Engineered imine reductase polypeptides and uses thereof

Abstract

The present disclosure provides polypeptides (e.g., IRED polypeptides), polynucleotides, expression vectors, and host cells comprising the same, methods of producing polypeptides (e.g., IRED polypeptides), and methods of catalyzing the reduction of an imine group.

Description

ENGINEERED IMINE REDUCTASE POLYPEPTIDES AND USES THEREOFFIELD

[0001] The present disclosure relates generally to engineered NAD(P)H-dependent imine reductase enzymes, useful in biocatalytic and synthetic processes involving, inter alia, selective imine reduction, as well as related polynucleotides, expression vectors, host cells, methods of production, and methods of catalyzing imine reduction.BACKGROUND OF THE INVENTION

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

[0003] Ketoreductases, also known as alcohol dehydrogenases, are enzymatic reducing agents, a specific class of enzymes that catalyze the selective reduction of ketones or aldehydes to alcohols. Enzymes belonging to the ketoreductase or carbonyl reductase class may be useful for the synthesis of optically active alcohols. Ketoreductase enzymes typically selectively convert a ketone or aldehyde substrate to the corresponding alcohol product, but these enzymes may also convert alcohols into the corresponding ketones or aldehydes in a reverse reaction. Enzymatic reduction of ketones and aldehydes requires participation of a co-factor that can act as an electron donor, while enzymatic oxidation of alcohols requires participation of a co-factor that can act as an electron acceptor. For example, the co-factor may be reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) for the reduction reaction, and nicotinamide adenine dinucleotide (NAD+) or nicotinamide adenine dinucleotide phosphate (NADP+) for the oxidation reaction. NADH and NADPH serve as electron donors, while NAD+ and NADP+ serve as electron acceptors.

[0004] Ketoreductase enzy mes are well known in nature, and numerous genes that encode ketoreductase enzymes and ketoreductase enzy me sequences have been reported. See, e.g., Candida magnoliae (Genbank Acc. No. JC7338; GI:11360538) Candida parapsilosis (Genbank Acc. No. 10 BAA24528.1; GL2815409), Sporobolomyces salmonicolor (Genbank Acc. No. AF 160799; GL6539734), and Rhodococcus erythropolis (Genbank Acc. No. AAN73270.1; GI: 34776951).

[0005] Ketoreductase enzymes are being used with increasing frequency to provide alternative synthetic pathways to key compounds. For example, Kosjek, B. et al. disclosed the asymmetric synthesis of a chiral precursor 4,4-dimethoxy-2H-pyran-3-ol with a ketoreductase. Organic Process Research & Development, 2008, 12. 584-588. When used, the ketoreductase enzymes may be provided as purified enzymes or as whole cells that express the desired ketoreductase. In view of their promise for improved synthetic pathways, there remains a need to identify additional ketoreductase enzymes that can be used to carry out certain chemical transformations on an industrial scale with increased efficiency and under non-physiological conditions.

[0006] Imine reductases (IREDs) are a class of enzymes that reduce imine functional groups to amines. For example, IREDs have been applied to the stereoselective synthesis of chiral or otherwise difficult to synthesize amines through asymmetric imine reduction and reductive amination. While amine dehydrogenases and transaminases have previously been exploited in the direct conversion of carbonyl-containing compounds to the corresponding chiral primary amines, only IREDs have been shown to catalyze reductive amination with multiple different amine nucleophiles. Asymmetric reductive amination requires high chemoselectivity to avoid direct reduction of the carbonyl starting material, and existing chemical catalyst systems that fulfill this requirement tend to be limited to a particular class of amine nucleophile, such as anilines or ammonia. There is a need in the art to identify additional imine reductase enzymes that can be used to carry out, under industrially applicable conditions, the synthesis of amines.SUMMARY

[0007] The present disclosure provides, inter alia, polypeptides (e.g., IRED polypeptides), polynucleotides encoding the same, expression vectors, host cells, methods of producing polypeptides, and methods of catalyzing the reduction of imine groups.

[0008] The present disclosure provides novel imine reductases useful in the selective oxidation of alcohols, including for example oxidation of primary alcohol side-chains of peptides. The imine reductases of the present disclosure may also be useful for catalyzing reactions in the process of generating macrocyclic peptides.

[0009] The present disclosure provides novel imine reductase enzymes useful in the selective reduction of imines, including for example to synthesize secondary amines. The imine reductase enzymes of the present disclosure may also be useful for catalyzing reactions in the process of generating macrocyclic peptides.

[0010] In some embodiments, the ketoreductases and imine reductase enzymes and reductase- catalyzed methods of producing provided herein are useful for production of macrocyclicpeptides, e.g., enlicitide. The structure of enlicitide is shown below as Formula 1. In some embodiments, A is decanoate.Formula 1

[0011] In one aspect, provided herein is an engineered polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 40, wherein the polypeptide comprises an amino acid substitution at one or more amino acid positions selected from 9, 32, 36, 37, 38, 39, 41, 47, 53, 54, 55, 57, 72, 74, 93, 102, 106, 108, 117, 123, 127, 129, 135, 136, 137,138, 139, 146, 156, 160, 164, 166, 174, 183, 184, 194, 196, 197, 198, 200, 207, 210, 211, 214,218, 220, 224, 226, 227, 229, 230, 232, 233, 235, 239, 240, 241, 242, 243, 244, 258, 264, 265,266, 267. 269, 278, 279, 281, 283, 285, 286, 292, and 293 and / or at least one amino acid insertion between positions 240 and 241 or between positions 288 and 289. wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 40. In some embodiments, the engineered polypeptide has imine reductase activity. In some embodiments, the engineered polypeptide has NADH-dependent or NAD(P)H-dependent imine reductase activity.

[0012] In another aspect, provided herein is an engineered polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 38, wherein the polypeptide comprises at least one amino acid substitution at one or more amino acid positions selected from 9, 32, 36. 37. 38, 39, 41, 47, 53, 54, 55, 57, 72, 74, 93, 102, 106, 108. 117, 123, 127, 129, 135,136, 137. 138, 139, 146. 156, 160. 164, 166, 174. 183, 184. 194, 196. 197, 198, 200. 207, 210.211, 214, 218, 220, 224, 226, 227, 229, 230, 232, 233, 235, 239, 240, 241, 242, 243, 244, 258,264, 265, 266, 267, 269, 278, 279, 281, 283, 285, 286, 292, and 293, and / or at least one amino acid insertion between positions 240 and 241, and 288 and 289, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38. In some embodiments, the26081 engineered polypeptide has imine reductase activity. In some embodiments, the engineered polypeptide has NADH-dependent or NAD(P)H-dependent imine reductase activity.

[0013] In another aspect, provided herein is an engineered polypeptide comprising at least 98% sequence identity to any one of SEQ ID NO: 42, 44, 46. 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70. 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94. 96. 98. 136, 138. 140, 142, 144. 146, 148. 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the engineered polypeptide has imine reductase activity. In some embodiments, the engineered polypeptide has NADH-dependent or NAD(P)H-dependent imine reductase activity.

[0014] In another aspect, provided herein is a polynucleotide comprising at least 80% sequence identity to any one of SEQ ID NO: 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97. 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153. 155, 157, 159. 161, 163. 165, 167, 169, 171, 173. 175, 177. 179, 181, 183. 185, 187. 189, or 191, wherein the polynucleotide does not comprise the sequence of SEQ ID NO: 37 or SEQ ID NO: 39. In some embodiments, the engineered polypeptide has imine reductase activity. In some embodiments, the engineered polypeptide has NADH-dependent or NAD(P)H- dependent imine reductase activity.

[0015] Also provided herein is an expression vector comprising at least one of the polynucleotides disclosed herein.

[0016] 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.

[0017] Also provided herein is a method of producing a polypeptide, the method compnsing culturing any one of the host cells disclosed herein under conditions such that the polypeptide encoded by the polynucleotide is produced.

[0018] Also provided herein is a method of producing secondary amine 4 in the following Scheme B. which comprises reacting substrate 3 in the following Scheme B with the polypeptide of any one of claims 1-26, wherein Scheme B is26081

[0019] In some embodiments, the method further comprises recovering secondary amine 4. In some embodiments, the polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168. 170, 172, 174, 176, 178. 180, 182, 184, 186, 188, 190, or 192.

[0020] 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

[0021] The present disclosure relates to engineered ketoreductase (KRED) polypeptides. As is described in the Examples, the present disclosure provides engineered NAD(P)+-dependent KRED polypeptides derived from wild-type Krypidia tusciae KRED, which exhibit improved enzyme properties relative to wild-type Krypidia tusciae KRED, and which were discovered through iterative rounds of directed evolution as described herein. For example, the novel engineered KRED polypeptides of the disclosure may exhibit one or more of the following improvements relative to a reference polypeptide (e.g., wild-type Krypidia tusciae KRED polypeptide): enzyme activity (e.g., improved activity in catalyzing alcohol oxidation), regioselectivity, affinity for substrate and cofactor, chemoselectivity, enzyme expression and / or solubility’ in E. coli, soluble enzyme expression, solvent stability / tolerance, and / or thermal stability.

[0022] The engineered ketoreductases may be useful for catalyzing reactions in the process of generating macrocyclic peptides. For example, the KRED polypeptides of the disclosure may selectively oxidize primary alcohol side-chains of peptides that can be further used in the synthesis of macrocyclic peptides. The KRED polypeptides of the disclosure may, for example,exhibit increased activity on substrate 1 ((l1S,l2S.l3S,9S,12S)-9-amino-12-((l-(6-aminohexyl)-5- fluoro-lH-indol-3-yl)methyl)-N-((2S,3R)-3-hydroxy-l-(((S)-l-((S)-2-((4- (hydroxymethyl)phenethyl)carbamoyl)-2-methylpyrrolidin-l-yl)-3-(4-methoxyphenyl)-l- oxopropan-2-y l)amino)- 1 -oxobutan-2-y l)-4, 10,13-trioxo-2-oxa-5, 11 -diaza- 1 (3 , 1 )-py rrolidina- 7(l,3)-benzenacyclotridecaphane-l2-carboxamide) in Scheme A below to provide the aldehyde 2, which in turn can spontaneously and reversibly cyclize to yield the imine 3 in Scheme A below.Scheme A

[0023] The present disclosure also relates to polynucleotides and expression vectors encoding the KRED polypeptides of the present disclosure, host cells comprising the polynucleotides or expression vectors, methods of producing the KRED polypeptides, and methods of catalyzing alcohol oxidation.

[0024] The present disclosure also relates to engineered imine reductase (IRED) polypeptides. As is described in the Examples, the present disclosure provides engineered NAD(P)H-dependent IRED polypeptides derived from wild-type Pseudogymnoascus sp. VKM F-4516 IRED and which were discovered through iterative rounds of directed evolution as described herein. For example, the novel engineered IRED polypeptides of the disclosure may exhibit one or more of the following improvements relative to a reference polypeptide (e.g., wild-type Pseudogymnoascus sp. VKM F-4516 IRED polypeptide): enzyme activity (e.g., improved activity in catalyzing imine reduction), regioselectivity, affinity for substrate and cofactor, chemoselectivity, enzyme expression and / or solubility in E. coli, soluble enzy me expression, solvent tolerance / stability, and / or thermal stability.

[0025] The engineered imine reductases may be useful for catalyzing reactions in the process of generating macrocyclic peptides. For example, the NAD(P)H-dependent imine reductase enzymes described herein can catalyze reduction of imines formed from the condensation of nucleophilic amino acid residue side-chains and other electrophilic amino acid residue side-chains in peptides. The 1RED polypeptides of the disclosure may. for example, exhibit increased activity on substrate 3 to selectively provide the secondary amine 4 in Scheme B below.Scheme B

[0026] The present disclosure also relates to polynucleotides and expression vectors encoding the IRED polypeptides of the present disclosure, host cells comprising the polynucleotides or expression vectors, methods of producing the IRED polypeptides, and methods of catalyzing imine reduction.

[0027] In some embodiments, the KRED and IRED polypeptides disclosed herein may be useful in the half reaction shown in Schemes A and B above and / or in a single-pot reaction with a KRED generating the imine substrate in situ as shown, for example, in Scheme C below. The KRED polypeptides of the disclosure may work with either NADP+ or NAD+ and the IRED polypeptides of the disclosure may work with either NADPH or NADH.Scheme C

[0028] The evolved enzymes of the disclosure have the advantage of being able to catalyze selective reactions on a large scale for production purposes. The use of enzymes as a replacement for more traditional synthetic approaches reduces waste and the use of potentially harsh or dangerous chemical reagents.Definitions

[0029] 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.

[0030] 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.

[0031] 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”.

[0032] 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).

[0033] 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.

[0034] 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 the 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.

[0035] 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 ofthe 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.

[0036] 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.

[0037] “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 KRED polypeptide variants of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, and 130 were obtained by artificially evolving over multiple generations the gene encoding the wildtype Krypidia tusciae KRED enzyme of SEQ ID NO: 2 or the wild-type Krypidia tuscicie KRED enzyme with an added C-terminal His tag (SEQ ID NO: 4). Thus, the evolved KRED variant enzymes are “derived from” the Krypidia tusciae KRED enzyme of SEQ ID NO: 2 or SEQ ID NO: 4. In another example, the IRED polypeptide variants of SEQ ID NOs: 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 136, 138, 140. 142, 144, 146, 148, 150. 152, 154, 156, 158, 160, 162, 164. 166, 168, 170, 172, 174. 176, 178, 180, 182, 184, 186, 188, 190, or 192 were obtained by artificially evolving over multiple generations the gene encoding the wild-type Pseudogymnoascus sp. VKM F-4516 IRED enzyme (SEQ ID NO: 38) or the wild-ty pe Pseudogymnoascus sp. VKM F-4516 IRED enzyme with an added C-terminal His tag (SEQ ID NO: 40). Thus, the evolved IRED variant enzymes are “derived from” the Pseudogymnoascus sp. VKM F-4516 IRED of SEQ ID NO: 38 or SEQ ID NO: 40.

[0038] As used herein, “reference sequence” refers to a defined sequence used as a basis for a sequence comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotide or amino 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 is26081 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 .

[0039] “Comparison window” refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acids residues wherein a sequence may be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids and wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The comparison window can be longer than 20 contiguous residues, and includes, optionally 30, 40, 50, 100, or longer windows.

[0040] 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.

[0041] 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.

[0042] 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 an26081 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.

[0043] As used herein, the terms “amino acid’’ or “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.

[0044] 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).

[0045] “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 hy drophilic 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).

[0046] “Acidic amino acid” or “acidic residue” refers to a hydrophilic amino acid or residue having a side chain exhibiting a pK 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).

[0047] “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).

[0048] “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 in26081 which the pair of electrons shared in common by two atoms is held more closely by one of the atoms. Genetically encoded polar amino acids include L-Asn (N), L-Gln (Q), L-Ser (S), and L- Thr (T).

[0049] ‘‘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).

[0050] “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.

[0051] 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.

[0052] “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).

[0053] 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).

[0054] 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.26081

[0055] 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 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).

[0056] “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).

[0057] 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 aromatic side chains 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 a9[ 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 hydrophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively.

[0058] 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.

[0059] “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.26081

[0060] 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.

[0061] 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.

[0062] 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.

[0063] “Ammo acid difference” or “residue difference” refers to a change in the ammo acid residue at a position of a polypeptide sequence relative to the amino acid residue at a corresponding position in a reference sequence. The positions of amino acid differences generally are referred to herein as “n,” where n refers to the corresponding position in the reference sequence upon which the residue difference is based. For example, a “residue difference at position 25 as compared to SEQ ID NO: 2” refers to a change of the amino acid residue at the polypeptide position corresponding to position 25 of SEQ ID NO: 2. Furthermore, in some instances, a polypeptide of the present invention can include one or more amino acid differences relative to a reference sequence, which may be indicated by a list of the specified positions where changes are made relative to a reference sequence. The present invention includes engineered polypeptide sequences comprising one or more amino acid differences that include either or both conservative and non-conservative amino acid substitutions.26081

[0064] "Codon optimized” refers to changes in the codons of the polynucleotide encoding a protein to those preferentially used in a particular organism such that the encoded protein is efficiently expressed in the organism of interest. Although the genetic code is degenerate in that most amino acids are represented by several codons, called “synonyms” or “synonymous” codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome. In some embodiments, the polynucleotides encoding the KRED or IRED enzymes may be codon optimized for optimal production from the host organism selected for expression.

[0065] “Preferred,” “optimal,” and “high codon usage bias codons” refers interchangeably to codons that are used at higher frequency in the protein coding regions than other codons that code for the same amino acid. The preferred codons may be determined in relation to codon usage in a single gene, a set of genes of common function or origin, highly expressed genes, the codon frequency in the aggregate protein coding regions of the whole organism, codon frequency in the aggregate protein coding regions of related organisms, or combinations thereof. Codons whose frequency increases with the level of gene expression are typically optimal codons for expression. A variety’ of methods are known for determining the codon frequency (e.g., codon usage, relative synonymous codon usage) and codon preference in specific organisms, including multivariate analysis, for example, using cluster analysis or correspondence analysis, and the effective number of codons used in a gene.

[0066] 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, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 74, 75, 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).

[0067] “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 actual26081 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.

[0068] 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.

[0069] 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.

[0070] 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.26081

[0071] “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, SEQ ID NON, SEQ ID NO: 38. or SEQ ID NO: 40), although in some embodiments, the reference enzy me 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), expanded or altered optimal chemical conditions for enzymatic activity7, thermal stability' (i.e., thermostability), chemical stability', solvent stability', solvent tolerance, soluble expression. pH activity profile, cofactor requirements, refractoriness to inhibitors (e.g.. product inhibition), regioselectivity, chemoselectivity, stereospecificity, and stereoselectivity (including enantioselectivity).

[0072] “Increased enzy matic activity ” refers to an improved property of the enzy mes, w hich 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 activity are provided in the Examples. Any property relating to enzyme activity may be affected, including the classical enzyme properties of Km, N ax, or kc«r, changes of which can lead to increased enzymatic activity. Improvements in enzyme activity' can be at least about 1.1 times the enzy matic activity' of the corresponding wild-type enzyme, to as much as 2 times, 5 times, 10 times, 20 times, 25 times, 50 times, 75 times, 100 times, 150 times, 200 times, 500 times, 1000 times, 3000 times, 5000 times, 7000 times, 10,000 times or more enzymatic activity than the reference enzyme, e.g., a naturalty occurring enzyme or another enzyme from which the polypeptides were derived. The term “fold” is also used in relation to the enzymatic 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 enzy me 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 kc^ / Km, is generally about 108to 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 enzy me. Enzy me activity can be measured by any suitable approach, e.g., an enzy me activity'26081 assay or by any of the 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.

[0073] “Substrate” in the context of a biocatalyst mediated process refers to the compound or molecule acted on by the biocatalyst.

[0074] “Product” in the context of a biocatalyst mediated process refers to the compound or molecule resulting from the action of the biocatalyst.

[0075] As used herein, a “vector” is a polynucleotide (e.g., DNA) construct for introducing a polynucleotide (e.g., 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.

[0076] “Control sequence” is defined herein to include all components, which are necessary7or advantageous for the expression of a polynucleotide and / or polypeptide of the present invention. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. The control sequences may include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a polypeptide.

[0077] “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.26081

[0078] 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.

[0079] As used herein with respect to polypeptides, the terms “expression” and “production” 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.

[0080] 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.

[0081] 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 a KRED polypeptide or an IRED 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.

[0082] The term “analogue” means a polypeptide having more than 70% sequence identity but less than 100% sequence identity (e.g., more than 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 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 norvaline, 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.

[0083] “Coding sequence” refers to that portion of a polynucleotide (e.g., a gene) that encodes an amino acid sequence of a polypeptide.26081

[0084] As used herein, a designation of * in reference to a polypeptide or a position in an amino acid sequence indicates the introduction of a stop codon (i.e., UAA, UAG and UGA in RNA and TAA, TAG and TGA in DNA) in the polynucleotide sequence corresponding to the indicated position in the polypeptide.

[0085] “Naturally occurring’7or “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-type polypeptide or polynucleotide sequences as identified herein may include a tag, such as a histidine (His) tag. For example, the Krypidia tusciae KRED 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 Krypidia tusciae KRED herein. Similarly, Pseudogymnoascus sp. VKM F-4516 RED sequence (SEQ ID NO: 40), which includes a C-terminal His tag (and thus is not found in nature), may be referred to as a wild-type Pseudogymnoascus sp. VKM F-4516 IRED herein. Herein, “wild-type” polypeptide or polynucleotide sequences may be denoted “WT.”

[0086] 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 present 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.

[0087] 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.

[0088] “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 both26081 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.

[0089] 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 sequence 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).

[0090] 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 homology 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 (see26081 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.

[0091] “Stereoselectivity” refers to the preferential formation in a chemical or enzy matic reaction of one stereoisomer over another. Stereoselectivity' can be partial, where the formation of one stereoisomer is favored over the other, or it may be complete where only one stereoisomer is formed. When the stereoisomers are enantiomers, the stereoselectivity is referred to as enantioselectivity, the fraction (typically reported as a percentage) of one enantiomer in the sum of both. It is commonly alternatively reported in the art (typically as a percentage) as the enantiomeric excess (EE) calculated therefrom according to the formula [major enantiomer - minor enantiomer] / [major enantiomer + minor enantiomer]. Where the stereoisomers are diastereoisomers, the stereoselectivity is referred to as diastereoselectivity, the fraction (typically’ reported as a percentage) of one diastereomer in a mixture of two diastereomers, commonly alternatively reported as the diastereomeric excess (DE). Enantiomeric excess and diastereomeric excess are types of stereomeric excess.

[0092] “Chemoselectivity” refers to the preferential formation in a chemical or enzymatic reaction of one product over another.

[0093] “Regioselectivity” or “regioselective reaction” refers to a reaction in which one direction of bond making or breaking occurs preferentially over all other possible directions. Reactions can be completely (100%) regioselective if the discrimination is complete or partially regioselective (x %, for example, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95% but less than 100%) if the product of reaction at one site predominates over the product of reaction at other sites.

[0094] “Conversion” refers to the enzy matic 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.

[0095] “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 enzy me.26081

[0096] “Solvent stable’7or “solvent tolerant” or “cosolvent stable” or “cosolvent tolerant” refers to a polypeptide that maintains similar activity (more than e.g., 60% to 80%) after exposure to varying concentrations (e.g., 5-99%) of a solvent or cosolvent (ethanol, isopropyl alcohol, dimethylsulfoxide (DMSO), tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butyl acetate, methyl tert-butyl ether, etc.) for a period of time (e.g., 0.5-24 hrs) compared to the untreated enzyme.

[0097] As used herein, the terms “biocatalysis,” “biocatalytic,” “biotransformation,” and “biosynthesis” refer to the use of enzymes to perform chemical reactions on organic compounds.

[0098] 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.

[0099] The terms “isolated” and “purified” are used to refer to a molecule (e.g., an isolated nucleic acid or polypeptide) or another component that is removed from at least one other component with which it is naturally associated. The term “purified” does not require absolute purity, rather it is intended as a relative definition.

[0100] The terms “ketoreductase,” “KRED,” “KRED enzymes,” “KRED polypeptides,” and “alcohol dehydrogenase” are used interchangeably herein to refer to a polypeptide having a capability of catalyzing the NAD(P)H-dependent reduction of a ketone or aldehyde by converting NAD(P)H to NAD(P)+ and the ketone or aldehyde to alcohol, or of catalyzing the NAD(P)+- dependent oxidation of an alcohol group by converting NAD(P)+ to NAD(P)H and the alcohol to the corresponding ketone or aldehyde. For example, the KRED enzy mes disclosed herein are capable of catalyzing the NAD(P)H-dependent reduction of ketones or aldehydes to alcohols or NAD(P)+-dependent oxidation of alcohols into the corresponding ketones or aldehydes in a reverse reaction. “KRED polypeptide” as used herein includes naturally occurring (wild-type) KRED polypeptides as well as non-naturally occurring engineered polypeptides generated by human manipulation. In an embodiment, a KRED polypeptide as disclosed herein may have an amino acid sequence selected from any one of SEQ ID NO: 4, 6, 8. 10. 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 100, 102, 104, 106, 108, 110, 1 12, 114, 116, 118, 120, 122, 124, 126, 128, or 130.

[0101] The term “ketoreductase activity” is used to refer to an enzymatic activity in which an alcohol is converted to ketone or aldehyde in the presence of a co-factor such as NAD+ or NAD(P)+.

[0102] The terms “imine reductase,” “IRED,” “IRED enzymes,” and “IRED polypeptides” are used interchangeably herein to refer to a polypeptide having a capability7of catalyzing the26081 reduction of an imine group. For example, the 1RED enzymes disclosed herein are capable of catalyzing the NAD(P)H-dependent reduction of an imine group by converting NAD(P)H to NAD(P)+ and the imine to amine. For example, the IRED enzy mes disclosed herein are capable of catalyzing an NAD(P)H-dependent reaction by converting NADPH to NADP+ or a NAD(P)H-dependent reaction by converting NADH to NAD+. For example. IRED enzymes disclosed herein are capable of catalyzing the NAD(P)H-dependent reduction of an imine group to a primary or secondary amine. The IRED enzymes disclosed herein are also capable of catalyzing the NAD(P)+-dependent oxidation of an amine group by converting NAD(P)+ to NAD(P)H and the amine to the imine. “IRED polypeptide” as used herein includes naturally occurring (wild-type) IRED polypeptides as well as non-naturally occurring engineered polypeptides generated by human manipulation. In an embodiment, an IRED polypeptide as disclosed herein may have an amino acid sequence selected from any one of SEQ ID NO: 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68. 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96. 98, 136, 138. 140, 142, 144, 146, 148. 150, 152. 154, 156, 158, 160, 162. 164, 166. 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192.

[0103] The term “imine reductase activity” is used to refer to an enzy matic activity' in which an imine group is converted to a primary or secondary amine in the presence of co-factor such as NADH or NAD(P)H.

[0104] The term “NAD(P)H” as used herein refers to cofactor reduced nicotinamide adenine dinucleotide (NADH) and / or cofactor reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H). The term “NAD(P)+” refers to cofactor(s) NAD(P)+ and / or NAD+. As used herein, the term “cofactor” refers to a non-protein compound that operates in combination with a ketoreductase. Cofactors suitable for use with the engineered KRED or IRED enzymes described herein include, but are not limited to, NAD(P)+ (nicotinamide adenine dinucleotide phosphate), NAD(P)H (the reduced form of NAD(P)+), NAD+ (nicotinamide adenine dinucleotide) and NADH (the reduced form of NAD+). A cofactor regeneration system can optionally be used to regenerate NAD(P)H from the oxidized NAD(P)+ or to regenerated NAD(P)+ from the reduced 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.KRED Polypeptides

[0105] This disclosure provides polypeptides (e.g., KRED polypeptides) capable of catalyzing the oxidation of an alcohol group to the corresponding ketone or aldehyde. In some embodiments, the KRED polypeptides described herein are capable of catalyzing the NAD(P)+-26081 dependent oxidation of an alcohol-side chain to the corresponding ketone or aldehyde. In some embodiments, the KRED polypeptides described herein are capable of catalyzing the NAD(P)+- dependent oxidation of an alcohol-side chain of a peptide that can be further used in the synthesis of macrocyclic and semi-macrocyclic peptides. The KRED polypeptides of the disclosure may work with either NADP+ or NAD+.

[0106] In some embodiments, the KRED polypeptides of the disclosure are capable of catalyzing reactions in the process of generating macrocyclic peptides.

[0107] In some embodiments, the KRED polypeptides described herein may be useful in the preparation of compounds such as compound 2 or 3 in Scheme A above. For example, in some embodiments, the KRED polypeptides described herein are capable of catalyzing the reaction shown in Scheme A above.

[0108] In some embodiments, the KRED poly peptides disclosed herein may be useful in a half reaction, such as the one shown in Scheme A above and / or may be useful in a single-pot reaction along with an IRED polypeptide of the disclosure w here the KRED generates the imine substrate in situ as shown, for example, in Scheme C above.

[0109] In certain embodiments, a polypeptide (e.g., a KRED 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.

[0110] In some embodiments, the KRED polypeptides described herein are a product of directed evolution from a wild-ty pe KRED sequence, which was itself identified by screening a panel of wild-type KRED polypeptides (i.e., a w ild-type KRED polypeptide as set forth in SEQ ID NO: 4, which includes a C-termmal His tag or a wild-type KRED polypeptide as set forth in SEQ ID NO: 2, which lacks a C-terminal His tag).

[0111] Enzyme properties for which improvements are desirable include, but are not limited to, enzymatic activity, expanded or altered optimal chemical conditions for enzymatic activity, thermal stability, substrate scope, chemical stability, pH activity profile, cofactor requirements, cofactor selectivity7, refractoriness to inhibitors (e.g., product inhibition), chemoselectivity, regioselectivity, stereospecificity7, stereoselectivity, solvent tolerance, and solvent stability7. The improvements can relate to a single enzy me property7, such as enzymatic activity, or a combination of different enzyme properties, such as enzymatic activity and thermal stability.

[0112] In some embodiments, the polypeptide (e.g., the KRED polypeptide) of the disclosure may demonstrate one or more improvements relative the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4, including, but not limited to, increases in enzy matic activity, enzyme expressionand / or solubility in E. colt, soluble enzyme expression, thermal stability, cofactor selectivity and affinity, regioselectivity, chemoselectivity, and / or solvent stability. In some embodiments, the polypeptide (e.g., the KRED 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, enzyme expression and / or solubility in E. coll, soluble enzyme expression, thermal stability', cofactor selectivity and affinity, regioselectivity, chemoselectivity, and / or solvent stability.

[0113] For example, in some embodiments, the polypeptide has one or more of the following properties relative to a reference polypeptide: a) increased activity in catalyzing the NAD(P)+- dependent oxidation of an alcohol group to the corresponding ketone or aldehyde; b) increased activity' in catalyzing the reduction of NAD(P)+ to NAD(P)H; c) increased thermostability; d) increased solvent tolerance / stability; e) increased enzyme expression and / or solubility; f) increased soluble enzyme expression; and / or g) increased substrate selectivity and affinity. In some embodiments, the polypeptide has the following properties relative to a reference polypeptide: a) increased activity in catalyzing the NAD(P)+-dependent oxidation of an alcohol group to the corresponding ketone or aldehyde, and b) increased activity in catalyzing the reduction of NAD(P)+ to NAD(P)H. In some embodiments, the polypeptide has the following properties relative to a reference polypeptide: a) increased activity in catalyzing the NAD(P)+- dependent oxidation of an alcohol group to the corresponding ketone or aldehyde; and b) increased thermostability'. In some embodiments, the polypeptide has the following properties relative to a reference polypeptide: a) increased activity in catalyzing the NAD(P)+-dependent oxidation of an alcohol group to the corresponding ketone or aldehyde; b) increased activity in catalyzing the reduction of NAD(P)+ to NAD(P)H; and c) increased thermostability. In some embodiments, the polypeptide has one or more of the following properties relative to a reference polypeptide: a) increased activity in catalyzing reaction of Scheme A above; b) increased activity in catalyzing the reduction of NAD(P)+ to NAD(P)H; and / or c) increased thermostability. In some embodiments, the polypeptide exhibits increased activity in catalyzing the NAD(P)+- dependent oxidation of a primary alcohol to the corresponding ketone or aldehyde. 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 is any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22. 24. 26, 28, 30, 32, 34, 36, 100, 102, 104, 106, 108, 110. 112, 114, 116, 118, 120, 122, 124, 126, 128, or 130. In some embodiments, the reference polypeptide is any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34. In someembodiments, the reference polypeptide is any one of SEQ ID NO: 2, 100, 102, 104, 106, 108. 110, 112, 114, 116, 118, 120, 122, 124, 126, or 128.

[0114] In some embodiments, the KRED 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 KRED 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 25- fold, about 50- fold, about 100- fold, about 150- fold, about 200- fold, about 400- fold, about 500-fold, about 700-fold, about 800-fold, about 900-fold, about 1000-fold, about 5000-fold, about 10.000-fold, or more than about 10,000-fold increased compared to the rate exhibited by the polypeptides of SEQ ID NO: 2 or SEQ ID NO: 4.

[0115] In some embodiments, the KRED polypeptides of the disclosure may demonstrate increased activity' in catalyzing the NAD(P)+-dependent oxidation of an alcohol group to the corresponding ketone or aldehyde, 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 25- fold, about 50- fold, about 100- fold, about 150- fold, about 200- fold, about 400- fold, about 500-fold, about 700-fold, about 800-fold, about 900-fold, about 1000-fold, about 5000-fold, about 10,000-fold, or more than about 10,000-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 KRED polypeptides of the disclosure may demonstrate increased activity in catalyzing the NAD(P)+- dependent oxidation of an alcohol group to the corresponding ketone or aldehyde, wherein the polypeptide has at least 1.1-fold, about 5-fold, about 10-fold, about 100-fold, or about 500-fold increased activity’ relative to a reference polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

[0116] In some embodiments, the KRED polypeptides of the disclosure may demonstrate increased activity in catalyzing the reduction of NAD(P)+ to NAD(P)H, 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 25- fold, about 50- fold, about 100- fold, about 150- fold, about 200- fold, about 400- fold, about 500-fold, about 700-fold, about 800-fold, about 900-fold, about 1000- fold, about 5000-fold, about 10,000-fold, or more than about 10,000-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 KRED polypeptides of the disclosure may demonstrate increased activity in catalyzing the reduction of NAD(P)+ to NAD(P)H, wherein the polypeptide has at least 1.1-fold, about 5-fold, about 10-fold, about 100-fold, or about 500-fold increased activity relative to a reference polypeptide comprising the amino acid sequence of SEQ ID NO: 226081 or SEQ ID NO: 4. For example, the KRED polypeptides of the disclosure may demonstrate increased activity in catalyzing the reduction of NAD(P)+ to NAD(P)H in the presence of an appropriate substrate (e.g., the substrates disclosed herein).

[0117] In some embodiments, the KRED polypeptides of the disclosure may demonstrate increased activity in catalyzing the reaction outlined in Scheme A above, 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 25- fold, about 50- fold, about 100- fold, about 150- fold, about 200- fold, about 400- fold, about 500-fold, about 700-fold, about 800-fold, about 900-fold, about 1000- fold, about 5000-fold, about 10,000-fold, or more than about 10,000-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 KRED polypeptides of the disclosure may demonstrate increased activity' in catalyzing the reaction outlined in Scheme A above, wherein the polypeptide has at least 1.1-fold, about 5-fold, about 10-fold, about 100-fold, or about 500-fold increased activity relative to a reference poly peptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

[0118] In some embodiments, the KRED polypeptides of the disclosure may demonstrate increased solvent 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, about 5000-fold, about 10,000-fold, or more than about 10,000- 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 KRED polypeptides of the disclosure may demonstrate increased solvent 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.

[0119] In some embodiments, the KRED polypeptides of the disclosure have been improved for thermostability. In some embodiments, the KRED polypeptides of the disclosure have been improved for thermostability and can maintain at least 50% enzymatic activity at temperatures of at least about 35 °C, 36 °C, 37°C, 38 °C, 39°C, 40 °C, 41 °C, 42°C, 43°C, 50°C or more degrees compared to a reference polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the KRED polypeptides of the disclosure can maintain at26081 least about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or more enzymatic activity at temperatures of at least about 35 °C, 36 °C, 37°C, 38 °C, 39°C, 40 °C, 41 °C, 42°C, 43°C, 50°C or more degrees compared to a reference polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 2 or SEQ ID NO: 4 when subjected to a heat challenge test for about 10, 20, 30, 40, 50, or 60 minutes. Thermostability may be assessed using any suitable approach, e.g., isothermal titration calorimetry (ITC).

[0120] In some embodiments, the KRED polypeptides of the disclosure may demonstrate increased enzyme expression and / or solubility, 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, about 5000-fold, about 10,000-fold, or more than about 10,000-fold increase in enzyme expression and / or solubility relative to a reference polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

[0121] In some embodiments, the KRED polypeptides of the disclosure may demonstrate increased soluble enzyme expression, 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, about 5000-fold, about 10,000-fold, or more than about 10,000-fold increase in soluble enzyme expression relative to a reference polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

[0122] In some embodiments, the KRED polypeptides have been improved for cofactor selectivity and / or affinity. In some embodiments, the KRED polypeptides may demonstrate increased selectivity and / or affinity for NADP+ cofactor over NAD+ cofactor relative to a reference polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

[0123] In some embodiments, a polypeptide (e.g., KRED polypeptide) of the disclosure is a polypeptide that comprises an amino acid sequence having at least 85% but less than 100% (e.g., 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., KRED polypeptide) of the disclosure is a polypeptide that comprises an amino26081 acid sequence having at least 85% but less than 100% (e.g., 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, wherein the polypeptide does not comprise the amino acid sequence of SEQ ID NO: 2.

[0124] 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 some embodiments, the ammo acid sequence difference(s) is / are conservative amino acid substitution(s). In other embodiments, the amino acid sequence difference(s) is / are non- conservative amino acid substitution(s). In some embodiments, the amino acid sequence differences are a combination of non-conservative and conservative amino acid substitutions. In some embodiments, the ammo acid sequence differences are insertions. In some embodiments, the amino acid sequence differences are deletions.

[0125] In some embodiments, provided herein is a polypeptide (e.g., a KRED polypeptide) comprising an amino acid sequence having at least 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 or SEQ ID NO: 4. In other words, provided herein is a polypeptide (e.g., a KRED polypeptide) comprising an amino acid sequence having at least 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 or SEQ ID NO: 4, wherein the polypeptide does not comprise SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., a KRED polypeptide) comprising an amino acid sequence having at least 85% but less than 100% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., a KRED polypeptide) comprising an amino acid sequence having at least 90% but less than 100% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., a KRED polypeptide) comprising an amino acid sequence having at least 91% but less than 100% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., a KRED polypeptide) comprising an amino acid sequence having at least 92% but less than 100% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., a KRED polypeptide) comprising an amino acid sequence having at least 93% but less than 100% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is26081 a polypeptide (e.g., a KRED polypeptide) comprising an amino acid sequence having at least 94% but less than 100% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., a KRED polypeptide) comprising an amino acid sequence having at least 95% but less than 100% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., a KRED polypeptide) comprising an amino acid sequence having at least 96% but less than 100% sequence identity’ to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., a KRED polypeptide) comprising an amino acid sequence having at least 97% but less than 100% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., a KRED polypeptide) comprising an amino acid sequence having at least 98% but less than 100% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, provided herein is a polypeptide (e.g., a KRED polypeptide) comprising an amino acid sequence having at least 99% but less than 100% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 4.

[0126] In some embodiments, provided herein is a polypeptide (e g., a KRED polypeptide) comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 4, wherein the polypeptide comprises at least one amino acid substitution at one or more positions selected from 2, 16, 18, 37, 38, 39, 42, 45, 57, 93, 95, 96, 106, 110, 120, 141, 143, 144, 145, 148, 150, 152, 157, 168, 171, 194, 195, 206, 217, 218, 246, and 251, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2 or SEQ ID NO: 4.

[0127] In some embodiments, the polypeptide comprises any number from 2 to 32 amino acid substitutions, each amino acid substitution at an amino acid position selected from 2, 16, 18, 37, 38, 39, 42, 45, 57, 93, 95, 96, 106, 1 10, 120, 141, 143, 144, 145, 148, 150, 152, 157, 168, 171, 194, 195, 206, 217, 218, 246, and 251, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2 or SEQ ID NO: 4.

[0128] In some embodiments, the polypeptide comprises one of the following substitutions or substitution sets: a) an amino acid substitution at position 148; b) an amino acid substitution set at positions 148, 157, and 246; c) an amino acid substitution set at positions 57, 141, 148, 157, 168, and 246; d) an amino acid substitution set at positions 38, 39, 42, 57, 141, 148, 157, 168, and 246; e) an amino acid substitution set at positions 16, 37, 38, 39, 42, 57, 141, 148, 157, 168, and 246; f) an amino acid substitution set at positions 16, 37, 38, 39, 42, 57, 93, 141, 143, 144, 148, 157, 168, 194, 195, and 246; g) an amino acid substitution set at positions 2, 16, 37, 38, 39, 42, 57, 93, 141, 143, 144, 148, 157, 168, 194, 195, and 246; h) an amino acid substitution set at26081 positions 57, 96, 106, 141, 144, 148, 157, 168, 194, and 246; i) an amino acid substitution set at positions 57, 93, 96, 106, 141, 144, 148, 157, 168, 194, and 246; j) an amino acid substitution set at positions 57, 93, 95, 96, 106, 141, 144, 148, 157, 168, 194, 195, 217, and 246; k) an amino acid substitution set at positions 18, 57, 93, 95, 96, 106, 141, 144, 148, 157, 168, 194, 195, 217, and 246; 1) an amino acid substitution set at positions 18, 57, 93, 95, 96, 106. 141, 143, 144, 148, 152, 157, 168, 171, 194, 195, 206, 217, 246, and 251; m) an amino acid substitution set at positions 18, 45, 57, 93, 95, 96, 106, 110, 141, 143, 144, 145, 148, 152, 157, 168, 171, 194, 195, 206, 217, 218, 246, and 251; n) an amino acid substitution set at positions 18, 45, 57. 93, 95, 96, 106, 110. 141, 143, 144, 145, 148. 150, 152, 157, 168, 171, 194, 195. 206, 217, 218, 246, and 251; or o) an amino acid substitution set at positions 18, 45, 57, 93, 95, 96, 106, 110, 120, 141, 143, 144, 145, 148, 150, 152, 157, 168, 171, 194, 195, 206, 217, 218, 246, and 251 ; wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the polypeptide comprises an amino acid substitution at position 148, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the polypeptide comprises an amino acid substitution set at positions 148, 157, and 246, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the polypeptide comprises an amino acid substitution set at positions 57, 141, 148, 157, 168, and 246, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the polypeptide comprises an amino acid substitution set at positions 38, 39, 42, 57, 141, 148, 157, 168, and 246, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the polypeptide comprises an amino acid substitution set at positions 16, 37, 38, 39, 42, 57, 141, 148, 157, 168, and 246, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the polypeptide comprises an amino acid substitution set at positions 16, 37, 38, 39. 42, 57, 93, 141, 143. 144, 148. 157, 168, 194, 195, and 246. wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the polypeptide comprises an amino acid substitution set at positions 2, 16, 37, 38, 39, 42, 57, 93, 141, 143, 144, 148, 157, 168, 194, 195, and 246, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the polypeptide comprises an amino acid substitution set at positions 57, 96, 106, 141, 144, 148, 157, 168, 194, and 246, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the26081 polypeptide comprises an amino acid substitution set at positions 57, 93, 96, 106, 141, 144, 148, 157, 168, 194, and 246, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the polypeptide comprises an amino acid substitution set at positions 57, 93, 95, 96, 106, 141, 144, 148, 157, 168, 194, 195, 217, and 246, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the polypeptide comprises an amino acid substitution set at positions 18, 57, 93, 95, 96, 106, 141, 144, 148, 157, 168, 194, 195, 217, and 246, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the polypeptide comprises an amino acid substitution set at positions 18, 57, 93, 95, 96, 106, 141, 143, 144, 148, 152, 157, 168, 171,194, 195, 206, 217, 246, and 251, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the polypeptide comprises an amino acid substitution set at positions 18, 45, 57, 93, 95, 96, 106, 110, 141, 143. 144, 145, 148. 152, 157. 168, 171, 194, 195, 206. 217, 218. 246, and 251 , wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the polypeptide comprises an amino acid substitution set at positions 18, 45, 57, 93, 95, 96, 106. 110, 141, 143, 144, 145, 148, 150, 152, 157, 168, 171, 194,195, 206. 217, 218, 246, and 251, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the polypeptide comprises an amino acid substitution set at positions 18, 45, 57, 93, 95, 96, 106, 110, 120, 141, 143, 144, 145, 148, 150, 152, 157, 168, 171, 194, 195, 206, 217, 218, 246, and 251, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2 or SEQ ID NO: 4.

[0129] In some embodiments, the polypeptide comprises one or more amino acid substitutions or amino acid substitution sets set forth in Table 1 or Table 2.

[0130] In some embodiments, the polypeptide comprises one of the following amino acid substitutions or amino acid substitution sets: a) a substitution at position G148L; b) a substitution set at positions G148L, A157V, and Y246L; c) a substitution set at positions T57V, S141G, G148L, A157V, T168V, and Y246L; d) a substitution set at positions V38R, L39S, E42A, T57V, S141G, G148L, A157V, T168V, and Y246L; e) a substitution set at positions R16S, D37G, V38R, L39S. E42A, T57V, S141G, G148L, A157V, T168V, and Y246L; I) a substitution set at positions R16S, D37G, V38R, L39S, E42A, T57V, I93C, S141G, I143V, Y144W, G148L, A157V, T168V, A194D, G195K, and Y246L; g) a substitution set at positions E2D, R16S, D37G, V38R, L39S, E42A, T57V, I93C, S141G, I143V, Y144W, G148L, A157V, T168V,26081A194D, G195K. and Y246L; h) a substitution set at positions T57V, M96Y, I106Q, S141G. Y144W, G148L, A157V, T168V, A194D, and Y246L; i) a substitution set at positions T57V, I93H, M96H, I106Q, S141G, Y144W, G148L, A157V, T168V, A194D, and Y246L; j) a substitution set at positions T57V, I93H, A95K, M96H, I106Q, S141G, Y144W, G148L, A157V, T168V, A194D, G195K. P217T, and Y246L; k) a substitution set at positions Q18L. T57V, I93H, A95K, M96H, I106Q, S141G, Y144W, G148L, A157V, T168V, A194D, G195K, P217T, and Y246L; 1) a substitution set at positions Q18L, T57V, I93H, A95K, M96H, I106Q, S141G, I143V, Y144W, G148L, A152G, A157V, T168V, V171I, A194D, G195K, N206H, P217T, Y246L, and E251N; m) a substitution set at positions Q18L, T45G. T57V, I93H. A95K, M96H, I106Q, V110T, S141G, I143V, Y144W, G145A, G148L, A152G, A157V, T168V, V171I, A194D, G195K, N206H, P217T, E218Q, Y246L, and E251N; n) a substitution set at positions Q18L, T45G, T57V, I93H, A95K, M96H, I106Q, V110T, S141G, I143V, Y144W, G145A, G148L, A152G, A157V. T168V, V171I, A194D, G195P. N206H, P217T, E218Q, Y246L, and E251N; o) a substitution set at positions Q18L, T45G, T57V, I93L, A95K, M96H, I106Q, VI 10T, S141G, I143V, Y144W, G145A, G148T, G150A, A152G, A157V, T168V, V171I, A194D, G195P, N206H, P217T, E218Q, Y246L, and E251N; or p) a substitution set at positions Q18L, T45G, T57V, I93L, A95K, M96H, I106Q, V110T, L120I, S141G, I143V. Y144W, G145A, G148T, G150A. A152G, A157V. T168V, V171I, A194D, G195P. N206H, P217T, E218Q, Y246L, and E251N; wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the polypeptide comprises a G148L substitution, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the polypeptide comprises G148L, Al 57V, and Y246L substitutions, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the polypeptide comprises T57V, S141G, G148L, A157V, T168V, and Y246L substitutions, w herein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the polypeptide comprises V38R, L39S, E42A, T57V, S141G, G148L, A157V, T168V, and Y246L substitutions, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the polypeptide comprises R16S, D37G, V38R, L39S, E42A, T57V, S141G, G148L, A157V, T168V, and Y246L substitutions, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the polypeptide comprises R16S, D37G, V38R, L39S, E42A, T57V, I93C, S141G, I143V, Y144W, G148L, A157V, T168V, A194D, G195K, and Y246L substitutions,26081 wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the polypeptide comprises E2D, R16S, D37G, V38R, L39S, E42A, T57V, I93C, S141G, I143V, Y144W, G148L, A157V, T168V, A194D, G195K, and Y246L substitutions, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the polypeptide comprises T57V, M96Y, I106Q, S141G, Y144W, G148L, A157V, T168V, A194D, and Y246L substitutions, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the polypeptide comprises T57V, I93H, M96H, I106Q, S141G. Y144W, G148L, A157V, T168V, A194D, and Y246L substitutions, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the polypeptide comprises T57V, I93H, A95K, M96H, I106Q, S141G, Y144W, G148L, A157V, T168V, A194D, G195K, P217T, and Y246L substitutions, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the polypeptide comprises Q18L, T57V, I93H, A95K, M96H, I106Q, S141G, Y144W, G148L, A157V, T168V, A194D, G195K, P217T, and Y246L substitutions, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the polypeptide comprises Q18L, T57V, I93H, A95K, M96H, I106Q, S141G, I143V, Y144W, G148L, A152G, A157V, T168V, V171I, A194D, G195K, N206H, P217T, Y246L, and E251N substitutions, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the polypeptide comprises Q18L, T45G, T57V, I93H, A95K, M96H, I106Q, V110T, S141G, I143V, Y144W, G145A, G148L, A152G, A157V. T168V. V1711. A194D, G195K. N206H, P217T, E218Q. Y246L. and E251N substitutions, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the polypeptide comprises Q18L, T45G, T57V, I93H, A95K, M96H, I106Q, V110T, S141G, I143V, Y144W, G145A, G148L, A152G, A157V, T168V. V171I. A194D, G195P, N206H, P217T, E218Q, Y246L. and E251N substitutions, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the polypeptide comprises Q18L, T45G, T57V, I93L, A95K, M96H, I106Q, V110T, S141G, I143V, Y144W, G145A, G148T, G150A, A152G, A157V, T168V, V171I, A194D, G195P, N206H, P217T, E218Q. Y246L, and E251N substitutions, wherein the ammo acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the polypeptide comprises Q18L, T45G, T57V, I93L, A95K, M96H, I106Q, V110T, L120I, S141G, I143V, Y144W,26081G145A, G148T. G150A. A152G, A157V. T168V, V171I, A194D, G195P. N206H, P217T, E218Q, Y246L, and E251N substitutions, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2 or SEQ ID NO: 4.

[0131] In some embodiments, the amino acid sequence comprises any one of SEQ ID NOs: 6, 8, 10, 12. 14. 16. 18. 20. 22, 24, 26, 28, 30, 32, 34, 36, 100. 102, 104. 106, 108, 110, 112, 114. 116, 118, 120, 122, 124, 126, 128, or 130. In some embodiments, the amino acid sequence consists of any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, or 130. In some embodiments, the amino acid sequence comprises any one of SEQ ID NO: 6. 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36. In some embodiments, the amino acid sequence comprises any one of SEQ ID NO: 100, 102, 104, 106, 108, 110, 112, 114, 1 16, 118, 120, 122, 124, 126, 128, or 130.

[0132] In some embodiments, provided herein is a polypeptide (e.g., a KRED polypeptide) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, or 130, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

[0133] In some embodiments, provided herein is a polypeptide (e.g., a KRED polypeptide) comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 100, 102, 104, 106, 108, 110, 112, 114, 116. 118, 120, 122, 124, 126. 128, or 130, 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., a KRED polypeptide) comprising an amino acid sequence having at least 92% sequence identity to any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, or 130, 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., a KRED polypeptide) comprising an amino acid sequence having at least 93% sequence identity to any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120. 122, 124, 126, 128, or 130, 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., a KRED polypeptide) comprising an amino acid sequence having at least 94% sequence identity' to any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 100, 102, 104,26081106, 108. 110, 112, 114. 116, 118. 120, 122, 124. 126, 128. or 130. 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., a KRED polypeptide) comprising an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28. 30, 32, 34, 36, 100, 102, 104, 106. 108, 110. 112, 114, 116. 118, 120. 122, 124, 126, 128, or 130, 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., a KRED polypeptide) comprising an amino acid sequence having at least 96% sequence identity to any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22. 24. 26. 28, 30, 32, 34, 36, 100, 102, 104, 106. 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, or 130, 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., a KRED polypeptide) comprising an amino acid sequence having at least 97% sequence identity to any one of SEQ ID NOs: 6. 8, 10, 12, 14, 16, 18. 20, 22, 24, 26, 28, 30, 32, 34. 36, 100, 102. 104, 106, 108, 110, 112. 114, 116. 118, 120, 122, 124, 126. 128, or 130, 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., a KRED polypeptide) comprising an amino acid sequence having at least 98% sequence identity to any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36. 100, 102, 104, 106, 108. 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, or 130, 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., a KRED polypeptide) comprising an amino acid sequence having at least 99% sequence identity to any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28. 30. 32. 34, 36, 100, 102. 104, 106, 108. 110, 112, 114. 116, 118. 120, 122, 124. 126, 128. or 130. 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., a KRED polypeptide) comprising the amino acid sequence of any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24. 26, 28, 30, 32, 34, 36, 100, 102, 104, 106, 108. 110, 112, 114. 116, 118. 120, 122, 124, 126, 128. or 130. Also provided herein is a polypeptide (e.g., a KRED polypeptide) consisting essentially of the amino acid sequence of any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, or 130. Also provided herein is a polypeptide (e.g., a KRED polypeptide) consisting of the amino acid sequence of any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, or 130.26081

[0134] In some embodiments, provided herein is a polypeptide (e.g., a KRED 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 or SEQ ID NO: 122, 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., a KRED polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 28 or SEQ ID NO: 122, 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., a KRED polypeptide) comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 28 or SEQ ID NO: 122, 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., a KRED polypeptide) comprising the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 122. Also provided herein is a polypeptide (e.g., a KRED polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 122. Also provided herein is a polypeptide (e.g., a KRED polypeptide) consisting of the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 122.

[0135] In some embodiments, provided herein is a polypeptide (e.g., a KRED 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 or SEQ ID NO: 124, 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., a KRED polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 30 or SEQ ID NO: 124, 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., a KRED polypeptide) comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 30 or SEQ ID NO: 124, 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., a KRED polypeptide) comprising the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 124. Also provided herein is a polypeptide (e.g., a KRED polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 124. Also provided herein is a polypeptide (e.g., a KRED polypeptide) consisting of the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 124.

[0136] In some embodiments, provided herein is a polypeptide (e.g., a KRED polypeptide) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,2608198%, 99%, or higher sequence identity to SEQ ID NO: 32 or SEQ ID NO: 126, 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., a KRED polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 32 or SEQ ID NO: 126, 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., a KRED polypeptide) comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 32 or SEQ ID NO: 126, 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., a KRED polypeptide) comprising the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 126. Also provided herein is a polypeptide (e.g., a KRED polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 126. Also provided herein is a polypeptide (e.g., a KRED polypeptide) consisting of the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 126.

[0137] In some embodiments, provided herein is a polypeptide (e g., a KRED 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: 34 or SEQ ID NO: 128, 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., a KRED polypeptide) comprising an amino acid sequence having at least 98% sequence identity’ to SEQ ID NO: 34 or SEQ ID NO: 128, 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., a KRED polypeptide) comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 34 or SEQ ID NO: 128, 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., a KRED polypeptide) comprising the amino acid sequence of SEQ ID NO: 34 or SEQ ID NO: 128. Also provided herein is a polypeptide (e.g.. a KRED polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 34 or SEQ ID NO: 128. Also provided herein is a polypeptide (e.g., a KRED polypeptide) consisting of the amino acid sequence of SEQ ID NO: 34 or SEQ ID NO: 128.

[0138] In some embodiments, provided herein is a polypeptide (e.g., a KRED 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: 36 or SEQ ID NO: 130, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some26081 embodiments, provided herein is a polypeptide (e.g., a KRED polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 36 or SEQ ID NO: 130, 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., a KRED polypeptide) comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 36 or SEQ ID NO: 130, 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., a KRED polypeptide) comprising the amino acid sequence of SEQ ID NO: 36 or SEQ ID NO: 130. Also provided herein is a polypeptide (e.g., a KRED polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 36 or SEQ ID NO: 130. Also provided herein is a polypeptide (e.g., a KRED polypeptide) consisting of the amino acid sequence of SEQ ID NO: 36 or SEQ ID NO: 130.

[0139] 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: 131). 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 consisting of SEQ ID NO: 131 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 some 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 LEHHHHHH (SEQ ID NO: 132). In some embodiments the His tag and the linker comprise the amino acid sequence of LNHHHHHH (SEQ ID NO: 198).

[0140] For example, a KRED 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: 4-36 may be modified to no longer include a C-terminal His tag having the amino acid sequence26081 of SEQ ID NO: 131. SEQ ID NO: 132, or SEQ ID NO: 198. For example, the polypeptide sequence of any one of even numbered SEQ ID NO: 100-130 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: 131, SEQ ID NO: 132, or SEQ ID NO: 198.

[0141] In some embodiments, the polypeptide is isolated.

[0142] In some embodiments, the polypeptide has ketoreductase activity.

[0143] In some embodiments, the polypeptide has NAD(P)+- dependent or NAD+-dependent ketoreductase activity'.

[0144] In some embodiments, the polypeptide has NAD(P)+- dependent ketoreductase activity.

[0145] In some embodiments, the polypeptide has NAD+-dependent ketoreductase activity.

[0146] 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.

[0147] In addition to the positions of residue differences specified above, any of the engineered KRED 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 in above and in Tables 1 and 2, e.g., residue positions other than 2, 16, 18, 37, 38, 39, 42, 45, 57, 93, 95, 96, 106, 110, 120, 141, 143, 144, 145, 148, 150, 152, 157, 168, 171, 194, 195, 206, 217. 218, 246, and 251. 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 KRED polypeptides selected from SEQ ID NOs: 6. 8, 10, 12, 14. 16. 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 100, 102, 104, 106. 108, 110, 112. 114, 116. 1 18, 120, 122. 124, 126. 128, or 130, 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, 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, 30, 35, 40, 45 or 50 residue positions. 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, 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: 4.26081

[0148] In some embodiments, the engineered KRED polypeptide can comprise a deletion at one or more amino acid positions as compared to any one of the engineered KRED polypeptides described herein, such as the exemplary7engineered polypeptides of SEQ ID NOs: 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122. 124, 126, 128. or 130. Thus, for each embodiment of the engineered KRED polypeptides of the invention, 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 10% 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 KRED polypeptides, where the associated functional activity and / or improved properties of the engineered KRED 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, 30, 35, 40, 45, or 50 amino 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.

[0149] In some embodiments, the engineered KRED polypeptide herein can have an amino acid sequence comprising an amino acid insertion as compared to any one of the engineered KRED polypeptides described herein, such as the exemplary7engineered polypeptides of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22. 24. 26, 28, 30, 32, 34, 36, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, or 130. 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 KRED described herein is maintained. The insertions can be to amino or carboxy terminus, or internal portions of the KRED polypeptide.

[0150] In some embodiments, the engineered KRED 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, 26, 28, 30, 32, 34, 36, 100, 102, 104, 106, 108, 110, 1 12, 114, 1 16, 118, 120, 122, 124, 126, 128, or 130, 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 has26081 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, 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, 1 1, 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.

[0151] Also provided herein are compositions comprising any of the KRED polypeptides disclosed herein. For example, the composition may include an effective amount of the KRED polypeptide for catalyzing the NAD(P)+ -dependent oxidation of an alcohol group to the corresponding ketone or aldehyde. In some embodiments, the composition may include an effective amount of the KRED polypeptide for catalyzing the NAD(P)+ -dependent oxidation of a primary alcohol to the corresponding ketone or aldehyde. In some embodiments, the composition may include an effective amount of the KRED polypeptide for catalyzing the NAD(P)+-dependent oxidation of an alcohol-side chain of a peptide that can be further used in the synthesis of macrocyclic and semi-macrocyclic peptides. In some embodiments, the composition may include an effective amount of the KRED polypeptide for catalyzing reactions in the process of generating macrocyclic peptides. The composition may include one or more carriers or diluents.IRED Polypeptides

[0152] This disclosure provides polypeptides (e.g., IRED polypeptides) capable of catalyzing the reduction of an imine to an amine. In some embodiments, the IRED polypeptides are capable of catalyzing the NAD(P)H-dependent reduction of an imine group by converting NAD(P)H to NAD(P)+ and the imine to amine. In some embodiments, the IRED polypeptides disclosed herein are capable of catalyzing the NAD(P)H-dependent reduction of an imine group to a primary or secondary amine. For example, in some embodiments, the NAD(P)H-dependent IRED polypeptides described herein can catalyze the reduction of imines formed from the condensation of nucleophilic amino acid residue side-chains and other electrophilic amino acid residue sidechains in peptides.

[0153] In some embodiments, the IRED polypeptides of the disclosure are capable of catalyzing reactions in the process of generating macrocyclic peptides.26081

[0154] In some embodiments, the IRED polypeptides of the disclosure may be useful in the preparation of compounds such as compound 4 in Scheme B above. For example, in some embodiments, the IRED polypeptides described herein are capable of catalyzing the reaction shown in Scheme B above. In some embodiments, the IRED polypeptides of the disclosure may exhibit increased activity on substrate 3 to selectively provide the secondary amine 4 in Scheme B above.

[0155] In some embodiments, the IRED polypeptides disclosed herein may be useful in the half reaction, such as the one shown in Scheme B above and / or may be useful in a single-pot reaction along with a KRED polypeptide of the disclosure where the KRED generates the imine substrate in situ as shown, for example, in Scheme C above.

[0156] In some embodiments, a polypeptide (e.g., an IRED 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.

[0157] In some embodiments, the IRED polypeptides described herein are a product of directed evolution from a wild-type IRED sequence, which was itself identified by screening a panel of wild-type IRED polypeptides((i.e., a wild-type IRED polypeptide as set forth in SEQ ID NO: 40, which includes a C-terminal His tag or a wild-type IRED polypeptide as set forth in SEQ ID NO: 38, which lacks a C-terminal His tag).

[0158] Enzyme properties for which improvements are desirable include, but are not limited to, enzymatic activity, expanded or altered optimal chemical conditions for enzymatic activity, thermal stability, substrate scope, chemical stability, pH activity profile, cofactor requirements, cofactor selectivity, refractoriness to inhibitors (e.g., product inhibition), chemoselectivity, regioselectivity, stereospecificity, stereoselectivity, solvent tolerance, and solvent stability. The improvements can relate to a single enz me property, such as enz matic activity, or a combination of different enzyme properties, such as enzymatic activity and thermal stability.

[0159] In some embodiments, the polypeptide (e.g.. the IRED polypeptide) of the disclosure may demonstrate one or more improvements relative the polypeptide of SEQ ID NO: 38 or SEQ ID NO: 40, including, but not limited to, increases in enzymatic activity, enzyme expression and / or solubility in E. coli, soluble enzyme expression, cofactor selectivity and affinity, thermal stability, solvent tolerance, and / or solvent stability. In some embodiments, the polypeptide (e.g., the IRED polypeptide) of the disclosure may demonstrate at least two improvements relative the polypeptide of SEQ ID NO: 38 or SEQ ID NO: 40, including, but not limited to, increases in26081 enzymatic activity, enzyme expression and / or solubility in A’, colt, soluble enz me expression, cofactor selectivity and affinity, thermal stability, solvent tolerance, and / or solvent stability.

[0160] For example, in some embodiments, the polypeptide has one or more of the following properties relative to a reference polypeptide: a) increased activity in catalyzing the NAD(P)H- dependent reduction of an imine to an amine; b) increased activity in catalyzing the oxidation of NAD(P)H to NAD(P)+; c) improved thermostability; d) improved solvent tolerance; d) increased solvent tolerance / stability; e) increased enz me expression and / or solubility; f) increased soluble enzyme expression; and / or g) increased substrate selectivity and affinity'. In some embodiments, the polypeptide has the following properties relative to a reference polypeptide: a) increased activity in catalyzing the NAD(P)H-dependent reduction of an imine to an amine; and b) improved thermostability. In some embodiments, the polypeptide has the following properties relative to a reference polypeptide: a) increased activity in catalyzing the NAD(P)H-dependent reduction of an imine to an amine; and b) improved organic solvent tolerance. In some embodiments, the polypeptide has the following properties relative to a reference polypeptide: a) increased activity in catalyzing the NAD(P)H-dependent reduction of an imine to an amine; b) increased activity in catalyzing the oxidation of NAD(P)H to NAD(P)+; and c) improved organic solvent tolerance. In some embodiments, the polypeptide has the following properties relative to a reference polypeptide: a) increased activity in catalyzing the NAD(P)H-dependent reduction of an imine to an amine; b) increased activity’ in catalyzing the oxidation of NAD(P)H to NAD(P)+; c) improved thermostability; and d) improved organic solvent tolerance. In some embodiments, the IRED polypeptides of the disclosure may demonstrate increased activity' in catalyzing the reaction outlined in Scheme B above relative to a reference polypeptide. In some embodiments, the reference polypeptide comprises the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the reference polypeptide is any one of SEQ ID NO: 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 136, 138, 140, 142, 144, 146, 148, 150, 152. 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174. 176, 178, 180. 182, 184. 186, 188, 190, or 192. In some embodiments, the reference polypeptide is any one of SEQ ID NO: 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, or 96. In some embodiments, the reference polypeptide is any one of SEQ ID NO: 38, 136, 138, 140, 142, 144. 146, 148, 150, 152, 154, 156, 158, 160. 162, 164, 166, 168, 170. 172, 174, 176, 178, 180, 182, 184. 186, or 188.

[0161] In some embodiments, the IRED polypeptides of the disclosure may demonstrate improvements in the rate of enzymatic activity, i.e., the rate of converting the substrate to the product, relative to a reference polypeptide. In some embodiments, the IRED polypeptides are26081 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 25- fold, about 50- fold, about 100- fold, about 150- fold, about 200- fold, about 400- fold, about 500-fold, about 700-fold, about 800-fold, about 900-fold, about 1000- fold, or more than about 1000- fold the rate exhibited by a reference polypeptide comprising the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 40.

[0162] In some embodiments, the IRED polypeptides of the disclosure may demonstrate increased activity in catalyzing the NAD(P)H-dependent reduction of an imine to an amine, 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 25- fold, about 50- fold, about 100- fold, about 150- fold, about 200- fold, about 400- fold, about 500-fold, about 700-fold, about 800-fold, about 900-fold, about 1000- fold, or more than about 1000- fold increased activity7relative to a reference polypeptide comprising the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the IRED polypeptides of the disclosure may demonstrate increased activity in catalyzing the NAD(P)H-dependent reduction of an imine to an amine, wherein the polypeptide has at least 1.1-fold, about 5-fold, about 10-fold, about 100-fold, or about 500-fold increased activity7relative to a reference polypeptide comprising the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 40.

[0163] In some embodiments, the IRED 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 25- fold, about 50- fold, about 100- fold, about 150- fold, about 200- fold, about 400- fold, about 500-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: 38 or SEQ ID NO: 40. In some embodiments, the IRED 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 1. 1 -fold, about 5-fold, about 10-fold, about 100-fold, or about 500-fold increased activity relative to a reference polypeptide comprising the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 40.

[0164] In some embodiments, the IRED polypeptides of the disclosure may demonstrate increased activity in catalyzing the reaction outlined in Scheme B above, 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 25- fold, about 50- fold, about 100- fold, about 150- fold, about 200- fold,26081 about 400- fold, about 500-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: 38 or SEQ ID NO: 40. In some embodiments, the IRED polypeptides of the disclosure may demonstrate increased activity in catalyzing the reaction outlined in Scheme B above, wherein the polypeptide has at least 1. 1 -fold, about 5-fold, about 10-fold, about 100-fold, or about 500-fold increased activity relative to a reference polypeptide comprising the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 40.

[0165] In some embodiments, the IRED 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: 38 or SEQ ID NO: 40. In some embodiments, the IRED 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 s tabi 1 ity relative to a reference polypeptide comprising the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 40.

[0166] In some embodiments, the IRED polypeptides of the disclosure have been improved for thermostability. In some embodiments, the IRED polypeptides of the disclosure have been improved for thermostability and can maintain at least 50% enzymatic activity at temperatures of at least about 35 °C, 36 °C, 37°C, 38 °C, 39°C, 40 °C, 41 °C, 42°C, 43°C, 50°C or more degrees compared to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the IRED polypeptides of the disclosure can maintain at least about 40%, about 50%, about 55%, about 60%, about 65%, about 70%. about 75%, about 80%, about 85%, about 90% or more enzymatic activity at temperatures of at least about 35 °C, 36 °C, 37°C, 38 °C, 39°C, 40 °C, 41 °C, 42°C, 43°C, 50°C or more degrees compared to any one of SEQ ID NO: 38 or SEQ ID NO: 40 when subjected to a heat challenge test for about 10, 20, 30, 40, 50, or 60 minutes.

[0167] In some embodiments, a polypeptide (e.g., IRED polypeptide) of the disclosure is a polypeptide that comprises an amino acid sequence having at least 70% but less than 100% (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than2608199% but less than 100%) sequence identity to SEQ ID NO: 38. In some embodiments, a polypeptide (e.g., IRED polypeptide) of the disclosure is a polypeptide that comprises an amino acid sequence having at least 70% but less than 100% (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or more than 99% but less than 100%) sequence identity to SEQ ID NO: 40, wherein the polypeptide does not comprise the amino acid sequence of SEQ ID NO: 40.

[0168] The difference(s) between the variant(s) and SEQ ID NO: 38 or SEQ ID NO: 40 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 some embodiments, the amino acid sequence difference(s) is / are conservative amino acid substitution(s). In other embodiments, the amino acid sequence difference(s) is / are non- conservative amino acid substitution(s). In some embodiments, the amino acid sequence differences are a combination of non-conservative and conservative amino acid substitutions. In some embodiments, the amino acid sequence differences are insertions. In some embodiments, the amino acid sequence differences are deletions.

[0169] In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. or more than 99% but less than 100% sequence identity to SEQ ID NO: 38 or SEQ ID NO: 40. In other words, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 38 or SEQ ID NO: 40, wherein the polypeptide does not comprise SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 70% but less than 100% sequence identity to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 75% but less than 100% sequence identity to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 80% but less than 100% sequence identity7to SEQ ID NO: 38 or SEQ ID NO: 40. In some26081 embodiments, provided herein is a polypeptide (e.g., an 1RED polypeptide) comprising an amino acid sequence having at least 85% but less than 100% sequence identity to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 90% but less than 100% sequence identity to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 91% but less than 100% sequence identity to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 92% but less than 100% sequence identity to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 93% but less than 100% sequence identity to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 94% but less than 100% sequence identity to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 95% but less than 100% sequence identity to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 96% but less than 100% sequence identity’ to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 97% but less than 100% sequence identity to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 98% but less than 100% sequence identity to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 99% but less than 100% sequence identity to SEQ ID NO: 38 or SEQ ID NO: 40.

[0170] In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity' to SEQ ID NO: 38 or SEQ ID NO: 40, wherein the polypeptide comprises an amino acid substitution at one or more positions selected from 9, 32, 36, 37, 38, 39, 41, 47, 53, 54, 55, 57, 72, 74, 93, 102, 106, 108, 117, 123, 127, 129, 135, 136, 137, 138, 139, 146, 156, 160, 164, 166, 174, 183, 184, 194, 196, 197, 198, 200, 207, 210, 211, 214, 218, 220, 224, 226, 227, 229, 230, 232, 233, 235, 239, 240, 241, 242, 243, 244,26081258, 264. 265, 266, 267. 269, 278. 279, 281, 283. 285, 286. 292, and 293, and / or at least one amino acid insertion between positions 240 and 241 or between positions 288 and 289, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40.

[0171] In some embodiments, the polypeptide comprises any number from 2 to 74 amino acid substitutions, each amino acid substitution at an amino acid position selected from 9, 32, 36, 37, 38, 39, 41, 47, 53, 54, 55, 57, 72, 74, 93, 102, 106, 108, 117, 123, 127, 129, 135, 136, 137, 138,139, 146, 156, 160, 164, 166, 174, 183, 184, 194, 196, 197, 198, 200, 207, 210, 211, 214, 218,220, 224. 226, 227, 229, 230, 232. 233, 235, 239, 240, 241, 242, 243. 244, 258, 264, 265, 266.267, 269, 278, 279, 281, 283, 285, 286, 292, and 293, and / or at least one amino acid insertion between positions 240 and 241 or between positions 288 and 289, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40.

[0172] In some embodiments, the polypeptide further comprises at least one amino acid insertion between positions 240 and 241 or between positions 288 and 289. wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40.

[0173] In some embodiments, the polypeptide comprises one or more amino acid substitutions or substitution sets set forth in Table 3 or Table 4. In some embodiments, the polypeptide comprises one or more amino acid substitutions or substitution sets set forth in Table 3. In some embodiments, the polypeptide comprises one or more amino acid substitutions or substitution sets set forth in Table 4.

[0174] In some embodiments, the polypeptide comprises one of the following amino acid substitutions, substitution sets, or substitution and insertion sets: i) an amino acid substitution at position 197; ii) amino acid substitutions at positions 32, 53, 197, 220, and 281; iii) amino acid substitutions at positions 32, 53, 137, 174, 196, 197, 214, 220, and 281; iv) amino acid substitutions at positions 32, 53, 137, 174, 196, 197, 214, 220, 241, and 281; v) amino acid substitutions at positions 32. 53. 127, 137. 174, 196, 197, 214, 220. 239, 241. 244, 278, 281. and292; vi) amino acid substitutions at positions 32, 53, 127, 137, 174, 196, 197, 214, 220, 239, 241,242, 244, 278, 281, and 292; vii) amino acid substitutions at positions 32, 53, 127, 136, 137, 146, 174, 196, 197, 198, 214, 220, 226, 229, 232, 239, 241, 242, 244, 266, 278, 281, and 292; viii) amino acid substitutions at positions 32, 53, 127, 136. 137, 146, 174, 196, 197. 198, 214. 220,229, 239, 241, 242, 244, 266, 278, 281, and 292; ix) amino acid substitutions at positions 32, 53,127, 136, 137, 146, 174, 194, 196, 197, 198, 214, 220, 229, 239, 241, 242, 244, 266, 278, 281, and 292; x) amino acid substitutions at positions 32, 53, 127, 136, 137, 146, 174, 196, 197, 198,26081214, 220. 229, 239, 241. 242, 244. 266, 278, 281. and 292; xi) amino acid substitutions at positions 32, 53, 74, 127, 136, 137, 146, 174, 183, 196, 197, 198, 214, 220, 229, 239, 241, 242, 244, 266, 278, 281, and 292; xii) amino acid substitutions at positions 32, 53, 74, 127, 136, 137, 146, 156. 174, 183, 196, 197, 198, 200, 214, 220, 229, 239, 241, 242, 244, 266, 278, 281, and 292; xiii) amino acid substitutions at positions 32. 53. 74. 106, 108. 127, 136, 137, 146, 156. 164, 174, 183, 196, 197, 198, 200, 211, 214, 220, 229, 239, 241, 242, 244, 266, 278, 281, and 292; xiv) substitutions at positions 32, 53, 74, 106, 127, 136, 137, 146, 156, 174, 183, 196, 197, 198, 200, 214, 220, 229, 239, 241, 242, 244, 258, 266, 278, 279, 281, and 292; xv) amino acid substitutions at positions 32, 36, 37, 38, 41, 53, 74, 106, 127, 136, 137, 146, 156. 174, 183, 196,197, 198, 200, 214, 220, 229, 239, 241, 242, 244, 258, 266, 278, 279, 281, and 292; xvi) amino acid substitutions at positions 32, 36, 37, 38, 41, 53, 74, 106, 127, 136, 137, 146, 156, 174, 183, 196, 197, 198, 200, 214, 220, 229, 235, 239, 241, 242, 244, 258, 266, 278, 279, 281, and 292; xvii) amino acid substitutions at positions 32, 36, 37, 38, 39, 41, 47, 53, 74, 102, 106, 127, 135, 136, 137. 146, 156, 166. 174, 183. 196, 197, 198, 200, 214. 220, 229. 235, 239, 241. 242, 244. 258, 266, 278, 279, 281, and 292; xviii) amino acid substitutions at positions 32, 36, 37, 38, 39, 41, 47, 53, 54, 74, 102, 106, 127, 129, 135, 136, 137, 146, 156, 160, 166, 174, 183, 196, 197,198, 200, 207, 210, 214, 218, 220, 229, 235, 239, 241, 242, 244, 258, 266, 278, 279, 281, and292; xix) amino acid substitutions at positions 32. 36, 37, 38, 39, 41, 47, 53, 54, 74, 102. 106,127, 129, 135, 136, 137, 146, 156, 160, 166, 174, 183, 196, 197, 198, 200, 207, 210, 214, 218,220, 229, 233, 235, 239, 241, 242, 243, 244, 258, 266, 278, 279, 281, and 292, an amino acid insertion between positions 240 and 241, and an amino acid insertion between positions 288 and 289; xx) amino acid substitutions at positions 32, 36, 37, 38, 39, 41, 47, 53, 54. 55. 57, 74, 102, 106, 117. 127, 129, 135. 136, 137. 146, 156, 160. 166, 174. 183, 196. 197, 198, 200. 207, 210.214, 218, 220, 226, 229, 233, 235, 239, 241, 242, 243, 244, 258, 266, 278, 279, 281, and 292, an amino acid insertion between positions 240 and 241, and an amino acid insertion between positions 288 and 289; xxi) amino acid substitutions at positions 32, 36, 37, 38, 39, 41. 47, 53,54. 55, 57, 74, 102, 106. 117, 127. 129, 135, 136, 137, 146. 156, 160. 166, 174, 183, 196, 197.198, 200, 207, 210, 214, 218, 220, 226, 229, 233, 235, 239, 241, 243, 244, 258, 266, 269, 278,279, 281, and 292, an amino acid insertion between positions 240 and 241, and an amino acid insertion between positions 288 and 289; xxii) amino acid substitutions at positions 32, 36, 37, 38, 39, 41, 47, 53, 54, 74, 102. 106, 117, 127, 129, 135. 136, 137, 146, 156, 160, 166, 174. 183, 184, 196, 197, 198, 200, 207, 210, 214, 218, 220, 224, 226, 229, 233, 235, 239, 241, 243, 244, 258, 266, 269, 278, 279, 281, and 292, an amino acid insertion between positions 240 and 241, and an amino acid insertion between positions 288 and 289; xxiii) amino acid substitutions at26081 positions 32. 39. 47, 53, 54, 74, 102. 106. 117, 127. 129, 135. 136, 137, 146. 156, 160. 166, 174.183, 184, 196, 197, 198, 200, 207, 210, 214, 218, 220, 224, 226, 229, 233, 235, 239, 241, 243,244, 258, 266, 269, 278, 279, 281, and 292, an amino acid insertion between positions 240 and 241, and an amino acid insertion between positions 288 and 289; xxiv) amino acid substitutions at positions 32. 39. 47. 53. 54, 74, 102, 106. 117, 127. 129, 135, 136. 137, 146. 156, 160, 166, 174, 183, 184, 196, 197, 198, 200, 207, 210, 214, 218, 220, 224, 226, 229, 233, 235, 239, 241, 243, 244, 258, 266, 269, 278, 279, 281, and 292, an amino acid insertion between positions 240 and 241, and an amino acid insertion between positions 288 and 289; xxv) amino acid substitutions at positions 32, 39, 47, 53, 54, 74, 102, 106, 117, 127. 129, 135. 136, 137, 146, 156, 160, 166, 174, 183, 184, 196, 197, 198, 200, 207, 210, 214, 218, 220, 224, 226, 229, 233, 235, 239, 241, 243, 244, 258, 266, 269, 278, 279, 281, 285, and 292, an amino acid insertion between positions 240 and 241, and an amino acid insertion between positions 288 and 289; xxvi) amino acid substitutions at positions 32, 53, 54, 74, 102, 106, 117, 123, 127, 129, 135, 136, 137, 138, 139, 146. 156, 160, 166. 174, 183. 184, 196, 197, 198, 200. 207, 210. 214, 218, 220. 224, 226. 227, 229, 230, 233, 235, 239, 241, 243, 244, 258, 266, 269, 278, 279, 281, 285, 286, and 292, an amino acid insertion between positions 240 and 241, and an amino acid insertion between positions 288 and 289; xxvii) amino acid substitutions at positions 9, 32, 53, 54, 74, 93, 102, 106, 117, 123. 127, 129, 135, 136, 137. 139, 146, 156, 160, 166, 174, 183. 184, 196, 197, 198, 200. 207, 210, 214, 218, 220, 224, 226, 227, 229, 230, 233, 235, 239, 241, 243, 244, 258, 264, 265, 266, 267, 269, 278, 279, 281, 285, 286, 292, and 293 an amino acid insertion between positions 240 and 241, and an amino acid insertion between positions 288 and 289; xxviii) amino acid substitutions at positions 9, 32, 53, 54. 74. 93, 102, 106, 117, 123, 127, 129, 135. 136, 137, 139, 146, 156. 160, 166, 174. 183, 184. 196, 197, 198. 200, 207. 210, 214. 218, 220, 224. 226, 227.229, 230, 233, 235, 239, 240, 241, 242, 243, 244, 258, 264, 265, 266, 267, 269, 278, 279, 281,285, 286, 292, and 293, an amino acid insertion between positions 240 and 241, and an amino acid insertion between positions 288 and 289; or xxix) amino acid substitutions at positions 9, 32,53. 54, 72, 93, 102, 106. 117, 123. 127, 129, 135, 136, 137. 139, 146. 156, 160, 166, 174, 183.184, 196, 197, 198, 200, 207, 210, 214, 218, 220, 224, 226, 227, 229, 230, 233, 235, 239, 240,241, 242, 243, 244, 258, 264, 265, 266, 267, 269, 278, 279, 281, 283, 285, 286, 292, and 293, an amino acid insertion between positions 240 and 241, and an amino acid insertion between positions 288 and 289, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40.

[0175] In some embodiments, the polypeptide comprises an amino acid substitution at position 197, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID26081NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions at positions 32, 53, 197, 220, and 281, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions at positions 32, 53, 137, 174, 196, 197. 214, 220, and 281. wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions at positions 32, 53, 137, 174, 196, 197, 214, 220, 241, and 281, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions at positions 32, 53, 127, 137, 174, 196, 197, 214, 220, 239, 241, 244, 278, 281, and 292, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the poly peptide comprises amino acid substitutions at positions 32, 53, 127, 137. 174, 196, 197, 214, 220, 239, 241. 242, 244, 278, 281, and 292, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions at positions 32, 53, 127, 136, 137, 146, 174, 196, 197, 198, 214, 220, 226, 229, 232, 239, 241, 242, 244, 266, 278, 281, and 292, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions at positions 32, 53, 127, 136, 137, 146, 174, 196, 197, 198, 214, 220, 229, 239, 241, 242, 244, 266, 278, 281, and 292, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions at positions 32. 53. 127, 136. 137, 146. 174. 194, 196. 197, 198. 214, 220, 229. 239, 241. 242, 244. 266, 278, 281, and 292, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions at positions 32, 53, 127, 136, 137, 146, 174, 196, 197, 198. 214, 220. 229, 239, 241. 242, 244. 266, 278, 281, and 292, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions at positions 32, 53, 74, 127, 136, 137, 146, 174, 183, 196, 197, 198, 214, 220, 229, 239, 241, 242, 244, 266, 278, 281, and 292, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions at positions 32, 53, 74, 127, 136, 137, 146, 156, 174, 183, 196, 197, 198, 200, 214, 220, 229, 239, 241, 242, 244, 266, 278, 281, and 292, wherein the amino acid positions of the26081 polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions at positions 32, 53, 74, 106, 108, 127, 136, 137, 146, 156, 164, 174, 183, 196, 197, 198, 200, 211, 214, 220, 229, 239, 241, 242, 244, 266, 278, 281, and 292, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions at positions 32, 53, 74, 106, 127, 136, 137, 146, 156, 174, 183, 196, 197, 198, 200, 214, 220, 229, 239, 241, 242, 244, 258, 266, 278, 279, 281, and 292, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions at positions 32, 36, 37, 38, 41, 53, 74, 106, 127, 136, 137, 146, 156, 174, 183, 196, 197, 198, 200, 214, 220, 229, 239, 241, 242, 244, 258, 266, 278, 279, 281, and 292, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions at positions 32. 36. 37, 38, 41, 53, 74, 106. 127, 136, 137, 146, 156. 174, 183. 196, 197, 198. 200, 214, 220, 229, 235, 239, 241, 242, 244, 258, 266, 278, 279, 281, and 292, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions at positions 32, 36, 37, 38, 39, 41, 47, 53, 74, 102. 106, 127, 135, 136, 137. 146, 156, 166, 174, 183, 196, 197. 198, 200, 214, 220, 229, 235, 239, 241, 242, 244, 258, 266, 278, 279, 281, and 292, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions at positions 32, 36, 37, 38, 39, 41, 47, 53, 54, 74, 102, 106, 127. 129, 135, 136, 137, 146, 156, 160. 166, 174. 183, 196, 197. 198, 200. 207, 210, 214. 218, 220. 229, 235. 239, 241, 242. 244, 258. 266, 278, 279, 281, and 292, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions at positions 32, 36, 37, 38, 39, 41, 47, 53, 54, 74, 102, 106. 127, 129. 135, 136, 137. 146, 156. 160, 166, 174, 183, 196. 197, 198. 200, 207, 210, 214, 218. 220, 229, 233, 235, 239, 241, 242, 243, 244, 258, 266, 278, 279, 281, and 292, an amino acid insertion between positions 240 and 241, and an amino acid insertion between positions 288 and 289, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions at positions 32, 36, 37, 38, 39, 41, 47, 53, 54, 55, 57, 74, 102, 106, 117, 127, 129, 135, 136, 137, 146, 156, 160, 166, 174, 183, 196, 197, 198, 200, 207, 210, 214, 218, 220, 226, 229, 233, 235, 239, 241, 242, 243, 244, 258, 266, 278, 279, 281, and 292, an amino acid insertion26081 between positions 240 and 241, and an insertion between positions 288 and 289, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions at positions 32, 36, 37, 38, 39, 41, 47, 53, 54, 55, 57, 74. 102, 106, 117, 127, 129, 135, 136, 137, 146, 156. 160, 166, 174. 183, 196. 197, 198, 200, 207, 210. 214, 218. 220, 226, 229, 233, 235. 239, 241, 243, 244, 258, 266, 269, 278, 279, 281, and 292, an amino acid insertion between positions 240 and 241, and an amino acid insertion between positions 288 and 289, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions at positions 32, 36, 37, 38, 39, 41, 47, 53, 54, 74, 102, 106, 117, 127, 129, 135, 136, 137, 146, 156, 160, 166, 174, 183, 184, 196, 197, 198, 200, 207, 210, 214, 218, 220, 224, 226, 229, 233, 235, 239, 241, 243, 244, 258, 266, 269, 278, 279, 281, and 292, an amino acid insertion between positions 240 and 241, and an amino acid insertion between positions 288 and 289, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions at positions 32, 39, 47, 53, 54, 74, 102, 106, 117, 127, 129, 135, 136, 137, 146, 156, 160, 166, 174, 183, 184, 196, 197, 198, 200, 207, 210, 214, 218, 220, 224, 226, 229, 233, 235, 239, 241, 243, 244, 258. 266, 269, 278, 279, 281. and 292, an amino acid insertion between positions 240 and 241, and an amino acid insertion between positions 288 and 289, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions at positions 32, 39, 47, 53, 54, 74, 102, 106, 117, 127. 129, 135, 136, 137, 146, 156, 160. 166, 174, 183, 184, 196. 197, 198. 200, 207, 210. 214, 218. 220, 224, 226. 229, 233. 235, 239. 241, 243, 244. 258, 266. 269, 278, 279, 281, and 292, an amino acid insertion between positions 240 and 241, and an amino acid insertion betw een positions 288 and 289, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions at positions 32, 39, 47, 53, 54, 74, 102, 106, 1 17, 127, 129, 135, 136, 137, 146, 156, 160, 166, 174, 183, 184, 196, 197, 198, 200, 207, 210, 214, 218, 220, 224, 226, 229, 233, 235, 239, 241, 243, 244, 258, 266, 269, 278, 279, 281, 285, and 292, an amino acid insertion between positions 240 and 241, and an insertion between positions 288 and 289, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions at positions 32, 53, 54, 74, 102, 106, 117, 123, 127, 129, 135, 136, 137, 138, 139, 146, 156, 160, 166, 174, 183, 184, 196, 197, 198, 200, 207,26081210, 214. 218, 220, 224. 226, 227. 229, 230, 233. 235, 239. 241, 243. 244, 258, 266. 269, 278.279, 281, 285, 286, and 292, an amino acid insertion between positions 240 and 241, and an amino acid insertion between positions 288 and 289, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions at positions 9, 32, 53, 54, 74, 93, 102, 106, 1 17, 123, 127, 129, 135, 136, 137, 139, 146, 156, 160, 166, 174, 183, 184, 196, 197, 198, 200, 207, 210, 214, 218, 220, 224, 226, 227, 229, 230, 233, 235, 239, 241, 243, 244, 258, 264, 265, 266, 267, 269, 278, 279, 281, 285, 286, 292, and 293 an amino acid insertion between positions 240 and 241, and an amino acid insertion between positions 288 and 289, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions at positions 9, 32, 53, 54, 74, 93, 102, 106, 117, 123, 127, 129, 135, 136, 137, 139, 146, 156, 160, 166, 174. 183, 184, 196, 197, 198, 200, 207, 210, 214, 218, 220, 224. 226, 227, 229, 230, 233, 235, 239. 240, 241, 242. 243, 244. 258, 264, 265. 266, 267. 269, 278. 279, 281, 285. 286, 292. and 293, an amino acid insertion between positions 240 and 241, and an amino acid insertion between positions 288 and 289, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions at positions 9, 32, 53, 54. 72. 93. 102, 106, 117,123, 127, 129, 135, 136, 137, 139, 146, 156, 160, 166, 174, 183, 184, 196, 197, 198, 200, 207,210, 214, 218, 220, 224, 226, 227, 229, 230, 233, 235, 239, 240, 241, 242, 243, 244, 258, 264,265, 266, 267, 269, 278, 279, 281, 283, 285, 286, 292, and 293, an amino acid insertion between positions 240 and 241, and an amino acid insertion between positions 288 and 289, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40

[0176] In some embodiments, the polypeptide comprises one of the following amino acid substitutions, substitution sets, and substitution and insertion sets: i) R197A; ii) I32V, V53A, R197A, L220Y, and K281G; iii) I32V, V53A, S137Y, Y174V. M196L, R197A, N214S. L220Y, and K281G; iv) I32V, V53A, S137Y, Y174V, M196L, R197A, N214S, L220Y, N241L, and K281G; v) I32V, V53A, T127S, S137Y, Y174V, M196L, R197A, N214S, L220Y, L239N, N241Q, G244A, V278I, K281G, and G292K; vi) I32V, V53A, T127S, S137Y, Y174V, M196L, R197A, N214S, L220Y, L239N. N241Q, P242T, G244A. V278I. K281G, and G292K; vii) I32V, V53A, T127S, S136A, S137Y, P146R, Y174V, M196L, R197A, R198C, N214S, L220C, A226I, G229R, D232K, L239N, N241M, P242T, G244A, C266M, V278I, K281G, and G292K; viii) I32V, V53A, T127S, S136A, S137Y, P146R, Y174V, M196L, R197A, R198C, N214S, L220C,26081G229R, L239N, N241M, P242T, G244A. C266M, V2781, K281G. and G292K; ix) 132V, V53A, T127S, S136A, S137Y, P146R, Y174V, A194Q, M196L, R197A, R198C, N214S, L220C, G229R, L239N, N241M, P242T, G244A, C266M, V278I, K281G, and G292K; x) I32V, V53A, T127S, S136A, S137Y, P146R, Y174V. M196L, R197A, R198C, N214S, L220C, G229R. L239N, N241M. P242T. G244A, C266I, V278I, K281G, and G292K; xi) I32V, V53Q, P74A, T127S, S136A, S137Y, P146R, Y174V, Y183F, M196L, R197A, R198C, N214S, L220C, G229R, L239N, N241M, P242T, G244S, C266I, V278I, K281G, and G292K; xii) I32V, V53Q, P74A, T127S, S136A, S137H, P146R, A156Q, Y174V, Y183F, M196L, R197A, R198C, G200K, N214S, L220C, G229R, L239N, N241M, P242T, G244S, C266I, V278I, K281G, and G292K; xin) I32V, V53Q, P74A, H106R, A108S, T127S, S136A, S137H, P146R, A156Q, M164V, Y174V, Y183F, M196L, R197A, R198C, G200K, G211L, N214S, L220C, G229R, L239N, N241M, P242T, G244S, C266I, V278I, K281G, and G292K; xiv) I32V, V53Q, P74A, H106R, T127S, S136A, S137H, P146R, A156Q, Y174V, Y183F, M196L, R197A, R198C, G200K, N214S, L220C. G229R, L239N, N241M, P242T, G244S, E258R, C266I, V278I, E279Q, K281G, and G292K; xv) I32V, T36P, P37D, T38D, S41D, V53Q, P74A, H106R, T127S, S136A, S137H, P146R, A156Q, Y174V, Y183F, M196L, R197A, R198C, G200K, N214S, L220C, G229R, L239N, N241M, P242T, G244S, E258R, C266I, V278I, E279Q, K281G, and G292K; xvi) I32V, T36P, P37D, T38D, S41D. V53Q, P74A, H106R, T127S, S136A, S137H, P146R, A156Q, Y174V, Y183F, M196L, R197A, R198C, G200K, N214S, L220C, G229R, S235R, L239N, N241M, P242T, G244S, E258K, C266I, V278I, E279Q, K281G, and G292K; xvii) I32V, T36P, P37D, T38D, A39M, S41D, S47R, V53Q, P74A, R102S, H106R, T127S, A135N, S136A. S137H, P146A, A156Q, T166P. Y174V, Y183F, M196L, R197A, R198C, G200K. N214S, L220C, G229R. S235R. L239N, N241M. P242T. G244S, E258K, C266I, V278I, E279Q, K281G, and G292K; xviii) I32V, T36P, P37D, T38D, A39M, S41D, S47R, V53Q, P54E, P74A, R102S, H106R, T127S, D129A, A135N, S136A, S137H, P146A, A156Q, A160K, T166P. Y174V, Y183F, M196L, R197A, R198C, G200K, A207S, E210T. N214S, A218E, L220C. G229R, S235Q, L239M. N241M, P242T, G244S. E258K, C266I, V278I, E279Q, K281G, and G292K; xix) I32V, T36P, P37D, T38D, A39M, S41D, S47R, V53Q, P54E, P74A, R102S, H106R, T127S, D129A, A135N, S136A, S137H, P146A, A156Q, A160K, T166P, Y174V, Y183F. M196L, R197A, R198C, G200K, A207S, E210T, N214S, A218E, L220C, G229R, R233T. S235Q, L239M, N241M, P242T, V243I, G244S, E258K. C266I, V278I, E279Q, K281G, and G292K, a proline insertion between positions 240 and 241, and a glycine insertion between positions 288 and 289; xx) I32V, T36P, P37D, T38D, A39M, S41D, S47R, V53Q, P54E, S55E, P57E, P74A, R102S, H106R, A117R, T127S, D129A, A135N,26081S136A, S137H, P146A, A156Q. A160K, T166P, Y174V. Y183F, M196L. R197A, R198C, G200K, A207S, E210T, N214S, A218E, L220C, A226K, G229R, R233T, S235Q, L239M, N241M, P242T, V243I, G244S, E258K, C266I, V278I, E279Q, K281G, and G292K, a proline insertion between positions 240 and 241, and a glycine insertion between positions 288 and 289; xxi) I32V, T36P. P37D, T38D, A39M, S41D. S47R, V53Q, P54E, S55E. P57E, P74A, R102S, H106R, A117R, T127S, D129A, A135N, S136A, S 137H, P146A, A156Q, A160K, T166P, Y174V, Y183F, M196L, R197A, R198C, G200K, A207S, E210T, N214S, A218E, L220C, A226K, G229R, R233T, S235Q, L239M, N241M, V243I, G244S, E258K, C266I, Y269L, V278L, E279Q, K281G. and G292K, a lysine insertion between positions 240 and 241, and a glycine insertion between positions 288 and 289; xxii) I32V, T36P, P37D, T38D, A39M, S41D, S47R, V53Q, P54E, P74A, R102S, H106R, A117R, T127S, D129A, A135N, S136A, S137H, P146A, A156Q, A160K, T166P, Y174V, Y183F, G184C, M196L, R197A, R198C, G200K, A207S, E210T, N214S, A218E, L220C. Q224M, A226K, G229R, R233T, S235Q, L239M, N241M, V243I, G244S, E258K. C266I. Y269L, V278I, E279Q, K281G. and G292K, a lysine insertion between positions 240 and 241, and a glycine insertion between positions 288 and 289; xxiii) I32V, A39M, S47R, V53Q, P54E, P74A, R102S, H106R, A117R, T127S, D129A, A135N, S136A, S137H, P146A, A156Q, A160K, T166P, Y174V, Y183F, G184C, M196L, R197A, R198C, G200K, A207S, E210T. N214S, A218E, L220C, Q224M, A226K. G229R, R233T, S235Q, L239M, N241M, V243I, G244S, E258K, C266I, Y269L, V278I, E279Q, K281G, and G292K, a lysine insertion between positions 240 and 241, and a glycine insertion between positions 288 and 289; xxiv) I32V, A39M, S47R, V53Q, P54E, P74A, R102S, H106R, A117R, T127S. D129A, A135N, S136A. S137H, P146A, A156Q. A160K, T166P, Y174V. Y183F, G184C, M196L. R197G, R198C, G200K. A207S, E210T, N214S, A218E. L220C. Q224M. A226K, G229R, R233T, S235Q, L239M, N241M, V243I, G244S, E258K, C266I, Y269L, V278I, E279Q, K281G, and G292K, a lysine insertion between positions 240 and 241, and a glycine insertion between positions 288 and 289; xxv) I32V, A39M, S47R, V53Q, P54E, P74A, R102S. H106R, Al 17R. T127S. D129A, A135N, S136A. S137H, P146A, A156Q. A160K, T166P, Y174V, Y183F, G184C, M196L, R197G, R198C, G200K, A207S, E210T, N214S, A218E, L220C, Q224M, A226K, G229R, R233T, S235Q, L239M, N241M, V243I, G244S, E258K, C266I, Y269L, V278I, E279Q, K281G, G285E, and G292K, an asparagine insertion between positions 240 and 241, and a glycine insertion between positions 288 and 289; xxvi) I32V, V53Q, P54E, P74A, R102S, H106R, A117R, G123A, T127S, D129A, A135N, S136A, S137H, L138M, F139Y, P146A, A156Q, A160K, T166P, Y174V, Y183F, G184C, M196L, R197G, R198K, G200K, A207S, E210T, N214S, A218E, L220C, Q224M, A226K, M227W,26081G229R, S230G, R233T. S235Q. L239M, N241M, V2431, G244S, E258K, C266I, Y269L.V278I, E279Q, K281G, G285M, G286V, and G292K, an asparagine insertion between positions 240 and 241, and a glycine insertion between positions 288 and 289; xxvii) I9V, I32V, V53Q, P54E, P74A, I93V, R102S, H106R, A117R, G123A, T127S, D129A, A135N, S136A, S137H, F139Y, P146A, A156Q, A160K, T166P, Y174V, Y183F. G184C, M196L. R197G, R198K, G200K, A207S, E210T, N214S, A218E, L220C, Q224M, A226K, M227W, G229R, S230G, R233T, S235Q, L239M, N241M, V243I, G244S, E258K, G264P, G265H, C266I, L267F, Y269L, V278I, E279Q, K281G, G285M, G286V, G292K, and L293V, an asparagine insertion between positions 240 and 241 and an alanine insertion between positions 288 and 289; xxviii) I9V, I32V, V53Q, P54E, P74A, I93V, R102S, H106R, A117R, G123A, T127S, D129A, A135N, S136A, S137H, F139Y, P146A, A156Q, A160K, T166P, Y174V, Y183F, G184C, M196L, R197G, R198K, G200K, A207S, E210T, N214S, A218E, L220C, Q224M, A226K, M227W, G229R, S230G, R233T, S235Q, L239M, G240P, N241M, P242T, V243I, G244S, E258K, G264P, G265H, C266I, L267F, Y269L. V278E E279Q. K281G, G285K. G286V, G292K. and L293V, a glycine insertion between positions 240 and 241 and an alanine insertion between positions 288 and 289; or xxix) I9V, I32V, V53Q, P54E, D72N, I93V, R102S, H106R, A117R, G123A, T127S, D129A, A135N, S136A, S137H, F139Y, P146A, A156Q, A160K, T166P, Y174V, Y183F, G184C. M196L, R197G, R198K, G200K, A207S, E210T. N214S, A218E, L220C, Q224M, A226K, M227W, G229R, S230G, R233T, S235Q, L239M, G240P, N241M, P242T, V243I, G244S, E258K, G264P, G265H, C266I, L267F, Y269L, V278I, E279Q, K281G, G283E, G285M, G286V, G292K, and L293V, a glycine insertion between positions 240 and 241 and an alanine insertion between positions 288 and 289, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40.

[0177] In some embodiments, the polypeptide comprises an amino acid substitution R197A, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions I32V, V53A. R197A, L220Y, and K281G, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions I32V, V53A, S137Y, Y174V, M196L, R197A, N214S, L220Y, and K281G, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises ammo acid substitutions I32V, V53A, S137Y, Y174V, M196L, R197A, N214S, L220Y, N241L, and K281G, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptidecomprises amino acid substitutions 132V, V53A, T127S, S137Y. Y174V, M196L, R197A. N214S, L220Y, L239N, N241Q, G244A, V278I, K281G, and G292K, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions I32V, V53A, T127S, S137Y, Y174V, M196L. R197A, N214S, L220Y. L239N, N241Q, P242T. G244A, V278I, K281G, and G292K, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions I32V, V53A, T127S, S136A, S137Y, P146R, Y174V, M196L, R197A, R198C. N214S, L220C, A226I, G229R, D232K, L239N, N241M, P242T, G244A, C266M, V278I, K281G, and G292K, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions I32V, V53A, T127S, S136A, S137Y, P146R, Y174V, M196L, R197A, R198C, N214S, L220C, G229R, L239N. N241M, P242T, G244A, C266M, V278I, K281G, and G292K, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions I32V, V53A, T127S, S136A, S137Y, P146R, Y174V, A194Q, M196L, R197A. R198C, N214S, L220C, G229R, L239N, N241M, P242T, G244A. C266M, V278I, K281G. and G292K, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions I32V, V53A, T127S, S136A, S137Y, P146R, Y174V, M196L, R197A, R198C, N214S, L220C, G229R, L239N, N241M, P242T. G244A, C266I, V278I, K281G, and G292K, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions I32V, V53Q, P74A, T127S, S136A, S137Y, P146R, Y174V, Y183F, M196L, R197A, R198C, N214S, L220C, G229R, L239N, N241M, P242T, G244S, C266I. V278I, K281G, and G292K. wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions I32V, V53Q, P74A, T127S, S136A, S137H, P146R, A156Q, Y174V, Y183F, M196L, R197A, R198C, G200K, N214S, L220C, G229R, L239N, N241M, P242T, G244S, C266I, V278I, K281G, and G292K, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions I32V, V53Q, P74A, H106R, A108S, T127S, S136A, S137H, P146R, A156Q, M164V, Y174V, Y183F, M196L, R197A, R198C, G200K, G211L, N214S, L220C, G229R, L239N, N241M,P242T, G244S, C2661, V2781, K281G, and G292K, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions I32V, V53Q, P74A, H106R, T127S, S136A, S137H, P146R, A156Q. Y174V, Y183F, M196L, R197A, R198C, G200K, N214S, L220C, G229R, L239N. N241M, P242T, G244S. E258R, C266I, V278I. E279Q, K281G, and G292K, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions I32V, T36P, P37D, T38D, S41D, V53Q, P74A, H106R, T127S. S136A, S137H, P146R, A156Q. Y174V, Y183F, M196L, R197A, R198C, G200K, N214S, L220C, G229R, L239N, N241M, P242T, G244S, E258R, C266I, V278I, E279Q, K281G, and G292K, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions I32V, T36P. P37D, T38D, S41D, V53Q, P74A, H106R, T127S. S136A, SI37H, P146R, A156Q. Y174V, Y183F, M196L, R197A, R198C. G200K, N214S, L220C, G229R, S235R, L239N, N241M, P242T, G244S, E258K, C266I, V278I, E279Q, K281G, and G292K, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions I32V, T36P. P37D, T38D, A39M, S41D. S47R, V53Q, P74A, R102S, H106R, T127S, A135N, S136A, S137H, P146A, A156Q, T166P, Y174V, Y183F, M196L, R197A, R198C, G200K, N214S, L220C, G229R, S235R, L239N, N241M, P242T, G244S, E258K, C266I, V278I, E279Q, K281G, and G292K, wherein the ammo acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises ammo acid substitutions 132V, T36P, P37D, T38D, A39M, S41D, S47R, V53Q, P54E, P74A, R102S, H106R, T127S, D129A, A135N, S136A, S137H, P146A, A156Q, A160K, T166P, Y174V, Y183F, M196L, R197A, R198C, G200K, A207S, E210T, N214S, A218E, L220C. G229R, S235Q, L239M, N241M, P242T, G244S, E258K, C266I. V278I. E279Q, K281G, and G292K, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions I32V, T36P, P37D, T38D, A39M, S41D, S47R, V53Q, P54E, P74A, R102S, H106R, T127S, D129A, A135N, S136A, S137H, P146A, A156Q, A160K, T166P, Y174V, Y183F. M196L, R197A, R198C, G200K, A207S, E210T, N214S, A218E, L220C, G229R, R233T, S235Q, L239M, N241M, P242T,V243I, G244S, E258K, C266I, V278I, E279Q, K281G, and G292K, a proline insertion between amino acid positions 240 and 241, and a glycine insertion between amino acid positions 288 and26081289, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions I32V, T36P, P37D, T38D, A39M, S41D, S47R, V53Q, P54E, S55E, P57E, P74A, R102S. H106R, A117R, T127S, D129A, A135N, S136A, S137H, P146A, A156Q, A160K, T166P. Y174V, Y183F, M196L, R197A, R198C. G200K, A207S, E210T, N214S. A218E, L220C, A226K, G229R, R233T, S235Q, L239M, N241M, P242T, V243I, G244S, E258K, C266I, V278I, E279Q, K281G, and G292K, a proline insertion between amino acid positions 240 and 241, and a glycine insertion between amino acid positions 288 and 289, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions I32V, T36P, P37D, T38D, A39M, S41D, S47R, V53Q, P54E, S55E, P57E, P74A, R102S, H106R, A117R, T127S, D129A, A135N, S136A, S137H, P146A, A156Q, A160K, T166P, Y174V, Y183F, M196L, R197A, R198C. G200K, A207S, E210T, N214S, A218E, L220C, A226K, G229R, R233T, S235Q, L239M. N241M, V243I, G244S, E258K. C266I. Y269L, V278L, E279Q. K281G, and G292K, a lysine insertion between amino acid positions 240 and 241, and a glycine insertion between amino acid positions 288 and 289, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions I32V, T36P. P37D, T38D, A39M, S41D, S47R, V53Q, P54E, P74A, R102S, H106R, A117R, T127S, D129A, A135N, S136A, S137H, P146A, A156Q, A160K, T166P, Y174V, Y183F, G184C, M196L, R197A, R198C, G200K, A207S, E210T, N214S, A218E, L220C, Q224M, A226K, G229R, R233T, S235Q, L239M, N241M, V243I, G244S, E258K, C266I, Y269L, V278I, E279Q, K281G, and G292K, a lysine insertion between amino acid positions 240 and 241, and a glycine insertion between amino acid positions 288 and 289, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions I32V, A39M, S47R, V53Q, P54E, P74A, R102S, H106R, A117R, T127S, D129A, A135N, S136A. S 137H. P146A, A156Q, A160K, T166P, Y174V, Y183F, G184C, M196L, R197A, R198C, G200K, A207S, E210T, N214S, A218E, L220C, Q224M, A226K, G229R, R233T, S235Q, L239M, N241M, V243I, G244S, E258K, C266I, Y269L, V278I, E279Q, K281G, and G292K, a lysine insertion between amino acid positions 240 and 241, and a glycine insertion between amino acid positions 288 and 289, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions I32V, A39M, S47R, V53Q, P54E, P74A, R102S, H106R, A117R, T127S, D129A, A135N,26081S136A, S137H, P146A, A156Q. A160K, T166P, Y174V. Y183F, G184C, M196L, R197G, R198C, G200K, A207S, E210T, N214S, A218E, L220C, Q224M, A226K, G229R, R233T, S235Q, L239M, N241M, V243I, G244S, E258K, C266I, Y269L, V278I, E279Q, K281G, and G292K, a lysine insertion between amino acid positions 240 and 241, and a glycine insertion between amino acid positions 288 and 289, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions I32V, A39M, S47R, V53Q, P54E, P74A, R102S, H106R, A117R, T127S, D129A, A135N, S136A, S137H, P146A, A156Q, A160K, T166P, Y174V, Y183F, G184C. M196L, R197G, R198C, G200K, A207S, E210T. N214S, A218E, L220C, Q224M, A226K, G229R, R233T, S235Q, L239M, N241M, V243I, G244S, E258K, C266I, Y269L, V278I, E279Q, K281G, G285E, and G292K, an asparagine insertion between amino acid positions 240 and 241, and a glycine insertion between amino acid positions 288 and 289, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions I32V, V53Q, P54E, P74A, R102S, H106R, A117R, G123A, T127S, D129A, A135N, S136A, S137H, L138M, F139Y, P146A, A156Q, A160K, T166P, Y174V, Y183F, G184C, M196L, R197G, R198K, G200K, A207S, E210T, N214S, A218E, L220C, Q224M, A226K, M227W, G229R, S230G. R233T, S235Q, L239M, N241M, V243I, G244S, E258K. C266I, Y269L, V278I, E279Q, K281G, G285M, G286V, and G292K, an asparagine insertion between amino acid positions 240 and 241, and a glycine insertion between amino acid positions 288 and 289, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions 19V. I32V, V53Q. P54E, P74A. 193V, R102S, H106R. A117R, G123A, T127S, D129A, A135N, S136A, S137H, F139Y, P146A, A156Q, A160K, T166P, Y174V, Y183F, G184C, M196L, R197G, R198K, G200K, A207S, E210T, N214S, A218E, L220C, Q224M, A226K, M227W, G229R, S230G, R233T, S235Q, L239M, N241M, V243I, G244S, E258K, G264P, G265H. C266I, L267F. Y269L, V278I. E279Q, K281G, G285M, G286V. G292K, and L293V, an asparagine insertion between amino acid positions 240 and 241 and an alanine insertion between amino acid positions 288 and 289, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions I9V, I32V, V53Q. P54E, P74A, I93V, R102S, H106R, A117R, G123A, T127S, D129A, A135N, S136A, S137H, F139Y, P146A, A156Q, A160K, T166P, Y174V, Y183F, G184C, M196L, R197G, R198K, G200K, A207S, E210T, N214S, A218E, L220C, Q224M, A226K, M227W, G229R, S230G, R233T,26081S235Q, L239M, G240P. N241M, P242T, V243I, G244S. E258K. G264P, G265H. C266I.L267F, Y269L, V278I, E279Q, K281G, G285K, G286V, G292K, and L293V, a glycine insertion between amino acid positions 240 and 241 and an alanine insertion between amino acid positions 288 and 289, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the polypeptide comprises amino acid substitutions I9V, I32V, V53Q, P54E, D72N, I93V, R102S, H106R, Al 17R, G123A, T127S, D129A, A135N, S136A, S137H, F139Y, P146A, A156Q, A160K, T166P, Y174V, Y183F, G184C, M196L, R197G, R198K, G200K, A207S, E210T, N214S, A218E, L220C, Q224M, A226K. M227W. G229R, S230G, R233T, S235Q. L239M, G240P, N241M, P242T, V243I, G244S, E258K, G264P, G265H, C266I, L267F, Y269L, V278I, E279Q, K281G, G283E, G285M, G286V, G292K, and L293V, a glycine insertion between amino acid positions 240 and 241 and an alanine insertion between amino acid positions 288 and 289, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38 or SEQ ID NO: 40.

[0178] In some embodiments, the amino acid sequence comprises any one of SEQ ID NOs: 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192. In some embodiments, the amino acid sequence consists of any one of SEQ ID NOs: 42. 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192. In some embodiments, the amino acid sequence comprises any one of SEQ ID NOs: 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66. 68. 70. 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, or 98. In some embodiments, the amino acid sequence comprises any one of SEQ ID NOs: 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192.

[0179] In some embodiments, provided herein is a polypeptide (e g., an IRED polypeptide) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to any one of SEQ ID NOs: 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182. 184, 186, 188, 190, or 192, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40.

[0180] In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 91% sequence identity' to any one of SEQ ID26081NOs: 42. 44. 46. 48, 50, 52, 54, 56, 58, 60, 62, 64, 66. 68. 70. 72. 74. 76. 78. 80. 82, 84, 86, 88, 90, 92, 94, 96, 98, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 92% sequence identity to any one of SEQ ID NOs: 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182. 184, 186, 188, 190, or 192, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 93% sequence identity to any one of SEQ ID NOs: 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98. 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156. 158, 160, 162. 164, 166. 168, 170, 172, 174, 176. 178, 180. 182, 184, 186. 188, 190. or 192, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 94% sequence identity to any one of SEQ ID NOs: 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66. 68. 70. 72. 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 42, 44, 46. 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e g., an IRED polypeptide) comprising an amino acid sequence having at least 96% sequence identity’ to any one of SEQ ID NOs: 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156. 158, 160, 162, 164, 166. 168, 170, 172, 174, 176, 178, 180. 182, 184, 186, 188, 190. or 192, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 97% sequence identity' to any one of SEQ ID26081NOs: 42. 44. 46. 48, 50, 52, 54, 56, 58, 60, 62, 64, 66. 68. 70. 72. 74. 76. 78. 80. 82, 84, 86, 88, 90, 92, 94, 96, 98, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 98% sequence identity to any one of SEQ ID NOs: 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182. 184, 186, 188, 190, or 192, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 99% sequence identity to any one of SEQ ID NOs: 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98. 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156. 158, 160, 162. 164, 166. 168, 170, 172, 174, 176. 178, 180. 182, 184, 186. 188, 190. or 192, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising the amino acid sequence of any one of SEQ ID NOs: 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82. 84. 86. 88, 90, 92, 94, 96, 98, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192. Also provided herein is a polypeptide (e.g., an IRED polypeptide) consisting essentially of the amino acid sequence of any one of SEQ ID NOs: 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68. 70. 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96. 98. 136, 138. 140, 142, 144, 146, 148. 150, 152. 154, 156, 158. 160, 162. 164, 166. 168. 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192. Also provided herein is a polypeptide (e.g., an IRED polypeptide) consisting of the amino acid sequence of any one of SEQ ID NOs: 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66. 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94. 96, 98, 136, 138. 140, 142. 144, 146, 148, 150, 152. 154, 156, 158, 160, 162. 164, 166. 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192.

[0181] In some embodiments, provided herein is a polypeptide (e.g., an IRED 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: 82 or SEQ ID NO: 176, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 82 or SEQ ID NO: 176,26081 wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 82 or SEQ ID NO: 176, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising the amino acid sequence of SEQ ID NO: 82 or SEQ ID NO: 176. Also provided herein is a polypeptide (e.g., an IRED polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 82 or SEQ ID NO: 176. Also provided herein is a polypeptide (e.g., an IRED polypeptide) consisting of the amino acid sequence of SEQ ID NO: 82 or SEQ ID NO: 176.

[0182] In some embodiments, provided herein is a polypeptide (e.g., an IRED 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: 84 or SEQ ID NO: 178, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 98% sequence identity’ to SEQ ID NO: 84 or SEQ ID NO: 178, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 84 or SEQ ID NO: 178, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising the amino acid sequence of SEQ ID NO: 84 or SEQ ID NO: 178. Also provided herein is a polypeptide (e.g., an IRED polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 84 or SEQ ID NO: 178. Also provided herein is a polypeptide (e.g., an IRED polypeptide) consisting of the amino acid sequence of SEQ ID NO: 84 or SEQ ID NO: 178.

[0183] In some embodiments, provided herein is a polypeptide (e.g., an IRED 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: 86 or SEQ ID NO: 180, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 98% sequence identity’ to SEQ ID NO: 86 or SEQ ID NO: 180, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising26081 an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 86 or SEQ ID NO: 180, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising the amino acid sequence of SEQ ID NO: 86 or SEQ ID NO: 180. Also provided herein is a polypeptide (e.g.. an IRED polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 86 or SEQ ID NO: 180. Also provided herein is a polypeptide (e.g., an IRED polypeptide) consisting of the amino acid sequence of SEQ ID NO: 86 or SEQ ID NO: 180.

[0184] In some embodiments, provided herein is a polypeptide (e.g., an IRED 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: 88 or SEQ ID NO: 182, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 88 or SEQ ID NO: 182, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 88 or SEQ ID NO: 182, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising the amino acid sequence of SEQ ID NO: 88 or SEQ ID NO: 182. Also provided herein is a polypeptide (e.g., an IRED polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 88 or SEQ ID NO: 182. Also provided herein is a polypeptide (e.g., an IRED polypeptide) consisting of the amino acid sequence of SEQ ID NO: 88 or SEQ ID NO: 182.

[0185] In some embodiments, provided herein is a polypeptide (e.g., an IRED 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: 90 or SEQ ID NO: 184, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 90 or SEQ ID NO: 184, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 90 or SEQ ID NO: 184, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO:2608140. In some embodiments, provided herein is a polypeptide (e.g., an 1RED polypeptide) comprising the amino acid sequence of SEQ ID NO: 90 or SEQ ID NO: 184. Also provided herein is a polypeptide (e.g., an IRED polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 90 or SEQ ID NO: 184. Also provided herein is a polypeptide (e.g., an IRED polypeptide) consisting of the amino acid sequence of SEQ ID NO: 90 or SEQ ID NO: 184.

[0186] In some embodiments, provided herein is a polypeptide (e.g., an IRED 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: 92 or SEQ ID NO: 186, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 92 or SEQ ID NO: 186, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 92 or SEQ ID NO: 186, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising the amino acid sequence of SEQ ID NO: 92 or SEQ ID NO: 186. Also provided herein is a polypeptide (e.g., an IRED polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 92 or SEQ ID NO: 186. Also provided herein is a polypeptide (e.g., an IRED polypeptide) consisting of the amino acid sequence of SEQ ID NO: 92 or SEQ ID NO: 186.

[0187] In some embodiments, provided herein is a polypeptide (e.g., an IRED 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: 94 or SEQ ID NO: 188, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 94 or SEQ ID NO: 188, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 94 or SEQ ID NO: 188, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising the amino acid sequence of SEQ ID NO: 94 or SEQ ID NO: 188. Also provided26081 herein is a polypeptide (e.g., an IRED polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 94 or SEQ ID NO: 188. Also provided herein is a polypeptide (e.g., an IRED polypeptide) consisting of the amino acid sequence of SEQ ID NO: 94 or SEQ ID NO: 188.

[0188] In some embodiments, provided herein is a polypeptide (e.g., an IRED 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: 96 or SEQ ID NO: 190, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 96 or SEQ ID NO: 190, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 96 or SEQ ID NO: 190, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising the amino acid sequence of SEQ ID NO: 96 or SEQ ID NO: 190. Also provided herein is a polypeptide (e.g., an IRED polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 96 or SEQ ID NO: 190. Also provided herein is a polypeptide (e.g., an IRED polypeptide) consisting of the amino acid sequence of SEQ ID NO: 96 or SEQ ID NO: 190.

[0189] In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 98 or SEQ ID NO: 192, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 98 or SEQ ID NO: 192, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 98 or SEQ ID NO: 192, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising the amino acid sequence of SEQ ID NO: 98 or SEQ ID NO: 192. Also provided herein is a polypeptide (e.g., an IRED polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 98 or SEQ ID NO: 192. Also provided herein is a polypeptide (e.g., an26081IRED polypeptide) consisting of the amino acid sequence of SEQ ID NO: 98 or SEQ ID NO: 192.

[0190] In some embodiments, provided herein is a polypeptide (e.g., an IRED 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: 94 or SEQ ID NO: 186, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 94 or SEQ ID NO: 186, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 94 or SEQ ID NO: 186, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, provided herein is a polypeptide (e.g., an IRED polypeptide) comprising the amino acid sequence of SEQ ID NO: 94 or SEQ ID NO: 186. Also provided herein is a polypeptide (e.g., an IRED polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 94 or SEQ ID NO: 186. Also provided herein is a polypeptide (e.g., an IRED polypeptide) consisting of the amino acid sequence of SEQ ID NO: 94 or SEQ ID NO: 186.

[0191] 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: 131). 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 consisting of SEQ ID NO: 131 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 some embodiments the amino acid linker comprises 1, 2, 3, 4, 5, or 6 amino acids.

[0192] For example, an IRED 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:2608140-98 may be modified to no longer include a C-terminal His tag having the amino acid sequence of SEQ ID NO: 131. For example, the polypeptide sequence of any one of even numbered SEQ ID NO: 136-190 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: 131.

[0193] In some embodiments, the polypeptide is isolated.

[0194] In some embodiments, the polypeptide has imine reductase activity.

[0195] 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.

[0196] In addition to the positions of residue differences specified above, any of the engineered IRED polypeptides disclosed herein can further comprise other residue differences relative to SEQ ID NO: 38 or SEQ ID NO: 40 at other residue positions than those of amino acid differences disclosed in Tables 3 and 4, e.g., residue positions other than 9, 32, 36, 37, 38, 39, 41, 47, 53, 54, 55, 57, 72, 74, 93, 102, 106, 108, 117, 123, 127, 129, 135. 136, 137, 138, 139, 146,156, 160. 164, 166, 174. 183, 184. 194, 196, 197, 198, 200. 207, 210. 211, 214, 218. 220, 224.226, 227, 229, 230, 232, 233, 235, 239, 240, 241, 242, 243, 244, 258, 264, 265, 266, 267, 269,278, 279, 281, 283, 285, 286, 292, and 293. 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 IRED polypeptides selected from SEQ ID NOs: 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156. 158, 160, 162, 164, 166. 168, 170, 172, 174, 176, 178, 180. 182, 184, 186, 188, 190. or 192, 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, 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: 38 or SEQ ID NO: 40. In some embodiments, the number of amino acid residue differences as compared to the reference sequence 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, 30, 35, 40, 45 or 50 residue positions. In some embodiments, the number of amino acid residue differences as compared to the reference sequence can be 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: 38 or SEQ ID NO: 40.26081

[0197] In some embodiments, the engineered IRED polypeptide can comprise a deletion at one or more amino acid positions as compared to any one of the engineered IRED polypeptides described herein, such as the exemplary7engineered polypeptides of SEQ ID NOs: 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74. 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 136, 138. 140, 142, 144, 146, 148. 150, 152, 154, 156, 158. 160, 162. 164, 166, 168, 170, 172. 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192. Thus, for each embodiment of the engineered IRED polypeptides of the invention, 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 10% 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 IRED polypeptides, where the associated functional activity and / or improved properties of the engineered IRED 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 ammo 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, 30, 35, 40, 45, or 50 amino acid residues. In some embodiments, the deletions can comprise deletions of 1, 2, 3, 4, 5, 6. 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 amino acid residues.

[0198] In some embodiments, the engineered IRED polypeptide herein can have an amino acid sequence comprising an insertion as compared to any one of the engineered IRED polypeptides described herein, such as the exemplary engineered polypeptides of SEQ ID NOs: 42, 44, 46, 48, 50. 52. 54. 56, 58, 60, 62, 64, 66, 68, 70, 72, 74. 76. 78. 80. 82. 84. 86, 88, 90, 92, 94, 96, 98, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192. 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 IRED described herein is maintained. The insertions can be to amino or carboxy terminus, or internal portions of the IRED polypeptide.

[0199] In some embodiments, the engineered IRED polypeptide herein can have an amino acid sequence comprising a sequence selected from SEQ ID NOs: 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 136, 138, 140, 142,26081144, 146. 148, 150, 152. 154, 156. 158, 160, 162. 164, 166. 168, 170. 172, 174, 176. 178, 180. 182, 184, 186, 188, 190, or 192, 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, 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.

[0200] Also provided herein are compositions comprising any of the polypeptides disclosed herein. For example, the composition may include an effective amount of the IRED polypeptide for catalyzing the reduction of an imine to an amine. In some embodiments, the composition may include an effective amount of the IRED polypeptide for catalyzing the NAD(P)H-dependent reduction of an imine group by converting NAD(P)H to NAD(P)+ and the imine to amine. In some embodiments, the composition may include an effective amount of the IRED polypeptide for catalyzing the NAD(P)H-dependent reduction of an imine group to a primary or secondary amine. In some embodiments, the composition may include an effective amount of the IRED polypeptide for catalyzing the reduction of imines formed from the condensation of nucleophilic amino acid residue side-chains and other electrophilic amino acid residue side-chains in peptides. In some embodiments, the composition may include an effective amount of the IRED polypeptide for catalyzing reactions in the process of generating macrocyclic peptides. The composition may include one or more carriers or diluents.

[0201] In some embodiments, the polypeptides of the invention 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.

[0202] 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.26081

[0203] 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, and can have swelling or non-swelling characteristics. A solid support can be configured in the form of a well, depression, or other container, vessel, feature, or location.

[0204] In some embodiments, the engineered KRED and / or IRED polypeptides of the present invention 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 facilitate the biocatalytic conversions according to Scheme A or Scheme B and after the reaction is complete are easily retained (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 KRED and / or IRED polypeptides of the present invention can be carried out using the same KRED and / or IRED polypeptides bound or immobilized on a solid support.

[0205] In some embodiments, the poly peptides 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.

[0206] In some embodiments, the kits of the present invention include arrays comprising a plurality of different KRED and / or IRED 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 plurality of polypeptides immobilized on solid supports can be configured on an array at various locations, addressable for robotic delivery of 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.26081Polynucleotides Encoding KRED and IRED Polypeptides

[0207] In another aspect, the present disclosure provides polynucleotides encoding the polypeptides (e.g., KRED polypeptides or IRED 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., KRED polypeptide or IRED polypeptide) can be introduced into appropriate host cells to express the corresponding polypeptide.

[0208] Because of the knowledge of the codons corresponding to the various amino acids, availability of 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 are 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., KRED polypeptides or IRED 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.

[0209] 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. coll.

[0210] In certain embodiments, all codons need not be replaced to optimize the codon usage of the polypeptide (e.g., KRED polypeptide or IRED 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., KRED polypeptides or IRED 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.

[0211] 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%, or2608199%) sequence identity to the polynucleotide sequence of SEQ ID NOs: 1, 3. 5, 7. 9, 1 1, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, or 129, 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 NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, or 129, wherein the polynucleotide does not comprise SEQ ID NO: 1 or SEQ ID NO: 3. In some examples, the polynucleotide sequence of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, or 35 is modified to no longer code for a C-terminal His tag having the amino acid sequence of SEQ ID NO: 131, SEQ ID NO: 132, or SEQ ID NO: 198. In some embodiments, 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.

[0212] 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 any one of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33. 35. 101, 103, 105, 107, 109, 11 1, 113, 115, 117, 119, 121, 123, 125, 127, or 129, 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 anyone of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33. 35. 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, or 129, 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 NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33. 35, 101, 103, 105, 107, 109, 111. 113, 115, 117. 119, 121. 123, 125, 127, or 129, 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 NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 101, 103, 105, 107, 109, 111. 113, 115, 117, 119, 121. 123, 125, 127, or 129, 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 NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 101, 103, 105, 107,26081109, 111, 113, 115, 117, 119, 121, 123, 125, 127, or 129, 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 NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33. 35, 101, 103, 105, 107, 109, 111. 113, 115, 117. 119, 121. 123, 125, 127, or 129, 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 NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 101, 103, 105, 107, 109, 111. 113, 115, 117, 119, 121. 123, 125, 127, or 129, 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 NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 101, 103, 105, 107, 109, 111. 113, 115, 117, 119, 121, 123, 125, 127, or 129, 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 NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, or 129, 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 NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, or 129, 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 NOs: 5, 7, 9, 1 1, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, or 129, 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% sequence identity to the polynucleotide sequence of any one of SEQ ID NOs: 5, 7, 9, 1 1, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, or 129, 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 NOs: 5, 7, 9, 11. 13. 15. 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 101, 103, 105, 107, 109, 111, 113, 115, 117, 1 19, 121, 123, 125, 127, or 129. In some embodiments, provided herein is a polynucleotide consisting of any one of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 101, 103, 105, 107, 109, 111, 113, 115, 117,26081119, 121, 123, 125, 127, or 129. In some embodiments, provided herein is a polynucleotide comprising any one of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, or 35. In some embodiments, provided herein is a polynucleotide consisting of any one of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31. 33, or 35. In some embodiments, provided herein is a polynucleotide comprising any one of SEQ ID NOs: 101, 103. 105, 107, 109. I l l, 113, 115, 117, 119, 121, 123, 125, 127, or 129. In some embodiments, provided herein is a polynucleotide consisting of any one of SEQ ID NOs: 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, or 129.

[0213] In another aspect, the present disclosure provides polynucleotides encoding the polypeptides (e.g., IRED 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.. IRED polypeptide) can be introduced into appropriate host cells to express the corresponding polypeptide.

[0214] Because of the knowledge of the codons corresponding to the various amino acids, availability of 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 are 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., IRED 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.

[0215] 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: 39 provided herein has been codon optimized for expression in E. coll.

[0216] In certain embodiments, all codons need not be replaced to optimize the codon usage of the polypeptide (e.g., IRED polypeptide) since the natural sequence will comprise preferred codons and because use of preferred codons may not be required for all amino acid residues.26081Consequently, codon optimized polynucleotides encoding the polypeptides (e.g., 1RED 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.

[0217] 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 NOs: 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173. 175, 177, 179, 181, 183. 185, 187, 189, or 191, wherein the polynucleotide does not comprise SEQ ID NO: 37 or SEQ ID NO: 39. 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 NOs: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61. 63, 65, 67. 69, 71. 73, 75, 77, 79, 81, 83, 85, 87, 89, 91. 93. 95. 97. 133, 135. 137, 139, 141. 143, 145. 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, or 191, wherein the polynucleotide does not comprise SEQ ID NO: 37 or SEQ ID NO: 39. In some examples, the polynucleotide sequence of SEQ ID NO: 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77. 79. 81. 83. 85, 87, 89, 91, 93, 95, or 97 is modified to no longer code for a C-terminal His tag having the amino acid sequence of SEQ ID NO: 131. In some embodiments, 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.

[0218] 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 any one of SEQ ID NOs: 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67. 69, 71, 73, 75, 77, 79, 81. 83, 85, 87, 89, 91, 93, 95, 97, 133. 135, 137. 139, 141, 143. 145, 147. 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, or 191, wherein the polynucleotide does not comprise SEQ ID NO: 37 or SEQ ID NO: 39. In some embodiments, provided herein is a polynucleotide comprising at least 80% sequence identity to the polynucleotide sequence of any one of SEQ ID NOs: 41, 43, 45, 47, 49, 51, 53. 55. 57. 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, or 191, wherein the polynucleotide does not comprise SEQ ID NO: 37or SEQ ID NO: 39. In some embodiments, provided herein is a polynucleotide compnsing at least 90% sequence identity to the polynucleotide sequence of any one of SEQ ID NOs: 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 133, 135, 137, 139, 141, 143, 145, 147, 149. 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171. 173, 175, 177. 179, 181. 183, 185, 187, 189, or 191, wherein the polynucleotide does not comprise SEQ ID NO: 37 or SEQ ID NO: 39. In some embodiments, provided herein is a polynucleotide comprising at least 91% sequence identity to the polynucleotide sequence of any one of SEQ ID NOs: 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 133, 135, 137. 139, 141, 143, 145, 147. 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, or 191, wherein the polynucleotide does not comprise SEQ ID NO: 37 or SEQ ID NO: 39. In some embodiments, provided herein is a polynucleotide comprising at least 92% sequence identity to the polynucleotide sequence of any one of SEQ ID NOs: 41, 43, 45, 47, 49, 51, 53. 55, 57, 59, 61. 63, 65. 67, 69, 71, 73, 75, 77, 79, 81, 83, 85. 87. 89. 91. 93. 95. 97, 133, 135. 137, 139. 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, or 191, wherein the polynucleotide does not comprise SEQ ID NO: 37 or SEQ ID NO: 39. In some embodiments, provided herein is a polynucleotide comprising at least 93% sequence identity to the polynucleotide sequence of any one of SEQ ID NOs: 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, or 191, wherein the polynucleotide does not comprise SEQ ID NO: 37 or SEQ ID NO: 39. In some embodiments, provided herein is a polynucleotide comprising at least 94% sequence identity to the polynucleotide sequence of any one of SEQ ID NOs: 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159. 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, or 191, wherein the polynucleotide does not comprise SEQ ID NO: 37 or SEQ ID NO: 39. In some embodiments, provided herein is a polynucleotide comprising at least 95% sequence identity' to the polynucleotide sequence of any one of SEQ ID NOs: 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 133, 135, 137, 139, 141, 143, 145. 147, 149, 151, 153, 155. 157, 159, 161, 163, 165, 167, 169. 171, 173, 175, 177, 179. 181, 183, 185, 187, 189, or 191, wherein the polynucleotide does not comprise SEQ ID NO: 37 or SEQ ID NO: 39. In some embodiments, provided herein is a polynucleotide comprising at least 96% sequence identity to the polynucleotide sequence of any one of SEQ ID NOs: 41, 43,45. 47. 49. 51, 53, 55, 57, 59, 61, 63, 65, 67, 69. 71. 73. 75. 77. 79. 81, 83, 85, 87, 89, 91, 93, 95, 97, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, or 191, wherein the polynucleotide does not comprise SEQ ID NO: 37 or SEQ ID NO: 39. In some embodiments, provided herein is a polynucleotide comprising at least 97% sequence identity to the polynucleotide sequence of any one of SEQ ID NOs: 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, or 191, wherein the polynucleotide does not comprise SEQ ID NO: 37 or SEQ ID NO: 39. In some embodiments, provided herein is a polynucleotide comprising at least 98% sequence identity to the polynucleotide sequence of any one of SEQ ID NOs: 41, 43, 45, 47, 49, 1, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 133, 135, 137, 139, 141, 143, 145. 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169. 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, or 191, wherein the polynucleotide does not comprise SEQ ID NO: 37 or SEQ ID NO: 39. In some embodiments, provided herein is a polynucleotide comprising at least 99% sequence identity to the polynucleotide sequence of any one of SEQ ID NOs: 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 133, 135. 137, 139, 141, 143, 145. 147, 149. 151, 153, 155, 157, 159. 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, or 191, wherein the polynucleotide does not comprise SEQ ID NO: 37 or SEQ ID NO: 39. In some embodiments, provided herein is a polynucleotide comprising any one of SEQ ID NOs: 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89. 91. 93, 95, 97, 133, 135. 137, 139, 141, 143, 145, 147. 149, 151, 153. 155, 157. 159, 161, 163. 165, 167. 169, 171. 173, 175, 177. 179, 181. 183, 185, 187, 189, or 191. In some embodiments, provided herein is a polynucleotide consisting any one of SEQ ID NOs: 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151. 153, 155, 157, 159. 161, 163, 165. 167, 169. 171, 173, 175, 177, 179. 181, 183. 185, 187, 189, or 191. In some embodiments, provided herein is a polynucleotide comprising any one of SEQ ID NOs: 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, or 97. In some embodiments, provided herein is a polynucleotide consisting any one of SEQ ID NOs: 41, 43, 45, 47, 49, 51, 53, 55, 57. 59. 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, or 97. In some embodiments, provided herein is a polynucleotide comprising any one of SEQ ID NOs: 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, or 191. In some26081 embodiments, provided herein is a polynucleotide consisting of any one of SEQ ID NOs: 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, or 191.

[0219] In various embodiments, an isolated polynucleotide encoding polypeptide (e.g., a KRED polypeptide or an IRED polypeptide) may be manipulated in a variety of ways to provide for expression of the 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.

[0220] 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.

[0221] 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.

[0222] 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 prokaryotic 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,26081 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., WO 96 / 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 -phosphoglycerate 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).

[0223] 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 enzyme polypeptide. Any suitable terminator that is functional in the host cell of choice finds use in the present disclosure. Exemplar)’ transcription terminators for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-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).

[0224] 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 3-26081 phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0225] 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 poly adenosine residues to transcribed mRNA). Any suitable polyadenylation sequence that is functional in the host cell of choice may 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 polyadenylation sequences for yeast host cells are known (See e.g., Guo and Sherman, Mol. Cell. Biol., 15:5983-5990, 1995).

[0226] 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 pathw ay 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 1 1837 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.26081

[0227] In some embodiments, regulator}- sequences are also utilized. These sequences facilitate the regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory' 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 regulatory sequences include, but are not limited to, the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter.

[0228] In another aspect, the present disclosure provides a recombinant expression vector comprising a polynucleotide encoding a polypeptide (e.g., a KRED polypeptide or an IRED 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 enzy me 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.

[0229] 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 ty pically 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.

[0230] 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 or26081 plasmids which together contain the total DNA to be introduced into the genome of the host cell, and / or a transposon is utilized.

[0231] 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, ADE2, 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 A. 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.

[0232] 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.

[0233] 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.

[0234] 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.

[0235] 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 integration26081 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 acid sequences. In other examples, the vector may be integrated into the genome of the host cell by non-homologous recombination.

[0236] 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 bactenal origins of replication are P15A ori or the origins of replication of plasmids pBR322, pUC19, pET30a(+), pACYC177 (which contains the P15A ori), or pACYC184 (which contains the P15A ori) permitting replication in A. coll, and pUBUO, 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 ARS1 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).

[0237] 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.

[0238] 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. coll 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).

[0239] Thus, in some embodiments, a vector comprising a sequence encoding at least one variant polypeptide (e.g., a KRED polypeptide or an IRED polypeptide) is transformed into a26081 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 grown in HTP media. Suitable media are available from various commercial suppliers or may be prepared according to published recipes (e.g., in catalogues of the American Type Culture Collection).Host Cells for Expression of KRED and IRED Polypeptides

[0240] The present disclosure also provides a host cell comprising a polynucleotide encoding a polypeptide (e.g., a KRED polypeptide or an IRED polypeptide) disclosed herein, or an expression vector comprising a polynucleotide encoding a polypeptide (e.g., a KRED polypeptide or an IRED 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.

[0241] Host cells for use in expressing the polypeptides described herein are well know n in the art and include, but are not limited to, prokaryotic cells (e.g., bacterial cells (e.g., E. coll, B. subtilis, B. licheniformis , B. megaterium, B. stearothermophilus , B. amyloliquefaciens , Lactobacillus kejir, 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 pastoris) or mammalian cells). Appropriate culture mediums and growth conditions for the above-described host cells are well known in the art.

[0242] Polynucleotides for expression of the polypeptides (e.g., KRED polypeptides or IRED 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.

[0243] 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, C ochliobolus,, Corynascus, Cryphonectria, Cryptococcus, Coprinus, Corioliis, Diplodia, Endothis, Fusarium, Gibberella, Gliocladium,26081Humicola, 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.

[0244] In some embodiments, the host cell is a yeast cell, including but not limited to cells of Candida, Hansenula, Saccharomyces , Schizosaccharomyces , Pichia, Kluyveromyces, or Yarrowia species. In some embodiments of the present disclosure, the yeast cell is Hansenula polymorpha, Saccharomyces cerevisiae. Saccharomyces carlsber gensis, 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.

[0245] 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, Rhodospir ilium, 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 an26081 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 / I. 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, B. megaterium, B. subtilis, B. lentus. B. circulans, B. pumihis, B. lautus, B.coagulans, B. brevis, B. flrmus, B. alkaophius. B. licheniformis. B. clausii, B. stear other mophilus, B. halodurans, orB. amyloliquefaciens). In some embodiments, the host cell is an industrial Bacillus strain including but not limited to B. subtilis, B. pumihis, B. licheniformis, B. megaterium, B. clausii, B. stear othermophilus, or B. amyloliquefaciens. In some embodiments, the Bacillus host cells are B. subtilis, B. licheniformis, B. megaterium, B. stear other mophilus, 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 coll 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 aStreptomyces 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 aZymomonas species (e.g., Z. mobilis, or Z. lipolytica).

[0246] Many prokaryotic 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).26081

[0247] 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 KRED or IRED variant(s) within the host cell and / or in the culture medium. In one 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 variety7of methods are know n 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).

[0248] 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.

[0249] 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 host26081 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.

[0250] 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 with the host cell 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.

[0251] In some embodiments, cells expressing a KRED polypeptide or an IRED 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 know n in the art of industrial microbiology.

[0252] 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 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.26081

[0253] In some embodiments, cell-free transcription and translation systems find use in producing the polypeptide(s) (e.g., KRED polypeptide(s) or IRED polypeptide(s)). Several systems are commercially available, and the methods are well-known to those skilled in the art.Methods of Evolving KRED and IRED polypeptides

[0254] Evolution (e.g., directed evolution) may be used to identify polypeptides (e.g., KRED polypeptides or IRED polypeptides) of the present disclosure. For example, in some embodiments, to make a KRED polypeptide of the present disclosure, a KRED polypeptide may be obtained (or derived) from any suitable bacterial species, e.g., Krypidia tusciae. In some embodiments, the parent polynucleotide sequence is codon optimized to enhance expression of the KRED 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, was codon optimized for expression in E. coll and the codon-optimized polynucleotide was cloned into an expression vector, placing the expression of the KRED gene under the control of the T7 promoter. The T7 polymerase needed to express the gene of interest may be under control of the lac promoter, and both the gene of interest and the T7 polymerase may be subject to lacl repression. In this example, the presence of IPTG activates the T7 polymerase production and eliminates the repression, resulting in production of the KRED gene. Clones expressing the active KRED in E. coli may be identified and the genes sequenced to confirm their identity.

[0255] Similarly, in some embodiments, to make an IRED polypeptide of the present disclosure, an IRED polypeptide may be obtained (or derived) from any suitable bacterial species, e.g., Pseudogymnoascus sp. VKM F-4516. In some embodiments, the parent polynucleotide sequence is codon optimized to enhance expression of the IRED polypeptide in a specified host cell (e g., E. coli). For example, as is described in Example 12, a parental polynucleotide sequence, designated as SEQ ID NO: 39, was codon optimized for expression in E. coli and the codon-optimized polynucleotide was cloned into an expression vector, placing the expression of the KRED gene under the control of the T7 promoter in a manner similar to that described for KRED expression above.

[0256] The polypeptides (e.g., KRED polypeptides or IRED polypeptides) of the disclosure may be obtained by subjecting a polynucleotide encoding a parent sequence to mutagenesis and / or directed evolution methods. An exemplary directed 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 used26081 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).

[0257] The clones obtained following mutagenesis treatment may be screened for KRED polypeptides having a desired improved enzyme property. Measuring enzyme activity from 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 increase (via an increase in absorbance or fluorescence) of NADH or NADPH concentration. In this reaction, the NAD+ or NADP+ is reduced by the ketoreductase as the ketoreductase oxidizes an alcohol group into the corresponding ketone or aldehyde. The rate of increase of NADH or NADPH concentration, as measured by the increase in absorbance or fluorescence, per unit time indicates the relative (enzymatic) activity of the ketoreductase polypeptide in a fixed amount of the lysate (or a lyophilized powder made therefrom). Where the improved enzyme property desired is thermal stabi 1 i ty, enzy me activity may be measured after subjecting the enzy me preparations to a defined temperature for a defined time and measuring the amount of enzyme activity' remaining after heat treatments. For example, a KRED activity assay as described in any of Examples 4-12 may be used.

[0258] Similarly, the clones obtained following mutagenesis treatment may be screened for IRED polypeptides having a desired improved enzyme property'. Measuring enzy me 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 imine reductase as the imine reductase reduces an imine group to an amine. 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 imine reductase polypeptide in a fixed amount of the lysate (or a lyophilized powder made therefrom). Where the improved enzyme property desired is thermal stability', enzy me activity' may be measured after subjecting the enzy me preparations to a defined temperature for a defined time and measuring the amount of enzyme activity remaining after heat treatments. For example, an IRED activity assay as described in any of Examples 14-26 may be used.26081

[0259] Where the improved enzyme 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, e.g., differential scanning colorimetry (DSC) a biochemical assay, or spectroscopy. Clones containing a polynucleotide encoding a KRED polypeptide or an IRED polypeptide may then be isolated, sequenced to identify the nucleotide sequence changes (if any), and used to express the enzyme in a host cell.

[0260] 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 enzymatic 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.

[0261] Polypeptides (e.g., KRED polypeptides or IRED 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, salting-out, 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.

[0262] Chromatographic techniques for isolation of the polypeptide (e g., KRED polypeptide or an IRED 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 w ill be apparent to those having skill in the art.26081

[0263] In some embodiments, affinity techniques may be used to isolate the improved polypeptides (e.g., KRED polypeptides or IRED 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 others 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 KRED polypeptide may be used.Methods of Using KRED Polypeptides

[0264] Also provided herein are methods of using a KRED polypeptide of the disclosure. In some examples, a KRED polypeptide of the disclosure is used in a method of catalyzing the oxidation of an alcohol group to the corresponding ketone or aldehyde. In some examples, a KRED polypeptide is used in a method of catalyzing the NAD(P)+-dependent oxidation of an alcohol group to the corresponding ketone. In some embodiments, the KRED polypeptides described herein are used in a method of catalyzing the NAD(P)+-dependent oxidation of an alcohol-side chain of a peptide that can be further used in the synthesis of macrocyclic and semimacrocyclic peptides. In some examples, a KRED polypeptide of the disclosure is used in a method of catalyzing reactions in the process of generating macrocyclic peptides.

[0265] In some examples, a KRED polypeptide of the disclosure is used in a method of catalyzing the NAD(P)+-dependent oxidation of substrate 1 in Scheme A above to provide the aldehyde 2, which in turn can spontaneously and reversibly cyclize to yield the imine 3 in Scheme A above. In some embodiments, the KRED polypeptides disclosed herein may be used in a single-pot reaction along with an IRED polypeptide of the disclosure where the KRED generates the imine substrate in situ as shown, for example, in Scheme C above.

[0266] For example, provided herein is a method of catalyzing the oxidation of an alcohol group to the corresponding ketone or aldehyde, the method comprising contacting a substrate with any one of the KRED polypeptides disclosed herein. In some embodiments, provided herein is a method of catalyzing the oxidation of an alcohol group to the corresponding ketone or aldehyde, the method comprising incubating a substrate in the presence of any one of the KRED polypeptides disclosed herein.

[0267] In some embodiments, the method of catalyzing the oxidation of an alcohol group to the corresponding ketone or aldehyde, comprises contacting a substrate with a KRED polypeptide as disclosed herein under reaction conditions suitable for converting the substrate to a desired product. In some embodiments, the method of catalyzing the oxidation of an alcohol group to the26081 corresponding ketone or aldehyde, comprises incubating a substrate in the presence of a KRED polypeptide as disclosed herein under reaction conditions suitable for converting the substrate to a desired product.

[0268] For example, provided herein is a method of catalyzing the oxidation of substrate 1 in Scheme A above to provide the aldehyde 2, the method comprising contacting the substrate 1 with any one of the KRED polypeptides disclosed herein. In some embodiments, provided herein is a method of catalyzing the oxidation of substrate 1 in Scheme A above to provide the aldehyde 2, the method comprising the method comprising incubating the substrate 1 with any one of the KRED polypeptides disclosed herein.

[0269] In some embodiments, the method of catalyzing the oxidation of substrate 1 in Scheme A above to provide the aldehyde 2 comprises contacting the substrate 1 with any one of the KRED polypeptides as disclosed herein under reaction conditions suitable for converting the substrate 1 into the aldehyde 2. In some embodiments, the method of catalyzing the oxidation of substrate 1 in Scheme A above to provide the aldehyde 2 comprises incubating the substrate 1 with any one of the KRED polypeptides as disclosed herein under reaction conditions suitable for converting the substrate 1 into the aldehyde 2.

[0270] Any KRED polypeptide or combination of KRED polypeptides disclosed herein may be used in the methods for catalyzing the oxidation an alcohol. As noted herein, in some embodiments, the KRED polypeptide can comprise an amino acid sequence that is at least about 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 conser ative substitutions.

[0271] For example, in some embodiments, the KRED 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, 32, 34, 36, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126. 128, or 130, wherein the polypeptide does not comprise SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the KRED 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, 32, 34, 36, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128,26081 or 130, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the KRED polypeptide comprises any of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, or 130. In some embodiments, the KRED polypeptide consists of any of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22. 24. 26. 28. 30, 32, 34, 36, 100, 102, 104, 106. 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, or 130.

[0272] In some embodiments, provided herein is a method of catalyzing the oxidation an alcohol, the method comprising contacting a substrate with any one of the KRED polypeptides disclosed herein, wherein at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the substrate is converted to the desired product.

[0273] In some embodiments, provided herein is a method of catalyzing the oxidation of substrate 1 in Scheme A above to provide the aldehyde 2, the method comprising contacting the substrate 1 with any one of the KRED polypeptides disclosed herein, wherein at least about 60%, 65%. 70%. 75%. 80%. 85%. 90%. 95%. 96%. 97%. 98%. 99%. or more of the substrate 1 is converted to the aldehyde 2 according to Scheme A above. In some embodiments, provided herein is a method of catalyzing the oxidation of substrate 1 in Scheme A above to provide the aldehyde 2, wherein at least about 90% (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%. 98%. 99%. or more) of the substrate 1 is converted to the aldehyde 2 according to Scheme A above.

[0274] As is known by those of skill in the art, KRED catalyzed reactions typically require a cofactor. Oxidation reactions catalyzed by the KRED enzy mes described herein also ty pically 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 KRED polypeptides described herein include, but are not limited to, NAD(P)+ (nicotinamide adenine dinucleotide phosphate), NAD(P)H (the reduced form of NAD(P)+), NAD+ (nicotinamide adenine dinucleotide) and NADH (the reduced form of NAD+).

[0275] In some embodiments, provided herein is a method of catalyzing the reduction of NAD(P) + to NAD(P)H, the method comprising contacting the NAD(P)+ with any one of the KRED 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)+ is converted to NAD(P)H. In some embodiments, provided herein is a method of catalyzing the reduction of NAD(P) + to NAD(P)H, 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)+ is converted to NAD(P)H.26081

[0276] The methods of the invention are generally carried out in a solvent. Suitable solvents include water, aqueous buffer solutions, organic solvents, polymeric solvents, and / or co-solvent systems, which generally comprise aqueous solvents, organic solvents and / or polymeric solvents. The aqueous solvent (water or aqueous co-solvent system) may be pH-buffered or unbuffered. In some embodiments, the methods using the engineered KRED polypeptides can be carried out in an aqueous co-solvent system comprising an organic solvent (e g., ethanol, isopropanol (IP A), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMAc), N- methyl-pyrrolidone (NMP), ethyl acetate, butyl acetate, 1 -octanol, heptane, octane, methyl t butyl ether (MTBE), toluene, and the like), ionic or polar solvents (e.g., l-ethyl-4-methylimidazolium tetrafluoroborate, l-butyl-3-methylimidazolium tetrafluoroborate, l-butyl-3-methylimidazolium hexafluorophosphate, glycerol, polyethylene glycol (PEG), and the like). In some embodiments, the co-solvent can be a polar solvent, such as a polyol, dimethylsulfoxide (DMSO), or lower alcohol. The non-aqueous co-solvent component of an aqueous co-solvent system may be miscible with the aqueous component, providing a single liquid phase, or may be partly miscible or immiscible with the aqueous component, providing two liquid phases. Generally, when an aqueous co-solvent system is employed, it is selected to be biphasic, wi th water dispersed in an organic solvent, or vice-versa. Exemplary aqueous co-solvent systems can comprise water and one or more co-solvents selected from an organic solvent, polar solvent, and polyol solvent. In general, the co-solvent component of an aqueous co-solvent system is chosen such that it does not adversely inactivate the KRED enzy me under the reaction conditions. Appropriate co-solvent systems can be readily identified by measuring the enzymatic activity of the specified engineered KRED enzyme with a defined substrate of interest in the candidate solvent system, utilizing an enzyme activity assay, such as those described herein.

[0277] In general, the ratio of water to organic solvent in the co-solvent system is typically in the range of from about 90: 10 to about 10:90 (v / v) organic solvent to water. In some embodiments, the ratio of water to organic solvent in the co-solvent system is in the range of from about 80:20 to about 20:80 (v / v) organic solvent to water. The co-solvent system may be pre-formed prior to addition to the reaction mixture, or it may be formed in situ in the reaction vessel.

[0278] In some embodiments of the method, the suitable reaction conditions comprise an aqueous co-solvent, where the co-solvent comprises DMSO at about 1% to about 50% (v / v). about 1 to about 40% (v / v), about 2% to about 40% (v / v), about 5% to about 30% (v / v), about 5% to about 30% (v / v), about 5% to about 25% (v / v), about 7% to about 20%, or about 8% to about 20%. In some embodiments of the method, the suitable reaction conditions can comprise26081 an aqueous co-solvent comprising DMSO at about 1% (v / v), about 5% (v / v), about 8% (v / v), about 10% (v / v), about 15% (v / v), about 20% (v / v), about 25% (v / v), about 30% (v / v), about 35% (v / v), about 40% (v / v), about 45% (v / v), or about 50% (v / v).

[0279] In some embodiments of the method, the suitable reaction conditions comprise an aqueous co-solvent, where the co-solvent comprises DMAc at about 1% to about 50% (v / v). about 1 to about 40% (v / v), about 2% to about 40% (v / v), about 5% to about 30% (v / v), about 7% to about 30% (v / v), about 7% to about 25% (v / v), or about 10% to about 20% (v / v). In some embodiments of the method, the suitable reaction conditions can comprise an aqueous co-sol vent comprising DMAc at about 1% (v / v), about 5% (v / v). about 10% (v / v). about 15% (v / v). about 20% (v / v), about 25% (v / v), about 30% (v / v), about 35% (v / v), about 40% (v / v), about 45% (v / v), or about 50% (v / v).

[0280] The aqueous solvent (water or aqueous co-solvent system) may be pH-buffered or unbuffered. In some embodiments, the method of catalyzing the oxidation of an alcohol group using a KRED of the disclosure is earned 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 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 are carried out at a pH of about 9 or below, e.g.. in a range of from about 7 to about 9 or in a range of from about 7.5 to about 9. Alternatively, the method may be carried out a neutral pH, i.e., about 7.

[0281] 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.

[0282] In some embodiments, the method of catalyzing the oxidation of an alcohol group using a KRED polypeptide of the disclosure 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 catalyzing the oxidation of an alcohol group using a KRED 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 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, from260817.5 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.

[0283] In some embodiments, the method of catalyzing the oxidation of an alcohol group of a substrate (e.g., substrate 1 in Scheme A above) to provide the ketone or aldehyde (e.g., compound 2 Scheme A above) 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 are performed at a temperature of from about 20 °C to about 30 °C, e.g., about 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, or 30 °C. In some embodiments, the methods are performed at a temperature of from about 25 °C to about 30 °C, e.g., about 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, or 30 °C.

[0284] In some embodiments, the method of catalyzing the oxidation of an alcohol group of a substrate (e.g., substrate 1 in Scheme A above) to provide the ketone or aldehyde (e.g., compound 2 Scheme A above) comprises contacting or incubating from about 0.01 to about 100 g / L, about 0. 1 to about 80 g / L, about 0.5 to about 75 g / L, about 0.5 to about 60 g / L, about 1 to about 50 g / L, about 1 to about 40 g / L, about 1 to about 30 g / L, about 1 to about 20 g / L, or about 5 to about 50 g / L of the substrate with any one of the KRED polypeptides disclosed herein or any combination of the polypeptides disclosed herein. In some embodiments, the method of catalyzing the oxidation of an alcohol group of a substrate comprises contacting or incubating about 0.5 g / L, about 1 g / L, about 5 g / L, about 10 g / L, about 15 g / L, about 20 g / L, about 25 g / L, about 30 g / L, about 35 g / L, about 40 g / L, about 45 g / L, about 50 g / L, about 55 g / L, or about 60 g / L of the substrate with any one of the KRED polypeptides disclosed herein or any combination of the polypeptides disclosed herein.

[0285] In some embodiments, the method of catalyzing the oxidation of an alcohol group of a substrate (e.g., substrate 1 in Scheme A above) to provide the ketone or aldehyde (e.g., compound 2 Scheme A above) is performed in the presence of from about 0.01% w / w to about 50% 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, 1.25% w / w, 1.5% 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, 15% w / w, 20% w / w, 25% w / w, 30% w / w, 35% w / w, 40% w / w, 45% w / w, or 50% w / w) of any one of the KRED polypeptides disclosed herein or any combination of the polypeptides disclosed herein. In some embodiments,26081 the engineered KRED polypeptides are present at about 0.01 g / L to about 50 g / L; about 0.05 g / L to about 50 g / L; about 0. 1 g / L to about 40 g / L; about 1 g / L to about 40 g / L; about 2 g / L to about 40 g / L; about 5 g / L to about 40 g / L; about 5 g / L to about 30 g / L; about 0. 1 g / L to about 10 g / L; about 0.5 g / L to about 10 g / L; about 1 g / L to about 10 g / L; about 0.1 g / L to about 5 g / L; about 0.5 g / L to about 5 g / L; or about 0. 1 g / L to about 2 g / L. In some embodiments, the KRED polypeptide is present at about 0.01 g / L, 0.05 g / L, 0.1 g / L, 0.2 g / L, 0.5 g / L, 1, 2 g / L, 5 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, or 50 g / L.

[0286] In some embodiments, the cofactor is selected from, but not limited to, NADP+ and NAD+. Accordingly, in some embodiments, the methods are carried out in presence of a cofactor selected from NADP+ and NAD+. In some embodiments, the electron acceptor is an NADP+ cofactor. In some embodiments, the electron acceptor is NAD+ cofactor. In some embodiments, the method can be carried out wherein the reaction conditions comprise an NAD+ or NADP+ cofactor concentration of about 0.01 to about 3 g / L, about 0.01 to about 2 g / L, about 0.01 to about 1 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, about 0.25 to about 0.8 g / L, about 0.5 to about 1.5 g / L, or about 0.5 to about 1.25 g / L. In some embodiments, the method is carried out under an NAD(P)+ concentration of about 2 g / L, about 1.5 g / L, about 1.25g / 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, or about 0.03 g / L.

[0287] The concentration of the substrate in the reaction mixtures can be varied, taking into consideration, for example, the desired amount of product, the effect of substrate concentration on enzyme activity, stability of enzyme under reaction conditions, and the percent conversion of the substrates to the product. In some embodiments, the suitable reaction conditions comprise a substrate compound loading of at least about 0.5 to about 200 g / L, 1 to about 200 g / L, 5 to about 150 g / L, about 10 to about 100 g / L, 20 to about 100 g / L or about 50 to about 100 g / L. In some embodiments, the suitable reaction conditions comprise loading of the substrate of at least about 0.5 g / L, at least about 1 g / L. at least about 5 g / L, at least about 10 g / L, at least about 15 g / L, at least about 20 g / L, at least about 30 g / L, at least about 50 g / L, at least about 75 g / L, at least about 100 g / L, at least about 150 g / L or at least about 200 g / L, or even greater. The values for substrate loadings provided he...

Claims

WHAT IS CLAIMED IS:

1. An engineered polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 40, wherein the polypeptide comprises at an amino acid substitution at one or more amino acid positions selected from 9, 32, 36, 37, 38, 39, 41, 47, 53, 54, 55, 57, 72, 74, 93, 102, 106, 108, 117, 123, 127, 129, 135, 136, 137, 138, 139, 146, 156, 160, 164, 166, 174, 183, 184, 194, 196, 197, 198, 200, 207, 210, 211, 214, 218, 220, 224, 226, 227, 229, 230, 232, 233, 235, 239, 240, 241, 242, 243, 244, 258, 264, 265, 266, 267, 269, 278, 279, 281, 283. 285, 286, 292, and 293 and / or at least one amino acid insertion between positions 240 and 241 or between positions 288 and 289, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 40.

2. An engineered polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 40, wherein the polypeptide comprises from 2 to 74 amino acid substitutions, each amino acid substitution at an amino acid position selected from 9, 32, 36, 37, 38, 39, 41, 47, 53, 54, 55, 57, 72, 74, 93, 102, 106, 108, 117, 123, 127, 129, 135, 136, 137, 138, 139, 146, 156, 160, 164, 166, 174, 183, 184, 194, 196, 197, 198, 200, 207, 210, 211, 214, 218, 220, 224. 226, 227, 229, 230, 232. 233, 235, 239, 240, 241, 242, 243. 244, 258, 264, 265, 266. 267, 269, 278, 279, 281, 283, 285, 286, 292, and 293, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 40.

3. The polypeptide of claim 2, wherein the polypeptide further comprises at least one amino acid insertion between positions 240 and 241 or between positions 288 and 289, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 40.

4. The polypeptide of any one of claims 1-3, wherein the polypeptide comprises one or more amino acid substitutions or amino acid substitution sets set forth in Table 4.

5. The polypeptide of any one of claims 1-4, wherein the amino acid sequence comprises any one of SEQ ID NO: 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, or 98.

6. The polypeptide of any one of claims 1-5, wherein the amino acid sequence comprises SEQ ID NO: 94.

7. The polypeptide of any one of claims 1-5, wherein the amino acid sequence comprises SEQ ID NO: 96.

8. The polypeptide of any one of claims 1-5, wherein the amino acid sequence comprises SEQ ID NO: 98.

9. An engineered polypeptide comprising an amino acid sequence having at least 70% sequence identity’ to SEQ ID NO: 38, wherein the polypeptide comprises an amino acid substitution at one or more amino acid positions selected from 9, 32, 36, 37, 38, 39, 41, 47, 53, 54, 55, 57, 72, 74, 93, 102, 106, 108, 117, 123, 127, 129, 135, 136, 137, 138, 139, 146, 156, 160, 164, 166, 174, 183, 184, 194, 196, 197, 198, 200, 207, 210, 211, 214, 218, 220, 224, 226, 227, 229, 230. 232, 233, 235, 239, 240, 241, 242, 243, 244, 258, 264, 265. 266, 267, 269, 278, 279, 281, 283, 285, 286, 292, and 293, and / or at least one amino acid insertion between positions 240 and 241, and 288 and 289, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38.

10. An engineered polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 38, wherein the polypeptide comprises from 1 to 75 amino acid substitutions, each amino acid substitution at an amino acid position selected from 9, 32, 36, 37, 38, 39, 41, 47, 53, 54, 55, 57, 72, 74, 93, 102, 106, 108, 117, 123, 127, 129, 135, 136, 137, 138, 139, 146. 156, 160, 164, 166, 174. 183, 184, 194, 196, 197, 198, 200. 207, 210, 211, 214, 218. 220, 224. 226, 227, 229. 230, 232. 233, 235, 239. 240, 241. 242, 243. 244, 258, 264. 265, 266. 267, 269, 278, 279, 281, 283, 285, 286, 292, and 293, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38.

11. The polypeptide of claim 10. wherein the polypeptide further comprises at least one amino acid insertion between positions 240 and 241 or between positions 288 and 289, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 38.

12. The polypeptide of any one of claims 9-11, wherein the polypeptide comprises one or more amino acid substitutions or amino acid substitution sets set forth in Table 4.

13. The polypeptide of any one of claims 9-12, wherein the amino acid sequence comprises any one of SEQ ID NOs: 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192.

14. The polypeptide of any one of claims 9-13, wherein the amino acid sequence comprises SEQ ID NO: 190.

15. The polypeptide of any one of claims 9-14, further comprising an epitope tag.

16. The polypeptide of claim 15, wherein the epitope tag is a His tag.

17. The polypeptide of claim 16, wherein the His tag comprises the amino acid sequence of HHHHHH (SEQ ID NO: 131).

18. The polypeptide of any one of claims 16-17, wherein the polypeptide comprises a His tag at the C -terminus.

19. An engineered polypeptide comprising at least 98% sequence identity to any one of SEQ ID NO: 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40.

20. An engineered polypeptide comprising at least 99% sequence identity to any one of SEQ ID NO: 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 136, 138, 140, 142, 144, 146. 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168. 170, 172, 174. 176, 178. 180, 182, 184, 186, 188. 190, or 192, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38 or SEQ ID NO: 40.

21. A polypeptide consisting of the amino acid sequence of any of SEQ ID NO: 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72.

74.

76. 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192.2608122. An engineered polypeptide comprising at least 80% sequence identity to any one of SEQ ID NO: 188, 190, or 192, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 38.

23. The polypeptide of any one of claims 1-22, which is isolated.

24. The polypeptide of any one of claims 1-23, wherein the polypeptide has imine reductase activity.

25. The polypeptide of claim 24, wherein the polypeptide has NADH-dependent or NAD(P)H-dependent imine reductase activity.

26. The polypeptide of any one of claims 1-25, 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: 38 or SEQ ID NO: 40: a) increased imine reductase activity wherein the polypeptide has at least about 103-fold, about 106-fold, about 109-fold, or about 1012-fold; b) improved enzyme expression and / or solubility; c) improved soluble enz me expression; d) improved thermostability; and / or e) improved cofactor affinity; and / or f) improved organic solvent tolerance.

27. A polynucleotide encoding at least one polypeptide set forth in any one of claims 1-26, wherein the polynucleotide does not comprise SEQ ID NO: 37 or SEQ ID NO: 39.

28. The polynucleotide of claim 27, wherein the polynucleotide is codon-optimized.

29. The polynucleotide of claim 27 or 28, wherein the polynucleotide comprises any one of SEQ ID NO: 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 133. 135, 137, 139, 141, 143. 145, 147, 149, 151, 153, 155, 157. 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, or 191.2608130. A polynucleotide comprising at least 80% sequence identity to any one of SEQ ID NO: 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169. 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, or 191, wherein the polynucleotide does not comprise the sequence of SEQ ID NO: 37 or SEQ ID NO: 39.

31. An expression vector comprising at least one polynucleotide sequence of any one of claims 27-30.

32. The expression vector of claim 31, wherein the polynucleotide sequence is operably linked to a control sequence.

33. The expression vector of claim 32, wherein the control sequence is a promoter.

34. The expression vector of claim 33, wherein the promoter is a heterologous promoter.

35. A host cell comprising the polynucleotide of any one of claims 27-30 or the expression vector of any one of claims 31-34.

36. The host cell of claim 35, wherein the host cell is prokaryotic or eukaryotic.

37. A method of producing a polypeptide, the method comprising culturing the host cell of claim 35 or 36 under conditions such that the polypeptide encoded by the polynucleotide is produced.

38. The method of claim 37, further comprising the step of recovering the polypeptide.

39. The method of claim 38, further comprising the step of purifying the polypeptide.

40. A method of catalyzing the reduction of an imine group in the presence of the polypeptide of any one of claims 1 -26.

41. A method of producing secondary amine 4 in the following Scheme B, which comprises reacting substrate 3 in the following Scheme B with the polypeptide of any one of claims 1-26, wherein Scheme B is42. The method of claim 41, further comprising recovering secondary amine 4.

43. The method of claim 41 or 42, w herein the polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156. 158, 160, 162, 164, 166. 168, 170, 172, 174, 176, 178, 180. 182, 184, 186, 188, 190, or 192.

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