Engineered thioesterase enzymes and uses thereof

Abstract

The present disclosure provides polypeptides (e.g., TE polypeptides), polynucleotides and expression vectors and host cells comprising the same, methods of producing polypeptides (e.g., TE polypeptides), and methods of catalyzing an amide bond formation.

Description

ENGINEERED THIOESTERASE ENZYMES AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 717,524, filed November 7, 2024. which is incorporated herein by reference in its entirety.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The contents of the electronic sequence listing (26078-WO-PCT_SL.xml; Size: 525,729 bytes; and Date of Creation: May 22, 2025) are herein incorporated by reference in their entirety.FIELD

[0003] The present disclosure relates to engineered thioesterase enzymes, useful in the generation of linear, semimacrocyclic, and macrocyclic peptides, as well as related polynucleotides, expression vectors, host cells, and methods of production.BACKGROUND

[0004] Enzy mes are protein molecules 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 carry out life processes. Enzymes display great specificity for their substrates and are not permanently modified by their participation in reactions. Because they are not changed during the reactions, enzymes may be cost effectively used as catalysts for a desired chemical transformation.

[0005] Thioesterases (TE) are a class of enzymes capable of catalyzing several chemical transformations, including, for example, thioester bond hydrolysis, macrocyclization of linear peptides, and intermolecular transacylation as components of diverse biosynthetic pathways. Thioesterases derived from non-ribosomal peptide synthesis (NRPS) pathways have found limited use in commercial applications given their dependence on thiol-activated or carrier protein-activated peptides, the hydrolytic instability of thioesters and the cost and odor of thiol precursors / byproducts. However, given their potential for selectivity, broad substrate scope, and catalytic diversity, such enzymes may be useful as commercial biocatalysts to carry out protecting group-free peptide-peptide coupling chemistries.

[0006] The present disclosure provides novel engineered NRPS thioesterases with the ability to catalyze amide bond formation, for example in the generation of macrocyclic and semi-macrocyclic peptides to improve their activity, substrate scope, regioselectivity, organic solvent tolerance, and / or thermostability.SUMMARY

[0007] The present disclosure provides, inter alia, polypeptides (e.g.. TE polypeptides), polynucleotides encoding the same, expression vectors, host cells, methods of producing polypeptides, and methods of catalyzing amide bond formation.

[0008] In some embodiments, the engineered TE polypeptides and TE-catalyzed methods (e.g., methods of catalyzing amide bond formation) provided herein are useful in the production of macrocyclic peptides, e.g., enlicitide. The structure of enlicitide is shown below as Formula IV. In some embodiments, A is decanoate.Formula IV

[0009] In one aspect, provided herein an engineered polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 6 or 378, wherein the polypeptide comprises an amino acid substitution at one or more amino acid positions selected from 4, 13, 16, 18, 20, 22, 23, 24, 25, 27, 30, 31, 36, 37, 49, 54, 66, 75, 79, 90, 91, 93, 98, 100, 104, 107, 112, 114, 118, 119, 123, 124, 125, 126, 127, 129, 130, 131, 132, 133, 134, 135, 139, 142, 146, 150, 157, 163, 167, 171, 172, 176, 187, 189, 190, 191, 192, 195, 196, 197, 199, 203, 208, 209. 210, 212, 217, 219, 221, 226, 228, 229, 234, 242, 245, and 249, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 6, and wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2.

[0010] Also provided herein is an engineered polypeptide comprising an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NO: 4. 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46. 48. 50. 52. 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 378, or 380, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2.

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

[0012] Also provided herein is a polynucleotide comprising at least 80% sequence identity to any one of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33. 35, 37, 39, 41, 43. 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67. 69. 71. 73. 75. 377, or 379, wherein the polynucleotide does not comprise SEQ ID NO: 1.

[0013] Also provided herein is engineered polypeptide comprising an amino acid sequence having at least 65% sequence identity to SEQ ID NO: 6 or 378, wherein the polypeptide comprises an amino acid substitution at one or more amino acid positions selected from 4, 10, 11, 12, 13, 15, 18, 20, 23, 24, 27, 31, 34, 35, 36, 37, 48, 62, 72, 93, 97, 98, 107, 108, 109, 111, 113, 114, 115, 120, 121, 123, 124, 125, 126, 127, 129, 132, 133, 134, 135, 138, 139, 144, 145, 146, 147, 148, 150, 154, 157, 158, 166, 167, 168, 172, 175, 183, 186, 187, 188, 189, 190, 192, 194, 195, 196. 199, 203, 205, 206, 212, 213, 214, 217, 221, 222, 226, 228. 229, 234, 238, 243, 245, and 247, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 6, and wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2.

[0014] Also provided herein is engineered polypeptide comprising at least 98% sequence identity to any one of SEQ ID NO: 16, 78, 90, 106, 114, 130, 140, 150, 162, 172, 188, 194, 204, 216, 230. 238, 248, 260, 272, 294. 296, 310, 322, 340, 352, 354, 360. 364, 366, 368, 370, 372. 374, 376, or 380 wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2.

[0015] Also provided herein is a polynucleotide comprising at least 80% sequence identity to any one of SEQ ID NO: 15, 77, 89, 105, 113, 129, 139, 149, 161, 171, 187, 193, 203, 215, 229, 237, 247. 259, 271, 293, 295, 309. 321, 339, 351, 353, 359, 363, 365. 367, 369, 371, 373, 375. or 379, wherein the polynucleotide does not comprise SEQ ID NO: 1.

[0016] Also provided herein is an expression vector comprising at least one of the polynucleotides disclosed herein.

[0017] 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.

[0018] Also provided herein is a method of producing a polypeptide, the method comprising culturing any one of the host cells disclosed herein under conditions such that the polypeptide encoded by the polynucleotide is produced.

[0019] Also provided herein is a method of catalyzing an amidation reaction for septapeptide formation as set forth in Table A, comprising contacting one or more of the substrates listed in Table A with any one of the polypeptides described herein.

[0020] Also provided herein is a method of producing product 3 in Scheme A below (see above Example 35), which comprises reacting substrate 1 and substrate 2 in Scheme A with any one of the polypeptides described herein.

[0021] 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

[0022] The present disclosure relates to engineered thioesterase polypeptides. As is described in the Examples, the present disclosure provides engineered thioesterase polypeptides derived from wild-type thioesterase domain of the Brevibacullus laterosporus non-ribosomal peptide synthetase (NRPS) polypeptide (SEQ ID NO: 2), which exhibit improved enzyme properties relative to the TE domain of wild-type Brevibacullus laterosporus NRPS polypeptide, and which were discovered through iterative rounds of directed evolution as described herein. For example, the novel engineered TE polypeptides of the disclosure may exhibit one or more of the following improvements relative to a reference polypeptide (e.g., TE domain of a wild-type Brevibacullus laterosporus NRPS polypeptide or a polypeptide of, e.g., SEQ ID NO: 2, 4, 6, 378, or 380): enzyme activity (e.g., improved activity in catalyzing an amide bond formation), thermal stability (i.e., thermostability), substrate tolerance, substrate scope, substrate selectivity, regioselectivity, chemoselectivity, organic solvent or cosolvent tolerance, reduction in sideproducts, refractoriness to inhibitors (e.g., reduction in product inhibition for any of the substrates), protein expression and / or solubility, and / or thermostability. The Examples describe the discovery of TE polypeptide variants with enhanced activity relative to the wild-type TE domain of a wild-type Brevibacullus laterosporus NRPS polypeptide.

[0023] The engineered thioesterases of the disclosure may be useful in biocatalytic and synthetic processes involving peptide-peptide coupling for the generation of linear, semimacrocyclic, and macrocyclic peptides. For example, the engineered TE polypeptides may be useful for amide bond formation between thio- or oxo-ester and amine moieties, such as those in peptides and / or macrocyclic peptides.

[0024] The present disclosure also relates to polynucleotides and expression vectors encoding the TE polypeptides of the present disclosure, host cells comprising the polynucleotides or expression vectors, methods of producing the TE polypeptides, and methods of catalyzing an amide bond formation.Definitions

[0025] 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.

[0026] 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.

[0027] 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”.

[0028] 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).

[0029] 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.

[0030] 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 tw o” 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.

[0031] 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.

[0032] 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.

[0033] “Derived from” as used herein in the context of enzymes, identifies the originating enzyme, and / or the gene encoding such enzyme, upon which the enzyme was based. For example, the TE polypeptide variants of even numbered SEQ ID NOs: 6-376 were obtained by artificially evolving over multiple generations the gene encoding the TE domain of wild-type Brevibacullus laterosporus NRPS polypeptide of SEQ ID NO: 2. Thus, the evolved TE variant enzymes are “derived from” the TE domain of wild-type Brevibacullus laterosporus NRPS polypeptide of SEQ ID NO: 2.

[0034] 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 is similar between the two sequences, and (2) may further comprise a sequence that is divergent between the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptide are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity’.

[0035] 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.

[0036] 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.

[0037] As used herein, the terms “protein,” “polypeptide,” and “peptide” are used interchangeably herein to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristoylation, ubiquitination, and the like). Included within this definition are D- and L-amino acids, and mixtures of D- and L-amino acids, as well as polymers comprising D- and L-amino acids, and mixtures of D- and L-amino acids. Proteins, polypeptides, and peptides may include a tag (e.g.. an epitope tag), such as a histidine tag.

[0038] 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.

[0039] 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 (lie 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).

[0040] "Hydrophilic amino acid” or "hydrophilic residue’7refers to an amino acid or residue having a side chain exhibiting a hydrophobicity of less than zero according to the normalized consensus hydrophobicity scale of Eisenberg et al., 1984, J. Mol. Biol. 179:125-142. Genetically encoded hydrophilic amino acids include L-Thr (T), L-Ser (S), L-His (H), L-Glu (E), L-Asn (N), L-Gln (Q). L-Asp (D). L-Lys (K), and L-Arg (R).

[0041] “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).

[0042] “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).

[0043] “Polar amino acid” or “polar residue” refers to a hydrophilic amino acid or residue having a side chain that is uncharged at physiological pH, but which has at least one bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms. Genetically encoded polar amino acids include L-Asn (N), L-Gln (Q), L-Ser (S), and L-Thr (T).

[0044] “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).

[0045] “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.

[0046] 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.

[0047] "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).

[0048] 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).

[0049] 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.

[0050] As used herein, “small amino acid” or “small residue” refers to an amino acid or residue having a side chain that is composed of a total of three or fewer carbon and / or heteroatoms (excluding the a carbon and hydrogens). The small amino acids or residues may be further categorized as aliphatic, non-polar, polar or acidic small amino acids or residues, in accordance with the above definitions. Genetically encoded small amino acids include L-Ala (A), L-Val (V), L-Cys (C), L-Asn (N), L-Ser (S), L-Thr (T), and L-Asp (D).

[0051] “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).

[0052] As used herein, “conservative amino acid substitution” refers to a substitution of a residue wdth 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 w ay of example and not limitation, in some embodiments, an amino acid with an aliphatic side chain is substituted with another aliphatic amino acid (e.g.. alanine, valine, leucine, and isoleucine); an amino acid with an hydroxyl side chain is substituted with another amino acid with an hydroxyl side chain (e.g., serine and threonine); an amino acid having an aromatic side chain is substituted with another amino acid having an aromatic sidechain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain is substituted with another amino acid with a basic side chain (e.g., lysine and arginine); an amino acid with an acidic side chain is substituted with another amino acid with an acidic side chain (e.g., aspartic acid and glutamic acid); and / or a hydrophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively.

[0053] As used herein, “non-conservative substitution” refers to substitution of an amino acid in the polypeptide with an amino acid with significantly differing side chain properties. Nonconservative substitutions may use amino acids between, 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.

[0054] ‘"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.

[0055] 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., enzyme). Deletions can comprise removal of 1 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, 7 or more amino acids, 8 or more amino acids, 9 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.

[0056] 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., enzy me). 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.

[0057] 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 aminoacid 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.

[0058] “Amino acid difference” or “residue difference” refers to a change 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. 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 disclosure 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 disclosure includes engineered polypeptide sequences comprising one or more amino acid differences that include either or both conservative and non-conservative amino acid substitutions.

[0059] 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, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 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 follow ed (e.g., in the foregoing example at each of positions 148, 157, and 246).

[0060] “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 actual 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 gapsare present, the numbenng 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.

[0061] 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.

[0062] 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 polypeptides can be an isolated polypeptide.

[0063] 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.

[0064] “Improved enzy me property” refers to any property of an enzy me that exhibits an improvement as compared to a reference enzyme. For the enzymes described herein, the comparison can be made to a wild-type enzyme (e.g., SEQ ID NO: 2) or to another improved enzy me derived from the wild-type enzy me (e.g., SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 52, 340,352, 354, 360, 378, 380, etc.). Enzyme properties for which improvement may be desirable include, but are not limited to, enzymatic activity (which may be expressed in terms of percent conversion of the substrate), thermal stability, soluble expression, substrate scope, substrate tolerance (e.g., higher tolerance to increased substrate loading), substrate selectivity, solvent or cosolvent tolerance, pH activity profile, reduction in side-products, refractoriness to inhibitors (e g., product inhibition), regioselectivity, chemoselectivity, stereospecificity and stereoselectivity (including enantioselectivity).

[0065] ‘‘Increased enzymatic activity” refers to an improved property of the enzymes, which can be represented by an increase in specific activity (e.g., amount, selectivity, or quantity of product produced, time of activity, and / or amount of product produced per time unit per weight of enzy me) 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 enzyme. 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 K™, Vm«, or kcat, changes of which can lead to increased enzymatic activity. Improvements in enzyme activity' can be at least about 1.1 times the enzymatic activity of the corresponding wild-type enzyme, at least about 1.1 times, at least about 1.2 times, at least about 1.5 times; at least about 2 times; at least about 5 times; at least about 10 times; at least about 20 times; at least about 25 times; at least about 50 times; at least about 75 times; at least about 100 times; at least about 150 times; at least about 200 times; at least about 500 times; at least about 1,000 times; at least about 3,000 times; at least about 5,000 times; at least about 7,000 times; at least about 10,000 times; at least about 50,000 times; at least about 100,0000 times; at least about 500,000 times; at least about 1,000,000 times or more enzymatic activity than the reference enzyme, e.g., a naturally occurring enzyme or another enzyme from which the polypeptides were derived. The term “fold” is also used in relation to the 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 2 to 100 times; 100 to 3,000 times; 3,000 to 7,000 times; 7,000 to 10,000 times; 10,000 to 50,000 times; 50,000 to 100,000 times; 100,000 to 500,000 times; 500,000 to 1,000,000 times; or more than 1,000,000 times greater than that of the parent enzyme. It is understood by the skilled artisan that the activity of any enzyme is diffusion limited such that the catalytic turnover rate cannot exceed the diffusion rate of the substrate, including any required cofactors. The theoretical maximum of the diffusion limit, or kcat / Km, is generally about 108to 109(XT's-1). Hence, any improvements in the enzy me activitywill have an upper limit related to the diffusion rate of the substrates acted on by the enzyme. Enzyme activity can be measured by any suitable approach, e.g., an enzyme activity assay or by any of 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.

[0066] 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.

[0067] As used herein with respect to polypeptides, the terms “expression” and “production” and “produce” includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from a cell.

[0068] 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.

[0069] 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 TE 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.

[0070] The term “analogue” means a polypeptide having more than 70% sequence identity but less than 100% sequence identity (e.g., more than 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 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 non aline, as well as naturally occurring amino acids. In some embodiments, analogues also include one or more D-amino acid residues and non-peptide linkages between two or more amino acid residues.

[0071] “Coding sequence” refers to that portion of a polynucleotide (e.g.. a gene) that encodes an ammo acid sequence of a polypeptide.

[0072] 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.

[0073] “Naturally occurring” or “wild-type” generally refers to a form found in nature. As used herein, a naturally occurring or w ild-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. Herein, “wild-type” polypeptide or polynucleotide sequences may be denoted “WT.”

[0074] “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 TE enzymes may be codon optimized for optimal production from the host organism selected for expression.

[0075] “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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] “Percentage of sequence identity,” “percent identity,” and “percent identical” are used herein to refer to comparisons between polynucleotide sequences or polypeptide sequences, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Determination of optimal alignment and percent sequence identity can be performed using the BLAST and BLAST 2.0 algorithms (see e.g., Altschul et al., 1990, J. Mol. Biol. 215: 403-410; and Altschul et al., 1977, Nucleic Acids Res. 3389-3402). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website.

[0080] 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).

[0081] Numerous other algorithms are available that function similarly to BLAST in providing percent identify for two sequences. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homolog}’ algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection (see generally, Cunent Protocols in Molecular Biolog}’, F. M. Ausubel et al., eds., Current Protocols, ajoint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)). Additionally, determination of sequence alignment and percent sequence identify can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison WI), using default parameters provided.

[0082] “Stereoselectivity” refers to the preferential formation in a chemical or enzymatic reaction of one stereoisomer over another. Stereoselectivity can be partial, where the formation of one stereoisomer is favored over the other, or it may be complete where only one stereoisomeris 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.

[0083] “Chemoselectivity” refers to the preferential formation in a chemical or enzymatic reaction of one product over one or more alternative potential products.

[0084] “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.

[0085] “Conversion” refers to the enzymatic transformation of a substrate to the corresponding product. “Percent conversion” refers to the percent of the substrate that is converted to the product within a period of time under specified conditions. Thus, for example, the “enzymatic activity” or “activity” of a polypeptide can be expressed as “percent conversion” of the substrate to the product.

[0086] “Thermostable” refers to a polypeptide that maintains similar activity (more than 60% to 80%, for example) after exposure to elevated temperatures (e.g., 40 °C to 80 °C) for a period of time (e g., 0.5 h to 24 h) compared to the untreated enzyme.

[0087] “Solvent stable” or “co-solvent stable” or “solvent tolerance” or “co-solvent tolerance” refers to a polypeptide that maintains similar activity (more than e g., 60% to 80%) after exposure to varying concentrations (e.g., 1-99%) of a solvent or co-solvent, (e.g., isopropyl alcohol, dimethylacetamide, dimethylsulfoxide, tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butylacetate, methyl tert-butylether, acetonitrile, etc.) for a period of time (e.g..0.5-24 hrs) compared to the untreated enzyme.

[0088] As used herein, the terms “biocatalysis,” “biocatalytic,” “biotransformation,” and “biosynthesis” refer to the use of enzymes to perform chemical reactions on organic compounds.

[0089] 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.

[0090] The terms “isolated” and “purified” are used to refer to a molecule (e.g., an isolated nucleic acid or polypeptide) or other component that is removed from at least one other component with which it is naturally associated. The term “purified” does not require absolute purity, rather it is intended as a relative definition.

[0091] The terms “thioesterase,” “TE,” “TE enzymes,” and “TE polypeptides” are used interchangeably herein to refer to polypeptides of the disclosure and include naturally occurring (wild-type) TE polypeptides as well as non-naturally occurring engineered polypeptides generated by human manipulation. In an embodiment, a TE polypeptide as disclosed herein may have an amino acid sequence selected from any one of even numbered SEQ ID NO: 2-380 or 4-380. In an embodiment, a TE polypeptide as disclosed herein may be an enzyme which is evolved away from its natural activity on r / zzo-ester substrates to effectively mediate reaction on, for example, an isopropyl ester form of a substrate compound. In an embodiment, a TE polypeptide as disclosed herein may be capable of catalyzing the formation of an amide bond.TE Polypeptides

[0092] This disclosure provides engineered thioesterase polypeptides (TE polypeptides). In some embodiments, the engineered TE polypeptides are capable of catalyzing an amide bond formation. In some embodiments, the TE polypeptides described herein are capable of catalyzing amide bond formation in macrocyclic and semi-macrocyclic peptides. In some embodiments, the TE polypeptides described herein are capable of catalyzing amide bond formation in macrocyclic and semi-macrocyclic peptide in a protective-group free manner. In some embodiments, the TE polypeptides described herein are capable of catalyzing amide bond formation in macrocyclic and semi-macrocyclic peptide in a protective-group free manner from activated aminoacylthioesters, aminoacyl-esters and cyclic peptides. In some embodiments, the engineered TE polypeptides are capable of catalyzing amide bond formation between thio- or oxo-ester and amine moieties, such as those in peptides and / or macrocyclic peptides. In embodiments, the TE polypeptides described herein can couple two peptides that can be further used in the synthesis of multimer peptides.

[0093] In some embodiments, the TE polypeptides of the disclosure are capable of catalyzing reactions in the process of generating macrocyclic peptides.

[0094] In some embodiments, the TE polypeptides described herein are capable of catalyzing one or more of the reactions shown in Table A and Scheme A in the Examples below.

[0095] In some embodiments, the TE polypeptides described herein may be useful in the preparation of compounds, including but not limited to, (l1< S',l2<S',l3S,9S,12< S)-9-amino-12-((l-(6-aminohexyl)-5-fluoro-17f-indol-3-yl)methyl)-A-((2< S’,3A)-3-hydroxy-l-(((> S)-l-((< S’)-2-((4-(hydroxymethy l)phenethyl)carbamoyl)-2-methylpyrrolidin- 1 -yl)-3-(4-methoxyphenyl)- 1 -oxopropan-2-yl)amino)- 1 -oxobutan-2-yl)-4, 10,13-trioxo-2-oxa-5, 11 -diaza- 1 (3, 1 )-py rrolidina-7(l,3)-benzenacyclotridecaphane-12-carboxamide [compound 3g of Table A] and 6-(((22?)-2-((2S)-2-(4-((3aS,27S,30S,41a,44S,44aS,47S,50S', Z)-30-amino-23-fluoro-47-((?)-l-hydroxyethyl)-50-(4-methoxybenzyl)-3a-methyl-4,29,39,45,48,51,53-heptaoxo- 2, 3, 3a,4, 5, 6, 7, 12, 14, 15, 16, 17, 18, 19, 26, 27, 28, 29,30, 31, 37, 38, 39, 40, 41a, 42, 44a, 45, 46, 47, 48, 49, 50, 51 -tetratriacontahy dro-4377-8, 11 -etheno-27,44-methano-20,25: 32,36-di(melherio)benzo| / |dipyrrolo|2. IAi:2’.3'- O][l]oxa[4, 14,20, 27,35, 38, 41, 44]octaazacy cloheptatetracontin- 13(177)-yl)-4-oxobutanamido)propanamido)-3-isopropoxy-3-oxopropyl)amino)-jV,jV,jV-trimethyl-6-oxohexan-1-aminium [WF+ACD in Scheme A], or of intermediates formed during preparation of such compounds.

[0096] In some embodiments, the TE polypeptides disclosed herein are capable of catalyzing the regioselective coupling of a diester to a diamine to synthesize a monoester. In some embodiments, the TE polypeptides disclosed herein are capable of catalyzing the regioselective coupling of a diester of formula (1) to a diamine of formula (2) to synthesize a monoester of formula (3) in reference to Scheme A below. In some embodiments, a diester of formula (1) is selected from bis-isopropyl ester, hexafluoro bis-isopropyl ester, tetrafluoro bis-isopropyl ester, or difluoro bis-isopropyl ester. In some embodiments, a di ester of formula (1) is bis-isopropyl ester. In some embodiments, the monoester of formula (3) is selected from isopropyl ester, trifluoro isopropyl ester, difluoro isopropyl ester, or monofluoro isopropyl ester. In some embodiments, the monoester of formula (3) is isopropyl ester.

[0097] In certain embodiments, a polypeptide (e.g., a TE 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-ty pe polypeptide that results in an improved enzyme property.

[0098] In some embodiments, the TE polypeptides described herein are a product of directed evolution from sequence of the TE domain of wild-type Brevibacullus laterosporus NRPS polypeptide (i.e., SEQ ID NO: 2).

[0099] Enzyme properties for which improvements are desirable include, but are not limited to, protein expression and / or solubility, enzymatic activity, thermal stability, substrate scope, substrate tolerance (e.g., higher tolerance to increased substrate loading), substrate selectivity,, substrate affinity, solvent and / or co-solvent tolerance, reduction in side-products, refractoriness to inhibitors (e.g., product inhibition), regioselectivity, and chemoselectivity. The improvements can relate to a single enzyme property, such as enzymatic activity, or a combination of different enzyme properties, such as enzymatic activity and stereoselectivity.

[0100] In some embodiments, the polypeptide (e.g., a TE polypeptide) of the disclosure may demonstrate one or more improvements relative a reference polypeptide. For example, in some embodiments, the polypeptide (e.g., the TE polypeptide) of the disclosure may demonstrate one or more improvements relative to a reference polypeptide as provided in any one of Examples 1-62 below. In some embodiments, the polypeptide (e.g., a TE polypeptide) of the disclosure may demonstrate one or more improvements relative a reference polypeptide including, but not limited to, increases in enzymatic activity, thermal stability, substrate scope, substrate tolerance (e.g., higher tolerance to increased substrate loading), substrate selectivity, solvent or cosolvent tolerance, reduction in side-products, refractoriness to inhibitors (e.g., product inhibition), substrate affinity, protein expression and / or solubility, regioselectivity, and chemoselectivity. In some embodiments, the polypeptide (e.g., a TE polypeptide) of the disclosure may demonstrate one or more improvements relative to the polypeptide of SEQ ID NO: 2, 4, 6, 16, 378, or 380, including, but not limited to, increases in enzymatic activity, thermal stability, substrate scope, substrate tolerance, substrate selectivity, solvent or cosolvent tolerance, reduction in sideproducts. refractoriness to inhibitors, substrate affinity, protein expression and / or solubility, regioselectivity, and chemoselectivity. In some embodiments, the polypeptide (e.g., a TE polypeptide) of the disclosure may demonstrate at least two improvements relative the polypeptide of SEQ ID NO: 2, 4, 6, 16, 378, or 380, including, but not limited to, increases in enzymatic activity, thermal stability, substrate scope, substrate tolerance, substrate selectivity, solvent or cosolvent tolerance, reduction in side-products, refractoriness to inhibitors, substrate affinity, protein expression and / or solubility, regioselectivity, and chemoselectivity’.

[0101] In some embodiments, the reference polypeptide is any one of even numbered SEQ ID NOs: 2-380. 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, 38, 40, 42, 44. 46. 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 378, or 380. In some embodiments, the reference polypeptide is any one SEQ ID NO: 2, 16, and even numbered SEQ ID NO: 78-380. In some embodiments, the reference polypeptide is any one of SEQ ID NO: 2, 4, 6, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,38. 40. 42. 44, 46, 48, 50, 52, 54, 56, 58, 60, 62. 64. 66. 68. 70. 72. 74, 76, 378, or 380. In some embodiments, the reference polypeptide is any one SEQ ID NO: 2, 16, 78, 90, 106, 114, 130, 140, 150, 162, 172, 188, 194, 204, 216, 230, 238, 248, 260, 272, 294, 296, 310, 322, 340, 352, 354, 360. or 380. In some embodiments, the reference polypeptide is any one of SEQ ID NO: 2, 4, 6, 16, 378. or 380. In some embodiments, the reference polypeptide is any one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 52, 340, 352, 354, 360, 378, or 380. In some embodiments, the reference polypeptide is any one of SEQ ID NO: 2, 6, or 378. In some embodiments, the reference polypeptide is any one of SEQ ID NO: 6, 16, 378, or 380. In some embodiments, the reference polypeptide is SEQ ID NO: 2 or SEQ ID NO: 6. In some embodiments, the reference polypeptide is SEQ ID NO: 2 or SEQ ID NO: 16. In some embodiments, the reference polypeptide is any one of SEQ ID NO: 16, 340, 352, 354, 360, or 380.

[0102] For example, in some embodiments, the polypeptide has one or more of the following properties relative to a reference polypeptide: a) increased enzyme activity (e.g., increased catalyzation of amide bond formation); b) increased substrate scope; c) increased regioselectivity; d) increased chemoselectivity; e) increased solvent or cosolvent tolerance; f) reduction in side-products; g) reduction in product inhibition; h) increased thermostability; i) increased tolerance to increased substrate loading,)) increased protein expression, k) increased protein solubility, and / or 1) increased substrate affinity. For example, in some embodiments, the polypeptide has the following properties relative to a reference polypeptide: a) increased enzyme activity (e.g., increased catalyzation of amide bond formation; and b) increased thermal stability. For example, in some embodiments, the polypeptide has the following properties relative to a reference polypeptide: a) increased enzyme activity (e.g., increased catalyzation of amide bond formation; b) increased chemoselectivity and / or regioselectivity; and c) increased tolerance to solvent or cosolvent. For example, in some embodiments, the polypeptide has the following properties relative to a reference polypeptide: a) increased enzyme activity (e.g., increased catalyzation of amide bond formation; b) increased chemoselectivity and / or regioselectivity; c) increased tolerance to solvent or cosolvent; and d) increased thermal stability. For example, in some embodiments, the polypeptide has the following properties relative to a reference polypeptide: a) increased enzy me activity (e.g., increased catalyzation of amide bond formation; and b) reduction in side-products.

[0103] In some embodiments, the reference polypeptide comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the reference polypeptide comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the reference polypeptide comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the reference polypeptidecomprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the reference polypeptide comprises the amino acid sequence of SEQ ID NO: 52. In some embodiments, the reference polypeptide comprises the amino acid sequence of SEQ ID NO: 340. In some embodiments, the reference polypeptide comprises the amino acid sequence of SEQ ID NO: 352. In some embodiments, the reference polypeptide comprises the amino acid sequence of SEQ ID NO: 354. In some embodiments, the reference polypeptide comprises the amino acid sequence of SEQ ID NO: 360. In some embodiments, the reference polypeptide comprises the amino acid sequence of SEQ ID NO: 378. In some embodiments, the reference polypeptide comprises the amino acid sequence of SEQ ID NO: 380.

[0104] In some embodiments, the TE polypeptide as disclosed herein may be an enzyme which is evolved away from its natural activity on thio-ester substrates to effectively mediate reaction on, for example, an isopropyl ester form of a substrate compound. In some embodiments, the TE polypeptide is evolved to recognize and couple an amine of an ambident nucleophile with greater than about 10: 1, about 20: 1, about 30: 1, about 40: 1, about 50: 1. about 60: 1, about 70: 1, about 80:1, about 90:1, about 95:1, about 97:1, about 98:1, or about 99:1 N. O selectivity as its exclusive coupling partner.

[0105] In some embodiments, the polypeptide has any of the improvements shown in any of Examples 2-62 relative to a reference sequence. For example, in some embodiments, the polypeptide has any of the improvements shown in any of Tables 2.1-62.1.

[0106] In some embodiments, the TE polypeptides of the disclosure may demonstrate improvements in tolerance to a solvent and / or cosolvent relative to a reference sequence. In some embodiments, the solvent or cosolvent is dimethylacetamide. In some embodiments, the polypeptide has improved tolerance to a dimethylacetamide solvent or cosolvent or impurities contained in dimethylacetamide solvent or cosolvent preparations.

[0107] In some embodiments, the TE polypeptides of the disclosure may demonstrate improvements relative to a reference sequence. In some embodiments, the TE polypeptides of the disclosure may demonstrate improvements in selectivity for a desired product. In some embodiments, the TE polypeptides of the disclosure may demonstrate improvements in substrate affinity. In some embodiments, the TE polypeptides of the disclosure may demonstrate improvements in activity in lower enzy me loading. In some embodiments, the TE polypeptides of the disclosure may demonstrate higher tolerance to increased substrate loading. In some embodiments, the TE polypeptides of the disclosure may demonstrate increased substrate selectivity. In some embodiments, the TE polypeptides of the disclosure may demonstrate increased selectivity for a desired amino alcohol product. In some embodiments, the TEpolypeptides of the disclosure may demonstrate a reduction in side products. In some embodiments, the TE polypeptides of the disclosure may demonstrate improved activity on ester substrates. In some embodiments, the TE polypeptides of the disclosure may demonstrate improved activity in the intermolecular ligation reaction from a thioesterase substrate. In some embodiments, the TE polypeptides of the disclosure may demonstrate improved activity in the intermolecular ligation reaction from an isopropyl ester substrate. In some embodiments, the TE polypeptides of the disclosure may demonstrate improved activity at higher substrate loading.

[0108] In some embodiments, the TE polypeptides of the disclosure may be engineered to be: a) active on less reactive hindered esters; b) tolerant of organic co-solvents; c) tolerant of high loadings of reactants; and / or d) more regioselective relative to a reference sequence. For example, the TE polypeptides of the disclosure may be more selective for regioisomer (3) over (4), (5), (6), (7), and (8) in Scheme A below.

[0109] In some embodiments, the TE polypeptides of the disclosure may demonstrate a reduction in side products relative to a reference sequence. In some embodiments, the TE polypeptides of the disclosure may be more selective for product (3g) in Table A below over products (A), (B), (C), (D), (E), (F), (G), (H), or (I) in Table B below.

[0110] In some embodiments, the engineered TE polypeptides of the disclosure can minimize the formation of substrate oligomers even at high substrate concentration and / or do not require protective group manipulations.

[0111] In some embodiments, the TE 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 TE polypeptides are capable of converting the substrate to the product at a rate that is at least about 1.1-fold; about 1.2-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 1,000-fold; about 50,000-fold; about 100,000 fold; about 500,000 fold; about 1,000,000-fold; or more than about 1,000,000-fold the rate exhibited by a reference polypeptide of any one of SEQ ID NO: 2, 4, 6, or 378.

[0112] In some embodiments, the TE polypeptides of the disclosure may demonstrate increased activity in amide bond formation, wherein the polypeptide has at least about 1.1-fold; about 1.2-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 1,000-fold; about 50,000-fold; about 100,000 fold; about 500,000 fold; about 1,000,000-fold; or more than about 1,000,000-fold increased activity relative to a reference polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 2. 4, 6, 16, 340. 352, 354, 360, 378, or 380.

[0113] In some embodiments, the TE polypeptides of the disclosure may demonstrate increased thermal stability (i.e., thermostability), wherein the polypeptide has at least about 1.1-fold, about 1.2-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 thermal stability relative to a reference polypeptide of any one of SEQ ID NO: 2, 4, 6, 16, 340, 352, 354, 360, 378, or 380.

[0114] In some embodiments, the TE polypeptides of the disclosure have been improved for thermostability. In some embodiments, the TE 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 any one of SEQ ID NO: 2, 4, 6, 16, 340, 352, 354, 360, 378, or 380. In some embodiments, the TE 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: 2, 4, 6, 16, 340, 352, 354, 360, 378, or 380 when subjected to a heat challenge test for about 10, 20, 30, 40, 50, or 60 minutes. In some embodiments, the TE polypeptides of the disclosure 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: 2, 4, 6, or 16 when subjected to a heat challenge test for about 60 minutes.

[0115] In some embodiments, the TE polypeptides of the disclosure may demonstrate increased chemoselectivity, wherein the polypeptide has at least about 1.1-fold; about 1.2-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 1,000-fold; about 50,000-fold; about 100,000 fold; about 500,000 fold; about 1,000,000-fold; or more than about 1,000,000-fold increased chemoselectivity relative to a reference polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 2, 4, 6, 16, 340, 352, 354, 360. 378, or 380.

[0116] In some embodiments, the TE polypeptides of the disclosure may demonstrate increased regioselectivity, wherein the polypeptide has at least about 1.1-fold; about 1.2-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 1,000-fold; about 50,000-fold; about 100,000 fold; about 500,000 fold; about 1,000,000-fold; or more than about 1.000,000-fold increased regioselectivity relative to a reference polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 2, 4, 6, 16, 340, 352, 354, 360, 378, or 380.

[0117] In some embodiments, the TE polypeptides of the disclosure may demonstrate increased tolerance to a solvent or cosolvent, wherein the polypeptide has at least about 1.1-fold; about 1.2-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 1,000-fold; about 50,000-fold; about 100,000 fold; about 500,000 fold; about 1,000.000-fold; or more than about 1,000.000-fold increased tolerance to a solvent or cosolvent relative to a reference polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 2, 4, 6, 16, 340, 352, 354, 360, 378, or 380.

[0118] In some embodiments, the TE polypeptides of the disclosure may demonstrate increased substrate selectivity, wherein the polypeptide has at least about 1.1-fold; about 1.2-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 1,000-fold; about 50,000-fold; about 100,000 fold; about 500,000 fold; about 1,000,000-fold; or more than about 1,000,000-fold increased substrate selectivity relative to areference polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 2, 4, 6, 16, 340, 352, 354, 360, 378, or 380.

[0119] In some embodiments, the TE polypeptides of the disclosure may demonstrate increased substrate affinity, wherein the polypeptide has at least about 1.1-fold; about 1.2-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 1,000-fold; about 50,000-fold; about 100,000 fold; about 500,000 fold; about 1,000,000-fold; or more than about 1,000,000-fold increased substrate affinity relative to a reference polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 2, 4, 6, 16, 340, 352, 354, 360, 378, or 380.

[0120] In some embodiments, the TE polypeptides of the disclosure may demonstrate increased protein expression and / or protein solubility, wherein the polypeptide has at least about 1.1-fold; about 1.2-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 1,000-fold; about 50,000-fold; about 100,000 fold; about 500,000 fold; about 1,000,000-fold; or more than about 1,000,000-fold increased protein expression and / or protein solubility relative to a reference polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 2, 4, 6. 16, 340, 352. 354, 360, 378, or 380.

[0121] In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%. or 99%, but less than 100% sequence identity SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%. 95%, 96%. 97%. 98%. or 99%, sequence identity to any one of SEQ ID NO: 2. 4, 6, 16, 340, 352, 354, 360, 378, or 380, wherein the polypeptide does not comprise the amino acid sequence of SEQ ID NO: 2.

[0122] The difference(s) between the variant(s) and a reference polypeptide (e.g., SEQ ID NO: 2, 4, 6, 16, 52, 378, 380, etc.) 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 nonconservative. 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.

[0123] In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%. 69%. 70%. 71%. 72%. 73%. 74%. 75%. 76%. 77%. 78%. 79%. 80%. 81%. 82%. 83%. 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to any one of SEQ ID NO: 2, 4, 6, 16, 52, 378, or 380, wherein the polypeptide does not comprise SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 65% sequence identity to any one of SEQ ID NO: 2, 4, 6, 16, 52, 378, or 380, wherein the polypeptide does not comprise SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., aTE polypeptide) comprising an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NO: 2, 4. 6, 16, 52, 378, or 380, wherein the polypeptide does not comprise SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 75% sequence identity to any one of SEQ ID NO: 2, 4, 6, 16, 52, 378, or 380, wherein the polypeptide does not comprise SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NO: 2, 4. 6, 16. 52.378, or 380, wherein the polypeptide does not comprise SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NO: 2, 4, 6, 16, 52, 378, or 380, wherein the polypeptide does not comprise SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NO: 2, 4, 6, 16, 52, 378, or 380, wherein the polypeptide does not comprise SEQ ID NO: 2. In some embodiments, provided herein is apolypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 91% sequence identity to any one of SEQ ID NO: 2, 4, 6, 16, 52, 378, or 380, wherein the polypeptide does not comprise SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 92% sequence identity to any one of SEQ ID NO: 2. 4, 6, 16, 52, 378. or 380. wherein the polypeptide does not comprise SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 93% sequence identity to any one of SEQ ID NO: 2, 4, 6, 16, 52, 378, or 380, wherein the polypeptide does not comprise SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 94% sequence identity to any one of SEQ ID NO: 2, 4, 6, 16, 52, 378, or 380, wherein the polypeptide does not comprise SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NO: 2, 4, 6, 16. 52.378, or 380, wherein the polypeptide does not comprise SEQ ID NO: 2 In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 96% sequence identity to any one of SEQ ID NO: 2, 4, 6, 16, 52, 378, or 380, wherein the polypeptide does not comprise SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 97% sequence identity to any one of SEQ ID NO: 2, 4, 6, 16, 52, 378, or 380, wherein the polypeptide does not comprise SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 98% sequence identity to any one of SEQ ID NO: 2, 4, 6, 16, 52. 378, or 380, wherein the polypeptide does not comprise SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 99% sequence identity’ to any one of SEQ ID NO: 2, 4, 6, 16, 52, 378, or 380, wherein the polypeptide does not comprise SEQ ID NO: 2

[0124] In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20. 22. 24. 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 90, 106, 114, 130, 140, 150, 162, 172, 188, 194, 204, 216, 230, 238, 248, 260, 272, 294, 296, 310, 322, 340, 352, 354, 360, 364, 366, 368, 370, 372, 374, 376, 378, or 380, wherein the polypeptidedoes not comprise SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid of any one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 90, 106. 114, 130, 140, 150, 162, 172, 188, 194, 204, 216, 230, 238. 248, 260, 272, 294. 296, 310, 322, 340, 352. 354, 360, 364, 366, 368. 370, 372. 374, 376, 378, or 380. In some embodiments, provided herein is a polypeptide (e g., a TE polypeptide) consisting of any one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 90, 106, 114, 130, 140, 150, 162, 172, 188. 194, 204, 216, 230, 238. 248, 260, 272, 294, 296, 310, 322. 340, 352, 354, 360, 364.366, 368, 370, 372, 374, 376, 378, or 380.

[0125] In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%. 76%. 77%. 78%. 79%. 80%. 81%. 82%. 83%. 84%. 85%. 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. or 99%, sequence identity to SEQ ID NO: 2, wherein the polypeptide comprises at least one amino acid substitution at position 82, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the polypeptide comprises an S82C substitution, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2.

[0126] In some embodiments, the polypeptide comprises at least one amino acid deletion. In some embodiments, the polypeptide comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid deletions. In some embodiments, the polypeptide comprises a deletion at amino acid positions 2, 3, 4, 5, 6, 7, and 8, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the polypeptide comprises an S82C substitution, and a deletion at positions 2, 3, 4, 5, 6, 7, and 8, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2.

[0127] In some embodiments, the polypeptide comprises at least one amino acid substitution at position 82, and at least one further amino acid substitution at one or more positions selected from 4, 13, 16, 18, 20, 22, 23, 24, 25, 27, 30, 31, 36, 37, 49, 54, 66, 75, 79, 90, 91, 93, 98, 100, 104, 107, 112, 114, 118, 119, 123, 124, 125, 126, 127, 129, 130, 131, 132, 133, 134, 135, 139, 142, 146. 150, 157, 163, 167, 171. 172, 176, 187, 189, 190, 191, 192. 195, 196, 197, 199, 203.208, 209, 210, 212, 217, 219, 221, 226, 228, 229, 234, 242, 245, and 249, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 6 or 378, and wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2.

[0128] In some embodiments, provided herein is a polypeptide (e.g., a TE 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: 6 or 378, and wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e g., a TE 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: 6 or 378, wherein the polypeptide comprises an amino acid substitution at one or more positions selected from 4, 13, 16, 18, 20, 22, 23, 24, 25, 27, 30, 31, 36, 37, 49, 54, 66, 75, 79, 90, 91, 93, 98, 100, 104, 107, 112, 114, 118, 119, 123, 124, 125, 126, 127, 129, 130, 131, 132, 133, 134, 135, 139, 142, 146, 150, 157, 163, 167, 171, 172, 176, 187, 189, 190. 191, 192, 195, 196, 197, 199, 203, 208, 209, 210, 212, 217. 219, 221, 226, 228, 229, 234, 242, 245, and 249, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 6 or 378, and wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2.

[0129] In some embodiments, the polypeptide comprises from 1 to 76 amino acid substitutions, each amino acid substitution at an amino acid position selected from 4, 13, 16, 18, 20, 22, 23, 24, 25, 27, 30, 31, 36, 37, 49, 54, 66, 75, 79, 90, 91, 93, 98, 100, 104, 107, 112, 114, 118, 119, 123, 124, 125, 126, 127, 129, 130, 131, 132, 133, 134, 135, 139, 142, 146, 150, 157, 163, 167, 171, 172, 176, 187, 189, 190, 191, 192, 195, 196, 197, 199, 203, 208, 209, 210, 212, 217, 219, 221, 226, 228. 229, 234, 242, 245, and 249, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 6 or 378. In some embodiments, the polypeptide comprises from 4 to 76 amino acid substitutions at four or more amino acid positions selected from 4, 13, 16, 18, 20, 22, 23, 24, 25, 27, 30, 31, 36, 37, 49, 54, 66, 75, 79, 90, 91, 93, 98, 100, 104, 107. 112, 114, 118, 119, 123, 124, 125, 126, 127, 129, 130, 131, 132, 133, 134, 135, 139, 142, 146. 150, 157, 163. 167, 171. 172, 176, 187, 189, 190. 191, 192. 195, 196, 197, 199, 203.208, 209, 210, 212, 217, 219, 221, 226, 228, 229, 234, 242, 245, and 249, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 6 or 378. In some embodiments, the polypeptide comprises at least 6 amino acid substitutions at six or more amino acid positions selected from 4. 13, 16, 18, 20, 22, 23, 24, 25, 27, 30, 31, 36, 37, 49. 54. 66. 75, 79, 90, 91, 93, 98, 100, 104, 107, 112, 114, 118, 119, 123, 124, 125, 126, 127, 129, 130, 131, 132, 133, 134, 135, 139, 142, 146, 150, 157, 163, 167, 171, 172, 176, 187, 189, 190, 191, 192, 195, 196, 197, 199, 203, 208, 209, 210, 212, 217, 219, 221, 226, 228, 229, 234, 242, 245, and249, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 6 or 378. In some embodiments, the polypeptide comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49. or 50 amino acid substitutions at 2, 3, 4. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16. 17. 18. 19. 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31. 32. 33.34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid positions selected from 4, 13, 16, 18, 20, 22, 23, 24, 25, 27, 30, 31, 36, 37, 49, 54, 66, 75, 79, 90, 91, 93, 98, 100, 104, 107, 112, 114, 118, 119, 123, 124, 125, 126, 127, 129, 130, 131, 132, 133, 134, 135, 139, 142, 146. 150, 157, 163, 167, 171. 172, 176, 187, 189, 190, 191, 192. 195, 196, 197, 199, 203.208, 209, 210, 212, 217, 219, 221, 226, 228, 229, 234, 242, 245, and 249, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 6 or 378.

[0130] In some embodiments, the polypeptide comprises an amino acid substitution at position 196, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: SEQ ID NO: 6 or 378.

[0131] In some embodiments, the polypeptide comprises amino acid substitutions or amino acid substitution sets at the following amino acid positions: i) 196; ii) 27, 125, 127, 129, 196, 199; iii) 27, 31, 107, 123, 125, 129, 196, 199, 228, 245; iv) 18, 20, 27, 31, 107, 123, 125, 129, 196, 199. 228, 234, 245; v) 18, 20, 23. 27. 31. 36, 107, 123. 125, 129, 132, 135, 167. 196, 199.226, 228, 234, 245; vi) 18, 20, 23, 27, 31, 36, 107, 123, 125, 129, 132, 135, 163, 167, 187, 196, 199, 203, 226, 228, 234, 245; vii) 16, 18, 20, 23, 27, 31, 36, 79, 107, 123, 125, 129, 132, 135, 142, 163, 167, 187, 196, 199, 203, 226, 228, 234, 245; viii) 16, 18, 20, 23, 27, 31, 36, 75, 79, 107, 123. 125, 129, 132, 133, 135. 142, 163, 167, 187, 196, 199, 203. 226, 228, 234, 245; ix) 16, 18. 20. 23. 25, 27, 31, 36, 79, 107. 123, 125, 129. 132, 135. 142, 163. 167, 187, 196. 197, 199.203, 226, 228, 234, 245; x) 16, 18, 20, 22, 23, 25, 27, 31, 36, 79, 107, 123, 125, 129, 132, 134, 135, 142, 163, 167, 187, 196, 197, 199, 203, 226, 228, 234, 245; xi) 13, 16, 18, 20, 22, 23, 25, 27, 31, 36, 79, 107, 123, 125, 129, 132, 134, 135, 142, 163, 167, 187, 196, 197, 199, 203, 226, 228, 234. 245; xii) 13, 16, 18, 20, 22, 23, 25, 27, 31, 36, 79. 107, 123, 125. 129, 132. 134, 135, 142, 163, 167, 172, 187, 189, 190, 196, 197, 199, 203, 226, 228, 234, 245; xiii) 13, 16, 18, 20, 22, 23, 25, 27, 31, 36, 79, 107, 123, 125, 129, 132, 134, 135, 142, 163, 167, 172, 187, 189, 190, 196, 197, 199, 203, 226, 228, 234, 245, 249; xiv) 13, 16, 18, 20, 22, 23, 25, 27, 31, 36, 79, 107, 112, 123. 125, 129, 132, 134, 135. 142, 163, 167, 172, 187, 189, 190. 196, 197, 199, 203, 226.228, 234, 245, 249; xv) 13, 16, 18, 20, 22, 23, 25, 27, 31, 36, 79, 107, 112, 123, 125, 129, 132, 134, 135, 142, 150, 163, 167C, 172, 187, 189, 190, 192, 196, 197, 199, 203, 208, 226, 228, 229, 234, 245, 249; xvi) 4, 13, 16, 18, 20, 22, 23, 24, 25, 27, 31, 36, 79, 90, 107, 112, 123, 124, 125,129, 132. 134, 135, 142. 150, 163. 167, 172, 187. 189, 190. 192, 196. 197, 199, 203. 208, 226.228, 229, 234, 245, 249; xvii) 4, 13, 16, 18, 20, 22, 23, 24, 25, 27, 30, 31, 36, 79, 90, 107, 112, 123, 124, 125, 129, 132, 134, 135, 142, 150, 163, 167, 172, 187, 189, 190, 192, 196, 197, 199, 203, 208. 226, 228, 229, 234, 245, 249; xviii) 4, 13, 16, 18, 20, 22, 23, 24, 25, 27, 30, 31, 36, 54, 79. 90, 93, 107, 112. 125, 129. 132, 134, 135, 142, 146. 150, 163, 167, 172, 187. 189, 190. 192, 196, 197, 199, 203, 208, 210, 212, 226, 228, 234, 242, 245, 249; xix) 4, 13, 16, 18, 20, 22, 23, 24, 25, 27, 30, 31, 36, 54, 79, 90, 93, 104, 107, 112, 125, 129, 132, 134, 135, 142, 146, 150, 163, 167, 171, 172, 187, 189, 190, 192, 196, 197, 199, 203, 208, 210, 212, 226, 228, 234, 242, 245, 249; xx) 4, 13, 16, 18, 20, 22, 23, 24, 25, 27, 30, 31, 36. 54. 79. 90, 93, 100, 104. 107, 112, 125, 129, 132, 134, 135, 142, 146, 150, 163, 167, 172, 187, 189, 190, 192, 196, 197, 199, 203, 208, 210, 212, 226, 228, 234, 242, 245, 249; xxi) 4, 13, 16, 18, 20, 22, 23, 24, 25, 27, 31, 36, 54, 66, 79, 90, 93, 100, 104, 107, 112, 125, 129, 132, 134, 135, 142, 146, 150, 163, 167, 172, 187, 189, 190, 192. 196, 197, 199, 203, 208, 210, 212, 226, 228, 234, 242, 245, 249; xxii) 4, 13. 16. 18, 20. 22, 23. 24, 25, 27, 30, 31, 36, 54, 79, 90, 93. 100, 104, 107. 112, 125. 129, 132, 134. 135, 142, 146, 150, 163, 167, 172, 187, 189, 190, 192, 196, 197, 199, 203, 208, 210, 212, 226, 228, 234, 242, 245, 249; or xxiii) 4, 13, 16, 18, 20, 22, 23, 24, 25, 27, 31, 36, 54, 66, 79, 90, 93, 100, 104, 107, 112, 125, 129, 132, 134, 135, 142, 146, 150, 163, 167, 172, 187, 189, 190, 192, 196, 197, 199. 203, 208, 210, 212, 226, 228, 234, 242, 245, 249, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 6 or 378.

[0132] In some embodiments, the polypeptide comprises at least one amino acid substitution selected from H4F, I13Q, V16M, S18C, T20V, I22R, G23M, A24G, H25N, T27K, K31P, I36L, E54D, E66K, N79G. N90K, H93K, L100V, Y104W, E107S, Q112L. F125V, G129L, N132P, M134E, F135W. L142A, V146R, F150A. L163T. N167V, E172T, E187D. E189F. N190D, A192L, A196R, E197R, W199K, V203L, S208T, Q210R, I212T, G226T, Q228A, S234R, E242Q, V245, and S249*, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 6 or 378. In some embodiments, the polypeptide comprises at least 2. 3, 4, 5, 6. 7, 8, 9. 10. 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22. 23. 24. 25. 26. 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 amino acid substitutions selected from H4F, I13Q, V16M, S18C, T20V, I22R, G23M, A24G, H25N, T27K, K31P, I36L, E54D, E66K, N79G, N90K, H93K, L100V, Y104W, E107S, Q112L, F125V, G129L, N132P, M134E. F135W, L142A, V146R, F150A, L163T, N167V. E172T, E187D, E189F, N190D, A192L, A196R, E197R, W199K, V203L, S208T, Q210R, I212T, G226T, Q228A, S234R, E242Q, V245, and S249*, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 6 or 378. In some embodiments, the polypeptidecomprises at least one amino acid insertion between amino acid positions 224 and 225 or between amino acid positions 225 and 226.

[0133] In some embodiments, the polypeptide comprises one of the following amino acid substitutions, amino acid substitution sets, and amino acid substitution and amino acid insertion sets: i) A196R; ii) T27K, F125V, E127L, G129L. A196R, W199K; iii) T27K, K31P. E107S, A123L, F125V, G129L, A196R, W199K, Q228A, V245G; iv) S18C, T20V, T27K, K31P, E107S, A123L, F125V, G129L, A196R, W199K, Q228A, S234R, V245G; v) S18C, T20V, G23L, T27K, K31P, I36L, E107S, A123L, F125V, G129L, N132P. F135W, N167A, A196R, W199K, G226T, Q228A, S234R, V245G; vi) S18C, T20V. G23L, T27K, K31P, I36L. E107S. A123L, F125V, G129L, N132P, F135W, L163T, N167S, E187K, A196R, W199K, V203L, G226T, Q228A, S234R, V245G; vii) V16L, S18C, T20V, G23L, T27K, K31P, I36L, N79G, E107S, A123L, F125V, G129L, N132P, F135W, L142R, L163T, N167S, E187K, A196R, W199K, V203L, G226T, Q228A, S234R, V245G; viii) V16L, S18C, T20V, G23L, T27K, K31P, I36L. C75S, N79G, E107S, A123L, F125V. G129L, N132P, E133P, F135W, L142R, L163W. N167S, E187K, A196R, W199K, V203L, G226T, Q228A, S234R, V245G: ix) V16L, S18C, T20V, G23L, H25N, T27K, K31P, I36L, N79G, E107S, A123L, F125V, G129L, N132P, F135W, L142R, L163T, N167S, E187K, A196R, E197R, W199K, V203L, G226T, Q228A, S234R. V245G; x) V16L, S18C. T20V, I22R. G23L, H25N, T27K. K31P, I36L, N79G, E107S, A123L, F125V, G129L, N132P, M134E, F135W, L142R, L163T, N167S, E187K, A196R, E197R, W199K, V203L, G226T, Q228A, S234R, V245G; xi) I13Q, V16L, S18C, T20V, I22R, G23L, H25N, T27K, K31P, I36L, N79G, E107S, A123L, F125V, G129L, N132P, M134E, F135W, L142R, L163T, N167S. E187K, A196R, E197R, W199K, V203L. G226T, Q228A, S234R. V245G; xii) I13Q. V16L, S18C, T20V. I22R. G23L, H25N, T27K, K31P, I36L. N79G, E107S, A123L, F125V, G129L, N132P, M134E, F135W, L142A, L163T, N167S, E172T, E187D, E189D, N190D, A196R, E197R, W199K, V203L, G226T, Q228A, S234R, V245G; xiii) I13Q, V16L, S18C, T20V, I22R, G23L, H25N, T27K, K31P, I36L. N79G, E107S, A123L, F125V, G129L, N132P, M134E, F135W, L142A. L163T, N167S, E172T, E187D. E189D, N190D, A196R, E197R, W199K, V203L, G226T, Q228A, S234R, V245G, S249*; xiv) I13Q, V16L, S18C, T20V, I22R, G23L, H25N, T27K, K31P, I36L, N79G, E107S, Q112L, A123L, F125V, G129L, N132P, M134E, F135W, L142A, L163T, N167S, E172T, E187D, E189D, N190D, A196R, E197R. W199K, V203L. G226T, Q228A, S234R, V245G, S249*; xv) I13Q, V16L, S18C, T20V, I22R, G23L, H25N, T27K, K31P, I36L, N79G, E107S, Q112L, A123L, F125V, G129L, N132P, M134E, F135W, L142A, F150H, L163T, N167C, E172T, E187D, E189K, N190D, A192L, A196R, E197R, W199K, V203L, S208T, G226T, Q228A, I229T,S234R, V245G, S249*; xvi) H4F. I13Q, V16L. S18C, T20V. 122R, G23L. A24G, H25N, T27K. K31P, I36L, N79G, N90K, E107S, Q112L, A123L, F124R, F125V, G129L, N132P, M134E, F135W, L142A, F150H, L163T, N167L, E172T, E187D, E189K, N190D, A192L, A196R, E197R. W199K, V203L, S208T, G226T, Q228A, I229T, S234R, V245G; xvii) H4F, I13Q, V16L, S18C. T20V, I22R. G23L, A24G, H25N, T27K. Q30Y, K31P, I36L, N79G, N90K.E107S, Q112L, A123L, F124R, F125V, G129L, N132P, M134E, F135W, L142A, F150H, L163T, N167L, E172T, E187D, E189K, N190D, A192L, A196R, E197R, W199K, V203L, S208T, G226T, Q228A, I229T, S234R, V245G, S249*: xviii) H4F, I13Q, V16L. S18C, T20V, I22R. G23L, A24G, H25N, T27K. Q30Y, K31P, I36L, E54D, N79G. N90K, H93K, E107S, Q112L, F125V, G129L, N132P, M134E, F135W, L142A, V146R, F150A, L163T, N167L, E172T, E187D, E189K, N190D, A192L, A196R, E197R, W199K, V203L, S208T, Q210R, I212T, G226T, Q228A, S234R, E242Q, V245G, S249*; xix) H4F, I13Q, V16L, S18C, T20V, I22R. G23L, A24G, H25N, T27K. Q30Y, K31P, I36L, E54D, N79G. N90K, H93K, Y104W, E107S, Q112L, F125V, G129L, N132P, M134E, F135W, L142A, V146R, F150A, L163T, N167L, N171H, E172T, E187D, E189K, N190D, A192L, A196R, E197R, W199K, V203L, S208T, Q210R, I212T, G226T, Q228A, S234R, E242Q, V245G, S249*; xx) H4F, I13Q, V16L, S18C, T20V. I22R, G23M, A24G H25N, T27K, Q30Y, K31P, I36L, E54D, N79G, N90K, H93K, L100V. Y104W, E107S, Q112L, F125V, G129L, N132P. M134E, F135W. L142A, V146R, F150A, L163T, N167L, E172T, E187D, E189F, N190D, A192L, A196R, E197R, W199K, V203L, S208T, Q210R, I212T, G226T, Q228A, S234R, E242Q, V245G, S249*; xxi) H4F, I13Q, V16L, S18C, T20V, I22R, G23M, A24G, H25N, T27K, K31P, I36L, E54D, E66K, N79G, N90K, H93K, L100V, Y104W, E107S, Q112L, F125V. G129L, N132P, M134E. F135W, L142A, V146R, F150A. L163T. N167L, E172T, E187D. E189F. N190D, A192L, A196R.E197R, W199K, V203L, S208T, Q210R, I212T, G226T, Q228A, S234R, E242Q, V245G, S249*; xxii) H4F, I13Q, V16L, S18C, T20V, I22R, G23M, A24G, H25N, T27K, Q30Y, K31P, I36L. E54D, N79G, N90K, H93K. L100V, Y104W, E107S, Q112L, F125V, G129L. N132P, M134E, F135W. L142A, V146R, F150A. L163T. N167L, E172T, E187D, E189F. N190D, A192L, A196R, E197R, W199K, V203L, S208T, Q210R, I212T, G226T, Q228A, S234R, E242Q, V245G, S249* and an amino acid insertion between amino acid positions 224 and 225; or xxiii) H4F, I13Q, V16M, S18C, T20V, I22R, G23M, A24G, H25N, T27K, K31P, I36L,E54D, E66K, N79G. N90K, H93K, L100V, Y104W, E107S, Q112L. F125V, G129L, N132P, M134E, F135W, L142A, V146R, F150A, L163T, N167V, E172T, E187D, E189F, N190D, A192L, A196R, E197R, W199K, V203L, S208T, Q210R, I212T, G226T, Q228A, S234R, E242Q, V245G, S249* and an amino acid insertion between amino acid positions 225 and 226,wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 6 or 378.

[0134] In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. or 99%, sequence identity to SEQ ID NO: 52, and wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%. 92%, 93%. 94%. 95%. 96%. 97%. 98%. or 99%, sequence identity to SEQ ID NO: 52, wherein the polypeptide comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 amino acid substitutions at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 amino acid positions selected from 4, 30, 37, 49, 91, 98, 100, 107, 114, 118, 119, 126. 127, 130, 131, 139, 157, 171, 176, 191, 195, 209, 212, 219, 226, 227, and 230, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 52, and wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2.

[0135] In some embodiments, the polypeptide comprises a substitution set at one of the following sets of positions: i) 100. 157, 171. 209, 212, 226; ii) 100. 157, 171. 209, 212, 221, 226, 227; iii) 100, 114, 118, 157, 171, 209, 212, 217, 219, 226, 227, 230; iv) 100, 114, 118, 157, 171, 209, 212, 226, 227, 230; v) 49, 100, 114, 118, 126, 130, 157, 171, 195, 209, 212, 219, 226, 227, 230; vi) 4, 49, 100, 114, 118, 126, 130, 157, 171, 195, 209, 212, 219, 226, 227, 230; vii) 4, 49, 91, 100, 114. 118, 119, 126, 127, 130, 139, 157. 171, 195, 209, 212, 219. 226, 227, 230; viii) 4, 37.49. 91. 100, 114. 118, 119. 126, 130, 139. 157, 171. 176, 195, 209, 212, 219. 226, 227. 230; ix) 4, 37, 49, 91, 100, 107, 114, 118, 119, 126, 130, 139, 157, 171, 172, 176, 195, 209, 212, 219, 226, 227, 230; x) 4, 37, 49, 91, 100, 107, 114, 118, 119, 126, 127, 130, 139, 157, 171, 172, 176, 195, 209.212, 219, 226, 227, 230; xi) 4. 37, 49, 91, 98, 100, 107, 114, 118, 119, 126, 127, 130, 139, 157. 171, 176, 191. 195, 209.212, 219, 226, 227, 230; orxii) 4, 30, 37, 49, 91, 98, 100. 107, 114, 118, 119, 126, 127, 130, 131, 139, 157, 171, 176, 191, 195, 209, 212, 219, 226, 227, 230, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 52.

[0136] In some embodiments, the polypeptide comprises at least one amino acid substitution selected from F4V, Y30L, H37N, A49E, Q91R, I98L, V100L, S107P, A114P, R118Q, K119A, T126A, E127D, T130A, H131N, R139L, Q157R, N171H, K176L, I191L, E195S, E209S, T212V, E219S, N226S, T227P, and I230V, wherein the amino acid positions of the polypeptideare numbered with reference to SEQ ID NO: 52, and wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 substitutions selected from F4V, Y30L, H37N, A49E, Q91R, I98L, V100L, S107P, A114P, R118Q. K119A, T126A, E127D, T130A, H131N, R139L, Q157R. N171H, K176L, I191L, E195S, E209S, T212V, E219S, N226S, T227P, and I230V, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 52, and wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2.

[0137] In some embodiments, the polypeptide comprises one of the following amino acid substitution sets: i) V100L, Q157R, N171H, E209S, T212V, N226A; ii) V100L, Q157R, N171H, E209S, T212V, S221R, N226S, T227P; iii) V100L, A114P, R118Q, Q157R, N171H, E209S, T212V, H217A, E219P, N226S, T227P, I230V; iv) V100L, A114P, R118Q, Q157R, N171H, E209S, T212V, N226S, T227P, I230V; v) A49E, V100L, A114P, R118Q, T126A, T130A, Q157R, N171H. E195S. E209S. T212V, E219S, N226S, T227P, I230V; vi) F4C, A49E, V100L, A114P, R118Q, T126A, T130A, Q157R, N171H, E195S, E209S, T212V, E219S, N226S, T227P, I230V; vii) F4C, A49E, Q91R, V100L, A114P, R118Q, K119A, T126A, E127Q, T130A, R139L, Q157R, N171H, E195S, E209S, T212V, E219S, N226S, T227P, I230V; viii) F4C, H37G, A49E, Q91R. V100L, A114P, R118Q. K119A, T126A, T130A. R139L, Q157R, N171H. K176C, E195S, E209S, T212V, E219S, N226S, T227P, I230V; ix) F4C, H37G, A49E, Q91R, V100L, S107P, A114P, R118Q, K119A, T126A, T130A, R139L, Q157R, N171H, T172V, K176C, E195S, E209S, T212V, E219S, N226S, T227P, I230V; x) F4C, H37G, A49E, Q91R, V100L, S107P, A114P, R118Q, K119A, T126V, E127D, T130A, R139L, Q157R, N171H, T172V, K176C, E195S, E209S, T212V, E219S, N226S, T227P, I230V; xi) F4V, H37R. A49E, Q91R, I98L, V100L, S107P, A114P, R118Q, K119A, T126V, E127D, T130A, R139L, Q157R, N171H, K176L, I191L, E195S, E209S, T212V, E219S, N226S, T227P, I230V; or xii) F4V, Y30L, H37N, A49E, Q91R, I98L, V100L, S107P, A114P, R118Q, K119A, T126A, E127D, T130A, H131N, R139L. Q157R, N171H, K176L. I191L, E195S. E209S. T212V, E219S, N226S, T227P, I230V, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 52.

[0138] In some embodiments, the polypeptide comprises one or more amino acid sequence differences (e.g., substitutions, substitution sets, deletions and / or insertions) set forth in Table C and / or any of Tables 2.1 to 34.1 and 37.2. In some embodiments, the polypeptide comprises one or more amino acid sequence differences (e.g., substitutions, substitution sets, deletions or insertions) set forth in Table C. In some embodiments, the polypeptide comprises one or moreamino acid sequence differences (e.g., substitutions, substitution sets, deletions or insertions) set forth in any of Tables 2.1 to 34.1.

[0139] In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or higher sequence identity to any one of even numbered SEQ ID NO: 2-76, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%. 87%, 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or higher sequence identity to any one of even numbered SEQ ID NO: 4-76, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2.

[0140] In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or higher sequence identity to any one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 378, or 380, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2.

[0141] In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 378, or 380, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., aTE polypeptide) comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 378, or 380, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 378, or 380, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 91% sequence identity to any one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 378, or 380, wherein the polypeptide does not comprise the sequenceof SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 92% sequence identity to any one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70. 72, 74, 76, 378, or 380, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 93% sequence identity’ to any one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 378, or 380, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 94% sequence identity’ to any one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 378, or 380. wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 378, or 380, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., aTE polypeptide) comprising an amino acid sequence having at least 96% sequence identity’ to any one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 378, or 380, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 97% sequence identity to any one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 378, or 380, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g.. a TE polypeptide) comprising an amino acid sequence having at least 98% sequence identity to any one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 378, or 380, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e g., a TE polypeptide) comprising an amino acid sequence having at least 99% sequence identity to any one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46. 48. 50. 52, 54, 56, 58, 60, 62, 64, 66, 68, 70. 72. 74. 76. 378, or 380, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2.

[0142] In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising any one of SEQ ID NO: 4. 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26. 28. 30, 32, 34, 36, 38. 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62. 64. 66. 68. 70. 72, 74, 76, 378, or 380. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 378, or 380. In some embodiments, provided herein is a polypeptide (e.g., aTE polypeptide) consisting essentially of the amino acid sequence of any one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 378, or 380. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) consisting of any one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30. 32. 34, 36, 38, 40, 42, 44, 46, 48, 50. 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74. 76. 378, or 380.

[0143] In some embodiments, provided herein is a polypeptide (e g., a TE polypeptide) comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 68, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 68, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 68, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 68. wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 99% sequence identity' to SEQ ID NO: 68, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising the amino acid sequence of SEQ ID NO: 68. Also provided herein is a polypeptide (e.g., a TE polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 68. Also provided herein is a polypeptide (e.g., a TE polypeptide) consisting of the amino acid sequence of SEQ ID NO: 68.

[0144] In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 70, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 70, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 70, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 70, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 70, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising the amino acid sequence of SEQ ID NO: 70. Also provided herein is a polypeptide (e.g., a TE polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 70. Also provided herein is a polypeptide (e.g., a TE polypeptide) consisting of the amino acid sequence of SEQ ID NO: 70.

[0145] In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%. 93%, 94%. 95%. 96%. 97%. 98%. 99%. or higher sequence identity to SEQ ID NO: 72, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e g., a TE polypeptide) comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 72, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g.. a TE polypeptide) comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 72, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 72. wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 72, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is apolypeptide (e.g., a TE polypeptide) comprising the amino acid sequence of SEQ ID NO: 72. Also provided herein is a polypeptide (e.g., a TE polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 72. Also provided herein is a polypeptide (e.g., a TE polypeptide) consisting of the amino acid sequence of SEQ ID NO: 72.

[0146] In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 74, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e g., a TE polypeptide) comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 74, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 74, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 74, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 74, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising the amino acid sequence of SEQ ID NO: 74. Also provided herein is a polypeptide (e.g., a TE polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 74. Also provided herein is a polypeptide (e.g., a TE polypeptide) consisting of the amino acid sequence of SEQ ID NO: 74.

[0147] In some embodiments, provided herein is a polypeptide (e g., a TE polypeptide) comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 76, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e g., a TE polypeptide) comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 76, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 76, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQID NO: 76, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 76, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising the amino acid sequence of SEQ ID NO: 76. Also provided herein is a polypeptide (e.g., a TE polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 76. Also provided herein is a polypeptide (e.g., a TE polypeptide) consisting of the amino acid sequence of SEQ ID NO: 76.

[0148] In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 6 or 378, and wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE 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: 6 or 378, wherein the polypeptide comprises an amino acid substitution at one or more positions selected from 4, 10, 11, 12, 13, 15, 18, 20, 23, 24, 27, 31, 34, 35, 36, 37, 48, 62, 72, 93, 97, 98, 107, 108, 109, 111, 113, 114, 115, 120, 121, 123, 124, 125, 126, 127, 129, 132, 133, 134, 135, 138, 139, 144, 145, 146, 147, 148, 150, 154, 157, 158, 166, 167, 168, 172, 175, 183, 186, 187, 188, 189. 190, 192, 194, 195, 196. 199, 203, 205, 206, 212, 213, 214. 217, 221, 222, 226, 228.229, 234, 238, 243, 245, and 247, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 6 or 378, and wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2.

[0149] In some embodiments, the polypeptide comprises from 1 to 85 substitutions, each amino acid substitution at an amino acid position selected from 4, 10, 11, 12, 13, 15, 18, 20, 23. 24. 27.31, 34, 35, 36, 37, 48, 62, 72, 93, 97, 98, 107, 108, 109, 111, 113, 114, 115, 120, 121, 123, 124, 125, 126, 127, 129, 132, 133, 134, 135, 138, 139, 144, 145, 146, 147, 148, 150, 154, 157, 158, 166, 167, 168, 172, 175, 183, 186, 187, 188, 189, 190, 192, 194, 195, 196, 199, 203, 205, 206, 212, 213. 214, 217, 221, 222, 226. 228, 229, 234, 238, 243, 245, and 247, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 6 or 378. In some embodiments, the polypeptide comprises from at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 85 amino acid substitutions at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20 to 85 amino acid positions selected from 4, 10, 11, 12, 13, 15, 18, 20, 23, 24, 27, 31, 34, 35, 36, 37, 48, 62, 72, 93, 97, 98, 107, 108, 109, 111, 113, 114, 115, 120, 121, 123, 124, 125, 126, 127, 129, 132, 133, 134, 135, 138, 139, 144, 145, 146, 147, 148, 150, 154, 157, 158, 166. 167, 168, 172, 175, 183, 186, 187, 188, 189, 190, 192, 194. 195, 196, 199, 203, 205, 206, 212. 213, 214, 217, 221, 222. 226, 228, 229, 234, 238. 243, 245. and 247, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 6 or 378.

[0150] In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%. 70%, 71%. 72%. 73%. 74%. 75%. 76%. 77%. 78%. 79%. 80%. 81%. 82%. 83%. 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 16 or 380, and wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 60%, 61%, 62%, 63%, 64%, 65%. 66%. 67%. 68%. 69%. 70%. 71%. 72%. 73%. 74%. 75%. 76%. 77%. 78%. 79%. 80%. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 16 or 380, wherein the polypeptide comprises an amino acid substitution at one or more positions selected from 4, 10, 11, 12, 13, 15, 18, 23, 24, 27, 34, 35, 37, 48, 62, 72, 93, 97, 98. 107, 108, 109, 111, 113. 114, 115, 120, 121, 124, 126, 127, 129, 133, 134, 138, 139, 144, 145, 146, 147, 148, 150, 154, 157, 158, 166, 168, 172, 175, 183, 186, 187, 188, 189, 190, 192, 194, 195, 196, 199, 203, 205, 206, 212, 213, 214, 217, 221, 222, 226, 229, 234, 238, 243, 245, and 247, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 16 or 380, and wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2.

[0151] In some embodiments, the polypeptide comprises from 1 to 76 substitutions, each amino acid substitution at an amino acid position selected from 4, 10, 11, 12, 13, 15, 18, 23, 24, 27, 34, 35, 37, 48, 62, 72, 93, 97, 98, 107, 108, 109, 111, 113, 114, 115, 120, 121, 124, 126, 127, 129, 133, 134. 138, 139, 144. 145, 146. 147, 148, 150, 154, 157. 158, 166. 168, 172, 175, 183, 186.187, 188, 189, 190, 192, 194, 195, 196, 199, 203, 205, 206, 212, 213, 214, 217, 221, 222, 226, 229, 234, 238, 243, 245, and 247, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 16 or 380. In some embodiments, the polypeptide comprises from 4 to 76 amino acid substitutions at four or more amino acid positions selected from 4, 10, 11, 12, 13, 15, 18, 23, 24, 27, 34, 35, 37, 48, 62, 72, 93, 97, 98, 107, 108, 109, 111, 113, 114, 115, 120, 121, 124, 126, 127, 129, 133, 134, 138, 139, 144, 145, 146, 147, 148, 150, 154, 157, 158, 166, 168, 172, 175, 183, 186, 187, 188, 189, 190, 192, 194, 195, 196, 199, 203,205, 206. 212, 213, 214. 217, 221. 222, 226, 229. 234, 238. 243, 245. and 247. wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 16 or 380. In some embodiments, the polypeptide comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27. 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41. 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid substitutions at 2, 3. 4, 5, 6. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid positions selected from 4, 10, 11, 12, 13, 15, 18, 23, 24, 27, 34, 35, 37, 48, 62, 72, 93, 97, 98, 107, 108, 109, 111, 113, 114, 115, 120, 121, 124. 126, 127, 129, 133, 134. 138, 139, 144, 145, 146, 147, 148. 150, 154, 157, 158, 166. 168, 172, 175, 183, 186, 187, 188, 189, 190, 192, 194, 195, 196, 199, 203, 205, 206, 212, 213, 214, 217, 221, 222, 226, 229, 234, 238, 243, 245, and 247, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 16 or 380.

[0152] In some embodiments, the polypeptide comprises at least one amino acid substitution selected from H4F, N10A. KI IQ. T12G, I13T, S15R. S18C, S18A, T20V. G23S, A24C, T27K. T27H, T27R, K31P, K34A, K34S, E35N, I36L, H37N, E48P, Q62A, M72V, H93R, S97G, I98L, I98V, E107S, E107H, K108A, E109T, E109V, El IIP, T113V, A114E, I115S, E120M, E120T, E120V, I121C. A123L, F124Y, F125V, T126S, T126G, E127K, G129L, G129I, N132P, E133G, E133R, M134I, M134S, M134V, M134N. F135W, T138R, R139T. L144P, F145L, V146G, T147S, N148S, F150A, S154D, Q157E, N158S, N158R, N158H, Y166L, N167A, Q168K, E172T, I175V, Q183S, Y186S, E187D, S188G, E189D, N190S, A192T, Y194L, E195V, A196R, A196V, W199K, W199V, V203Y, A205T, A205Q, Q206D, I212F, N213G, Y214H, H217T, S221T, K222R, G226T. Q228A, I229A, I229M, I229V, I229L, S234R, S234G, S234E, S238Q, 1243Y, V245G, V245P, K247S, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 6 or 378. In some embodiments, the polypeptide comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38. 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid substitutions selected from H4F. N10A, KI IQ, T12G, I13T, S15R. S18C, S18A. T20V, G23S, A24C, T27K, T27H, T27R, K31P, K34A, K34S, E35N, I36L, H37N, E48P, Q62A, M72V, H93R, S97G, I98L, I98V, E107S, E107H, K108A, E109T, E109V, El IIP, T113V, Al 14E, I115S, E120M, E120T, E120V, I121C, A123L, F124Y, F125V, T126S, T126G, E127K, G129L, G129I, N132P, E133G, E133R, M134I. M134S, M134V, M134N, F135W, T138R, R139T, L144P, F145L, V146G, T147S, N148S, F150A, S154D, Q157E, N158S, N158R, N158H, Y166L, N167A, Q168K, E172T, I175V, Q183S, Y186S, E187D, S188G, E189D, N190S, A192T, Y194L, E195V, A196R, A196V, W199K, W199V, V203Y, A205T, A205Q,Q206D, 1212F, N213G, Y214H. H217T, S221T, K222R. G226T. Q228A, 1229 A, I229M, I229V, I229L, S234R, S234G, S234E, S238Q, I243Y, V245G, V245P, K247S, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 6 or 378.

[0153] In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising any one of SEQ ID NO: 16, 78, 90, 106, 114, 130, 140. 150, 162. 172, 188, 194. 204, 216, 230, 238, 248, 260, 272, 294, 296, 310, 322, 340, 352, 354, 360, or 380.

[0154] In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%. 70%, 71%. 72%. 73%. 74%. 75%. 76%. 77%. 78%. 79%. 80%. 81%. 82%. 83%. 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 16, wherein the polypeptide comprises at least one amino acid substitution at one or more positions selected from 250, 251, 252, 253, 254, and 255, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 16. In some embodiments, the polypeptide comprises at least 1-6 amino acid substitutions at 1-6 amino acid positions selected from 250, 251, 252, 253, 254, and 255. In some embodiments, the polypeptide comprises a substitution at each of amino acid positions 250, 251, 252, 253, 254, and 255. In some embodiments, the polypeptide comprises at least one amino acid substitution selected from H250G, H251S, H252G. H253S, H254G, and H255S. In some embodiments, the polypeptide comprises at least 1-6 amino acid substitutions selected from H250G, H251S, H252G, H253S, H254G, and H255S. In some embodiments, the polypeptide comprises the following amino acid substitution set: H250G, H251S, H252G, H253S, H254G, and H255S.

[0155] In some embodiments, the polypeptide comprises at least one amino acid insertion. In some embodiments, the polypeptide comprises an insertion of at least one amino acid between amino acid positions 1 and 2. In some embodiments, the polypeptide comprises an insertion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 amino acids between amino acid positions 1 and 2. In some embodiments, the polypeptide comprises an insertion of an amino acid sequence of SEQ ID NO: 383 between amino acid positions 1 and 2.

[0156] In some embodiments, provided herein is a polypeptide (e g., a TE polypeptide) comprising any one of SEQ ID NO: 364, 366, 368, 370, 372, 374, or 376. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising any one of SEQ ID NO: 340, 352. 354, 360, 364, 366, 368. 370, 372, 374, or 376.

[0157] In some embodiments, the polypeptide comprises one or more amino acid sequence differences (e.g., substitutions, substitution sets, deletions and / or insertions) set forth in Table D and / or any ofTables 37.2 or 38.1 to 61.1. In some embodiments, the polypeptide comprises oneor more amino acid sequence differences (e.g., substitutions, substitution sets, deletions or insertions) set forth in Table D. In some embodiments, the polypeptide comprises one or more amino acid sequence differences (e.g., substitutions, substitution sets, deletions or insertions) set forth in any of Tables 37.2 or 38.1 to 61.1.

[0158] In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 16 and any one of even numbered SEQ ID NO: 78-380, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to any one of even numbered SEQ ID NO: 78-376, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 95% sequence identity to any one of even numbered SEQ ID NO: 78-376, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 99% sequence identity to any one of even numbered SEQ ID NO: 78-376, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2.

[0159] In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%. 93%, 94%. 95%. 96%. 97%. 98%. 99%. or higher sequence identity to any one of SEQ ID NO: 16. 78. 90. 106, 114. 130, 140, 150. 162, 172. 188, 194. 204, 216, 230, 238, 248, 260, 272, 294, 296, 310, 322, 340, 352, 354, 360, 364, 366, 368, 370, 372, 374, 376, or 380, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2.

[0160] In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NO: 16, 78, 90, 106, 114, 130, 140, 150, 162, 172, 188, 194, 204, 216, 230, 238, 248, 260, 272, 294, 296, 310, 322, 340, 352, 354, 360, 364, 366, 368, 370, 372, 374, 376, or 380, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NO: 16, 78, 90, 106, 114, 130, 140, 150, 162, 172, 188, 194, 204, 216, 230, 238, 248, 260, 272, 294, 296, 310, 322, 340, 352, 354, 360, 364, 366, 368, 370, 372, 374, 376, or 380, wherein the polypeptide does not comprise the sequence ofSEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NO: 16, 78, 90, 106, 114, 130, 140, 150, 162, 172, 188, 194, 204, 216, 230, 238, 248, 260, 272, 294, 296. 310, 322, 340, 352, 354, 360, 364, 366, 368, 370, 372, 374. 376, or 380, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e g., a TE polypeptide) comprising an amino acid sequence having at least 91% sequence identity to any one of SEQ ID NO: 16, 78, 90, 106, 114, 130, 140, 150, 162, 172, 188, 194, 204, 216, 230, 238, 248, 260, 272, 294, 296, 310, 322, 340, 352, 354, 360, 364, 366, 368. 370, 372, 374, 376, or 380, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., aTE polypeptide) comprising an amino acid sequence having at least 92% sequence identity to any one of SEQ ID NO: 16, 78, 90, 106, 114, 130, 140, 150, 162, 172, 188, 194, 204, 216, 230, 238, 248, 260, 272, 294, 296. 310, 322, 340, 352, 354, 360, 364, 366, 368, 370, 372, 374. 376, or 380, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 93% sequence identity to any one of SEQ ID NO: 16, 78, 90, 106, 114, 130, 140, 150, 162, 172, 188, 194, 204, 216, 230, 238, 248, 260, 272, 294, 296, 310, 322, 340, 352, 354, 360, 364, 366, 368. 370, 372, 374, 376, or 380, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., aTE polypeptide) comprising an amino acid sequence having at least 94% sequence identity to any one of SEQ ID NO: 16, 78, 90, 106, 114, 130, 140, 150, 162, 172, 188, 194, 204, 216, 230, 238, 248, 260, 272, 294, 296. 310, 322, 340, 352, 354. 360, 364, 366, 368, 370, 372, 374. 376, or 380, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NO: 16, 78, 90, 106, 114, 130, 140, 150, 162, 172, 188. 194, 204, 216, 230, 238, 248, 260, 272, 294, 296, 310, 322, 340, 352, 354, 360, 364, 366, 368. 370, 372, 374. 376, or 380, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., aTE polypeptide) comprising an amino acid sequence having at least 96% sequence identity to any one of SEQ ID NO: 16, 78, 90, 106, 114, 130, 140, 150, 162, 172, 188, 194, 204, 216, 230, 238, 248, 260, 272, 294, 296. 310, 322, 340, 352, 354. 360, 364, 366, 368, 370, 372, 374. 376, or 380, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 97% sequence identity to any one of SEQ ID NO: 16, 78, 90, 106, 114, 130, 140, 150, 162,172, 188. 194, 204, 216. 230, 238. 248, 260, 272. 294, 296. 310, 322. 340, 352, 354. 360, 364.366, 368, 370, 372, 374, 376, or 380, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., aTE polypeptide) comprising an amino acid sequence having at least 98% sequence identity to any one of SEQ ID NO: 16, 78, 90, 106, 114, 130. 140, 150, 162, 172, 188. 194, 204. 216, 230, 238, 248, 260. 272, 294, 296, 310, 322, 340, 352, 354, 360, 364, 366, 368, 370, 372, 374, 376, or 380, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 99% sequence identity to any one of SEQ ID NO: 16, 78, 90, 106. 114, 130, 140, 150, 162.172, 188, 194, 204, 216, 230, 238, 248, 260, 272, 294, 296, 310, 322, 340, 352, 354, 360, 364, 366, 368, 370, 372, 374, 376, or 380, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2.

[0161] In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising SEQ ID NO: 16 and any one of even numbered SEQ ID NO: 78-376. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising any one of even numbered SEQ ID NO: 78-376. In some embodiments, provided herein is a polypeptide (e.g., aTE polypeptide) comprising any one of SEQ ID NO: 16, 78, 90, 106, 114, 130, 140, 150.162, 172. 188, 194, 204, 216, 230. 238, 248, 260, 272, 294, 296, 310. 322, 340, 352, 354, 360.364, 366, 368, 370, 372, 374, 376, or 380. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising any one of SEQ ID NO: 78, 90, 106, 114, 130, 140, 150, 162, 172, 188, 194, 204, 216, 230, 238, 248, 260, 272, 294, 296, 310, 322, 340, 352, 354, 360. 364, 366, 368, 370, 372. 374, or 376. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) consisting essentially of the amino acid sequence of any one of SEQ ID NO: 16, 78, 90, 106, 114, 130, 140, 150, 162, 172, 188, 194, 204, 216, 230, 238, 248, 260, 272, 294, 296, 310, 322, 340, 352, 354, 360, 364, 366, 368, 370, 372, 374, 376, or 380. In some embodiments, provided herein is a polypeptide (e g., a TE polypeptide) consisting of any one of SEQ ID NO: 16, 78, 90, 106. 114, 130. 140, 150, 162, 172, 188. 194, 204. 216, 230, 238.248, 260, 272, 294, 296, 310, 322, 340, 352, 354, 360, 364, 366, 368, 370, 372, 374, 376, or 380.

[0162] In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%. 90%, 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or higher sequence identity to SEQ ID NO: 340, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 340. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 80% sequence identity' to SEQ ID NO: 340, wherein thepolypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 340, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 98% sequence identity’ to SEQ ID NO: 340, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 340, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising the amino acid sequence of SEQ ID NO: 340. Also provided herein is a polypeptide (e.g., a TE polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 340. Also provided herein is a polypeptide (e.g., a TE polypeptide) consisting of the amino acid sequence of SEQ ID NO: 340.

[0163] In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 352, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 352, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 352, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 352, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 99% sequence identity’ to SEQ ID NO: 352, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising the amino acid sequence of SEQ ID NO: 352. Also provided herein is a polypeptide (e.g., a TE polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 352. Also provided herein is a polypeptide (e.g., a TE polypeptide) consisting of the amino acid sequence of SEQ ID NO: 352.

[0164] In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or higher sequence identity to SEQ ID NO: 354, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 354, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e g., a TE polypeptide) comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 354, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 354, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 354, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising the amino acid sequence of SEQ ID NO: 354. Also provided herein is a polypeptide (e.g., a TE polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 354. Also provided herein is a polypeptide (e.g., a TE polypeptide) consisting of the amino acid sequence of SEQ ID NO: 354.

[0165] In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 360, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 360. wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 360, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 360, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 99% sequence identity’ to SEQ ID NO: 360, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising the amino acid sequence of SEQ ID NO: 360. Also provided herein is a polypeptide (e.g., a TE polypeptide) consisting essentially ofthe amino acid sequence of SEQ ID NO: 360. Also provided herein is a polypeptide (e.g., a TE polypeptide) consisting of the amino acid sequence of SEQ ID NO: 360.

[0166] In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or higher sequence identity to SEQ ID NO: 364, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 364, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 364, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 364, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 364, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising the amino acid sequence of SEQ ID NO: 364. Also provided herein is a polypeptide (e.g., a TE polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 364. Also provided herein is a polypeptide (e.g., a TE polypeptide) consisting of the amino acid sequence of SEQ ID NO: 364.

[0167] In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 366, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 366, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 366, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 366, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an aminoacid sequence having at least 99% sequence identity to SEQ ID NO: 364, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising the amino acid sequence of SEQ ID NO: 366. Also provided herein is a polypeptide (e.g., a TE polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 366. Also provided herein is a polypeptide (e.g., a TE polypeptide) consisting of the amino acid sequence of SEQ ID NO: 366.

[0168] In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%. 90%, 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or higher sequence identity to SEQ ID NO: 368, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 368, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 368, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 368, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 368, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising the amino acid sequence of SEQ ID NO: 368. Also provided herein is a polypeptide (e.g., a TE polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 368. Also provided herein is a polypeptide (e g., a TE polypeptide) consisting of the amino acid sequence of SEQ ID NO: 368.

[0169] In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 370, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 370, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 370, wherein the polypeptide does not comprise thesequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 370, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 99% sequence identity’ to SEQ ID NO: 370, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising the amino acid sequence of SEQ ID NO: 370. Also provided herein is a polypeptide (e.g., a TE polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 370. Also provided herein is a polypeptide (e.g., a TE polypeptide) consisting of the amino acid sequence of SEQ ID NO: 370.

[0170] In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%. 97%. 98%. 99%. or higher sequence identity to SEQ ID NO: 372, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 372, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 372, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 372, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 372, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising the amino acid sequence of SEQ ID NO: 372. Also provided herein is a polypeptide (e.g., a TE polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 372. Also provided herein is a polypeptide (e g., a TE polypeptide) consisting of the amino acid sequence of SEQ ID NO: 372.

[0171] In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 374, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising anamino acid sequence having at least 80% sequence identity to SEQ ID NO: 374. wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 374, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 374, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 99% sequence identity’ to SEQ ID NO: 374. wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising the amino acid sequence of SEQ ID NO: 374. Also provided herein is a polypeptide (e.g., a TE polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 374. Also provided herein is a polypeptide (e g., a TE polypeptide) consisting of the amino acid sequence of SEQ ID NO: 374.

[0172] In some embodiments, provided herein is a polypeptide (e g., a TE polypeptide) comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 376, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 376, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 376, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 376, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 376, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide) comprising the amino acid sequence of SEQ ID NO: 376. Also provided herein is a polypeptide (e.g., a TE polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 376. Also provided herein is a polypeptide (e.g., a TE polypeptide) consisting of the amino acid sequence of SEQ ID NO: 376.

[0173] In some embodiments, provided herein is a polypeptide (e.g., a TE polypeptide), wherein the polypeptides 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: 381). In some embodiments, the polypeptide comprises an epitope tag at the C-terminus. In some embodiments, the polypeptide comprises a His tag at the C-terminus. In some embodiments, the polypeptide comprises a His tag comprising of SEQ ID NO: 381 at the C-terminus. In some embodiments, the polypeptide consisting of a His tag comprising of SEQ ID NO: 381 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 His tag and the linker comprise the amino acid sequence of GSHHHHHH (SEQ ID NO: 382).

[0174] For example, a TE 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: 4-30 and 78-376 may be modified to no longer include a C-terminal His tag having the amino acid sequence of SEQ ID NO: 381 or SEQ ID NO: 382. For example, the polypeptide sequence of any one of even numbered SEQ ID NO: 32-76, 378, and 380 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: 381 or SEQ ID NO: 382.

[0175] In some embodiments, the amino acid residues used to determine the percent identity listed above includes the sequence of a hexahistadine tag (SEQ ID NO: 381) (e.g., including GSHHHHHH (SEQ ID NO: 382)).

[0176] In some embodiments, the amino acid residues used to determine the percent identity listed above does not include the sequence of a hexahistadine tag (SEQ ID NO: 381) (e.g., does not include GSHHHHHH (SEQ ID NO: 382)).

[0177] In some embodiments, the polypeptide is isolated.

[0178] In some embodiments, the polypeptide is capable of catalyzing an amide bond formation.

[0179] 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.

[0180] In addition to the positions of residue differences specified above, any of the engineered TE polypeptides disclosed herein can further comprise other residue differences relative to any of SEQ ID NO: 2, 4, 6, 16, 52, 378, or 380 at other residue positions than those of amino acid differences disclosed above and in the Examples. 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 reactions. Accordingly, in some embodiments, in addition to the amino acid residue differences present in any one of the engineered TE polypeptides selected from even numbered SEQ ID NO: 4-380, 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, 4, 6, 16, 52, 378, or 380. In some embodiments, the number of amino acid residue differences as compared to the reference sequence can be at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45 or 50 residue positions. In some embodiments, the number of amino acid residue differences as compared to 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 and / or non-conservative changes.

[0181] In some embodiments, the engineered TE polypeptide can comprise a deletion at one or more amino acid positions as compared to any one of even numbered SEQ ID NO: 4-380. Thus, for each embodiment of the engineered TE polypeptides of the disclosure, the amino acid sequence can comprise a deletion of one or more ammo acids, 2 or more amino acids, 3 or more amino acids, 4 or more amino acids, 5 or more amino acids, 6 or more amino acids, 8 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, up to 15% of the total number of amino acids, up to 20% of the total number of amino acids, or up to 30% of the total number of amino acids of the TE polypeptides, where the associated functional activity and / or improved properties of the engineered TE described herein is maintained. In some embodiments, the number of deletions can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50 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 ammo acid residues.

[0182] In some embodiments, the engineered TE polypeptides provided herein can comprise an insertion as compared to any one of the engineered TE polypeptides described herein, such as theexemplary engineered polypeptides of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16. 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 90, 106, 114, 130, 140, 150, 162, 172, 188, 194, 204, 216, 230, 238, 248, 260, 272, 294, 296, 310, 322, 340. 352, 354, 360, 364, 366, 368, 370, 372, 374, 376, 378, or 380. 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 TE described herein is maintained. The insertions can be to amino or carboxy terminus, or internal portions of the TE polypeptide.

[0183] In some embodiments, the engineered TE polypeptide herein can have an amino acid sequence comprising a sequence selected from SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48. 50. 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76. 78, 90, 106, 114. 130, 140. 150, 162, 172, 188, 194. 204, 216, 230, 238, 248. 260, 272. 294, 296, 310, 322, 340, 352, 354, 360, 364, 366, 368, 370, 372, 374, 376, 378, or 380, and optionally one or several (e.g., up to 3, 4, 5, or up to 10) amino acid residue deletions, insertions and / or substitutions. In some embodiments, the amino acid sequence has optionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8. 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, or 1-50 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the number of amino acid sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the amino acid sequence has optionally 1. 2, 3, 4, 5, 6, 7, 8.9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the substitutions can be conservative or nonconservative substitutions.

[0184] Also provided herein are compositions comprising any of the polypeptides disclosed herein. For example, the composition may include an effective amount of the polypeptide for catalyzing an amide bond formation. In some embodiments, the composition may include an effective amount of the polypeptide for catalyzing amide bond formation in macrocyclic and semi-macrocyclic peptides. The composition may include one or more carriers or diluents.

[0185] In some embodiments, the polypeptides of the disclosure can be in the form of fusion polypeptides in which the engineered polypeptides are fused to other polypeptides, such as, by way of example and not limitation, antibody tags (e.g., myc epitope), purification sequences (e.g., His tags for binding to metals), and cell localization signals (e.g., secretion signals). Thus,the engineered polypeptides described herein can be used with or without fusions to other polypeptides.

[0186] 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.

[0187] 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.

[0188] In some embodiments, the engineered TE polypeptides of the present disclosure can be immobilized on a solid support such that they retain their improved activity’, stereoselectivity’, and / or other improved properties relative to the reference engineered polypeptide. In some embodiments, the immobilized polypeptides can be retained after a desired reaction (e.g., by’ retaining beads on which polypeptide is immobilized) and then reused or recycled in subsequent reactions. Such immobilized enzyme processes allow for further efficiency and cost reduction. Accordingly, it is further contemplated that any of the methods of using the TE polypeptides of the present disclosure can be carried out using the same TE polypeptides bound or immobilized on a solid support.

[0189] In some embodiments, the polypeptides described herein can be provided in the form of kits. The enzymes in the kits may be present individually or as a plurality of enzymes. The kits can further include reagents for carrying out the enzymatic reactions, substrates for assessing the activity of enzymes, as well as reagents for detecting the products. The kits can also include reagent dispensers and instructions for use of the kits.

[0190] In some embodiments, the kits of the present disclosure include arrays comprising a plurality of different TE 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 poly peptides immobilized onsolid 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.Polynucleotides Encoding TE Polypeptides

[0191] In another aspect, the present disclosure provides polynucleotides encoding the polypeptides (e.g., TE 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., TE polypeptide) can be introduced into appropriate host cells to express the corresponding polypeptide.

[0192] 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 ammo 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., TE 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.

[0193] In various embodiments, the codons are preferably selected to be suitable for the host cell in which the polypeptide is being produced. For example, preferred codons used in bacteria are used to express the gene in bacteria; preferred codons used in yeast are used for expression in yeast; and preferred codons used in mammals are used for expression in mammalian cells. By way of example, the polynucleotide of SEQ ID NO: 3 provided herein has been codon optimized for expression in E. coli.

[0194] In certain embodiments, all codons need not be replaced to optimize the codon usage of the polypeptide (e.g., TE 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., TE 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.

[0195] 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 odd numbered SEQ ID NO: 1-379, wherein the polynucleotide does not comprise SEQ ID NO: 1. 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 odd numbered SEQ ID NO: 3-379, wherein the polynucleotide does not comprise SEQ ID NO: 1. In some embodiments, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the polynucleotide sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 89, 105, 113, 129, 139, 149, 161, 171. 187, 193, 203, 215, 229, 237, 247, 259, 271, 293, 295, 309. 321, 339, 351, 353, 359, 363, 365, 367, 369, 371, 373, 375, 377, or 379, wherein the polynucleotide does not comprise SEQ ID NO: 1.

[0196] 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 odd numbered SEQ ID NO: 1-75, wherein the polynucleotide does not comprise SEQ ID NO: 1. In some embodiments, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the polynucleotide sequence of SEQ ID NO: 3, 5. 7, 9, 11. 13. 15. 17, 19, 21, 23, 25, 27, 29, 31, 33, 35. 37. 39. 41. 43. 45. 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 377, or 379, wherein the polynucleotide does not comprise SEQ ID NO: 1.

[0197] 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 NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 377, or 379, wherein the polynucleotide does not comprise SEQ ID NO: 1. In some embodiments, provided herein is a polynucleotide comprising at least 80% sequence identity to the polynucleotide sequence of any one of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 377, or 379, wherein the polynucleotide does notcomprise SEQ ID NO: 1. In some embodiments, provided herein is a polynucleotide comprising at least 90% sequence identity to the polynucleotide sequence of any one of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 377, or 379, wherein the polynucleotide does not comprise SEQ ID NO: 1. In some embodiments, provided herein is a polynucleotide comprising at least 91% sequence identity to the polynucleotide sequence of any one of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 377, or 379, wherein the polynucleotide does not comprise SEQ ID NO: 1. In some embodiments, provided herein is a polynucleotide comprising at least 92% sequence identity to the polynucleotide sequence of any one of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 377, or 379, wherein the polynucleotide does not comprise SEQ ID NO: 1. In some embodiments, provided herein is a polynucleotide comprising at least 93% sequence identity to the polynucleotide sequence of any one of SEQ ID NO: 3, 5, 7, 9, 11. 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 377, or 379, wherein the polynucleotide does not comprise SEQ ID NO: 1. In some embodiments, provided herein is a polynucleotide comprising at least 94% sequence identity to the polynucleotide sequence of any one of SEQ ID NO: 3, 5, 7, 9, 11. 13. 15. 17. 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 377, or 379, wherein the polynucleotide does not comprise SEQ ID NO: 1. In some embodiments, provided herein is a polynucleotide comprising at least 95% sequence identity to the polynucleotide sequence of any one of SEQ ID NO: 3, 5, 7, 9, 11. 13. 15. 17, 19, 21, 23, 25, 27. 29. 31. 33, 35, 37, 39, 41, 43, 45, 47, 49, 51. 53. 55. 57. 59. 61. 63, 65, 67, 69, 71, 73, 75, 377, or 379, wherein the polynucleotide does not comprise SEQ ID NO: 1. In some embodiments, provided herein is a polynucleotide comprising at least 96% sequence identity to the polynucleotide sequence of any one of SEQ ID NO: 3, 5, 7, 9, 11. 13, 15, 17, 19, 21, 23, 25, 27. 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51. 53. 55. 57. 59. 61, 63, 65, 67, 69, 71, 73, 75, 377, or 379, wherein the polynucleotide does not comprise SEQ ID NO: 1. In some embodiments, provided herein is a polynucleotide comprising at least 97% sequence identity' to the polynucleotide sequence of any one of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51. 53. 55. 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 377, or 379, wherein the polynucleotide does not comprise SEQ ID NO: 1. In some embodiments, provided herein is a polynucleotide comprising at least 98% sequence identity' to the polynucleotide sequence of any one of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27. 29. 31. 33, 35, 37, 39, 41, 43, 45, 47, 49, 51. 53. 55. 57. 59. 61. 63, 65, 67, 69, 71, 73, 75, 377, or 379, wherein the polynucleotide does not comprise SEQ ID NO: 1. In some embodiments, provided herein is a polynucleotide comprising at least 99% sequence identity to the polynucleotide sequence of any one of SEQ ID NO: 3, 5, 7, 9, 11. 13, 15, 17, 19, 21, 23, 25, 27. 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51. 53. 55. 57. 59. 61, 63, 65, 67, 69, 71, 73, 75, 377, or 379, wherein the polynucleotide does not comprise SEQ ID NO: 1. In some embodiments, provided herein is a polynucleotide comprising any one of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 377. or 379. In some embodiments, provided herein is a polynucleotide consisting of any one ofSEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 377, or 379.

[0198] 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 odd numbered SEQ ID NO: 1-15 and 77-379, wherein the polynucleotide does not comprise SEQ ID NO: 1. 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 odd numbered SEQ ID NO: 77-375, wherein the polynucleotide does not comprise SEQ ID NO: 1. In some embodiments, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the polynucleotide sequence of SEQ ID NO: 15, 77, 89, 105, 113, 129. 139, 149, 161. 171, 187. 193, 203, 215. 229, 237. 247, 259. 271, 293, 295. 309, 321.339, 351, 353, 359, 363, 365, 367, 369, 371, 373, 375, or 379, wherein the polynucleotide does not comprise SEQ ID NO: 1.

[0199] 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 NO: 15, 77, 89, 105, 113, 129, 139, 149, 161, 171, 187, 193, 203, 215, 229, 237, 247, 259, 271, 293, 295, 309, 321, 339, 351, 353, 359, 363, 365, 367, 369, 371, 373, 375, or 379, wherein the polynucleotide does not comprise SEQ ID NO: 1. In some embodiments, provided herein is a polynucleotide comprising at least 80% sequence identity to the polynucleotide sequence of any one of SEQ ID NO: 15, 77, 89, 105, 113, 129, 139, 149, 161, 171, 187, 193, 203, 215, 229, 237, 247, 259, 271, 293, 295, 309, 321, 339, 351, 353, 359, 363, 365, 367, 369,371, 373, 375, or 379, wherein the polynucleotide does not comprise SEQ ID NO: 1. In some embodiments, provided herein is a polynucleotide comprising at least 90% sequence identity to the polynucleotide sequence of any one of SEQ ID NO: 15, 77, 89, 105, 113, 129, 139, 149, 161, 171, 187. 193, 203, 215, 229, 237, 247, 259, 271, 293, 295, 309, 321. 339, 351, 353, 359, 363, 365, 367. 369, 371, 373. 375, or 379, wherein the polynucleotide does not comprise SEQ ID NO: 1. In some embodiments, provided herein is a polynucleotide comprising at least 91% sequence identity7to the polynucleotide sequence of any one of SEQ ID NO: 15, 77, 89, 105, 113, 129, 139, 149, 161, 171, 187, 193, 203, 215, 229, 237, 247, 259, 271, 293, 295, 309, 321, 339, 351, 353, 359. 363, 365, 367, 369, 371. 373, 375, or 379, wherein the polynucleotide does not comprise SEQ ID NO: 1. In some embodiments, provided herein is a polynucleotide comprising at least 92% sequence identity to the polynucleotide sequence of any one of SEQ ID NO: 15, 77, 89, 105, 113, 129, 139, 149, 161, 171, 187, 193, 203, 215, 229, 237, 247, 259, 271, 293, 295, 309, 321. 339, 351, 353, 359, 363, 365, 367, 369, 371, 373, 375, or 379, wherein the polynucleotide does not comprise SEQ ID NO: 1. In some embodiments, provided herein is a polynucleotide comprising at least 93% sequence identity to the polynucleotide sequence of any one of SEQ ID NO: 15, 77, 89, 105, 113, 129, 139, 149, 161, 171, 187, 193, 203, 215, 229, 237, 247, 259, 271, 293, 295, 309, 321, 339, 351, 353, 359, 363, 365, 367, 369, 371, 373, 375, or 379, wherein the polynucleotide does not comprise SEQ ID NO: 1. In some embodiments, provided herein is a polynucleotide comprising at least 94% sequence identity to the polynucleotide sequence of any one of SEQ ID NO: 15, 77, 89, 105, 113, 129, 139, 149, 161, 171, 187, 193, 203, 215, 229, 237, 247, 259, 271, 293, 295, 309, 321, 339, 351, 353, 359, 363, 365, 367, 369, 371, 373. 375, or 379, wherein the polynucleotide does not comprise SEQ ID NO: 1. In some embodiments, provided herein is a polynucleotide comprising at least 95% sequence identity to the polynucleotide sequence of any one of SEQ ID NO: 15, 77, 89, 105, 113, 129, 139, 149, 161, 171, 187, 193, 203, 215, 229, 237, 247, 259, 271, 293, 295, 309, 321, 339, 351, 353, 359, 363, 365, 367, 369, 371, 373, 375, or 379, wherein the polynucleotide does not comprise SEQ ID NO: 1. In some embodiments, provided herein is a polynucleotide comprising at least 96% sequence identity’ to the polynucleotide sequence of any one of SEQ ID NO: 15, 77, 89, 105, 113, 129, 139, 149, 161, 171, 187, 193, 203, 215, 229, 237, 247, 259, 271, 293, 295, 309, 321, 339, 351, 353, 359, 363, 365, 367, 369, 371, 373, 375, or 379, wherein the polynucleotide does not comprise SEQ ID NO: 1. In some embodiments, provided herein is a polynucleotide comprising at least 97% sequence identity to the polynucleotide sequence of any one of SEQ ID NO: 15, 77, 89, 105, 113, 129, 139, 149, 161, 171, 187, 193, 203, 215, 229, 237, 247, 259, 271, 293, 295, 309, 321, 339, 351, 353, 359, 363, 365, 367, 369, 371, 373, 375, or 379, wherein thepolynucleotide does not comprise SEQ ID NO: 1. In some embodiments, provided herein is a polynucleotide comprising at least 98% sequence identity to the polynucleotide sequence of any one of SEQ ID NO: 15, 77, 89, 105, 113, 129, 139, 149, 161, 171, 187, 193, 203, 215, 229, 237, 247, 259. 271, 293, 295, 309, 321, 339, 351, 353, 359, 363, 365, 367. 369, 371, 373, 375, or 379, wherein the polynucleotide does not comprise SEQ ID NO: 1. In some embodiments, provided herein is a polynucleotide comprising at least 99% sequence identity to the polynucleotide sequence of any one of SEQ ID NO: 15, 77, 89, 105, 113, 129, 139, 149, 161, 171, 187, 193, 203, 215, 229, 237, 247, 259, 271, 293, 295, 309, 321, 339, 351, 353, 359, 363, 365, 367, 369, 371, 373. 375, or 379, wherein the polynucleotide does not comprise SEQ ID NO: 1. In some embodiments, provided herein is a polynucleotide comprising any one of SEQ ID NO: 15, 77, 89, 105, 113, 129, 139, 149, 161, 171, 187, 193, 203, 215, 229, 237, 247, 259, 271, 293, 295, 309, 321, 339, 351, 353, 359, 363, 365, 367, 369, 371, 373, 375, or 379. In some embodiments, provided herein is a polynucleotide consisting of any one of SEQ ID NO: 15. 77. 89, 105, 113, 129, 139. 149, 161, 171. 187, 193. 203, 215, 229. 237, 247. 259, 271. 293, 295, 309. 321, 339.351, 353, 359, 363, 365, 367, 369, 371, 373, 375, or 379.

[0200] In some embodiments, the polynucleotide sequence of any of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 77, 89, 105, 113, 129, 139, 149. 161, 171, 187, 193, 203, 215, 229, 237. 247, 259, 271, 293, 295. 309, 321, 339, 351, 353, or 359, may be modified to no longer code for a C-terminal His tag. 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.

[0201] In various embodiments, an isolated polynucleotide encoding polypeptide (e.g.. a TE 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 know n 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 Biology7, Ausubel. F. ed., Greene Pub. Associates, 1998, updates to 2006.

[0202] 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 maybe desirable or necessary depending on the expression vector utilized. Techniques for modifying polynucleotides and nucleic acid sequences utilizing recombinant DNA methods are well known in the art.

[0203] 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.

[0204] 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 stearothermophihis 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, 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 Eusarium 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 known in the art (see e.g., Romanos et al., Yeast 8:423-488. 1992).

[0205] 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 acidsequence encoding the enzyme polypeptide. Any suitable terminator that is functional in the host cell of choice finds use in the present disclosure. Exemplary transcription terminators for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus 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).

[0206] 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-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0207] 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. Exemplary 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 Aspergi llus 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).

[0208] 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 encodesthe secreted polypeptide. Alternatively, in some embodiments, the 5' end of the coding sequence contains a signal peptide coding region that is foreign to the coding sequence. Any suitable signal peptide coding region that directs the expressed polypeptide into the secretory pathway of a host cell of choice finds use for expression of the engineered polypeptide(s). Effective signal peptide coding regions for bacterial host cells are the signal peptide coding regions include, but are not limited to, those obtained from the genes for Bacillus NC1B 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis betalactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus sub tills 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.

[0209] In some embodiments, regulatory 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.

[0210] In another aspect, the present disclosure provides a recombinant expression vector comprising a polynucleotide encoding a polypeptide (e.g., a TE polypeptide), and one or more expression regulating regions such as a promoter, a terminator, a replication origin, a leader sequence, a signal peptide, or a regulatory sequence, depending on the type of host into which it is to be introduced. In some embodiments, one or more nucleic acid and control sequences as described herein are joined together to produce recombinant expression vectors that include one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the enzyme polypeptide at such sites. Alternatively, in some embodiments, a nucleic acid sequence of the present disclosure is expressed by inserting the nucleic acidsequence 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.

[0211] The recombinant expression vector may be any suitable vector (e.g., a plasmid or virus), that can be subjected to recombinant DNA procedures and bring about the expression of the enzyme polynucleotide sequence. The choice of the vector typically depends on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

[0212] In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, such as a plasmid, an extra-chromosomal element, a minichromosome, or an artificial chromosome). The vector may contain any means for assuring self-replication. In some embodiments, the vector, when introduced into the host cell, is integrated into the genome of the host cell and replicates together with the chromosome(s) into which it has been integrated. Furthermore, in some embodiments, a single vector or plasmid, or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, and / or a transposon is utilized.

[0213] 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, thec / a / 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.

[0214] 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 thepolynucleotide(s) or expression vector(s) of the present disclosure are well known in the art and described herein.

[0215] 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.

[0216] 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.

[0217] In some embodiments, the expression vectors contain additional nucleic acid sequences for directing integration by homologous recombination into the genome of the host cell. The additional nucleic acid sequences enable the vector to be integrated into the host cell genome at a precise location(s) in the chromosome(s) of the host cell. To increase the likelihood of integration at a precise location, the integrational elements preferably contain a sufficient number of nucleotides, such as 100 to 10.000 base pairs, preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000 base pairs, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may include any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding nucleic acid sequences. In other examples, the vector may be integrated into the genome of the host cell by non-homologous recombination.

[0218] For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. Examples of bacterial origins of replication are P15A ori or the origins of replication of plasmids pBR322, pUC19. pET30a(+), pACYC177 (which contains the P15A ori), or pACYC184 (which contains the P15A ori) permitting replication in E. coli, and pUBl 10, pE194, or pTA1060 permitting replication in Bacillus. Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication. ARS1, ARS4, the combination of ARS 1 and CEN3, and the combination of ARS4 and CEN6. The origin of replication may be one having a mutation which makes its functioning temperature-sensitive in the host cell (See e.g., Ehrlich, Proc. Natl. Acad. Sci. USA 75:1433, 1978).

[0219] 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.

[0220] Many of the expression vectors for use in the present disclosure are commercially available. Suitable commercial expression vectors include, but are not limited to. NOVAGEN® pET E. coli T7 expression vectors, such as pET30a(+) vector (Millipore Sigma) and the p3xFLAG® expression vectors (Sigma- Aldrich Chemicals). Other suitable expression vectors include, but are not limited to, pBluescriptll SK(-) and pBK-CMV (Stratagene), and plasmids derived from pBR322 (Gibco BRL), pUC (Gibco BRL), pREP4, pCEP4 (Invitrogen) or pPoly (See e.g., Lathe et al., Gene 57:193-201, 1987).

[0221] Thus, in some embodiments, a vector comprising a sequence encoding at least one variant polypeptide (e.g., a TE polypeptide) is transformed into a host cell in order to allow propagation of the vector and expression of the variant polypeptide(s). In some embodiments, the transformed host cell described above is cultured in a suitable nutrient medium under conditions permitting the expression of the variant polypeptides(s). Any suitable medium useful for culturing the host cells finds use in the present disclosure, including, but not limited to minimal or complex media containing appropriate supplements. In some embodiments, host cells are 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 TE Polypeptides

[0222] The present disclosure also provides a host cell comprising a polynucleotide encoding a polypeptide (e.g., a TE polypeptide) disclosed herein, or an expression vector comprising a polynucleotide encoding a polypeptide (e.g., a TE 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.

[0223] Host cells for use in expressing the polypeptides described herein are well known in the art and include, but are not limited to, prokaryotic cells (e.g., bacterial cells (e.g., E. coli, B.subtilis, B. licheniformis, B. megaterium, B. stearothermophilus, B. amyloliquefaciens.Lactobacillus 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 grow th conditions for the above-described host cells are well known in the art.

[0224] Polynucleotides for expression of the polypeptides (e.g., TE 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.

[0225] In some embodiments, the host cell is a filamentous fungal host cell. For example, in some embodiments, the filamentous fungal host cells are of any suitable genus and species, including, but not limited to Achlya, Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Cephalosporium, Chrysosporium, Cochliobolus, Corynascus, Cryphonectria, Cryptococcus, Coprinus, Coriolus, Diplodia, Endothis, Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea, Myceliophthora, Mucor, Neurospora, Penicillium, Podospora, Phlebia, Piromyces, Pyricularia, Rhizomucor, Rhizopus, Schizophyllum, Scytalidium, Sporotrichum, Talaromyces, Thermoascus, Thielavia. Trametes, Tolypocladium. Trichoderma, Verticillium, and / or Volvariella, and / or teleomorphs, or anamorphs, and synonyms, basionyms, or taxonomic equivalents thereof.

[0226] 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 carlsbergensis, Saccharomyces diastaticus, Saccharomyces norbensis, Saccharomyces kluyveri, Schizosaccharomyces pombe, Pichia pastoris, Pichia finlandica, 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.

[0227] 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, Methylobactenum. Methylobacterium. Mycobacterium, Neisseria, Pantoea, Pseudomonas. Prochlorococcus. Rhodobacter, Rhodopseudomonas, Rhodopseudomonas, Roseburia, Rhodospirillum, Rhodococcus, Scenedesmus, Streptomyces, Streptococcus, Synecoccus, Saccharomonospora. Staphylococcus, Serratia, Salmonella. Shigella, Thermoanaerobacterium, Tropheryma, Tularensis, Temecula, Thermosynechococcus, Thermococcus. Ureaplasma.Xanthomonas, Xylella. Yersinia and Zymomonas. In some embodiments, the host cell is a species of Agrobacterium, Acinetobacter, Azobacter, Bacillus, Bifidobacterium, Buchnera, Geobacillus, Campylobacter, Clostridium, Corynebacterium, Escherichia, Enterococcus, Erwinia, Flavobacterium, Lactobacillus, Lactococcus, Pantoea, Pseudomonas, Staphylococcus.Salmonella, Streptococcus, Streptomyces, or Zymomonas. In some embodiments, the bacterial host strain is non-pathogenic to humans. In some embodiments the bacterial host strain is an industrial strain. Numerous bacterial industrial strains are known and suitable in the present disclosure. In some embodiments of the present disclosure, the bacterial host cell is an Agrobacterium species (e.g., A. radiobacter, A. rhizogenes, andd. 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. paraffineus, 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. pumilus, B. lautus, B.coagulans, B. brevis, B. firmus. B. alkaophius, B. licheniformis, B. clausii, B. stearothermophilus, B. halodurans, or B. amyloliquefaciens). In some embodiments, the host cell is an industrial Bacillus strain including but not limited to B. subtilis, B. pumilus, B. licheniformis, B. megaterium, B. clausii, B. stearothermophilus, or B. amyloliquefaciens. In some embodiments, the Bacillus host cells are B. subtilis. B. licheniformis, B. megaterium. B. stearothermophilus. and / or B. amyloliquefaciens. In some embodiments, the bacterial host cell is aClostridium species (e.g., C. acetobutylicum, C. tetani E88, C. lituseburense, C. saccharobutylicum, C. perfringens, and C. beijerinckii). In some embodiments, the bacterial host cell is a Corynebacterium species (e.g., C. glutamicum and C. acetoacidophilum). In some embodiments the bacterial host cell is an Escherichia species (e.g., E. coli). In some embodiments, the host cell is Escherichia coli W3110. In some embodiments the host is Escherichia coli BL21 or BL21(DE3). In some embodiments, the bacterial host cell isan Erwinia species (e.g.. E. uredovora, E. carotovora, E. ananas, E. herbicola, E. punctata, and E. terreus). In some embodiments, the bacterial host cell is aPantoea species (e g., P. citrea or P. agglomerans). In some embodiments the bacterial host cell is a Pseudomonas species (e.g., P. putida, P. aeruginosa, P. mevalonii, and P. sp. D-01 10). In some embodiments, the bacterial host cell is a Streptococcus species (e.g., S. equisimiles, S. pyogenes, and < S’. uberis). In some embodiments, the bacterial host cell is a Streptomyces species (e.g., S. ambofaciens, S. achromogenes, S. avermitilis, S. coelicolor, S. aureofaciens, S. aureus, S. fungicidicus, S. griseus, or S. lividans). In some embodiments, the bacterial host cell is aZymomonas species (e.g., Z. mobilis, or Z. lipolytica').

[0228] 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).

[0229] 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 TE 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 variety of 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 etal.. EMS Microbiol. Lett.. 220:141-8, 2003; and Firon et al.. Eukary. Cell 2:247-55, 2003, both of which are incorporated by reference).

[0230] 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.

[0231] In some embodiments, the present disclosure provides a method of producing a polypeptide, the method comprising culturing a host cell comprising at least one polynucleotide encoding at least one polypeptide of the present disclosure under conditions such that the polypeptide encoded by the polynucleotide is produced. In some embodiments, the present disclosure provides a method of producing a polypeptide, the method comprising culturing a host cell comprising at least one polynucleotide encoding at least one polypeptide of the present disclosure under conditions such that the polypeptide encoded by the polynucleotide is produced, further comprising the step of recovering the polypeptide. In some embodiments, the present disclosure provides a method of producing a polypeptide, the method comprising culturing a host cell comprising at least one polynucleotide encoding at least one polypeptide of the present disclosure under conditions such that the polypeptide encoded by the polynucleotide is produced, further comprising the step of recovering the polypeptide, and further comprising the step of purifying the polypeptide.

[0232] 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.

[0233] In some embodiments, cells expressing a TE 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-batchfermentations are common and well known in the art. Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth. Continuous fermentation systems strive to maintain steady state growth conditions. Methods for modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology.

[0234] 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.

[0235] In some embodiments, cell-free transcription and translation systems find use in producing the polypeptide(s) (e.g., TE polypeptide(s)). Several sy stems are commercially available, and the methods are well-known to those skilled in the art.Methods of Evolving TEs

[0236] Evolution (e.g., directed evolution) may be used to identify polypeptides (e.g., TE polypeptides) of the present disclosure. For example, in some embodiments, to make aTE polypeptide of the present disclosure, a TE polypeptide may be obtained (or denved) from any suitable bacterial species, e g., Brevibacullus laterosporus. In some embodiments, the parent polynucleotide sequence is codon optimized to enhance expression of the TE polypeptide in a specified host cell (e.g., E. coli). For example, as is described in Example 1, a parental polynucleotide sequence, designated as SEQ ID NO: 1, which encodes SEQ ID NO: 2. was codon optimized for expression in E. coli, additional changes were made (e.g., substitution S82C, and C-terminal hexahistadine tag (SEQ ID NO: 381) with linker was added), and the resulting polynucleotide (SEQ ID NO: 3) was cloned into an a pET30(+) vector, and expressed in E. coli strain BL-21(DE3) under the control of the T7 promoter. Clones expressing the active TE in A. coli may be identified and the genes sequenced to confirm their identify.

[0237] The polypeptides (e.g., TE polypeptides) of the disclosure may be obtained by subjecting a polynucleotide encoding a parent sequence to mutagenesis and / or directed evolutionmethods. 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 used include, among others, staggered extension process (StEP), in vitro recombination (Zhao et al.. 1998. Nat. Biotechnol. 16:258-261), mutagenic polymerase chain reaction (PCR) (Caldwell et al., 1994, PCR Methods Appl.3: S136-S140), and cassette mutagenesis (Black et al., 1996, Proc. Natl. Acad. Sci. USA 93:3525-3529).

[0238] The clones obtained following mutagenesis treatment may be screened for TE 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. For example, TE activity7assays as described in the Examples may be used.

[0239] Where the improved enzy me property desired is thermal stability, enzyme activity may be measured after subjecting the enzyme preparations to a defined temperature and measuring the amount of enzy me activity' remaining after heat treatments. Any suitable approach may be used, e.g., differential scanning colorimetry (DSC) a biochemical assay, or spectroscopy. Clones containing a polynucleotide encoding a TE polypeptide may then be isolated, sequenced to identify the nucleotide sequence changes (if any), and used to express the enzyme in a host cell.

[0240] 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.

[0241] Polypeptides (e.g., TE 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.

[0242] Chromatographic techniques for isolation of the polypeptide (e.g., TE polypeptide) include, among others, reverse phase chromatography high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, and affinity chromatography. Conditions for purifying a particular enzyme will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, and the like, and will be apparent to those having skill in the art.

[0243] In some embodiments, affinity techniques may be used to isolate the improved polypeptides (e.g., TE 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 TE polypeptide may be used.Methods of Using TE Polypeptides

[0244] Also provided herein are methods of using a polypeptide (e.g., a TE polypeptide) of the disclosure. In some examples, a polypeptide (e.g.. a TE polypeptide) of the disclosure is used in a method of catalyzing an amide bond formation. For example, provided herein is a method of catalyzing an amide bond formation in the presence of a polypeptide of the disclosure.

[0245] In some examples, a polypeptide (e.g., a TE polypeptide) of the disclosure is used in a method of catalyzing amide bond formation in macrocyclic and semi-macrocyclic peptides. In some examples, a polypeptide (e.g., a TE polypeptide) of the disclosure is used in a method of catalyzing amide bond formation in macrocyclic and semi-macrocyclic peptide in a protective-group free manner. In some examples, a polypeptide (e.g., a TE polypeptide) of the disclosure is used in a method of catalyzing amide bond formation in macrocyclic and semi-macrocyclic peptide in a protective-group free manner from activated aminoacyl-thioesters, aminoacyl-esters and cyclic peptides. In some examples, a polypeptide (e.g., a TE polypeptide) of the disclosure is used in a method of catalyzing reactions in the process of generating macrocyclic peptides.

[0246] In some examples, a polypeptide (e.g., a TE polypeptide) of the disclosure is used in a method of catalyzing one or more of the reactions shown in Table A and Scheme A in the Examples below.

[0247] In some embodiments, the TE polypeptides described herein may be useful in the preparation of compounds, including but not limited to. (l1<S’,l2<S’,l3S,9,12<S’)-9-amino-12-((l-(6-aminohexyl)-5-fluoro-l / f-indol-3-yl)methyl)-A-((25,3A)-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-12-carboxamide [compound 3g of Table A] and 6-(((2A)-2-((2S)-2-(4-((3aS’,27S,30S,41aS’,445,44aS 475,50AZ)-30-amino-23-fluoro-47-((7?)-l-hydroxyethyl)-50-(4-methoxybenzyl)-3a-methyl-4,29,39,45,48,51,53-heptaoxo- 2, 3, 3a,4, 5, 6, 7, 12, 14, 15, 16, 17, 18, 19, 26, 27, 28, 29, 30, 31, 37, 38, 39, 40, 41a, 42, 44 a, 45, 46, 47, 48, 49, 50, 51 -tetratriacontahy dro-43 / 7-8, 11 -etheno-27,44-methano-20,25: 32,36-di(metheno)benzo[r]dipyrrolo[2,l-:2',3'- O]

[0001] oxa[4, 14,20,27,35,38,41,44] octaazacy cloheptatetracontin- 13 ( 17 )-y 1 )-4 -oxobutanamido)propanamido)-3-isopropoxy-3-oxopropyl)amino)-A, A, A-trimethyl-6-oxohexan-1-aminium [WF+ACD in Scheme A], or of intermediates formed during preparation of such compounds.

[0248] In some examples, a polypeptide (e.g., a TE polypeptide) of the disclosure is used in a method of catalyzing the regioselective coupling of a diester to a diamine to synthesize a monoester. In some examples, a polypeptide (e.g., a TE polypeptide) of the disclosure is used in a method of catalyzing the regioselective coupling of a diester of formula (1) to a diamine of formula (2) to synthesize a monoester of formula (3) in reference to Scheme A below. In some embodiments, a diester of formula (1) is selected from bis-isopropyl ester, hexafluoro bisisopropyl ester, tetrafluoro bis-isopropyl ester, or difluoro bis-isopropyl ester. In some embodiments, a diester of formula (1) is bis-isopropyl ester. In some embodiments, the monoester of formula (3) is selected from isopropyl ester, trifluoro isopropyl ester, difluoro isopropyl ester, monofluoro isopropyl or ester. In some embodiments, the monoester of formula (3) is isopropyl ester.

[0249] For example, provided herein is a method of catalyzing an amide bond formation, the method comprising contacting a substrate with any one of the polypeptides (e.g., TE polypeptides) disclosed herein. In some embodiments, provided herein is a method of catalyzing an amide bond formation, the method comprising incubating a substrate in the presence of any one of the polypeptides (e.g., TE polypeptides) disclosed herein.

[0250] In some embodiments, the method of catalyzing an amide bond formation, comprises contacting a substrate with a polypeptide (e.g., a TE polypeptide) as disclosed herein under reaction conditions suitable for converting the substrate to a desired product. In some embodiments, the method of catalyzing an amide bond formation, comprises incubating a substrate in the presence of a polypeptide (e.g., a TE polypeptide) as disclosed herein under reaction conditions suitable for converting the substrate to a desired product.

[0251] In some embodiments, the substrate is any of the substrates listed in Table A below. In some embodiments, the substrate is (1g) and / or (2g). In some embodiments, the substrates are (1g) and (2g). In some embodiments, the product is any of the products listed in Table A and / or Table B. In some embodiments, the product is any of the products listed in Table A. In some embodiments, the product is (3g) in Table A.

[0252] In some embodiments, the polypeptides (e.g., TE polypeptides) of the disclosure catalyze the formation of product with formula (3g) of Table A at concentrations of substrate (1g) of Table A of at least about 0.5 mM, 1 mM, 3 mM. 10 mM, or more with a percent conversion of at least about 0.001%, about 0.01%, about 0.1%, about 1%, about 10%, about 40%, about 60%, about 80% or about at least 95% in a reaction time less than about 96 hours, about 48 hours, about 20 hours, about 10 hours or less. In some embodiments, the polypeptides (e.g., TE polypeptides) of the disclosure catalyze the formation of product with formula (3g) at concentrations of substrate (2g) of at least about 0.5 mM, 1 mM, 3 mM, 10 mM, 30 mM, 50 mM or more with a percent conversion of at least about 0.001%, about 0.01%, about 0.1%, about 1%, about 10%, about 40%, about 60%, about 80% or about at least 95% in a reaction time less than about 96 hours, about 48 hours, about 20 hours, about 10 hours or less. Suitable reaction conditions under which the above-stated improved properties of the engineered thioesterase enzymes are further described in Examples 2-34.

[0253] In some embodiments, the substrate is (1) and / or (2) in Scheme A below. In some embodiments, the substrate is (1) and (2) in Scheme A below. In some embodiments, the product is any of (3), (4). (5), (6), (7), or (8) in Scheme A below. In some embodiments, the product is (3) in Scheme A.

[0254] In some embodiments, the polypeptides (e.g., TE polypeptides) of the disclosure catalyze the formation of product with formula (3) of Scheme A at concentrations of substrate (1) of Scheme A of at least about 0.5 mM, 1 mM. 3 mM, 10 mM, or more with a percent conversion of at least about 0.001%, about 0.01%, about 0.1%, about 1%, about 10%, about 40%, about 60%, about 80% or about at least 95% in a reaction time less than about 96 hours, about 48 hours, about 20 hours, about 10 hours or less. In some embodiments the engineered thioesteraseenzymes catalyze the formation of product with formula (3) at concentrations of substrate (2) of at least about 0.5 mM, 1 mM, 3 mM, 10 mM, 30 mM. 50 mM or more with a percent conversion of at least about 0.001%, about 0.01%, about 0.1%, about 1%, about 10%, about 40%, about 60%, about 80% or about at least 95% in a reaction time less than about 96 hours, about 48 hours, about 20 hours, about 10 hours or less. Suitable reaction conditions under which the above-stated improved properties of the engineered thioesterase enzymes are further described in Examples 37-62.

[0255] Any engineered polypeptide (e.g., TE polypeptide) or combination of polypeptides disclosed herein may be used in the methods for catalyzing an amide bond formation. As noted herein, in some embodiments, the engineered polypeptide (e.g., TE polypeptide) comprises an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a reference sequence comprising the sequence of SEQ ID NO: 2, 4. 6, 16, 52, 378, or 360, wherein the polypeptide does not comprise SEQ ID NO: 2. In some embodiments, these polypeptides can have one or more modifications to the amino acid sequence of SEQ ID NO: 2, 4, 6, 16, 52, 378, or 360,. The modifications can include substitutions, deletions, and insertions. The substitutions can be non-conservative substitutions, conservative substitutions, or a combination of nonconservative and conservative substitutions.

[0256] For example, in some embodiments, the polypeptide (e.g., TE 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: 4, 6, 8, 10, 12. 14. 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44. 46. 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, or 78, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide comprises an amino acid sequence having at least about 98% sequence identity to any one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44. 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72. 74, 76, 78, 90, 106, 114, 130, 140. 150, 162. 172, 188, 194, 204, 216. 230, 238, 248, 260, 272, 294, 296, 310, 322, 340, 352, 354, 360, 364, 366, 368, 370, 372, 374, 376, 378, or 380, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide comprises any of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48. 50. 52. 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 90, 106, 114, 130, 140, 150, 162, 172, 188, 194, 204, 216, 230, 238, 248, 260, 272, 294, 296, 310, 322, 340, 352, 354, 360, 364, 366, 368, 370, 372, 374, 376, 378, or 380. In some embodiments, the polypeptide consists of any of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24. 26. 28. 30, 32, 34, 36, 38, 40, 42, 44, 46, 48. 50. 52. 54. 56. 58. 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 90, 106, 114, 130, 140, 150, 162, 172, 188, 194, 204, 216, 230, 238, 248, 260, 272, 294, 296, 310, 322, 340, 352, 354, 360, 364, 366, 368, 370, 372, 374, 376, 378, or 380.

[0257] For example, in some embodiments, the polypeptide (e.g., TE 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: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 378, or 380, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide comprises an amino acid sequence having at least about 98% sequence identity to any one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 378, or 380, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide comprises any of SEQ ID NO: 4, 6, 8, 10, 12. 14, 16. 18, 20, 22, 24, 26, 28, 30, 32, 34, 36. 38. 40. 42. 44. 46. 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 378, or 380. In some embodiments, the polypeptide consists of any of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 378, or 380.

[0258] For example, in some embodiments, the polypeptide (e.g.. TE 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: 16, 78, 90, 106, 114, 130, 140, 150, 162, 172, 188, 194, 204, 216, 230, 238, 248, 260, 272, 294, 296, 310, 322. 340, 352, 354, 360, 364. 366, 368, 370, 372, 374, 376, or 380, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide comprises an amino acid sequence having at least about 98% sequence identity to any one of SEQ ID NO: 16, 78, 90, 106, 114, 130, 140, 150, 162, 172, 188, 194, 204, 216, 230, 238, 248, 260, 272. 294, 296, 310, 322, 340, 352, 354, 360, 364, 366, 368, 370, 372, 374, 376, or 380, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide comprises any of SEQ ID NO: 16, 78, 90, 106, 114, 130, 140, 150, 162, 172, 188, 194, 204, 216, 230, 238, 248, 260, 272, 294, 296, 310, 322, 340, 352, 354, 360, 364, 366, 368, 370, 372, 374, 376, or 380. In some embodiments, the polypeptide consists of any of SEQ ID NO: 16, 78, 90. 106, 114, 130, 140, 150. 162, 172, 188, 194, 204, 216, 230.238, 248, 260, 272, 294, 296, 310, 322, 340, 352, 354, 360, 364, 366, 368, 370, 372, 374, 376, or 380.

[0259] In some embodiments, provided herein is a method of catalyzing an amide bond formation, the method comprising contacting a substrate with any one of the polypeptides (e.g.,TE 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.

[0260] The TE-catalyzed reactions described herein are generally carried out in a solvent. Suitable solvents include water, organic solvents (e.g., acetonitrile, methanol, isopropanol, ethyl acetate, butyl acetate, 1 -octanol, heptane, octane, methyl t-butyl ether (MTBE), dimethyl sulfoxide (DMSO), dimethylacetamide (DMAc), toluene, and the like), and ionic liquids (e g., 1-ethyl 4-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium hexafluorophosphate, and the like). In some embodiments, aqueous solvents, including water and aqueous cosolvent systems, are used.

[0261] Exemplary aqueous cosolvent systems have water and one or more organic solvent. In general, an organic solvent component of an aqueous cosolvent system is selected such that it does not completely inactivate the TE enzyme. Appropriate cosolvent systems can be readily identified for example, by measuring the enzymatic activity of the specified engineered TE enzyme with a defined substrate of interest in the candidate solvent system, utilizing an enzyme activity assay such as those described herein, or with an SDS-PAGE protein gel analysis.

[0262] The organic solvent component of an aqueous cosolvent 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 cosolvent system is employed, it is selected to be biphasic, with water dispersed in an organic solvent, or vice-versa. Generally, when an aqueous cosolvent system is utilized, it is desirable to select an organic solvent that can be readily separated from the aqueous phase. In general, the ratio of water to organic solvent in the cosolvent system is typically in the range of from about 90: 10 to about 10:90 (v / v) organic solvent to water or from about 80:20 to about 20:80 (v / v) organic solvent to water. The cosolvent system may be pre-formed prior to addition to the reaction mixture, or it may be formed in situ in the reaction vessel.

[0263] The aqueous solvent (water or aqueous cosolvent system) may be pH-buffered or unbuffered. In some embodiments, the method of catalyzing the amide bond formation is carried out at a pH of about 10 or below. e g., in a range of from about 5 to about 10. In some embodiments, the method of catalyzing the amide bond formation is 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 method of catalyzing the amide bond formation is carried out at a pH of about 8.5 or below, e.g., in a range of from about 5 to about 8.5, in a range of from about 6 to about 8.5, in a range of from about 7 to about 8.5, or in a range of from about 7.5 to about 8.5. In some embodiments, the method of catalyzing the amide bond formation is carried out at a pH of about 8 or below, e.g., in a range offrom about 5 to about 8 or in a range of from about 6 to about 8. The method may also be carried out in a range of from about 6 to about 8. The method may also be carried out at a pH of about 7.8 or below, or 7.5 or below. Alternatively, the method may be carried out a neutral pH, i.e., about 7.

[0264] During the course of the reaction, 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.

[0265] In some embodiments, the method 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 method of the disclosure is 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 method of the disclosure is performed at a pH of from 7 to 7.5, from 7 to 8, from 7 to 9, from 7.5 to 8.5, from 7.5 to 9. from 7.5 to 8, from 8 to 9, from 8.5 to 9, from 9 to 11, from 9 to 10.5, from 9 to 10, from 9 to 9.5, from 9.5 to 11, from 9.5 to 10.5, from 9.5 to 10, from 10 to 11, or from 10 to 10.5.

[0266] In some embodiments, the method of the disclosure 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 method of the disclosure is performed at a temperature of from about 20 °C to about 40 °C, e.g., about 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C. 27 °C, 28 °C, 29 °C, 30 °C, about 31 °C. 33 °C, 33 °C, 34 °C. 35 °C, 36 °C. 37 °C, 38 °C, 39 °C. or 40 °C. In some embodiments, the method of the disclosure is performed at a temperature of from about 10 °C to about 30 °C, e g., about 10°C, 11°C, 12°C, 13°C, 14°C, 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, or 30 °C.

[0267] In some embodiments, the method of the disclosure comprises contacting or incubating from about 0.1 g / L to about 75 g / L, 0.1 g / L to about 70 g / L, 0.1 g / L to about 65 g / L, 0.1 g / L to about 60 g / L, 0.1 g / L to about 50 g / L, 0.5 g / L to about 75 g / L, 0.5 g / L to about 70 g / L, 0.5 g / L to about 65 g / L, 0.5 g / L to about 60 g / L, 0.5 g / L to about 50 g / L, 1 g / L to about 75 g / L, 1 g / L toabout 70 g / L, 1 g / L to about 65 g / L, 1 g / L to about 60 g / L, 1 g / L to about 50 g / L. 5 g / L to about 75 g / L, 10 g / L to about 60 g / L, or about 20 g / L to about 60 g / L of a relevant substrate with any one of the polypeptides (e.g., TE polypeptides) disclosed herein or any combination of the polypeptides disclosed herein. In some embodiments, the method of the disclosure comprises contacting or incubating from about 0.5 g / L to about 60 g / L (e.g.. 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 a relevant substrate with any one of the polypeptides (e.g., TE polypeptides) disclosed herein or any combination of the polypeptides disclosed herein.

[0268] In some embodiments, the method of the disclosure 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, 2% w / w, 3% w / w, 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 polypeptides (e.g., TE polypeptides) disclosed herein or any combination of the polypeptides disclosed herein. In some embodiments, the TE 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 TE 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.

[0269] Any of the methods disclosed herein may further comprise filtering the product. Any suitable approach may be used for filtration. In some embodiments, the pH is adjusted to from about 6 to about 8 (e.g., about 6.0. 6.1, 6.2, 6.3, 6.4. 6.5, 6.6, 6.7, 6.8. 6.9, 7.0, 7.1, 7.2. 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0).

[0270] In some embodiments, the method of the disclosure comprises contacting or incubating a relevant substrate with the polypeptide (e.g., TE polypeptide) for any suitable period of time, e.g., about 1 min, 5 min, 10 min. 15 min, 20 min, 25 min, 30 min, 1 h, 2 h, 3 h. 4 h, 5 h, 6 h, 7 h.8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, 25 h, 26 h, 27 h, 28 h, 29 h, 30 h, 31 h, 32 h, 33 h, 34 h, 35 h, 36 h, 37 h, 38 h, 39 h, 40 h, 41 h, 42 h, 43 h, 44 h, 45 h, 46 h, 47 h, 48 h, 36 h, 60 h, or longer. In some embodiments, the period of timeis about 1 min to about 1 h, about 1 min to about 30 mm, about 1 min to about 10 min, about 5 min to about 30 min, about 10 min to about 30 min, about 30 min to about 1 h, about 1 h to about 36 h, about 1 h to about 24 h, about 1 h to 23 h, about 1 h to about 22 h, about 1 h to about 21 h, about 1 h to about 20 h, about 1 h to about 19 h, about 1 h to about 18 h, about 1 h to about 17 h. about 1 h to about 16 h, about 1 h to about 15 h. about 1 h to about 14 h, about 1 h to about 13 h. about 1 h to about 12 h, about 1 h to about 11 h, about 1 h to about 10 h, about 1 h to about 9 h, about 1 h to about 8 h, about 1 h to about 7 h, about 1 h to about 6 h, about 1 h to about 5 h, about 1 h to about 4 h, about 1 h to about 3 h, about 1 h to about 2 h, about 5 h to about 36 h, about 5 h to about 24 h, about 5 h to 23 h, about 5 h to about 22 h, about 5 h to about 21 h, about 5 h to about 20 h, about 5 h to about 19 h, about 5 h to about 18 h, about 5 h to about 17 h, about 5 h to about 16 h, about 5 h to about 15 h, about 5 h to about 14 h, about 5 h to about 13 h, about 5 h to about 12 h, about 5 h to about 11 h, about 5 h to about 10 h, about 5 h to about 9 h, about 5 h to about 8 h, about 5 h to about 7 h, about 5 h to about 6 h, about 10 h to about 36 h, about 10 h to about 24 h, about 10 h to 23 h, about 10 h to about 22 h, about 10 h to about 21 h, about 10 h to about 20 h, about 10 h to about 19 h, about 10 h to about 18 h, about 10 h to about 17 h, about 10 h to about 16 h, about 10 h to about 15 h, about 10 h to about 14 h, about 10 h to about 13 h, about 10 h to about 12 h, about 10 h to about 11 h, about 12 h to about 36 h, about 12 h to about 24 h, about 12 h to 23 h, about 12 h to about 22 h. about 12 h to about 21 h, about 12 h to about 20 h, about 12 h to about 19 h, about 12 h to about 18 h, about 12 h to about 17 h, about 12 h to about 16 h, about 12 h to about 15 h, about 12 h to about 14 h, about 12 h to about 13 h, about 18 h to about 36 h, about 18 h to about 24 h, about 18 h to 23 h, about 18 h to about 22 h, about 18 h to about 21 h, about 18 h to about 20 h, or about 18 h to about 19 h.EXAMPLES

[0271] The following examples are meant to be illustrative and should not be construed as further limiting. The contents of the figures and all references, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference.

[0272] Tables A and B provide example substrates and products for reactions that may be catalyzed by the TE polypeptides disclosed. In particular, the substrates and products in Table A and Table B are referenced in Examples 1 - 34.

[0273] Throughout the disclosure and examples, abbreviations and acronyms may be used with the following meanings unless otherwise indicated: 2-CB = 2-carboxybenzaldehyde; DMAc = N, -dimethylacetamide; DMSO = dimethyl sulfoxide; EPPS = 4-(2-hydroxyethyl)-l-piperazinepropanesulfonic acid; HEPES = 4-(2-hy droxy ethyl)- 1 -piperazineethanesulfonic acid; / Pr = isopropyl; MOPS = 3-(N-morpholino)propanesulfonic acid; TE = thioesterase; IPTG = Isopropyl B-D-l -thiogalactopyranoside; OD600 = Optical density at 600 nm; HTP = high-throughput; nt = nucleotide; aa= amino acid; ACD =((3aS.27S.30S,41aS,44S,44aS,47S.50S, Z)-30-amino-23-fluoro-47-((R)-l -hy droxy ethyl)-50-(4-methoxybenzyl)-3a-methyl-l,2,3,3a,6,7,12,13,14,15,16,17,18,19,27,28,30,31,37,38,41a,44a,46,47,49,50-hexacosahydro-43H-8,ll-etheno-27,44-methano-20,25:32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl:2',3'-tl][l]oxa[4,14.20,27,35.38,41,44]octaazacycloheptatetracontine- 4,29,39,45,48,5 l,53(5H,26H,40H,42H)-heptaone salt; ACW = 6-(((2R)-2-((2R)-2-(4-((3aS,27S,30S,41aS,44S,44aS,47S,50S, Z)-30-amino-23-fluoro-47-((R)-l-hy droxy ethyl)-50-(4-methoxybenzyl)-3a-methyl-4,29,39,45,48,51,53-heptaoxo- 2, 3, 3a,4, 5, 6, 7, 12, 14, 15, 16.17, 18, 19, 26, 27.28, 29, 30, 31, 37, 38, 39, 40.41a, 42, 44a.45, 46, 47, 48, 49.50, 51 -tetratriacontahy dro-43H-8, 11 -etheno-27,44-methano-20,25: 32,36-di(metheno)benzo[r]dipyrrolo[2,l-kl:2',3'-tl][l]oxa[4,14,20,27,35,38,41,44]octaazacycloheptatetracontin-13(lH)-yl)-4-oxobutanamido)propanamido)-3-isopropoxy-3-oxopropyl)amino)-N, N. N-trimethyl-6-oxohexan-1-aminium salt WF-OEt: (9R,12R)-12-(isopropoxycarbonyl)-N, N, N,9-tetramethyl-4,7,10,15-tetraoxo-3-oxa-8,l l,14-triazaicosan-20-aminium salt; WF-OiPr =((9R,12R)-12-(isopropoxycarbonyl)-N, N, N,2,9-pentamethyl-4,7,10,15-tetraoxo-3-oxa-8,ll,14-triazaicosan-20-aminium salt; WF-OiPrF2 = (9R,12R)-l,l-difluoro-12-(isopropoxycarbonyl)-N, N, N.2,9-pentamethyl-4,7,10,15-tetraoxo-3-oxa-8,l l,14-triazaicosan-20-aminium salt; WF-0iPrF3 =((9R, 12R)- 1,1,1 -tri fluoro- 12-(isopropoxy carbonyl)-N, N, N,2,9-pentamethyl-4,7, 10, 15-tetraoxo-3-oxa-8,ll,14-triazaicosan-20-aminium salt; WF-SiPr =((9R,12R)-12-(isopropoxycarbonyl)-N, N, N,2,9-pentamethyl-4,7,10,15-tetraoxo-3-thia-8,l l,14-triazaicosan-20-aminium salt; and WF-SNAc = (12R.15R)-15-(isopropoxycarbonyl)-N, N. N,12-tetramethyl-2,7.10,13,18-pentaoxo-6-thia-3, 11, 14, 17-tetraazatricosan-23-aminium salt.Example 1: Synthesis, Optimization, and Assay of Thioesterase enzymes.

[0274] This example describes methods to synthesize, optimize, and assay thioesterase enzymes for macrocyclization activity, and the composition of optimized enzymes.Gene synthesis and optimization:

[0275] The polynucleotide sequence (SEQ ID NO: 1) encoding the wild-type thioesterase (TE) domain of the Brevibacullus laterosporus non-ribosomal peptide synthetase polypeptide (SEQ ID NO: 2) was identified in silica as a component of the full polynucleotide sequence of GenBank accession # WP_003339348 based on a BLAST / clustering approach applied to the sequences of known non-ribosomal peptide synthetase polypeptide thioesterases. SEQ ID NO: 1 was then codon optimized for expression in E. call, the nucleophilic serine residue in the corresponding polypeptide was swapped for a cysteine (S82C), and a C-terminal hexahistadine tag (SEQ ID NO: 381) with a linker (SEQ ID NO: 382) was added. This resulted in the polynucleotide of SEQ ID NO: 3 and the polypeptide of SEQ ID NO: 4. The gene (SEQ ID NO: 3) was cloned into the pET30(+) vector, and expressed in E. coli strain BL-21(DE3) under the control of the T7 promoter.HTP Growth. Expression, and Lysate Preparation:

[0276] Monoclonal cell populations were used to inoculate 400 pL cultures of Zym-5052 (Teknova, Hollister, CA; part # 3S2000) autoinduction medium supplemented with 30 pg / mL kanamycin sulfate. Cells were grown overnight at 25-30 °C with shaking, followed by centrifugal harvesting. The cell pellets were resuspended in a lysis buffer of sodium phosphate pH 7.5 containing lysozy me and polymyxin b sulfate, and mixed by shaking for one hour. This lysate was then clarified by centrifugation.Production of Shake Flask Powders (SFP):

[0277] Monoclonal cell populations were used to inoculate 200 mL - 1.6 L cultures Zym-5052 autoinduction medium supplemented with 30 pg / mL kanamycin sulfate in a baffled culture flask. Cells were grown overnight at 25-30 °C with shaking, followed by centrifugal harvesting. Cell pellets were frozen at -80 °C overnight, then thawed and resuspended in 5mL / g sodium phosphate buffer pH 7.5. The resuspended cell pellets were lysed by sonication or microfluidization. The resulting lysate was frozen at -80 °C and lyophilized.Assay method for high-throughput thioesterase activity:

[0278] To 100 pL of lysate from high-throughput growth described above, 5 pL was added of a 40 mg / mL solution of compound la in DMSO. The reaction was incubated at 25 °C with shaking overnight, then quenched with acetonitrile and analyzed by HPLC-MS.High-throughput (HTP) analytical methodInstrument: Agilent 1290 Infinity II equipped with an Agilent 6130 Quadrupole MS Column: Acquity UPLC BEH Shield RP 18 1.7pm 2.1 mm x 50mm (Waters, #186002853) Column temperature: 55°CFlow rate: 1.2 mL / minuteDetection wavelength: 210 nm / ESI positiveMobile Phase A: 0.1% formic acid in waterMobile Phase B: 0.1% formic acid in acetonitrileIdentification of starting point for evolution:

[0279] Activity was detected for SEQ ID NO: 4. Under these conditions, 0.3% of the la was converted to 3a, while another 46.0% was converted to the acid I. The polypeptide SEQ ID NO:4 is therefore a novel biocatalytic reagent for the production of 3a from la, and the encoding polynucleotide SEQ ID NO: 3 was selected for further directed evolution.Example 2: (Rdl). Enzyme variants of SEQ ID NO: 4

[0280] In this example, directed evolution of SEQ ID NO: 4 for improved expression and stability and the resulting improved variants are described. Directed evolution was carried out by constructing libraries of variant genes with truncated N-termini. These libraries were plated to form single colonies, which were grown and screened using the high-throughput growth and expression method described in Example 1 and the high-throughput (HTP) assay described below.HTP assay for intramolecular thioesterase activity:

[0281] Cell pellets from a 400 pL culture of sequence variants was lysed with a 200 pL solution containing lysozyme and polymyxin B sulfate in HEPES buffer. 15 pL of the resulting lysate was transferred to a 50 pL reaction containing 10 pL of 5 mg / mL compound la in 25% DMSO. Reactions were incubated at 25 °C overnight and analyzed by the HTP analytical method as described in Example 1 or similar.

[0282] Engineered polypeptides with 1.2-fold hydrolysis of la to I relative to the parent polypeptide are listed in Table 2.1 and were re-expressed as shake flask powders as described in Example 1.Table 2.1 Variants and Conversion

[0283] These variants with the deletions listed in Table 2.1 produced I from la, and these engineered thioesterase enzymes provide new biocatalytic reagents for use in the hydrolysis reaction of la to I. The variant with seven truncations (SEQ ID NO: 6), had the highest activity. Thus, the encoding polynucleotide (SEQ ID NO: 5) was selected for further directed evolution.Example 3 (Rd2): Enzyme variants of SEQ ID NO: 6

[0284] In this example, directed evolution of SEQ ID NO: 6 for improved catalytic activity and the resulting improved variants are described. Directed evolution was carried out by constructing libraries of variant genes in which positions associated with the predicted active site of the enzyme were subjected to mutagenesis. These libraries were plated to form single colonies,which were grown and screened using the high-throughput growth and expression method described in Example 1 and the high-throughput assay described below-.HTP assay for intramolecular thioesterase activity:

[0285] Cell pellets from a 400 pL culture of sequence variants was lysed with a 200 pL solution containing lysozyme and polymyxin B sulfate in HEPES buffer. 20 pL of the resulting lysate was transferred to a 50 pL reaction containing 10 pL of 5 mg / mL compound la in 75% DMSO. Reactions were incubated at 30 °C overnight and analyzed by the HTP analytical method as described in Example 1 or similar.

[0286] Engineered polypeptides with >2-fold conversion relative to the parent polypeptide are listed in Table 3.1 and were re-expressed as shake flask powders as described in Example 1.Table 3.1 Variants and Conversion

[0287] Variants with mutations shown in Table 3.1 produced 3a from la, and these engineered thioesterase enzymes provide new biocatalytic reagents for use in new methods for the thioesterase reaction of la to 3a. The variant with the mutation A196R (SEQ ID NO: 8), had the highest activity. Thus, the encoding polynucleotide (SEQ ID NO: 7) was selected for further directed evolution.Example 4 (Rd3): Enzy me variants of SEQ ID NO: 8

[0288] In this example, directed evolution of SEQ ID NO: 8 for improved catalytic activityproperty and the resulting improved variants are described. Directed evolution was carried out by constructing libraries of variant genes in which positions associated with the predicted active site and surface of the enzyme were subjected to mutagenesis. These libraries were plated to formsingle colonies, which were grown and screened using the high-throughput growth and expression method described in Example 1 and the high-throughput assay described below.HTP assay for intramolecular thioesterase activity:

[0289] Cell pellets from a 400 pL culture of sequence variants was lysed with a 200 pL solution containing lysozyme and polymyxin B sulfate in sodium phosphate buffer. 10 pL of the resulting lysate was transferred to a 50 pL reaction containing 10 pL of 5 mg / mL compound la in 75% DMSO. Reactions were incubated at 30 °C overnight and analyzed by the HTP analy tical method as described in Example 1 or similar.

[0290] Engineered polypeptides with >3-fold conversion relative to the parent polypeptide are listed in Table 4.1 and were re-expressed as shake flask powders as described in Example 1.Table 4.1 Variants and Conversion

[0291] Variants with mutations shown in Table 4.1 produced 3a from la, and these engineered thioesterase enzymes provide new biocatalytic reagents for use in new methods for the intramolecular amidation reaction of la to 3a. The variant with mutations T27K, Fl 25V, E127L, G129L, and W199K (SEQ ID NO: 10) had the highest activity. Thus, the encoding polynucleotide (SEQ ID NO: 9) was selected for further directed evolution.Example 5 (Rd4): Enzy me variants of SEQ ID NO: 10

[0292] In this example, directed evolution of SEQ ID NO: 10 for improved catalytic activity and stability and the resulting improved variants are described. Directed evolution was carried out by constructing libraries of variant genes in which positions associated with the predicted surface and core of the protein were subjected to mutagenesis. These libraries were plated toform single colonies, which were grown and screened using the high-throughput growth and expression method described in Example 1 and the high-throughput assay described below.HTP assay for intermolecular thioesterase activity:

[0293] Cell pellets from a 400 pL culture of sequence variants was lysed with a 200 pL solution containing lysozyme and polymyxin B sulfate in sodium phosphate buffer. 10 pL of the resulting lysate was transferred to a 50 pL reaction containing 10 pL of 5 mg / mL compound lb in 75% DMSO and 10 pL 5 mg / mL 2b in DMSO. Reactions were incubated at 30 °C overnight and analyzed by the HTP analytical method as described in Example 1 or similar.

[0294] Engineered polypeptides with >2-fold conversion relative to the parent polypeptide are listed in Table 5.1 and were re-expressed as shake flask powders as described in Example 1.Table 5.1 Variants and Conversion

[0295] Variants with mutations shown in Table 5.1 produced 3b from lb and 2b. and these engineered thioesterase enzymes provide new biocatalytic reagents for use in new methods for the thioesterase reaction of lb and 2b to 3b. The variant with mutations K31P, E107S, A123L, L127E, Q228A, and V245G (SEQ ID NO: 12), had the highest activity. Thus, the encoding polynucleotide (SEQ ID NO: 11) was selected for further directed evolution.Example 6 (Rd5): Enzyme variants of SEQ ID NO: 12

[0296] In this example, directed evolution of SEQ ID NO: 12 for improved catalytic activity and stability7and the resulting improved variants are described. Directed evolution was carried out by constructing libraries of variant genes in which positions associated with the predicted active site and core of the protein were subjected to mutagenesis. These libraries were plated to form single colonies, which were grown and screened using the high-throughput growth and expression method described in Example 1 and the high-throughput assay described below.HTP assay for intermolecular thioesterase activity:

[0297] Cell pellets from a 400 pL culture of sequence variants was lysed with a 200 pL solution containing lysozy me and polymyxin B sulfate in potassium phosphate buffer. 10 pL of the resulting lysate was transferred to a 50 pL reaction containing 15 pL of a solution of 4.5 mg / mL lb and 4.5 mg / mL 2b in DMSO. Reactions were incubated at 30 °C overnight and analyzed by the HTP analytical method as described in Example 1 or similar.

[0298] Engineered polypeptides with >2.4-fold conversion relative to the parent polypeptide are listed in Table 6.1 and were re-expressed as shake flask powders as described in Example 1.Table 6.1 Variants and Conversion

[0299] Variants with the mutations in Table 6.1 produced 3b from lb and 2b, and these engineered thioesterase enzymes provide new biocatalytic reagents for use in new methods for the intermolecular reaction of lb and 2b to 3b. The variant with mutations S18C, T20V, and S234R (SEQ ID NO: 14), had the highest activity. Thus, the encoding polynucleotide (SEQ ID NO: 13) was selected for further directed evolution.Example 7 (Rd6): Enzyme variants of SEQ ID NO: 14

[0300] In this example, directed evolution of SEQ ID NO: 14 for improved activity on ester substrates and the resulting improved variants are described. Directed evolution was carried out by constructing libraries of variant genes across the entire gene sequence were subjected to mutagenesis. These libraries were plated to form single colonies, which were grown and screenedusing the high-throughput growth and expression method described in Example 1 and the high-throughput assay described below.HTP assay for intramolecular esterase macrocyclization activity:

[0301] Cell pellets from a 400 pL culture of sequence variants was lysed with a 200 pL solution containing 1 mg / mL lysozyme and 0.5 mg / mL polymyxin B sulfate in 50 mM MOPS buffer pH 7.5. 20 pL of the resulting lysate was transferred to a 50 pL reaction containing 2 mg / mL compound Id and 8% (v / v) DMSO. Reactions were incubated at 30 °C overnight and analyzed by the HTP analytical method as described in Example 1 or similar.

[0302] Engineered polypeptides with >0.020% conversion are listed in Table 7.1 and were reexpressed as shake flask powders as described in Example 1.Table 7.1 Variants and Conversion

[0303] Variants with mutations shown in Table 7.1 produced 3d from Id, and these engineered thioesterase enzymes provide new biocatalytic reagents for use in new methods for the transamidation reaction of 3d to Id. The variant with mutations G23L; I36L; N132P; F135W; N167A; G226T (SEQ ID NO: 16), had the highest activity. Thus, the encoding polynucleotide (SEQ ID NO: 15) was selected for further directed evolution.Example 8 (Rd7): Enzyme variants of SEQ ID NO: 16

[0304] In this example, directed evolution of SEQ ID NO: 16 for improved activity' in the intermolecular ligation reaction from a thioesterase substrate and the resulting improved variants are described. Directed evolution was carried out by constructing libraries of variant genes in which positions associated with the enzyme active site were subjected to mutagenesis. These libraries were plated to form single colonies, which w ere grown and screened using the high-throughput growth and expression method described in Example 1 and the high-throughput assay described below.HTP assay for intermolecular thioesterase activity:

[0305] Cell pellets from a 400 pL culture of sequence variants were lysed with a 200 pL solution containing lysozyme and polymyxin B sulfate in 3-(N-morpholino)propanesulfonic acid (MOPS) buffer. 15 pL of the resulting lysate was transferred to a 50 pL reaction containing 2.14 mg / mL lb and 2.14 mg / mL 2b in 25% DMSO. Reactions were incubated at 35 °C overnight and analyzed by the HTP analytical method as described in Example 1 or similar.

[0306] Engineered polypeptides with >2-fold conversion relative to the parent polypeptide are listed in Table 8.1 and were re-expressed as shake flask powders as described in Example 1.Table 8.1 Variants and Conversion

[0307] Variants with mutations shown in Table 8.1 produced 3b from lb and 2b. and these engineered thioesterase enzymes provide new biocatalytic reagents for use in new methods for the intermolecular reaction of lb and 2b to 3b. The variant with mutations L163T, A167S, E187K, V203L (SEQ ID NO: 18), had the highest activity. Thus, the encoding polynucleotide (SEQ ID NO: 17) was selected for further directed evolution.Example 9 (Rd8): Enzyme variants of SEQ ID NO: 18

[0308] In this example, directed evolution of SEQ ID NO: 18 for improved activity' in the intermolecular ligation reaction from a thioesterase substrate and reduced production of the off-target compound H and the resulting improved variants are described. Directed evolution was carried out by constructing libraries of variant genes in which positions not associated with theenzyme active site were subjected to mutagenesis. These libraries were plated to form single colonies, which were grown and screened using the high-throughput growth and expression method described in Example 1 and the high-throughput assay described below.HTP assay for intermolecular thioesterase activity:

[0309] Cell pellets from a 400 pL culture of sequence variants were lysed with a 200 pL solution containing lysozyme and polymyxin B sulfate in 3-(N-morpholino)propanesulfonic acid (MOPS) buffer. 15 pL of the resulting lysate was transferred to a 50 pL reaction containing 2.14 mg / mL le and 2.14 mg / mL 2e in 25% DMSO. Reactions were incubated at 30 °C overnight and analyzed by the HTP analytical method as described in Example 1 or similar.

[0310] Engineered polypeptides with >30% conversion relative of le to 3e are listed in Table 9.1 and were re-expressed as shake flask powders as described in Example 1.Table 9.1 Variants and Conversion

[0311] Variants with mutations shown in Table 8.1 produced 3e from le and 2e, and these engineered thioesterase enzymes provide new biocatalytic reagents for use in new methods for the intermolecular reaction of le and 2e to 3e. The variant with mutations V16L; N79G; L142R (SEQ ID NO: 20), had the highest activity. Thus, the encoding polynucleotide (SEQ ID NO: 19) was selected for further directed evolution.Example 10 (Rd9): Enzyme variants of SEQ ID NO: 20

[0312] In this example, directed evolution of SEQ ID NO: 20 for improved activity in the intermolecular ligation reaction from an isopropyl ester substrate and the resulting improved variants are described. Directed evolution was earned out by constructing hbranes of variant genes in which positions spatially adjacent to those already mutated were subjected to mutagenesis. These libraries were plated to form single colonies, which were grown and screenedusing the high-throughput growth and expression method described in Example 1 and the high-throughput assay described below.HTP assay for intermolecular thioesterase activity:

[0313] Cell pellets from a 400 pL culture of sequence variants were lysed with a 200 pL solution containing lysozyme and polymyxin B sulfate in 3-(N-morpholino)propanesulfonic acid (MOPS) buffer. 20 pL of the resulting lysate w as transferred to a 50 pL reaction containing 1.67 mg / mL 1g and 3.33 mg / mL 2g in 5% DMSO. Reactions were incubated at 30 °C overnight and analyzed by the HTP analytical method as described in Example 1 or similar.

[0314] Engineered polypeptides with >2-fold improvement over SEQ ID NO: 20 for the conversion of 1g to 3g are listed in Table 10.1 and were re-expressed as shake flask powders as described in Example 1.Table 10.1 Variants and Conversion

[0315] Variants with mutations shown in Table 10.1 produced 3g from 1g and 2g, and these engineered thioesterase enzymes provide new biocatalytic reagents for use in new methods for the intermolecular reaction of 1g and 2g to 3g. The variant with mutations H25N; E197R (SEQ ID NO: 24), had the highest activity. Thus, the encoding polynucleotide (SEQ ID NO: 23) was selected for further directed evolution.

[0316] Additionally, variants containing the C75S reversion were constructed and assayed as above. Instead of 3g, these polypeptides produced H, and those with >5-fold increase in the production of H from 1g and 2g compared to SEQ ID NO: 20 are listed in Table 10.2.Table 10.2 Variants and Conversion

[0317] Variants with mutations shown in Table 10.2 produced regioisomer H from 1g and 2g, and these engineered thioesterase enzymes provide new biocatalytic reagents for use in new methods for the intermolecular reaction of 1g and 2g to H. The variant with mutations C75S; E133P; T163W (SEQ ID NO: 22), from polynucleotide SEQ ID NO: 21, had the highest activity.Example 11 (RdlO): Enzyme variants of SEQ ID NO: 24

[0318] In this example, directed evolution of SEQ ID NO: 24 for improved activity in the intermolecular ligation reaction from an isopropyl ester substrate and the resulting improved variants are described. Directed evolution w as carried out by constructing libraries of variant genes in which positions spatially adjacent to those already mutated were subjected to mutagenesis. These libraries were plated to form single colonies, which were grown and screened using the high-throughput growth and expression method described in Example 1 and the high-throughput assay described below.HTP assay for intermolecular thioesterase activity:

[0319] Cell pellets from a 400 pL culture of sequence variants were lysed with a 200 pL solution containing lysozyme and polymyxin B sulfate in triethanolamine (TEoA) buffer. 20 pL of the resulting lysate was transferred to a 50 pL reaction containing 1.67 mg / mL 1g and 3.33 mg / mL 2g. Reactions were incubated at 35 °C overnight and analyzed by the HTP analytical method as described in Example 1 or similar.

[0320] Engineered polypeptides with >1.5-fold improvement over SEQ ID NO: 24 for the conversion of 1g to 3g are listed in Table 11.1 and were re-expressed as shake flask powders as described in Example 1.Table 11.1 Variants and Conversion_

[0321] Variants with mutations shown in Table 11.1 produced 3g from 1g and 2g, and these engineered thioesterase enzymes provide new biocatalytic reagents for use in new methods for the intermolecular reaction of 1g and 2g to 3g. The variant with mutations I22R and M134E (SEQ ID NO: 26), had the highest activity. Thus, the encoding polynucleotide (SEQ ID NO: 25) was selected for further directed evolution.Example 12 (Rdl 1): Enzyme variants of SEQ ID NO: 26

[0322] In this example, directed evolution of SEQ ID NO: 26 for improved thermostability and activity in the intermolecular ligation reaction from an isopropyl ester substrate and the resulting improved variants are described. Directed evolution was carried out by constructing libraries of variant genes in which positions in the first half of the protein were subjected to mutagenesis. These libraries were plated to form single colonies, which were grown and screened using the high-throughput growth and expression method described in Example 1 and the high-throughput assay described below.HTP assay for intermolecular thioesterase activity:

[0323] Cell pellets from a 400 pL culture of sequence variants were lysed with a 200 pL solution containing lysozyme and polymyxin B sulfate in triethanolamine (TEoA) buffer, and heated at 45 °C for 1 hour. 20 pL of the resulting lysate was transferred to a 50 pL reaction containing 1.67 mg / mL 1g and 3.33 mg / mL 2g. Reactions were incubated at 35 °C overnight and analyzed by the HTP analytical method as described in Example 1 or similar.

[0324] A variant with the mutation 113Q (SEQ ID NO: 28) produced >3-fold more 3g from 1g and 2g relative to SEQ ID NO: 26, and this engineered thioesterase enzymes provides a new biocatalytic reagent for use in new methods for the intermolecular reaction of 1g and 2g to 3g.Thus, the encoding polynucleotide (SEQ ID NO: 27) was selected for further directed evolution.Example 13 (Rdl2): Enzyme variants of SEQ ID NO: 28

[0325] In this example, directed evolution of SEQ ID NO: 28 for improved thermostability as well as activity in the intermolecular ligation reaction from an isopropyl ester substrate withimproved volumetric efficiency and the resulting improved variants are described. Directed evolution was carried out by constructing libraries of variant genes in which positions in the second half of the protein were subjected to mutagenesis. These libraries were plated to form single colonies, which were grown and screened using the high-throughput growth and expression method described in Example 1 and the high-throughput assay described below.HTP assay for intermolecular thioesterase activity:

[0326] Cell pellets from a 400 pL culture of sequence variants were lysed with a 200 pL solution containing lysozyme and polymyxin B sulfate in triethanolamine (TEoA) buffer and heated to 48 °C for one hour. 20 pL of the resulting lysate was transferred to a 50 pL reaction containing 16.67 mg / rnL 1g and 25 mg / mL 2g. Reactions were incubated at 25 °C overnight and analy zed by the HTP analytical method as described in Example 1 or similar.

[0327] Engineered polypeptides with >1.5% conversion under these conditions for the conversion of 1g to 3g are listed in Table 13.1 and were re-expressed as shake flask powders as described in Example 1.Table 13.1 Variants and Conversion

[0328] Variants with mutations shown in Table 13.1 produced 3g from 1g and 2g, and these engineered thioesterase enzymes provide new biocatalytic reagents for use in new methods for the intermolecular reaction of 1g and 2g to 3g. The variant with mutations R142A, E172T. K.187D, E189D, N190D (SEQ ID NO: 30), had the highest activity. Thus, the encoding polynucleotide (SEQ ID NO: 29) was selected for further directed evolution.Example 14 (Rdl3): Enzyme variants of SEQ ID NO: 30

[0329] In this example, directed evolution of SEQ ID NO: 30 for improved activity in the intermolecular ligation reaction from an isopropyl ester substrate with improved volumetric efficiency and the resulting improved variants are described. Directed evolution was carried out by constructing libraries of variant genes in which positions in surface and core of the proteinwere subjected to mutagenesis. These libraries were plated to form single colonies, which were grown and screened using the high-throughput growth and expression method described in Example 1 and the high-throughput assay described below.HTP assay for intermolecular thioesterase activity:

[0330] Cell pellets from a 400 pL culture of sequence variants were lysed with a 200 pL solution containing lysozyme and polymyxin B sulfate in triethanolamine (TEoA) buffer at 35 °C. 20 pL of the resulting lysate was transferred to a 50 pL reaction containing 16.67 mg / mL 1g and 25 mg / mL 2g. Reactions were incubated at 20 °C overnight and analyzed by the HTP analytical method as described in Example 1 or similar.

[0331] Engineered polypeptides with 1.5 improvement over SEQ ID NO: 30 under these conditions for the conversion of 1g to 3g are listed in Table 14.1 and were re-expressed as shake flask powders as described in Example 1.Table 14.1 Variants and Conversion

[0332] Variants with mutations shown in Table 14.1 produced 3g from 1g and 2g, and these engineered thioesterase enzymes provide new biocatalytic reagents for use in new methods for the intermolecular reaction of 1g and 2g to 3g. The variant with mutation K249* (SEQ ID NO:32) had the highest activity. Thus, the encoding polynucleotide (SEQ ID NO: 31) was selected for further directed evolution.Example 15 (Rdl4): Enzyme variants of SEQ ID NO: 32

[0333] In this example, directed evolution of SEQ ID NO: 32 for improved thermostability' as well as activity in the intermolecular ligation reaction from an isopropyl ester substrate with improved volumetric efficiency and the resulting improved variants are described. Directed evolution was carried out by constructing libraries of variant genes in which positions in the active site of the protein w ere subjected to mutagenesis. These libraries were plated to formsingle colonies, which were grown and screened using the high-throughput growth and expression method described in Example 1 and the high-throughput assay described below.HTP assay for intermolecular thioesterase activity:

[0334] Cell pellets from a 400 pL culture of sequence variants were lysed with a 200 pL solution containing lysozyme and polymyxin B sulfate in triethanolamine (TEoA) buffer and heated to 52 °C for one hour. 20 pL of the resulting lysate was transferred to a 50 pL reaction containing 25 mg / mL 1g and 25 mg / mL 2g. Reactions were incubated at 20 °C overnight and analyzed by the HTP analytical method as described in Example 1 or similar.

[0335] Engineered polypeptides with 1.5 fold improvement over SEQ ID NO: 32 under these conditions for the conversion of 1g to 3g are listed in Table 15.1 and w ere re-expressed as shake flask pow ders as described in Example 1.Table 15.1 Variants and Conversion

[0336] Variants with mutations shown in Table 15.1 produced 3g from 1g and 2g, and these engineered thioesterase enzymes provide new7biocatalytic reagents for use in new7methods for the intermolecular reaction of 1g and 2g to 3g. The variant with mutation Q112L (SEQ ID NO: 34), had the highest activity. Thus, the encoding polynucleotide (SEQ ID NO: 33) was selected for further directed evolution.Example 16 (Rdl5): Enzyme variants of SEQ ID NO: 34

[0337] In this example, directed evolution of SEQ ID NO: 34 for improved thermostability as well as activity in the intermolecular ligation reaction from an isopropyl ester substrate with improved volumetric efficiency and the resulting improved variants are described. Directedevolution was carried out by constructing libraries of variant genes in which positions in the active site of the protein were subjected to mutagenesis. These libraries were plated to form single colonies, which were grown and screened using the high-throughput growth and expression method described in Example 1 and the high-throughput assay described below.HTP assay for intermolecular thioesterase activity:

[0338] Cell pellets from a 400 pL culture of sequence variants were lysed with a 200 pL solution containing lysozyme and polymyxin B sulfate in triethanolamine (TEoA) buffer and heated to 54 °C for one hour. 20 pL of the resulting lysate was transferred to a 50 pL reaction containing 41.67 mg / mL 1g and 31.25 mg / mL 2g. Reactions were incubated at 20 °C overnight and analyzed by the HTP analytical method as described in Example 1 or similar.

[0339] Engineered polypeptides with 1.5 fold improvement over SEQ ID NO: 34 under these conditions for the conversion of 1g to 3g are listed in Table 16.1 and were re-expressed as shake flask powders as described in Example 1.Table 16.1 Variants and Conversion

[0340] Variants with mutations shown in Table 16.1 produced 3g from 1g and 2g, and these engineered thioesterase enzymes provide new biocatalytic reagents for use in new methods for the intermolecular reaction of 1g and 2g to 3g. The variant with mutations F150H, S167C, D189K, A192L, S208T, and I229T (SEQ ID NO: 36), had the highest activity. Thus, the encoding polynucleotide (SEQ ID NO: 35) was selected for further directed evolution.Example 17 (Rdl6): Enzyme variants of SEQ ID NO: 36

[0341] In this example, directed evolution of SEQ ID NO: 36 for improved thermostability as well as activity and selectivity in the intermolecular ligation reaction from an isopropyl ester substrate with improved volumetric efficiency and the resulting improved variants are described. Directed evolution was carried out by constructing libraries of variant genes in which positions on the surface of the protein were subjected to mutagenesis. These libraries were plated to form single colonies, which were grown and screened using the high-throughput growth and expression method described in Example 1 and the high-throughput assay described below.HTP assay for intermolecular thioesterase activity:

[0342] Cell pellets from a 400 pL culture of sequence variants were lysed with a 200 pL solution containing lysozyme and polymyxin B sulfate in triethanolamine (TEoA) buffer and heated to 57 °C for one hour. 20 pL of the resulting lysate was transferred to a 50 pL reaction containing 50 mg / mL 1g and 37.5 mg / rnL 2g. Reactions were incubated at 20 °C overnight and analyzed by the HTP analytical method as described in Example 1 or similar.

[0343] Engineered polypeptides with selective production of 3g over B of at least 2.5:1 under these conditions are listed in Table 17.1 and were re-expressed as shake flask powders as described in Example 1.Table 17.1 Variants and Conversion

[0344] Variants with mutations shown in Table 16.1 selectively produced 3g over B from 1g and 2g, and these engineered thioesterase enzymes provide new biocatalytic reagents for use in new methods for the intermolecular reaction of 1g and 2g to 3g. The variant with mutations H4F, A24G, N90K, F124R, and C167L (SEQ ID NO: 38), had the greatest selectivity. Thus, the encoding polynucleotide (SEQ ID NO: 37) was selected for further directed evolution.Example 18 (Rdl7): Enzyme variants of SEQ ID NO: 38

[0345] In this example, directed evolution of SEQ ID NO: 38 for improved activity in the intermolecular ligation reaction from an isopropyl ester substrate with improved volumetric efficiency and selectivity', and the resulting improved variants are described. Directed evolution was carried out by constructing libraries of variant genes. These libraries were plated to form single colonies, which were grown and screened using the high-throughput growth and expression method described in Example 1 and the high-throughput assay described below.HTP assay for intermolecular thioesterase activity:

[0346] Cell pellets from a 400 pL culture of sequence variants were lysed with a 300 pL solution containing lysozyme and polymyxin B sulfate in triethanolamine (TEoA) buffer and heated to 55 °C for one hour. 20 pL of the resulting lysate was transferred to a 50 pL reaction containing 66.67 mg / mL 1g and 16.67 mg / mL 2g. Reactions were incubated at 20 °C overnight and analyzed by the HTP analytical method as described in Example 1 or similar.

[0347] Engineered polypeptides with 1.5 fold improvement over SEQ ID NO: 38 under these conditions for the conversion of 1g to 3g are listed in Table 18.1 and were re-expressed as shake flask powders as described in Example 1.Table 18.1 Variants and Conversion

[0348] Variants with mutations shown in Table 18.1 produced 3g from 1g and 2g, and these engineered thioesterase enzymes provide new biocatalytic reagents for use in new methods for the intermolecular reaction of 1g and 2g to 3g. The variant with mutation Q30Y (SEQ ID NO: 40), had the highest activity. Thus, the encoding polynucleotide (SEQ ID NO: 39) was selected for further directed evolution.Example 19 (Rdl8): Enzyme variants of SEQ ID NO: 40

[0349] In this example, directed evolution of SEQ ID NO: 40 for improved thermostability as well as activity in the intermolecular ligation reaction from an isopropyl ester substrate, and theresulting improved variants are described. Directed evolution was carried out by constructing libraries of variant genes. These libraries were plated to form single colonies, which were grown and screened using the high-throughput growth and expression method described in Example 1 and the high-throughput assay described below.HTP assay for intermolecular thioesterase activity:

[0350] Cell pellets from a 400 pL culture of sequence variants were lysed with a 200 pL solution containing lysozyme and polymyxin B sulfate in triethanolamine (TEoA) buffer and heated to 52 °C for one hour. 20 pL of the resulting lysate was transferred to a 50 pL reaction containing 50 mg / mL 1g and 37.5 mg / mL 2g. Reactions were incubated at 20 °C overnight and analyzed by the HTP analytical method as described in Example 1 or similar.

[0351] Engineered polypeptides with 4-fold improvement over SEQ ID NO: 40 under these conditions for the conversion of 1g to 3g are listed in Table 19.1 and were re-expressed as shake flask powders as described in Example 1.Table 19.1 Variants and Conversion

[0352] Variants with mutations shown in Table 19.1 produced 3g from 1g and 2g, and these engineered thioesterase enzymes provide new biocatalytic reagents for use in new methods for the intermolecular reaction of 1g and 2g to 3g. The variant with mutations E54D, H93K, L123A, R124F. V146R, H150A, Q210R, I212T; T229I, and E242Q (SEQ ID NO: 42), had the highestactivity. Thus, the encoding polynucleotide (SEQ ID NO: 41) was selected for further directed evolution.Example 20 (Rdl9): Enzyme variants of SEQ ID NO: 42

[0353] In this example, directed evolution of SEQ ID NO: 42 for improved activity in the intermolecular ligation reaction from an isopropyl ester substrate, and the resulting improved variants are described. Directed evolution was carried out by constructing libraries of variant genes. These libraries were plated to form single colonies, which were grown and screened using the high-throughput growth and expression method described in Example 1 and the high-throughput assay described below.HTP assay for intermolecular thioesterase activity:

[0354] Cell pellets from a 400 pL culture of sequence variants were lysed with a 200 pL solution containing lysozyme and polymyxin B sulfate in triethanolamine (TEoA) buffer and heated to 54 °C for 45 minutes. 20 pL of the resulting lysate was transferred to a 50 µL reaction containing 33.33 mg / mL 1g and 16.67 mg / mL 2g. Reactions were incubated at 20 °C overnight and analyzed by the HTP analytical method as described in Example 1 or similar.

[0355] Engineered polypeptides with 2.5-fold improvement over SEQ ID NO: 42 under these conditions for the conversion of 1g to 3g are listed in Table 20.1 and were re-expressed as shake flask powders as described in Example 1.Table 20.1 Variants and Conversion

[0356] Variants with mutations shown in Table 20.1 produced 3g from 1g and 2g, and these engineered thioesterase enzymes provide new biocatalytic reagents for use in new methods for the intermolecular reaction of 1g and 2g to 3g. The variant with mutations L23M, L100V,Y104W, and K.189F (SEQ ID NO: 46), had the highest activity. Thus, the encoding polynucleotide (SEQ ID NO: 45) was selected for further directed evolution.Example 21 (Rd20): Enzyme variants of SEQ ID NO: 46

[0357] In this example, directed evolution of SEQ ID NO: 46 for activity in the intermolecular ligation reaction from an isopropyl ester substrate, and the resulting improved variants are described. Directed evolution was carried out by constructing libraries of variant genes wherein positions corresponding to the core of the protein were subjected to mutagenesis. These libraries were plated to form single colonies, which were grown and screened using the high-throughput growth and expression method described in Example 1 and the high-throughput assay described below.HTP assay for intermolecular thioesterase activity:

[0358] Cell pellets from a 400 pL culture of sequence variants were lysed with a 200 pL solution containing lysozyme and polymyxin B sulfate in triethanolamine (TEoA) buffer and heated to 52 °C for 1 hour. 20 pL of the resulting lysate was transferred to a 50 pL reaction containing 58.33 mg / mL 1g, 25 mg / mL 2g, and 1.82 mg / mL 2-carboxybenzaldehyde (2-CB). Reactions were incubated at 20 °C overnight and analyzed by the HTP analytical method as described in Example 1 or similar.

[0359] Engineered polypeptides with 2-fold improvement over SEQ ID NO: 46 under these conditions for the conversion of 1g to 3g are listed in Table 21.1 and were re-expressed as shake flask powders as described in Example 1.Table 21.1 Variants and Conversion

[0360] V ariants with mutations shown in Table 21.1 produced 3g from 1g and 2g, and these engineered thioesterase enzymes provide new biocatalytic reagents for use in new methods for the intermolecular reaction of 1g and 2g to 3g. The variant with mutations Y30Q and E66K (SEQ ID NO: 48), had the highest activity. Thus, the encoding polynucleotide (SEQ ID NO: 47) was selected for further directed evolution.

[0361] Random sequence insertions at selected positions were also screened using the conditions above, and variants showing 3-fold improvement over SEQ ID NO: 46 under these conditions for the conversion of 1g to 3g are listed in Table 21.2.Table 21.2 Variants and Conversion

[0362] Variants with mutations shown in Table 21.2 produced 3g and H from 1g and 2g, and these engineered thioesterase enzymes provide new biocatalytic reagents for use in new methods for the intermolecular reaction of 1g and 2g to 3g or H. The variant with insertion -225T (SEQ ID NO: 50), encoded by the polynucleotide SEQ ID NO: 49, had the highest production of H.Example 22 (Rd21): Enzyme variants of SEQ ID NO: 48

[0363] In this example, directed evolution of SEQ ID NO: 48 for improved thermostability, selectivity for 3g, and activity at higher loading of 1g and the resulting improved variants are described. Directed evolution was carried out by constructing libraries of variant genes in which positions associated with surface residues and structural deviations from modeling were subjected to mutagenesis. These libraries were plated to form single colonies, which were grown and screened using the high-throughput growth and expression method described in Example 1 and the high-throughput assay described below.HTP assay for thioesterase activity’:

[0364] Cell pellets (4000 rpm, 15 minutes, 4°C centrifugation) from a 400 pL culture of sequence variants were lysed with 200 pL of lysis buffer solution (0.5 mg / mL polymyxin B sulfate, 0.5 mg / mL lysozyme, 4 mM MgCl₂, and 1500 U / mL DNase I in 20 mM triethanolamine pH 7.5). Lysis was performed in the shaker at 35°C and 1000 rpm for 45 min. The resulting lysate was clarified (4000 rpm, 10 minutes, 4°C centrifugation) and heat treated in the shaker at 50°C and 1000 rpm for 1 hour followed by another centrifugation step to clarify the lysate. 20 pL of the clarified, heat-treated lysate was transferred to a 50 pL reaction containing 30 mg / mL 1g, 5.5 mg / mL 2g, and 0.5 mg / mL 2-carboxybenzaldehyde (2-CB) at a final pH of 7.5. Reactions were incubated in the shaker at 20°C and 1000 rpm overnight and analyzed by the HTP analytical method as described in Example 1 or similar.

[0365] Engineered polypeptides with >l-fold conversion relative to the parent polypeptide are listed in Table 22.1 and were re-expressed as shake flask powders as described in Example 1.Table 22.1 Variants and Conversion

[0366] Variants with mutations shown in Table 22.1 produced 3g from 1g and 2g, and these engineered thioesterase enzymes provide new biocatalytic reagents for use in new methods for the transamidation reaction of 1g and 2g. The variant with mutations L16M; L167V1-226N (SEQ ID NO: 52) had the highest selectivity, thermostability, and tolerance to increased 1g loadingwhich causes substrate inhibition. Thus, the encoding polynucleotide (SEQ ID NO: 51) was selected for further directed evolution.Example 23 (Rd22): Enzyme variants of SEQ ID NO: 52

[0367] In this example, directed evolution of SEQ ID NO: 52 for improved thermostability, selectivity for the desired amino alcohol product, and activity at higher loading of 1g and the resulting improved variants are described. Directed evolution was carried out by constructing libraries of variant genes in which positions associated with surface residues and positions spatially proximal to the -226N insertion mutation of the three-dimensional structure of SEQ ID NO: 52 were subjected to mutagenesis. These libraries were plated to form single colonies, which were grown and screened using the high-throughput growth and expression method described in Example 1 and the high-throughput assay described below.HTP assay for thioesterase activity:

[0368] Cell pellets (4000 rpm, 15 minutes, 4°C centrifugation) from a 400 pL culture of sequence variants were lysed with 200 pL of lysis buffer solution (0.5 mg / mL polymyxin B sulfate, 0.5 mg / mL lysozyme, 4 mM MgCl₂, and 1500 U / mL DNase I in 20 mM triethanolamine pH 7.5). Lysis was performed in the shaker at 35°C and 1000 rpm for 45 min. The resulting lysate was clarified (4000 rpm, 10 minutes, 4°C centrifugation) and heat treated in the shaker at 52°...

Claims

WHAT IS CLAIMED IS:

1. An engineered polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 6 or 378, wherein the polypeptide comprises an amino acid substitution at one or more amino acid positions selected from 4, 13, 16, 18, 20, 22, 23, 24, 25, 27, 30, 31, 36, 37, 49, 54, 66, 75, 79, 90, 91, 93, 98, 100, 104, 107, 112, 114, 118, 119, 123, 124, 125, 126, 127, 129, 130, 131, 132, 133, 134, 135, 139, 142, 146, 150, 157, 163, 167, 171, 172, 176, 187, 189, 190, 191, 192, 195, 196, 197, 199, 203, 208, 209, 210, 212, 217, 219, 221, 226, 228, 229. 234, 242, 245, and 249, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 6, and wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2.

2. The polypeptide of claim 1, wherein the polypeptide comprises from 4 to 76 amino acid substitutions, each amino acid substitution at an amino acid position selected from 4, 13, 16, 18, 20, 22, 23, 24, 25, 27, 30, 31, 36, 37, 49, 54, 66, 75, 79, 90, 91, 93, 98, 100, 104, 107, 112, 114, 118, 119, 123, 124, 125, 126, 127, 129, 130, 131, 132, 133, 134, 135, 139, 142, 146, 150, 157, 163, 167, 171, 172, 176, 187, 189, 190, 191, 192, 195, 196, 197, 199, 203, 208, 209, 210, 212, 217, 219. 221, 226, 228, 229, 234. 242, 245, and 249. wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 6.

3. The polypeptide of claim 1 or 2, wherein the polypeptide comprises an amino acid substitution at position 196.

4. The polypeptide of any one of claims 1-3, wherein the polypeptide comprises at least one ammo acid substitution selected from H4F, I13Q, V16M, S18C, T20V, I22R, G23M, A24G, H25N, T27K, K31P, I36L. E54D, E66K, N79G, N90K, H93K, L100V. Y104W, E107S, Q112L, F125V, G129L, N132P, M134E, F135W, L142A. V146R, F150A, L163T, N167V, E172T, E187D, E189F, N190D, A192L, A196R, E197R, W199K, V203L, S208T, Q210R, I212T, G226T, Q228A, S234R, E242Q, V245G, and S249*, wherein * indicates the introduction of a stop codon in the polynucleotide sequence corresponding to the indicated position in the polypeptide.

5. The polypeptide of any one of claims 1-4, further comprising at least one amino acid insertion between amino acid positions 224 and 225 or between amino acid positions 225 and 226.

6. The polypeptide of any one of claims 1-5, where in the polypeptide has at least 80% sequence identity to SEQ ID NO: 52, wherein the polypeptide comprises at least four amino acid substitutions, each amino acid substitution at an amino acid position selected from 4, 30, 37, 49, 91, 98, 100, 107, 114, 118, 119, 126, 127, 130, 131, 139, 157, 171, 176, 191, 195, 209, 212, 219, 226, 227. and 230, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 52.

7. The polypeptide of claim 6, wherein the polypeptide comprises at least two amino acid substitutions selected from F4V. Y30L, H37N, A49E. Q91R, I98L. V100L, S107P, A114P, R118Q, K119A. T126A. E127D, T130A, H131N. R139L, Q157R, N171H, K176L, I191L, E195S, E209S, T212V, E219S, N226S, T227P, and I230V.

8. The polypeptide of any one of claims 1-7, wherein the polypeptide comprises one or more amino acid sequence differences set forth in any one of Table C and / or Tables 2.1 to 34.1.

9. The polypeptide of any one of claims 1-8, wherein the amino acid sequence comprises any one of SEQ ID NO: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70.

72. 74, or 76.

10. The polypeptide of any one of claims 1-9, wherein the amino acid sequence comprises SEQ ID NO: 72.

11. The polypeptide of any one of claims 1-9, wherein the amino acid sequence comprises SEQ ID NO: 74.

12. The polypeptide of any one of claims 1-9, wherein the amino acid sequence comprises SEQ ID NO: 76.

13. An engineered polypeptide comprising an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34.

36.

38. 40, 42, 44, 46, 48, 50, 52, 54, 56, 58.

60.

62.

64.

66.

68. 70, 72, 74, 76, 378. or 380. wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2.

14. The polypeptide of claim 13. wherein the amino acid sequence has at least 99% sequence identity to any one of SEQ ID NO: 4, 6, 8, 10.

12.

14.

16. 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 378, or 380.

15. The polypeptide of claim 13 or 14, comprising any of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42.

44.

46. 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 378, or 380.

16. The polypeptide of any one of claims 1-15, which is isolated.

17. The polypeptide of any one of claims 1-16, wherein the polypeptide is capable of catalyzing an amide bond formation.

18. The polypeptide of any one of claims 1-17, wherein the polypeptide has one or more of the following properties relative to a reference polypeptide comprising the amino acid sequence of SEQ ID NO: 2:a) increased enzyme activity;b) increased substrate scope;c) increased regioselectivity;d) increased chemoselectivity;e) increased solvent or cosolvent tolerance;f) reduction in side-products;g) reduction in product inhibition;h) increased thermostability;i) increased tolerance to increased substrate loading;j) increased protein expression;k) increased protein solubility7; and / orl) increased substrate affinity.

19. A polynucleotide encoding at least one polypeptide set forth in any one of claims 1-18, wherein the polynucleotide does not comprise SEQ ID NO: 1.

20. The polynucleotide of claim 19, wherein the polynucleotide is codon-optimized.

21. The polynucleotide of claim 19 or 20, wherein the polynucleotide comprises any one of SEQ ID NO: 3, 5, 7, 9.

11.

13.

15. 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41.43.

45. 47.49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 377, or 379.

22. A polynucleotide comprising at least 80% sequence identity to any one of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33.

35. 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 377, or 379, wherein the polynucleotide does not comprise SEQ ID NO: 1.

23. An engineered polypeptide comprising an amino acid sequence having at least 65% sequence identity to SEQ ID NO: 6 or 378, wherein the polypeptide comprises an amino acid substitution at one or more amino acid positions selected from 4, 10, 11, 12, 13, 15, 18, 20, 23, 24. 27, 31, 34, 35, 36, 37, 48, 62, 72, 93, 97, 98, 107, 108, 109, 111, 113, 114, 115, 120, 121, 123, 124, 125, 126, 127, 129, 132, 133, 134, 135, 138, 139, 144, 145, 146, 147, 148, 150, 154, 157, 158. 166, 167, 168, 172, 175. 183, 186, 187, 188, 189, 190, 192. 194, 195, 196, 199, 203.205, 206, 212, 213, 214, 217, 221, 222, 226, 228, 229, 234, 238, 243, 245, and 247, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 6, and wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2.

24. The polypeptide of claim 23 comprising an ammo acid sequence having at least 65% sequence identity to SEQ ID NO: 16 or 380 wherein the polypeptide comprises from 4 to 76 substitutions, each amino acid substitution at an amino acid position selected from 4, 10, 11, 12, 13, 15, 18, 23, 24, 27, 34, 35, 37, 48, 62, 72, 93. 97, 98, 107, 108, 109, 111, 113, 114, 115, 120, 121, 124. 126, 127, 129. 133, 134. 138, 139, 144, 145, 146. 147, 148. 150, 154, 157, 158, 166.168, 172, 175, 183, 186, 187, 188, 189, 190, 192, 194, 195, 196, 199, 203, 205, 206, 212, 213, 214, 217, 221, 222, 226, 229, 234, 238, 243, 245, and 247, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 16, and wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2.

25. The polypeptide of any claim 23 or 24, wherein the polypeptide comprises at least six amino acid substitutions selected from H4F, N10A, KI IQ, T12G, I13T, S15R, S18C, S18A,T20V, G23S, A24C. T27K, T27H, T27R. K31P, K34A, K34S. E35N, 136L, H37N, E48P. Q62A, M72V, H93R, S97G, I98L, I98V, E107S, E107H, K108A, E109T, E109V, El IIP, T113V, A114E, I115S, E120M, E120T, E120V, I121C, A123L, F124Y, F125V, T126S, T126G, E127K, G129L, G129I, N132P, E133G, E133R, M134I. M134S, M134V, M134N, F135W, T138R, R139T, L144P, F145L, V146G, T147S. N148S. F150A, S154D, Q157E, N158S. N158R, N158H, Y166L, N167A, Q168K, E172T, I175V, Q183S, Y186S, E187D, S188G, E189D, N190S, A192T, Y194L, E195V, A196R, A196V, W199K, W199V, V203Y, A205T, A205Q, Q206D, I212F, N213G, Y214H, H217T, S221T, K222R, G226T, Q228A, 1229 A, I229M, I229V, I229L, S234R, S234G. S234E, S238Q, I243Y, V245G, V245P. K247S, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 6.

26. The polypeptide of any one of claims 23-25, wherein the polypeptide comprises one or more amino acid sequence differences set forth in any one of Table D and / or any of Tables 37.2 or 38.1 to 61.1.

27. The polypeptide of any one of claims 23-25, wherein the amino acid sequence comprises any one of SEQ ID NO: 78, 90, 106. 114, 130, 140, 150, 162, 172, 188, 194, 204, 216, 230, 238.248, 260. 272, 294, 296, 310, 322. 340, 352, 354, 360, or 380.

28. The polypeptide of any one of claims 23-26, further comprising the following amino acid substitution set: H250G, H251S, H252G, H253S, H254G, and H255S.

29. The polypeptide of any one of claims 23-27, further comprising an amino acid insertion of SEQ ID NO: 383 between amino acid positions 1 and 2.

30. The polypeptide of any one of claims 23-29, wherein the amino acid sequence comprises any one of SEQ ID NO: 364, 366, 368, 370. 372, 374, or 376.

31. An engineered polypeptide comprising at least 95% sequence identity to any one of even numbered SEQ ID NO: 78-376, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2.

32. An engineered polypeptide comprising at least 98% sequence identity to any one of SEQ ID NO: 16, 78, 90, 106, 114, 130, 140, 150, 162, 172, 188, 194, 204, 216, 230, 238, 248, 260,272, 294. 296, 310, 322. 340, 352. 354, 360, 364. 366, 368. 370, 372. 374, 376, or 380, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2.

33. A polypeptide comprising the amino acid sequence of any of SEQ ID NO: 16, 78, 90, 106, 114. 130, 140, 150. 162, 172. 188, 194, 204, 216, 230. 238, 248. 260, 272, 294, 296, 310.322, 340, 352, 354, 360, 364, 366, 368, 370, 372, 374, 376, or 380.

34. The polypeptide of any one of claims 23-33, which is isolated.

35. The polypeptide of any one of claims 23-34, wherein the polypeptide is capable of catalyzing an amide bond formation.

36. The polypeptide of any one of claims 23-35. wherein the polypeptide has one or more of the following properties relative to a reference polypeptide comprising the amino acid sequence of SEQ ID NO: 2:a) increased enzyme activity;;b) increased substrate scope;c) increased regioselectivity;d) increased chemoselectivity;e) increased solvent or cosolvent tolerance;f) reduction in side-products;g) reduction in product inhibition;h) increased thermostability;i) increased tolerance to increased substrate loading;j) increased protein expression;k) increased protein solubility; and / orl) increased substrate affinity.

37. A polynucleotide encoding at least one polypeptide set forth in any one of claims 23-36, wherein the polynucleotide does not comprise SEQ ID NO: 1.

38. The polynucleotide of claim 37, wherein the polynucleotide is codon-optimized.

39. The polynucleotide of claim 37 or 38, wherein the polynucleotide comprises SEQ ID NO: 15, 77, 89, 105, 113, 129, 139, 149, 161, 171, 187, 193, 203, 215, 229, 237, 247, 259, 271, 293, 295, 309, 321, 339, 351, 353, 359, 363, 365, 367, 369, 371, 373, 375, or 381.

40. A polynucleotide comprising at least 80% sequence identity to any one of SEQ ID NO: 15, 77, 89, 105, 113, 129, 139, 149, 161, 171, 187, 193, 203, 215, 229, 237, 247, 259, 271, 293, 295, 309, 321, 339, 351, 353, 359, 363, 365, 367, 369, 371, 373, 375, or 381, wherein the polynucleotide does not comprise SEQ ID NO: 1.

41. An expression vector comprising at least one polynucleotide sequence of any one of claims 19-22 or 36-39.

42. The expression vector of claim 41. wherein the polynucleotide sequence is operably linked to a control sequence.

43. The expression vector of claim 42, wherein the control sequence is a promoter.

44. The expression vector of claim 43. wherein the promoter is a heterologous promoter.

45. A host cell comprising the polynucleotide of any one of claims 19-22 or 37-40 or the expression vector of any one of claims 41-44.

46. The host cell of claim 45, wherein the host cell is prokaryotic or eukaryotic.

47. A method of producing a polypeptide, the method comprising culturing the host cell of claim 45 or 46 under conditions such that the polypeptide encoded by the polynucleotide is produced.

48. The method of claim 47, further comprising the step of recovering the polypeptide.

49. The method of claim 48, further comprising the step of purifying the polypeptide.

50. A method of catalyzing an amide bond formation in the presence of the polypeptide of any one of claims 1-18 or 23-36.

51. A method of catalyzing an amidation reaction for septapeptide formation as set forth in Table A, comprising contacting one or more of the substrates listed in Table A with the polypeptide of any one of claims 1-18.

52. A method of producing product 3 in the following Scheme A. which comprises reacting Substrate 1 and Substrate 2 in Scheme A with the polypeptide of any one of claims 23-36, wherein Scheme A is

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