, an open-reading frame encoding LacI, a pBR322 origin of replication and a KanR selection marker. The cloned gene was propagated into E. coli DH5a strain, sequence verified and26079 transformed into E. colt BL21(DE3). Likewise, genes of engineered NAD(P)H-dependent lactate dehydrogenases (e.g., SEQ ID NOs: 5, 7, 9, 11, 13, 15, and 17) were cloned into pET30a(+) vector and transformed into E. coli BL21(DE3) cells.Example 2: Enzyme preparation for well-plate reactions

[0231] pET30a(+) plasmid bearing a gene encoding NAD(P)H-dependent lactate dehydrogenase was transformed into E. coli BL21(DE3) and recovered in SOC (super optimal broth with catabolite repression) medium. A portion of the transformed cells were spread onto LB-agar plates supplemented with 50 pg / mL kanamycin and 1% (w / v) glucose and grown overnight at 30 °C. The following day, individual colonies were inoculated into 96-well deepwell plates containing 0.2 mL per well of Luria-Bertani (LB) Broth medium (10 mg / mL Tryptone, 5 mg / mL Yeast Extract, 10 mg / mL NaCl, 50 pg / mL Kanamycin). Plates were incubated at 30 °C shaking at 200 RPM overnight. The following day, the saturated culture optical density at 600 nm (OD600) was used to inoculate (10 pL inoculum) an expression culture in deep-well 96-well plates containing TB (Terrific Broth) medium (390 pL / well of 12 mg / mL Trytone, 24 mg / mL Yeast Extract, 4 mg / mL Glycerol, 54 mM K2HPO4, 1 .2 mM KH2PO4, 50 pg / mL Kanamycin.). Cells were grown at 30 °C at 400 RPM to an OD600 of approximately 0.65 and then the shaker temperature was lowered to 20 °C and protein expression was induced by adding isopropyl P-D-l -thiogalactopyranoside (IPTG) (0.1 mM final concentration). Expression was allowed to proceed at this condition for 20 h at which time cells were pelleted by centrifugation at 4000 g for 15 min and the supernatant discarded. Pelleted cells were frozen / thawed once and resuspended in lysis buffer (0.1 mL / well of 100 mM EPPS buffer pH 8.5, 1 mg / mL lysozyme, 0.5 mg / mL polymyxin B sulfate. 1 mM MgCh and 3 U / mL DNasel). The resulting cell-suspension was shaken at 800 RPM for 2 h at 25 °C, after which cell-debris was pelleted by centrifugation (3400 x g, 10 minutes, 4 C) and clarified lysate was used in subsequent well-plate reactions.Example 3: Enzy me preparation for vial and larger-scale reactions

[0232] Twenty7microliters of a glycerol stock of E. coli BL21(DE3) containing the NAD(P)H- dependent lactate dehydrogenase gene of interest in pET30a(+) vector was inoculated in 25 mL of LB broth supplemented with 30 pg / mL kanamycin and 1% (w / v) glucose. Cells were grown for 18-24 h at 30 °C and 250 RPM. The following day, 100 mL of TB medium supplemented with 50 pg / mL kanamycin was inoculated with the overnight culture and then grown at 30 °C shaking at 250 RPM until the OD600 reached approximately 0.7 at which time the incubator26079 temperature was reduced to 25 °C and protein production was induced by adding 1PTG (0. 1 mM final concentration). Expression was continued for 18-22 h at this condition. The following day, biomass was pelleted by centrifugation at (3400 x g, 10 minutes, 4 °C) and the supernatant discarded, cells were washed (0.5% NaCl in water), re-centrifuged (3400 x g, 10 min, 4 °C) and then resuspended in lysis buffer (20 mL, 100 mM EPPS buffer pH 8.5) and were then lysed by sonication (500 W for 15 minutes, 2 seconds on / 4 seconds off). The resulting lysate was clarified by centrifugation (18,000 x g, 20 minutes at 4 °C). The clarified lysate was frozen and lyophilized. Powders were used in subsequent vial reactions as detailed in Example 4 below.Example 4: Evolution and screening of polypeptides derived from SEQ ID NO: 4 for the NADH- dependent reduction of a-ketoglutarate

[0233] NAD(P)H-dependent lactate dehydrogenase enzy mes derived from SEQ ID. NO: 4 were evolved from the polynucleotide encoding SEQ ID NO: 3. Libraries of engineered polypeptides were generated using techniques such as site saturation mutagenesis and combinatorial libraries. Specifically, site-saturation libraries were constructed in 3 groups of 96-positions in SEQ ID NO: 4. The first set of amino acid positions mutated using the technique of site saturation mutagenesis were the following: 18, 49, 52, 53. 56. 57, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99, 100, 101, 102, 103, 104, 105, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 148, 149, 150, 151, 152, 153, 154, 155, 157, 177, 178, 179, 180, 181, 182, 183, 184, 185,186, 187, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,235, 236. 237, 238, 239, 240, 242, 261, 274, 275, 276, 301, 304, 305. 308, 309, 312, 313, 316.NAD(P)H-dependent lactate dehydrogenase enzymes derived from SEQ ID. NO: 4 were screened in a 96-well plate spectrophotometric assay using the loss of 340 nm absorbance resulting from NAD(P)H-dependent reduction of a-ketoglutarate. To prepare this assay, lysates containing engineered lactate dehydrogenases were generated as described in Example 2, and then preincubated for 18-22 h at 30 °C in the presence of cosolvent (dimethylacetamide (DMAc), 15 vol%). Following this overnight incubation, lysates were then dosed (final concentration 5 vol% lysate) into a solution containing 100 mM EPPS pH 8.5, 40 mM a-ketoglutarate and 1 mM NADH. To measure the extent of the reaction, the absorbance was measured at time zero and at 30 minutes. Variants that effected the greatest loss in absorbance at 340 nm over the 30 minute interval were judged to be better catalysts of the desired reaction and were further scaled up to shake-flasks as described in Example 3.

[0234] To assay the resulting enzy me powders, the powders were first dissolved in water to a concentration of 1 mg / mL in water. To an aliquot (40 pL) of this enzy me solution was then26079 added DMAc (15 vol% final concentration) and EPPS buffer pH 8.5 (100 mM final concentration) in a total volume of 50 pL. This solution was then aged overnight at 30 °C. The following day, the enzyme solution was dosed into a reaction mixture containing 100 mM EPPS buffer pH 8.5, 0.5 mM NADH, and 20 mM a-ketoglutarate and the progress of the reaction was monitored spectrophotometrically via loss of absorbance at 340 nm for 10 minutes. Powders were also assayed for activity in the NADPH-dependent reduction of a-ketoglutarate, which was done in an identical fashion as described above except that NADPH was substituted for NADH in the spectrophotometric assay. This first round of evolution led to the identification of the polypeptide with SEQ ID NO: 6 encoded by polynucleotide sequence SEQ ID NO: 5.Combinatorial libraries were then generated to create variants of SEQ ID NO: 6 composed of beneficial mutations that had been identified in the context of the first round of evolution. Additional site saturation mutagenesis libraries were constructed to generate variants of SEQ ID NO: 6 with single mutations at the following positions: 2, 3, 4, 5, 6, 7, 8, 17, 29, 33, 40, 41, 43, 44. 45, 48, 60, 62, 63, 65, 67, 70, 71, 72, 73, 76. 78. 106, 107, 110. I l l, 113. 114, 116. 118, 136,137, 138, 140, 142, 143, 144, 145, 159, 164, 165, 167, 171, 189, 200, 201, 204, 206, 207, 208,210, 211, 212, 215, 216, 245, 248, 252, 253, 265, 269, 271, 272, 284, 287, 288, 291, 293, 295,296, 297, 299, 300, 303, 307, 310, 311, 314, 315, 317, 318, 319, 320, 321, 322, 323, 324, 325,326, 327. 328. An additional site saturation mutagenesis library was constructed to generate variants of SEQ ID NO: 6 with single mutations at the following positions: 9, 10, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23, 24, 25, 28, 30, 31, 32, 34, 38, 39, 42, 46, 51, 55, 58, 59, 61, 64, 66, 68, 69, 75, 77, 83, 84, 108, 115, 117, 119, 120, 139, 141, 156, 160, 161, 163, 166, 168, 169, 170, 172, 174. 175, 176, 188, 190, 191. 192, 193, 195, 196, 197, 198, 199. 202, 203, 205, 209, 213.214, 217. 250, 251, 254. 255, 257. 258, 259, 263. 266, 267. 268, 270. 273, 282, 283. 286, 289.290, 292, 294, 298, 302, 306. Polypeptides with SEQ ID NO: 8, 10, 12, 14, 16, and 18 were identified from several rounds of evolution for improvements in enzy matic activity' (e.g., reduction of a-ketoglutarate) under similar conditions as described above, with the only- differences being that the DMAc concentration was increased (from 15 vol% to 20 vol%) during the preincubation step, the assay time was extended (from 10 minutes to 30 minutes), lysate concentration was varied over the range from 0.25 vol% to 5 vol%, and / or HEPES buffer pH 7.5 was substituted for EPPS buffer pH 8.5. The results are shown in Tables 1 and 2.Table 1. Relative activities of NAD(P)H-dependent lactate dehydrogenases in the reduction of a- ketoglutarate26079Table 2. Relative nicotinamide cofactor selectivities for NAD(P)H dependent engineered lactate dehydrogenases in the reduction of a-ketoglutarate26079(a) The relative nicotinamide cofactor selectivities are based on measurement of the initial rate of a -ketoglutarate reduction using NADH or NADPH. The ratio is then taken by dividing the initial rate of a -ketoglutarate reduction with NADH by the initial rate of a -ketoglutarate reduction with NADPH.(b) + corresponds to a ratio between 1 and 100++ corresponds to a ratio between 100 and 500 +++ corresponds to a ratio greater than 500

[0235] The disclosed subject matter is not to be limited in scope by the specific embodiments and examples described herein. Indeed, various modifications of the disclosure in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[0236] All references (e.g.. publications or patents or patent applications) cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) w as specifically and individually indicated to be incorporated by reference in its entirety' for all purposes. Other embodiments are within the following claims.26079260792607926079260792607926079260792607926079

Claims

WHAT IS CLAIMED IS:

1. An engineered polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 4, wherein the polypeptide comprises an amino acid substitution at one or more amino acid positions selected from 12, 88, 98, 99, 166, 199, 205. 229, and 308, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 4.

2. The polypeptide of claim 1, wherein the polypeptide comprises 2. 3, 4, 5, 6, 7, 8, or 9 substitutions, each amino acid substitution at an amino acid position selected from 12, 88, 98, 99, 1 6, 199, 205, 229, and 308, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 4.

3. The polypeptide of claim 1 or 2, wherein the polypeptide comprises: a) an amino acid substitution at amino acid position 88; b) an amino acid substitution set at amino acid positions 88, 98, 99, and 229; c) an amino acid substitution set at amino acid positions 88, 229, and 308; d) an amino acid substitution set at amino acid positions 88 and 308; e) an amino acid substitution set at amino acid positions 88, 98, and 308; or f) an amino acid substitution set at amino acid positions 12, 88, 98, 166, 199, 205, 229, and 308; wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 4.

4. The polypeptide of any one of claims 1-3, wherein the polypeptide comprises one of the following amino acid substitutions or amino acid substitution sets: a) Q88L; b) Q88L, V98T, G99D, and I229K; c) Q88L, I229R, and E308V; d) Q88L and E308I; e) Q88L, I229K, and E308V; f) Q88L, V98I, and E308V; or g) L12V, Q88L, V98I, L166M, L199C, D205L, I229R, and E308V;- 79 -wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 4.

5. The polypeptide of any one of claims 1-4, wherein the amino acid sequence comprises SEQ ID NO: 6.

6. The polypeptide of any one of claims 1-4, wherein the amino acid sequence comprises SEQ ID NO: 8.

7. The polypeptide of any one of claims 1-4, wherein the amino acid sequence comprises SEQ ID NO: 10.

8. The polypeptide of any one of claims 1-4, wherein the amino acid sequence comprises SEQ ID NO: 12.

9. The polypeptide of any one of claims 1-4, wherein the amino acid sequence comprises SEQ ID NO: 14.

10. The polypeptide of any one of claims 1-4, wherein the amino acid sequence comprises SEQ ID NO: 16.

11. The polypeptide of any one of claims 1 -4, wherein the amino acid sequence comprises SEQ ID NO: 18.

12. An engineered polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2, wherein the polypeptide comprises an amino acid substitution at one or more amino acid positions selected from 12, 88, 98, 99, 166, 199, 205. 229, and 308, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2.

13. The polypeptide of claim 12. wherein the polypeptide comprises 2, 3. 4, 5, 6, 7, 8, or 9 amino acid substitutions, each amino acid substitution at an amino acid position selected from 12, 88, 98, 99, 166, 199, 205, 229, and 308, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2.

14. The polypeptide of claim 12 or 13, wherein the polypeptide comprises: a) an amino acid substitution at amino acid position 88; b) an amino acid substitution set at amino acid positions 88, 98, 99, and 229; c) an amino acid substitution set at amino acid positions 88. 229, and 308; d) an amino acid substitution set at amino acid positions 88 and 308; e) an amino acid substitution set at amino acid positions 88, 98, and 308; or f) an amino acid substitution set at amino acid positions 12, 88, 98, 166, 199, 205, 229, and 308; wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2.

15. The polypeptide of any one of claims 12-14. wherein the polypeptide comprises one of the following amino acid substitutions or amino acid substitution sets: a) Q88L; b) Q88L, V98T, G99D, and I229K; c) Q88L, I229R, and E308V; d) Q88L and E308I; e) Q88L, I229K, and E308V; f) Q88L, V98I, and E308V; or g) L12V, Q88L, V98I, L166M, L199C, D205L, I229R, and E308V; wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2.

16. The polypeptide of any one of claims 12-15, wherein the amino acid sequence comprises any one of SEQ ID NO: 20, 22, 24, 26, 28, 30, or 32.

17. The polypeptide of any one of claims 12-16, wherein the amino acid sequence consists of any one of SEQ ID NO: 20, 22, 24, 26, 28, 30, or 32.

18. The polypeptide of any one of claims 12-17. further comprising an epitope tag.

19. The polypeptide of claim 18, wherein the epitope tag is a His tag.2607920. The polypeptide of claim 19, wherein the His tag comprises the amino acid sequence of HHHHHH (SEQ ID NO: 33) or the amino acid sequence of LEHHHHHH (SEQ ID NO: 34).

21. The polypeptide of any one of claims 18-20, wherein the polypeptide comprises a His tag at the C-terminus.

22. An engineered polypeptide comprising an amino acid sequence having at least 98% sequence identity to any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

23. The polypeptide according to claim 22, wherein the amino acid sequence has at least 99% sequence identity to any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32.

24. The polypeptide according to claim 22 or 23, consisting of any of one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32.

25. An engineered polypeptide comprising an amino acid sequence having at least 98% sequence identity’ to amino acid residues 1-328 of any one of SEQ ID NO:

6. 8, 10, 12.

14.

16. or 18, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

26. The polypeptide of any one of claims 1-25, which is isolated.

27. The polypeptide of any one of claims 1-26, which is a lactate dehydrogenase, optionally wherein the polypeptide is capable of catalyzing the NAD(P)H-dependent reduction of pyruvate to L- or D-lactate and / or the NAD(P)H-dependent reduction of a-ketoglutarate to 2- hydroxy glutarate.

28. The polypeptide of any one of claims 1-27, wherein the polypeptide has one or more of the following properties relative to a reference polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4: a) increased activity in the NAD(P)H-dependent conversion of 2-oxoglutarate to 2- hydroxy glutarate, optionally wherein the polypeptide has at least about 1.1 -fold, about 5-fold, about 10-fold, about 100-fold, or about 500-fold increased activity; b) increased activity7in catalyzing the oxidation of NAD(P)H to NAD(P)+; and / or26079 c) increased stability.

29. A polynucleotide encoding at least one polypeptide according to any one of claims 1-28, wherein the polynucleotide does not comprise SEQ ID NO: 1 or SEQ ID NO: 3.

30. The polynucleotide of claim 29, wherein the polynucleotide is codon-optimized.

31. A polynucleotide comprising at least 80% sequence identity to any one of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23.

25. 27, 29, or 31.

32. The polynucleotide of any one of claims 29-31, wherein the polynucleotide comprises any one of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, or 31.

33. An expression vector comprising at least one polynucleotide sequence of any one of claims 29-32.

34. The expression vector of claim 33, wherein the polynucleotide sequence is operably linked to a control sequence.

35. The expression vector of claim 34, wherein the control sequence is a promoter.

36. The expression vector of claim 35. wherein the promoter is a heterologous promoter.

37. A host cell comprising the polynucleotide of any one of claims 29-32 or the expression vector of any one of claims 33-36.

38. The host cell of claim 37, wherein the host cell is prokaryotic or eukaryotic.

39. A method of producing a polypeptide, the method comprising culturing the host cell of claim 37 or 38 under conditions such that the polypeptide encoded by the polynucleotide is produced.

40. The method of claim 39, further comprising the step of recovering the polypeptide.

41. The method of claim 40. further comprising the step of purifying the polypeptide.

42. A method of catalyzing the NAD(P)H-dependent reduction of 2-oxoglutarate to 2- hydroxyglutarate, the method comprising contacting the 2-oxoglutarate with a polypeptide of any one of claims 1-28.

43. The method of claim 42, further comprising isolating the 2 -hydroxy glutarate.

44. A method of catalyzing the oxidation of NAD(P)H to NAD(P)+, the method comprising contacting the NAD(P)H with a polypeptide of any one of claims 1-28.

45. The method of claim 44, wherein the method comprises contacting the NAD(P)H with a polypeptide of any one of claims 1-28 in the presence of 2-oxoglutarate.

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