Engineered macrocyclases and methods of using the same

Abstract

The present disclosure provides polypeptides (e.g., macrocyclase polypeptides), polynucleotides and expression vectors comprising the same, methods of producing polypeptides (e.g., macrocyclase polypeptides), and methods of use thereof (e.g., for generation of macrocyclic peptides).

Description

ENGINEERED MACROCYCLASES AND METHODS OF USING THE SAMEFIELD OF THE INVENTION

[0001] This disclosure relates generally to engineered macrocyclases, useful in biocatalytic and synthetic processes involving peptide-peptide coupling and cyclization for the generation of macrocyclic peptides.BACKGROUND OF THE INVENTION

[0002] Enzymes are protein molecules used in nature to catalyze many biological processes. Enzymes in nature display a high degree of efficiency and great specificity for their cognate substrates, making them ideal catalysts for carrying biological functions with high fidelity. Macrocyclase enzymes in particular are commonly found in nature in the biosynthetic pathways of nonribosomal peptides. This class of enzymes is capable of catalyzing amide or ester bond formation through generation of peptidyl-enzyme intermediates that are subsequently attacked by a nucleophile such as an amino or hydroxyl substituent on the substrate molecule or on another peptide molecule. This biocatalytic transformation is particularly attractive due to its regioselectivity and the lack of need of protecting group manipulations.

[0003] Nonribosomal peptide synthetase (NRPS) macrocyclases have found limited use in commercial applications given their dependence on compatible thiol-activated or carrier protein- activated peptides. However, given how selective this class of enzymes can be, they present as excellent catalysts to use in protecting group-free chemistries, simplifying the number of synthetic steps involved in the synthesis of peptides.

[0004] Therefore, there is a need for improved macrocyclases and methods of using the same.SUMMARY OF THE INVENTION

[0005] The present disclosure provides, inter alia, polypeptides (e.g., macrocyclases), polynucleotides encoding the same, expression vectors, host cells, methods of producing polypeptides, and methods of use thereof. The summary' of the technology described above is non-limiting and other features and advantages of the technology' will be apparent from the follow ing detailed description, and from the claims.

[0006] In some embodiments, the macrocyclases and macrocyclase-catalyzed methods of formation of an amide bond or an ester bond provided herein are useful in the production of macrocyclic peptides, e.g., enlicitide. The structure of enlicitide is shown below as Formula VII. In some embodiments, A is decanoate.Formula VII

[0007] In one aspect, provided herein is an engineered polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 6, wherein the polypeptide comprises an amino acid substitution at one or more amino acid positions selected from 5, 6, 7, 8, 9, 11, 12, 14, 15, 17, 18, 19, 20, 31, 45, 59, 80, 82, 87, 89, 94, 115, 119, 122, 151, 163, 164, 172, 204, 212. 218, 220, 223, 238, 244. 245, 250, 257, 258, 259, 267, 269. 275, 281, 284, 285, 294, 299, 300, 301, 303, 313, 325, 327, 328, 351, 354, 387, 389, 390, 391, 399, 405, 406, 407, 408, 410, 418, 429, 432, 433, 434, 436, and 448, 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.

[0008] In some embodiments, the polypeptide comprises from 4 to 74 substitutions, each amino acid substitution at an amino acid position selected from 5, 6, 7, 8, 9, 1 1 , 12, 14, 15, 17, 18, 19, 20, 31, 45, 59, 80, 82, 87, 89, 94, 115, 119, 122, 151, 163, 164, 172, 204, 212, 218, 220, 223, 238, 244, 245, 250, 257, 258, 259, 267, 269, 275, 281, 284, 285, 294, 299, 300, 301, 303, 313, 325, 327. 328, 351, 354, 387, 389. 390, 391, 399, 405, 406, 407, 408. 410, 418, 429, 432, 433. 434, 436, and 448.

[0009] In some embodiments, the polypeptide comprises at least one amino acid substitution selected from Y5I, A6E, D7K, P8T, A9D, Cl IP, G12K, G14I, D15P, A17T, P18D, V19L, G20V, D31G, L45T. R59G, A80M, L82F, D87R, D89L, R94T, S115L, A119L, D122G. A151S, A163R, L164T, P172K, F204L, A212N, T218G, P220Q, L223M, L238C, D244H. E245D, D250T, P257N, D258S, A259I, T267L, G269T, L275F, P281L, G284A, R285V, L294V, T299S, L300R, D301E, A303K, R313T, T325K, Q327Y, A328S, P351I, A354P, L387M, G389N, G390Q, A391Q, A399E. S405V, R406K, G407P, A408F, F410V, A418N, S429G. T432L, L433W, L434V, E436M, and S448H.

[0010] In some embodiments, the polypeptide comprises from 4 to 74 amino acid substitutions selected from Y5I, A6E, D7K, P8T, A9D, CUP, G12K, G14I, D15P, A17T, P18D, V19L, G20V, D31G, L45T, R59G, A80M, L82F, D87R, D89L, R94T, S115L, A119L, D122G, A151S, A163R, L164T, P172K, F204L, A212N, T218G, P220Q, L223M, L238C, D244H, E245D, D250T, P257N, D258S, A259I, T267L, G269T. L275F, P281L, G284A, R285V. L294V, T299S, L300R, D301E, A303K, R313T, T325K, Q327Y, A328S, P351I, A354P, L387M, G389N, G390Q, A391Q, A399E, S405V, R406K, G407P, A408F, F410V, A418N, S429G, T432L, L433W, L434V, E436M, and S448H.

[0011] In some embodiments, the polypeptide comprises all of the following amino acid substitutions: Y5I, A6E, D7K, P8T, A9D, CUP, G12K, G14I, D15P, A17T, P18D, V19L, G20V, D31G, L45T, R59G, A80M, L82F, D87R, D89L, R94T, S115L, A119L, D122G, A151S, A163R, L164T, P172K, F204L, A212N, T218G, P220Q, L223M, L238C, D244H, E245D, D250T, P257N, D258S, A259I, T267L, G269T. L275F, P281L, G284A, R285V. L294V, T299S, L300R, D301E, A303K. R313T. T325K, Q327Y. A328S, P351I. A354P. L387M, G389N. G390Q, A391Q, A399E, S405V, R406K, G407P, A408F, F410V, A418N, S429G, T432L, L433W, L434V, E436M, and S448H.

[0012] In some embodiments, the polypeptide further comprises an amino acid insertion between amino acid positions 390 and 391, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 6.

[0013] In some embodiments, the amino acid insertion comprises the amino acid sequence of GS.

[0014] In some embodiments, the polypeptide comprises one or more amino acid sequence differences set forth in any one of Tables 1.4, 1.5, 2.1, 3.1, 4.1, 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, 12.1, 13.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1, 20.1, 21.1, 22.1, 23.1, 24.1, 25.1, or 26.1.

[0015] In some embodiments, the amino acid sequence comprises any one of SEQ ID NO: 73, 70, 8, 10. 12, 14, 16, 18. 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 41, 44, 46, 48, 51, 54, 57, 61, 64, or 67.

[0016] In some embodiments, the amino acid sequence comprises SEQ ID NO: 73.

[0017] In some embodiments, the amino acid sequence comprises SEQ ID NO: 70.

[0018] In another aspect, provided herein is an engineered polypeptide comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 73.

[0019] In some embodiments, the amino acid sequence has at least 99% sequence identity to SEQ ID NO: 73.26080

[0020] In some embodiments, the polypeptide comprises the ammo acid sequence of SEQ ID NO: 73.

[0021] In another aspect, provided herein is an engineered polypeptide comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%. 98%. 99%. or higher sequence identity to amino acid residues 1-449 of SEQ ID NO: 73.

[0022] In some embodiments, the polypeptide is isolated.

[0023] In some embodiments, the polypeptide is capable of cataly zing an amide bond formation or an ester bond formation.

[0024] In some embodiments, 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 activity7; b) increased substrate scope; c) increased regioselectivity; d) increased chemoselectivity; e) increased solvent or cosolvent tolerance; and / or f) increased thermostability.

[0025] In another aspect, provided herein is a polynucleotide encoding at least one polypeptide as provided herein, wherein the polynucleotide does not comprise SEQ ID NO: 1.

[0026] In some embodiments, the polynucleotide is codon-optimized.

[0027] In some embodiments, the polynucleotide comprises any one of SEQ ID NO: 7. 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 40, 42, 43, 45, 47, 49, 50, 52, 53, 55, 56, 58,59, 60, 62, 63, 65, 66, 68, 69, 71, or 72.

[0028] In another aspect, provided herein is a polynucleotide comprising at least 80% sequence identity to any one of SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33. 35. 37, 39,40. 42. 43. 45, 47, 49, 50, 52, 53, 55, 56, 58, 59. 60. 62. 63. 65. 66. 68, 69, 71, or 72. wherein the polynucleotide does not comprise SEQ ID NO: 1.

[0029] In some embodiments, the polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 71 or 72.

[0030] In some embodiments, the polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 68 or 69.

[0031] In another aspect, provided herein is an expression vector comprising at least one polynucleotide sequence provided herein.

[0032] In some embodiments, the polynucleotide sequence is operably linked to a control sequence.

[0033] In some embodiments, the control sequence is a promoter.

[0034] In some embodiments, the promoter is a heterologous promoter.26080

[0035] In another aspect, provided herein is a host cell comprising any one of the polynucleotides disclosed herein or any one of the expression vectors disclosed herein.

[0036] In some embodiments, the host cell is prokaryotic or eukaryotic.

[0037] In another aspect, 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.

[0038] In some embodiments, the method further comprises a step of recovering the polypeptide.

[0039] In some embodiments, the method further comprises a step of purifying the polypeptide.

[0040] In another aspect, provided herein is a method of catalyzing an amide bond formation or an ester bond in the presence of any one of the polypeptides disclosed herein.

[0041] In some embodiments, the method produces a cy clic peptide.DETAILED DESCRIPTION OF THE INVENTION

[0042] The present disclosure relates to engineered macrocyclase enzymes. Described herein is the use of protein engineering to improve enzyme activity, substrate scope, selectivity, organic solvent tolerance and thermostability of several macrocyclases with the ability to form 11-mer polypeptides or 2b. Such enzymes maybe useful in the preparation of complex biological compounds or their intermediates. Such enzymes may be particularly useful in synthetic processes that may be used as part of the preparation of 6-({[(HS,17S,20S,23S,27S,39S,42S,63S,66R)-47-Fluoro-20-[(lR)-l-hydroxyethyl]-17-[(4- methoxylphenyl)methyl]-l l,63-dimethyl-10,16,19,22,30,40,58,61,64,67,70-undecaoxo-28-oxa- 1,9,15.18,21.24,31,41.51,62.65,68- dodecaazanonacyclo[37.18.11.23,6.124,42.133,37.144,51.011,15.023,37.045,50]triheptaconta- 3, 5, 33(71), 34, 36, 44(60)45, 47,49, 72-decaen-66-yl]methyl}amino)-N,N,N-trimethyl-6-oxohexan- 1-aminium decanoate [2b] or of intermediates formed during preparation of such compounds.

[0043] The present disclosure relates to NRPS macrocyclase enzymes capable of catalyzing amide bond formation in macrocycles or 11-mer peptides in a protecting group-free manner from activated aminoacyl-thioesters, aminoacyl-esters and cyclic peptides. In embodiments, the NRPS macrocyclase enzymes described herein can couple two peptides that can be further used in the synthesis of multimer peptides or can macrocyclize linear peptides. In some embodiments, the NRPS macrocyclase enzymes maybe useful in the preparation of compounds such as 3e or 2b.

[0044] As is described in the Examples, the present disclosure provides engineered macrocyclase polypeptides discovered through iterative rounds of directed evolution from a wild-type macrocyclase polypeptide from Streptomyces sp. KCB13F003 (SEQ ID NO: 2) which exhibited improved enzyme properties relative to wild-type Streptomyces sp. KCB13F003 NRPS macrocyclase, and which were discovered through iterative rounds of directed evolution as described herein.

[0045] The macrocyclase polypeptides of the present disclosure provide significant advantages. For example, the engineered macrocyclase polypeptides of the disclosure may exhibit one or more of the following improvements relative to a reference polypeptide (e.g., wild-ty pe Streptomyces sp. KCB13F003 NRPS macrocyclase polypeptide or a polypeptide of, e.g., SEQ ID NO: 4, 6. or 48): 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 side products, 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 macrocyclase polypeptide variants with enhanced activity relative to the wildtype Streptomyces sp. KCB13F003 NRPS macrocyclase polypeptide. As one example, the engineered macrocyclase polypeptide having the amino acid sequence of SEQ ID NO: 73 had a 436 million-fold improvement in enzyme activity compared to the corresponding wild-type sequence of SEQ ID NO: 6.Definitions

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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).

[0050] As used herein, the terms "at least one” item or “one or more” item each include a single item selected from a list as well as combinations of two or more items selected from a list (e g., a list of mutations). Similarly, the terms “at least two” items and “two or more” items each include combinations of tw o items selected from a list as well as combinations of three or more items selected from a list.

[0051] All ranges disclosed herein are inclusive of the recited endpoint and independently combinable (for example, the range of “from 50 mg to 500 mg” is inclusive of the endpoints, 50 mg and 500 mg, and all the intermediate values). The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value; they are sufficiently imprecise to include values approximating these ranges and / or values.

[0052] 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.

[0053] “Derived from” as used herein in the context of enzymes, identifies the originating enzyme, and / or the gene encoding such enzy me, upon which the enzy me was based. For example, the engineered macrocyclase enzyme of SEQ ID NO: 73 was obtained by artificially evolving over multiple generations the gene encoding the wild-type NRPS macrocyclase enzyme of SEQ ID NO: 6. Thus, the evolved engineered macrocyclase enzyme of SEQ ID NO: 73 is “derived from” the wild-ty pe NRPS macrocyclase of SEQ ID NO: 6.

[0054] As used herein, “polynucleotide” and “nucleic acid’ refer interchangeably 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' deoxy ribonucleotides. While the nucleosides will ty pically be linked together via standard phosphodiester linkages, the polynucleotides may include one or more non-standard linkages. The polynucleotide may be single-stranded or double-stranded, or the polynucleotide may include both single-stranded regions and doublestranded regions. Moreover, w hile a polynucleotide will typically7be composed of the naturally occurring encoding nucleobases (i.e., adenine, guanine, uracil, thymine, and cytosine), it mayinclude 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] The abbreviations used for the genetically encoded amino acids are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartate (Asp or D), cysteine (Cys or C), glutamate (Glu or E), glutamine (Gin or Q), histidine (His or H), isoleucine (He or I), leucine (Leu or L). lysine (Lys or K), methionine (Met or M). phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Vai or V).

[0059] “Hydrophilic amino acid” or “hydrophilic residue” refers to an amino acid or residue having a side chain exhibiting a hydrophobicity of less than zero according to the normalized consensus hydrophobicity scale of Eisenberg et al., 1984, J. Mol. Biol. 179: 125-142. Genetically encoded hydrophilic amino acids include L-Thr (T), L-Ser (S), L-His (H), L-Glu (E), L-Asn (N), L-Gln (Q), L-Asp (D), L-Lys (K), and L-Arg (R).

[0060] "Acidic amino acid7’ or "acidic residue” refers to a hydrophilic amino acid or residue having a side chain exhibiting a pKa value of less than about 6 when the amino acid is included in a peptide or polypeptide. Acidic amino acids typically have negatively charged side chains at physiological pH due to loss of a hydrogen ion. Genetically encoded acidic amino acids include L-Glu (E) and L-Asp (D).

[0061] “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).

[0062] “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).

[0063] “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).

[0064] “Aromatic amino acid” or “aromatic residue” refers to a hydrophilic or hy drophobic amino acid or residue having a side chain that includes at least one aromatic or heteroaromatic ring. Genetically encoded aromatic ammo 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.

[0065] 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.

[0066] “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).26080

[0067] 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).

[0068] 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.

[0069] 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 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).

[0070] “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).

[0071] As used herein, “conservative amino acid substitution” refers to a substitution of a residue with a different residue having a similar side chain, and thus typically involves substitution of the amino acid in the polypeptide with amino acids within the same or similar defined class of amino acids. By way of example and not limitation, in some embodiments, an amino acid with an aliphatic side chain is substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with an hydroxyl side chain is substituted with another amino acid with an hydroxyl side chain (e.g., serine and threonine); an amino acid having aromatic side chains is substituted with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain is substituted with another amino acid with 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.26080

[0072] As used herein, "non -conservative substitution7’ 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 affects (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.

[0073] "‘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.

[0074] 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, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids making up the reference polypeptide while retaining activity (e.g., enzymatic activity ) and / or retaining the improved properties of an evolved polypeptide. Deletions can be directed to the internal portions and / or terminal portions of the polypeptide. In various embodiments, the deletion can comprise a continuous segment or can be discontinuous. Deletions are typically indicated by in amino acid sequences.

[0075] As used herein, “insertion” refers to modification to a polypeptide by addition of one or more amino acids relative to a reference polypeptide (e.g., enzyme). Insertions can be in the internal portions of the polypeptide, or to the carboxy or amino terminus. Insertions as used herein include fusion proteins. The insertion can be a contiguous segment of amino acids or separated by one or more of the amino acids in the naturally occurring polypeptide.

[0076] As used herein with respect to amino acid sequences, a “substitution” refers to a difference in the amino acid residue at a position of a polypeptide sequence relative to the amino acid residue at a corresponding position in a reference sequence. In some instances, the present disclosure provides specific amino acid differences denoted by the conventional notation “AnB,” where A is the single letter identifier of the residue in the reference sequence, n is the number of the residue position in the reference sequence, and B is the single letter identifier of the residue substitution in the sequence of the engineered polypeptide.26080

[0077] The term "ammo acid substitution set” or “substitution set” refers to a group of amino acid substitutions in a polypeptide sequence, as compared to a reference sequence. For example, a substitution set may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 74, 75, 80, or more amino acid substitutions. When used in reference to a polypeptide comprising a substitution set (e.g., a polypeptide comprising “a substitution set at positions 148, 157, and 246”), the polypeptide would comprise (and the substitution set would include) substitutions at each of the positions that followed (e.g., in the foregoing example at each of positions 148, 157, and 246).

[0078] “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 gaps are present, the numbering of the residue in the given amino acid or polynucleotide sequence is made with respect to the reference sequence to which it has been aligned.

[0079] 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.

[0080] 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.

[0081] 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 species26080 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.

[0082] "‘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 is generally made to the wild-type enzyme, although in some embodiments, the reference enzyme can be another improved enzyme. Enzy me 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’, stability under high ammonia concentration, soluble expression, pH activity profile, cofactor requirements, refractoriness to inhibitors (e.g., product inhibition), stereospecificity', and stereoselectivity (including enantioselectivity).

[0083] “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 Km. Nmax,changes of which can lead to increased enzymatic activity'. Improvements in enzy me activity' can be from about 1.5 times the enzymatic activity of the corresponding wild-type enzyme, to as much as 2 times, 5 times, 10 times, 20 times, 25 times, 50 times, 75 times, 100 times, 150 times, 200 times, 500 times, 1000 times, 3000 times, 5000 times, 7000 times, 8000 times, 9000 times, or more enzymatic activity' than the reference enzy me, e.g., a naturally occurring enzy me or another enzy me from which the26080 polypeptides were derived. In some examples, the enzyme exhibits improved enzymatic activity in the range of 100 to 3000 times, 3000 to 7000 times, 3000 to 9000 times, more than 7000 times, or more than 9000 times greater than that of the parent enzy me. It is understood by the skilled artisan that the activity of any enzy me is diffusion limited such that the catalytic turnover rate cannot exceed the diffusion rate of the substrate, including any required cofactors. The theoretical maximum of the diffusion limit, or kc«r / Km, is generally about 108to 109(M"1s"1). Hence, any improvements in the enzyme activity will have an upper limit related to the diffusion rate of the substrates acted on by the enzyme. Enzyme activity' can be measured by any suitable approach, e.g., an enzyme activity assay or by any of 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 enzyme 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 enzy me produced by the host cells and present in the lysates.

[0084] 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.

[0085] As used herein with respect to polypeptides, the terms “expression” and “production” includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from a cell.

[0086] 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.26080

[0087] 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 macrocyclase 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.

[0088] The term “analogue” means a polypeptide having more than 70% sequence identity but less than 100% sequence identity (e.g., more than 75%, 78%, 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity ) with a reference polypeptide. In some embodiments, “analogues” means polypeptides that contain one or more non-naturally occurring amino acid residues including, but not limited, to homoarginine, ornithine and norvaline, as well as naturally occurring amino acids. In some embodiments, analogues also include one or more D-amino acid residues and non-peptide linkages between two or more amino acid residues.

[0089] “Coding sequence” refers to that portion of a polynucleotide (e.g., a gene) that encodes an amino acid sequence of a polypeptide.

[0090] “Naturally occurring” or “wild-ty pe” refers to a form found in nature. For example, a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a source in nature and that has not been intentionally modified by human manipulation, with the sole exception that wild-type polypeptide or polynucleotide sequences as identified herein may include a tag, such as a histidine (His) tag. For example, the macrocyclase sequence of SEQ ID NO: 4, which includes an N-terminal His tag (and thus is not found in nature), may be referred to as a wild-type macrocyclase herein. Herein, “wild-type” polypeptide or polynucleotide sequences may be denoted “WT.”

[0091] “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.

[0092] 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.26080

[0093] 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.

[0094] 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. Exemplary7selectable markers are described herein.

[0095] “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 identity7. Determination of optimal alignment and percent sequence identity7can 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.

[0096] 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 hits26080 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).

[0097] 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, Current Protocols in Molecular Biology', 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.

[0098] "‘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 stereoisomer is formed. When the stereoisomers are enantiomers, the stereoselectivity is referred to as enantioselectivify, 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.26080

[0099] “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 “enzy matic activity” or “activity” of a polypeptide can be expressed as “percent conversion” of the substrate to the product.

[0100] “Thermostable” refers to a polypeptide that maintains similar activity (more than 60% to 80%, for example) after exposure to elevated temperatures (e.g., 40 °C to 80 °C) for a period of time (e g., 0.5 h to 24 h) compared to the untreated enzy me.

[0101] As used herein, the terms “biocatalysis,” “biocatalytic.” “biotransformation.” and “biosynthesis” refer to the use of enzymes to perform chemical reactions on organic compounds.

[0102] 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.

[0103] The terms “isolated” and “punfied” are used to refer to a molecule (e.g., an isolated nucleic acid or polypeptide) or other component that is removed from at least one other component with which it is naturally associated. The term “purified” does not require absolute purity', rather it is intended as a relative definition.

[0104] The terms “macrocyclase enzyme” or “macrocyclase polypeptide” refer to a polypeptide having a capability of catalyzing an intramolecular amide or ester bond formation in the generation of macrocyclic peptides. In some embodiments, a macrocyclase enzyme is useful in the synthesis of 2b and / or its intermediates. Macrocyclase polypeptide as used herein includes naturally occurring (wild-type) macrocyclase polypeptides as well as non-naturally occurring engineered polypeptides generated by human manipulation. An exemplary wild-type macrocyclase enzyme known as Ulml6 is described, e g., in SEQ ID NO: 1 (nucleic acid sequence), SEQ ID NO: 2 (amino acid sequence), Genbank Accession ID ATU31793.1, and Uniprot ID A0A2D3E317. In an embodiment, a macrocyclase enzyme as disclosed herein may have an amino acid sequence selected from any one of SEQ ID NO: 8, 10, 12, 14, 16, 18. 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 41, 44, 46, 48, 51, 54, 57, 61, 64, 67, 70, or 73.Engineered Macrocyclase Polypeptides

[0105] This disclosure provides polypeptides (e.g., macrocyclase polypeptides) capable of catalyzing amide bond formation in the generation of macrocyclic peptides useful in the synthesis of 2b and / or its intermediates. Such compounds may be useful as active pharmaceutical ingredients or in the synthesis of their intermediates.26080

[0106] In certain embodiments, a polypeptide (e.g., a macrocyclase polypeptide) described herein has an amino acid sequence comprising one or more amino acid differences as compared to a reference amino acid sequence of a wild-type polypeptide that result in an improved enzyme property.

[0107] In some embodiments, the NRPS macrocylase enzymes described herein are the product of directed evolution from a wild-type NRPS macrocyclase from Streptomyces sp. K.CB13F003 described in literature by Son et al. (J. Nat. Prod. 2017, 80, 3025-3031) and Matsuda, Zhai, et al. (Nat. Catal. 2020, 3, 507-515). Such protein is encoded by a polynucleotide of SEQ ID NO: 1 and a polypeptide sequence as set forth below in SEQ ID NO: 2:MHGDYADPADCGAGDGAPVGLDLDRLARDCDVVGGQLALHHQGTLTTWEFGTEEHA GGRPVHVGSAFPYGSVTKAFTATAVLQLAGDGDLDLDRPVRELLPEAEAEAEIEVEAGS GTGARADGGHPALAATLRQLLSHTAGLPSDHDDERAPSLRRWLTGFLALPVGPWPAPG SFSYSNVGYGIAGRVVEAVTGLTWSEAVRDFLLHPLGTAITVLPTDPGSLPAGGLAGSAA DLVRLGRLHLDEPGDPDLARLADPDALREMARPTAGADPFGLADGWGPGLGRFGPAGNRWLGHDGTLDGATCHLRIHPGRGTVVALTTNSPTGQALWDAVVDALRDADIDVGVH RPAPPPAIAAAAFADCTGTYRNGDLAVTVGIDGPYLVLELPGGARELAQPLAHRTFSSRG AGFLGRFVTDARSDAVHALQYSGRTLLREAGESRRARSH (SEQ ID NO: 2).

[0108] In some examples, the polynucleotide of the wild-type NRPS macrocyclase enzyme described herein and used to discover the desired reactions has been codon optimized for production in specific hosts such as E. coli and a short poly-His polypeptide has been added at the N-terminus. Such construct is encoded by polynucleotide of SEQ ID NO: 3 and a polypeptide sequence as set forth below in SEQ ID NO: 4:MGSSHHHHHHSSGHGDYADPADCGAGDGAPVGLDLDRLARDCDVVGGQLALHHQGT LTTWEFGTEEHAGGRPVHVGSAFPYGSVTKAFTATAVLQLAGDGDLDLDRPVRELLPE AEAEAEIEVEAGSGTGARADGGHPALAATLRQLLSHTAGLPSDHDDERAPSLRRWLTGF LALPVGPWPAPGSFSYSNVGYGIAGRVVEAVTGLTWSEAVRDFLLHPLGTAITVLPTDP GSLPAGGLAGSAADLVRLGRLHLDEPGDPDLARLADPDALREMARPTAGADPFGLADGWGPGLGRFGPAGNRWLGHDGTLDGATCHLRIHPGRGTVVALTTNSPTGQALWDAVVD ALRDADIDVGVHRPAPPPAIAAAAFADCTGTYRNGDLAVTVGIDGPYLVLELPGGAREL AQPLAHRTFSSRGAGFLGRFVTDARSDAVHALQYSGRTLLREAGESRRARSH (SEQ ID NO: 4).

[0109] In other examples, the polynucleotide of the wild-type NRPS macrocyclase enzyme described herein and subsequently used for directed evolution has been codon optimized for production in specific hosts such as E. coli and a short poly-His polypeptide added at the C-26080 terminus. Such construct is encoded by polynucleotide of SEQ ID NO: 5 and a polypeptide sequence as set forth below in SEQ ID NO: 6: MHGDYADPADCGAGDGAPVGLDLDRLARDCDVVGGQLALHHQGTLTTWEFGTEEHA GGRPVHVGSAFPYGSVTKAFTATAVLQLAGDGDLDLDRPVRELLPEAEAEAEIEVEAGS GTGARADGGHPALAATLRQLLSHTAGLPSDHDDERAPSLRRWLTGFLALPVGPWPAPG SFSYSNVGYGIAGRVVEAVTGLTWSEAVRDFLLHPLGTAITVLPTDPGSLPAGGLAGSAA DLVRLGRLHLDEPGDPDLARLADPDALREMARPTAGADPFGLADGWGPGLGRFGPAG NRWLGHDGTLDGATCHLRIHPGRGTVVALTTNSPTGQALWDAVVDALRDADIDVGVH RPAPPPAIAAAAFADCTGTYRNGDLAVTVGIDGPYLVLELPGGARELAQPLAHRTFSSRG AGFLGRFVTDARSDAVHALQYAGRTLLREAGESRRARSHGSHHHHHH (SEQ ID NO: 8)

[0110] In embodiments, the NRPS macrocyclase enzy mes described herein are the result of directed evolution from an NRPS macrocyclase having the amino acid sequence of SEQ ID NO: 6. In embodiments, such enzymes are capable of cyclizing peptides of Formula I to produce cyclic peptides of Formula II as depicted in Table 1. 1. In particular embodiments, SEQ ID NO: 6 showed no detectable activity for compounds from entry C and therefore directed evolution was necessary'.[OHl] In embodiments, NRPS macrocyclase enzymes of the disclosure may demonstrate improvements relative to the NRPS macrocyclase enzyme of SEQ ID NO: 6. such as increases in enzyme activity, regioselectivity, thermostability, organic cosolvent tolerance, and reduction in competing side reactions. The improvements can relate to a single enzy me property, such as enzymatic activity, or a combination of different enzyme properties, such as enzy matic activity' and regioselectivity’.

[0112] In embodiments, NRPS macrocyclase enzymes of the disclosure may demonstrate increased enzyme activity relative to a reference polypeptide (e.g., the NRPS macrocyclase enzyme of SEQ ID NO: 6), e.g., 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 1000-fold, 10,000-fold, 100.000-fold, 1.000,000-fold. 10.000,000-fold, or higher enzyme activity relative to the reference polypeptide. Enzy me activity may be measured using any suitable approach, including any approach described herein, e.g., as described in the Examples.

[0113] In some embodiments, NRPS macrocyclase enzy mes of the disclosure may demonstrate improved activity in the conversion of various peptidyl-thioesters and peptidyl-esters, including the conversion of compound 1c to the cyclic compound 2b as depicted in Table 1.1, Entry C relative to a reference polypeptide (e.g., the NRPS macrocyclase enzyme of SEQ ID NO: 6), e.g., 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-26080 fold, 50-fold. 60-fold, 70-fold. 80-fold, 90-fold. 100-fold. 1000-fold, 10,000-fold, lOO.OOO-fold. 1,000,000-fold, 10,000,000-fold, or higher enzyme activity relative to the reference polypeptide.

[0114] In some embodiments, NRPS macrocyclase enzymes of the disclosure may demonstrate improved activity in the coupling of various peptidyl-esters to other peptides, including the conversion of compound le and 2e to the cyclic compound 3e, as depicted in Table 1.2, Entry E relative to a reference polypeptide (e.g., the NRPS macrocyclase enzyme of SEQ ID NO: 6), e.g., 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40- fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 1000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold, or higher enzyme activity relative to the reference polypeptide.

[0115] In embodiments, NRPS macrocyclase enzymes of the disclosure may demonstrate increased regioselectivity relative to a reference polypeptide (e.g., the NRPS macrocyclase enzy me of SEQ ID NO: 6), e.g., 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold. 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 1000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold, or higher regioselectivity relative to the reference polypeptide. Regioselectivity may be measured using any suitable approach, including any approach described herein, e.g., as described in the Examples.

[0116] In embodiments, NRPS macrocyclase enzymes of the disclosure may demonstrate increased thermostability’ relative to a reference polypeptide (e.g., the NRPS macrocyclase enzyme of SEQ ID NO: 6), e.g., a melting temperature (Tm) that is increased by 1 °C, 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 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, or higher relative to the reference polypeptide. Thermostability may be measured using any suitable approach, including any approach described herein, e.g., as described in the Examples. In some embodiments, thermostability is measured using isothermal titration calorimetry (ITC).

[0117] The present disclosure provides numerous exemplary’ NRPS macrocyclases capable of generating cyclic peptides shown in Table 1.1. Those exemplary' polypeptides were evolved from SEQ ID NO: 6 and exhibit improved properties, particularly improved activity in the conversion of various peptidyl-thioesters and peptidyl-esters, including the conversion of compound 1c to the cyclic compound 2b as depicted in Table 1.1, Entry C. The present disclosure also provides numerous exemplary NRPS macrocyclases capable of generating coupled peptides shown in Table 1.2. Those exemplary polypeptides were evolved from SEQ ID NO: 6 and exhibit improved properties, particularly improved activity in the coupling of various peptidyl-esters to other peptides, including the conversion of compound le and 2e to the cyclic compound 3e, as depicted in Table 1.2, Entry’ E. Selectivity was also evolved to perform the reactions depicted in26080Tables 1.1 and 1.2 in the presence of competing reagents from process streams, shown in Table 1.3. These exemplary engineered NRPS macrocyclase enzymes having macrocyclizing or intermolecular coupling activity have amino acid sequences that include two or more residue differences as compared to SEQ ID NO: 6 as depicted in the accompanying sequence listing with sequence identifiers SEQ ID NO: 8, 10, 12, 14, 16, 18. 20, 22, 24, 26, 28, 30. 32. 34. 36. 38. 41, 44, 46, 48, 51, 54, 57, 61, 64, 67, 70, or 73.2608026080

[0118] Table 1.4 below provides exemplary7engineered NRPS macrocyclase polypeptides with the ability to form macrocyclic peptides. Each row7lists multiple SEQ ID NOs, with the last number referring to the amino acid sequence that may be provided by one or more nucleotide sequences. For example, the amino acid sequence of SEQ ID NO: 44 is encoded by SEQ ID NO: 42 and SEQ ID NO: 43. The residue differences are based on comparison to a reference sequence such as of SEQ ID NO: 6, an NRPS macrocyclase derived from Streptomyces sp. KCB13F003 that differs from the naturally occurring enzyme (SEQ ID NO: 2) in having a codon optimized sequence for ty, coli expression and a short hexahistidine tag (SEQ ID NO: 76) at the C-terminus. The column listing the number of mutations (i.e., residue changes) refers to the number of amino acid substitutions as compared to the NRPS thioesterase enzy me of SEQ ID NO: 6. Polypeptide SEQ ID NO: 48 contains a two-residue insertion and all subsequent polypeptides derived from this lineage identified by screening for Reaction tyRxn”) C are listed in Table 1.5 relative to SEQ ID NO: 48.260802608026080260802608026080

[0119] Table 1.6 provides a summary of mutations present in the amino acid sequences of SEQ ID NO: 48 onwards compared to SEQ ID NO: 6.Table 1.6: Summary of Amino Acid Differences Relative to SEQ ID NO: 626080

[0120] In some embodiments, a polypeptide (e.g., macrocyclase polypeptide) of the disclosure is a polypeptide that comprises an amino acid sequence having 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 SEQ ID NO: 2, 4, or 6.

[0121] These differences between these variants and SEQ ID NO:2, 4, or 6 can be amino acid insertions, deletions, substitutions, or any combinations of such changes. In some embodiments, the amino acid sequence differences can comprise non-conservative, conservative, as well as a combination of non-conservative and conservative amino acid substitutions. In some embodiments, the amino acid sequence differences are conservative amino acid substitutions. In other embodiments, the amino acid sequence differences are non-conservative amino acid substitutions.

[0122] In some embodiments, the amino acid sequence comprises or consists of any amino acid sequence set forth in Table 1 .4 or Table 1 .5.

[0123] In some embodiments, the amino acid sequence comprises or consists of any one of SEQ ID NO: 8, 10, 12, 14, 16, 18. 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 41, 44. 46, 48, 51, 54, 57, 61, 64, 67, 70, or 73. In some embodiments, the amino acid sequence comprises SEQ ID NO:2608073. In some embodiments, the amino acid sequence consists of SEQ ID NO: 73. In some embodiments, the amino acid sequence comprises SEQ ID NO: 70. In some embodiments, the amino acid sequence consists of SEQ ID NO: 70.

[0124] For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 8. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 8. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 8. Also provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 8. Also provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 8.

[0125] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%. 93%. 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 10. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 10. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 10. Also provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 10. Also provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 10.

[0126] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%. 92%. 93%. 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 12. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 12. Namely, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 12. Moreover, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 12. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 12.

[0127] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 14. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at26080 least 99% sequence identity to SEQ ID NO: 14. Specifically, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 14. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 14. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 14.

[0128] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 16. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 16. Notably, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 16. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 16. Moreover, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 16.

[0129] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%. 93%, 94%, 95%. 96%, 97%, 98%, 99%. or higher sequence identity to SEQ ID NO: 18. For instance, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 18. Moreover, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 18.Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 18. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 18.

[0130] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%. 91%. 92%. 93%. 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 20. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 20. Specifically, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 20. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 20. Furthermore, provided herein is a26080 polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 20.

[0131] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%. 91%. 92%. 93%. 94%, 95%, 96%, 97%, 98%, 99%. or higher sequence identity to SEQ ID NO: 22. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 22. Namely, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 22. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 22. Moreover, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 22.

[0132] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%. 91%. 92%. 93%. 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 24. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 24. Specifically, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 24. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 24. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 24.

[0133] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%. 92%. 93%. 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 26. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 26. Notably, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 26. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 26. Moreover, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 26.

[0134] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 28. For instance, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at26080 least 99% sequence identity to SEQ ID NO: 28. Moreover, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 28. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 28. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 28.

[0135] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 30. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 30. Specifically, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 30. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 30. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 30.

[0136] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%. 91%. 92%. 93%. 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 32. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity7to SEQ ID NO: 32. Notably, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 32. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 32. Moreover, provided herein is a polypeptide (e g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 32.

[0137] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%. 91%. 92%. 93%. 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity7to SEQ ID NO: 34. For instance, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 34. Moreover, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 34. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 34. Furthermore, provided herein is a26080 polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 34.

[0138] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%. 91%. 92%. 93%. 94%, 95%, 96%, 97%, 98%, 99%. or higher sequence identity to SEQ ID NO: 36. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 36. Specifically, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 36. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 36. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 36.

[0139] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%. 93%. 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 38. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 38. Specifically, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 38. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 38. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 38.

[0140] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 41. For instance, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 41. Moreover, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 41. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 41. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 41.

[0141] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,2608099%, or higher sequence identity to SEQ ID NO: 44. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 44. Specifically, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 44. Furthermore, provided herein is a polypeptide (e.g.. a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 44. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 44.

[0142] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 46. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 46. Specifically, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 46. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 46. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 46.

[0143] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 48. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 48. Specifically, provided herein is a polypeptide (e g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 48. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 48. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 48.

[0144] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 51. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 51. Specifically, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 48.26080Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 51. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 51.

[0145] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 54. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 54. Specifically, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 54. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 54. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 54.

[0146] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 57. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 57. Specifically, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 57. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 57. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 57.

[0147] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%. 91%. 92%. 93%. 94%, 95%, 96%, 97%, 98%, 99%. or higher sequence identity to SEQ ID NO: 61. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 61. Specifically, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 61. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 61. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 61.26080

[0148] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 64. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 64. Specifically, provided herein is a polypeptide (e g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 64. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 64. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 64.

[0149] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 67. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 67. Specifically, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 67. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 67. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 67.

[0150] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%. 91%. 92%. 93%. 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 70. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 70. Specifically, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 70. Furthermore, provided herein is a polypeptide (e.g.. a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 70. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 70.

[0151] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 73. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at26080 least 99% sequence identity to SEQ ID NO: 73. Specifically, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 73. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 73. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 73.

[0152] In some embodiments, the polypeptide further comprises a tag (e.g., an epitope tag). Any suitable tag may be used, e.g., a His tag, a FLAG tag, a fluorescent protein tag (e.g., green fluorescent protein (GFP), yellow fluorescent protein (YFP), or red fluorescent protein (RFP)), a hemagglutinin (HA) tag, an ALFA-tag, a V5-tag, a Myc-tag, a SPOT-tag, a T7-tag, or an NE-tag. In some embodiments, the epitope tag is a His tag. In some embodiments, the His tag comprises the amino acid sequence of GSHHHHHH (SEQ ID NO: 74). In some embodiments, the polypeptide comprises an N-terminal methionine residue, and the epitope tag is inserted immediately following the N-terminal methionine residue, e.g., relative to a reference sequence.

[0153] In some embodiments, the polypeptide comprises one or more substitutions set forth in any one of Tables 1.4 or 1.5.

[0154] In some embodiments, the polypeptide does not comprise the sequence of SEQ ID NO: 2, 4, or 6.

[0155] In another embodiment, provided herein is an engineered polypeptide comprising at least 98% sequence identity to any one of SEQ ID NO: 8, 10, 12, 14, 16, 18. 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 41, 44, 46, 48, 51, 54, 57, 61, 64, 67, 70, or 73, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2, 4, or 6.

[0156] 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: 76) (e.g., including GSHHHHHH (SEQ ID NO: 74) or GHHHHHHH (SEQ ID NO: 75)).

[0157] 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: 76) (e.g., including GSHHHHHH (SEQ ID NO: 74) or GHHHHHHH (SEQ ID NO: 75)).

[0158] In some embodiments, the polypeptide is isolated.

[0159] In some embodiments, the polypeptide is a macrocyclase (e.g., a NRPS macrocyclase).

[0160] 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.26080

[0161] Also provided herein are compositions comprising any of the polypeptides disclosed herein. For example, the composition may include an effective amount of the polypeptide. The composition may include one or more carriers or diluents.Polynucleotides Encoding Macrocvclase Polypeptides

[0162] In another aspect, the present disclosure provides polynucleotides encoding the polypeptides (e.g., macrocyclase polypeptides, such as NRPS macrocyclase enzy mes) 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., macrocyclase polypeptide) can be introduced into appropriate host cells to express the corresponding polypeptide.

[0163] Because of the knowledge of the codons corresponding to the various amino acids, availability of a protein sequence provides a description of all the polynucleotides capable of encoding the subject. The degeneracy of the genetic code, where the same amino acids are encoded by alternative or synonymous codons allows an extremely large number of nucleic acids to be made, all of which encode the polypeptides (e.g., macrocyclase 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.

[0164] 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: 1 has been codon optimized for expression in E. coli to yield SEQ ID NO: 3 and SEQ ID NO: 5.

[0165] In certain embodiments, all codons need not be replaced to optimize the codon usage of the polypeptide (e.g., macrocyclase 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.,26080 macrocyclase 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.

[0166] Provided herein is a polynucleotide that encodes a polypeptide comprising an amino acid sequence selected from SEQ ID NO: 8, 10, 12, 14, 16, 18. 20, 22, 24, 26, 28, 30, 32, 34, 36, 38. 41, 44, 46, 48, 51, 54, 57, 61, 64, 67, 70, or 73.

[0167] For example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23. 25. 27, 29, 31, 33, 35, 37, 39, 40, 42, 43, 45, 47, 49, 50. 52. 53.55, 56, 58, 59, 60, 62, 63, 65, 66, 68, 69, 71, or 72. In some examples, the polynucleotide sequence is modified to no longer code for an N-terminal His tag or C-terminal His tag.

[0168] For example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 3.In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 3. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 3.

[0169] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%. 81%. 82%. 83%. 84%. 85%. 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%.96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 5. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 5. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 5.

[0170] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%. 81%. 82%. 83%. 84%. 85%. 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%.96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 7. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 7. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 7.

[0171] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 9. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 9. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 9.

[0172] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity' to the polynucleotide sequence of SEQ ID26080NO: 11. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 11. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 11.

[0173] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 13. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 13. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 13.

[0174] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%. 82%, 83%. 84%. 85%. 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 15. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 15. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 15.

[0175] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%. 81%. 82%. 83%. 84%. 85%. 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%.96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 17. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 17. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 17.

[0176] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 19. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 19. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 19.

[0177] In another example, provided herein is a polynucleotide compnsing 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 identity7to the polynucleotide sequence of SEQ ID NO: 21. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 21. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 21.

[0178] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity' to the polynucleotide sequence of SEQ ID NO: 23. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 23. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 23.

[0179] In another example, 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%,2608096%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 25. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 25. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 25.

[0180] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%. 81%. 82%. 83%. 84%. 85%. 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 27. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 27. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 27.

[0181] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 29. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 29. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 29.

[0182] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 31. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 31. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 31.

[0183] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 33. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 33. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 33.

[0184] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 35. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 35. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 35.

[0185] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%, 99%. or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 37. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 37. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 37.26080

[0186] In another example, provided herein is a polynucleotide compnsing 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 identity7to the polynucleotide sequence of SEQ ID NO: 39. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 39. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 39.

[0187] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity' to the polynucleotide sequence of SEQ ID NO: 40. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 40. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 40.

[0188] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 42. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 42. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 42.

[0189] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%.96%. 97%. 98%. 99%. or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 43. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 43. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 43.

[0190] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%. 85%, 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 45. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 45. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 45.

[0191] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%. 81%. 82%. 83%. 84%. 85%. 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%.96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 47. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 47. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 47.

[0192] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID26080NO: 49. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 49. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 49.

[0193] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 50. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 50. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 50.

[0194] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%. 82%, 83%. 84%. 85%. 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 52. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 52. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 52.

[0195] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%. 81%. 82%. 83%. 84%. 85%. 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 53. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 53. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 53.

[0196] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 55. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 55. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 55.

[0197] In another example, provided herein is a polynucleotide compnsing 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 identity7to the polynucleotide sequence of SEQ ID NO: 56. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 56. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 56.

[0198] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity' to the polynucleotide sequence of SEQ ID NO: 58. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 58. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 58.

[0199] In another example, 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%,2608096%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 59. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 59. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 59.

[0200] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%. 81%. 82%. 83%. 84%. 85%. 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 60. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 60. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 60.

[0201] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 62. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 62. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 62.

[0202] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 63. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 63. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 63.

[0203] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 65. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 65. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 65.

[0204] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 66. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 66. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 66.

[0205] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%, 99%. or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 68. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 68. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 68.26080

[0206] In another example, provided herein is a polynucleotide compnsing 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 identity7to the polynucleotide sequence of SEQ ID NO: 69. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 69. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 69.

[0207] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity' to the polynucleotide sequence of SEQ ID NO: 71. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 71. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 71.

[0208] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 72. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 72. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 72.

[0209] In various embodiments, an isolated polynucleotide encoding polypeptide (e.g., a macrocyclase polypeptide) may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the isolated polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. Techniques for modifying polynucleotides and nucleic acid sequences utilizing recombinant DNA methods are well known in the art. For example, guidance is provided in Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3rdEd., Cold Spring Harbor Laboratory’ Press; and Current Protocols in Molecular Biology, Ausubel. F. ed., Greene Pub. Associates, 1998, updates to 2006.

[0210] In some embodiments, an isolated polynucleotide encoding any of the polypeptides herein is manipulated in a variety of ways to facilitate expression of the polypeptide. In some embodiments, the polynucleotides encoding the polypeptides comprise expression vectors where one or more control sequences is present to regulate the expression of the polynucleotides and / or polypeptides. Manipulation of the isolated polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector utilized. Techniques for modifying polynucleotides and nucleic acid sequences utilizing recombinant DNA methods are well known in the art.

[0211] In some embodiments, the control sequences include among others, promoters, leader sequences, polyadenylation sequences, propeptide sequences, signal peptide sequences, and transcription terminators.26080

[0212] 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. coll lac operon, Streptomyces coelicolor agarase gene (dagA). Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokary otic beta-lactamase gene (see e.g., Villa-Kamaroff et al.. Proc. Natl Acad. Sci. USA 75: 3727-3731, 1978), as well as the tac promoter (see e.g., DeBoer et al., Proc. Natl Acad. Sci. USA 80: 21-25, 1983). Exemplary promoters for filamentous fungal host cells, include, but are not limited to, promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alphaamylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum try psin-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-phosphogly cerate kinase. Other useful promoters for yeast host cells are known in the art (see e.g., Romanos et al., Yeast 8:423-488, 1992).

[0213] In some embodiments, the control sequence is a suitable transcription terminator sequence (i.e., a sequence recognized by a host cell to terminate transcription). In some embodiments, the terminator sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the enzyme polypeptide. Any suitable terminator that is functional in the host cell of choice finds use in the present invention. Exemplary7transcription 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 cerevisiae26080 glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are known in the art (see e.g., Romanos et al., supra).

[0214] 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. Exemplar.- leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, and Aspergillus nidulans triose phosphate isomerase. Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3- phosphogly cerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0215] In some embodiments, the control sequence is a polyadenylation sequence (i.e., a sequence operably linked to the 3' terminus of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA). Any suitable polyadenylation sequence that is functional in the host cell of choice may be used in the present disclosure. Exemplary poly adenylation sequences for filamentous fungal host cells include, but are not limited to. the genes fo Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase. Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase. Useful polyadenylation sequences for yeast host cells are known (see e g., Guo and Sherman, Mol. Cell. Biol., 15:5983-5990, 1995).

[0216] In some embodiments, the control sequence is a signal peptide (i.e., a coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell’s secretory' pathway). In some embodiments, the 5' end of the coding sequence of the nucleic acid sequence inherently contains a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide. Alternatively, in some embodiments, the 5' end of the coding sequence contains a signal peptide coding region that is foreign to the coding sequence. Any suitable signal peptide coding region that directs the expressed polypeptide into the secretory- pathway of a host cell of choice finds use for expression of the engineered polypeptide(s). Effective signal peptide coding regions for bacterial host cells are the signal peptide coding regions include, but are not limited to, those obtained from the genes for Bacillus NC1B 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-26080 lactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are known in the art (see e.g., Simonen and Palva, Microbiol. Rev., 57: 109-137, 1993). In some embodiments, effective signal peptide coding regions for fdamentous 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.

[0217] 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.

[0218] In another aspect, the present disclosure provides a recombinant expression vector comprising a polynucleotide encoding a polypeptide (e.g., a macrocyclase 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 t pe 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, the nucleic acid sequence of the present invention is expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In some embodiments involving the creation of the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

[0219] 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 compatibility726080 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.

[0220] 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 alternative embodiments, the vector 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 polynucleotide(s) (e.g., DNA) to be introduced into the genome of the host cell, and / or a transposon is utilized.

[0221] In some embodiments, the expression vector contains one or more selectable markers, which permit easy selection of transformed cells. Examples of bacterial selectable markers include, but are not limited to, the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers, which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase; e.g., from A. nidulans or A. orzyae). argB (ornithine carbamoyltransferases), bar (phosphinothricin acetyltransferase; e.g., from S. hygroscopicus), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase; e.g., from A. nidulans or A. orzyae), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof.

[0222] 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 examples, the at least one polynucleotide is part of an expression vector. Host cells suitable for use in expressing the polypeptides encoded by the polynucleotide(s) or expression vector(s) of the present disclosure are well known in the art and described herein.

[0223] In some embodiments, the expression vectors of the present disclosure contain one or more element 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 the26080 polypeptide or any other element of the vector for integration of the vector into the genome by homologous or nonhomologous recombination.

[0224] In some alternative 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, 400 to 10,000 base pairs, or 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.

[0225] 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, pACYC177 (which contains the P15A ori), or pACYC184 (which contains the P15A ori) permitting replication in E. coll, 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 ARS1 and CEN3, and the combination of ARS4 and CEN6. The origin of replication may be one having a mutation which makes its functioning temperature-sensitive in the host cell (see e.g., Ehrlich, Proc. Natl. Acad. Sci. USA 75: 1433, 1978).

[0226] In some embodiments, more than one copy of a polynucleotide sequence of the present invention 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.

[0227] Many of the expression vectors for use in the present invention are commercially available. Suitable commercial expression vectors include, but are not limited to, NOVAGEN® pET E. coll T7 expression vectors (Millipore Sigma) and the p3xFLAG® expression vectors26080(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).

[0228] Thus, in some embodiments, a vector comprising a sequence encoding at least one variant polypeptide (e.g., macrocyclase 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 invention, 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 Macrocyclase Polypeptides

[0229] In another aspect, the present disclosure provides a host cell comprising a polynucleotide encoding a polypeptide (e.g., a macrocyclase polypeptide) disclosed herein, or an expression vector comprising a polynucleotide encoding a polypeptide (e.g., a macrocyclase 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. Suitable 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 growth conditions for the above-described host cells are well known in the art.

[0230] Polynucleotides for expression of the polypeptides (e.g., macrocyclase 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.26080

[0231] 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.

[0232] 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 invention, the yeast cell is Hansenula polymorpha, Saccharomyces cerevisiae, Saccharomyces carlsber gensis, 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.

[0233] In some other 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 invention, including but not limited to Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Acinetobacter , Acidothermus, Arthrobacter, Azobacter, Bacillus, Bifidobacterium, Brevibacterium, Butyrivibrio, Buchnera, Campestris, Camplyobacter, Clostridium, Corynebacterium, Chromatium, Coprococcus, Escherichia, Enterococcus, Enterobacter, Erwinia, Fusobacterium, Faecalibacterium, Francisella, Flavobacterium, Geobacillus, Haemophilus, Helicobacter. Klebsiella.Lactobacillus. Lactococcus, Ilyobacter, Micrococcus, Microbacterium, Mesorhizobium, Methylobacterium, Methylobacterium, Mycobacterium, Neisseria, Pantoea, Pseudomonas, Prochlorococcus, Rhodobacter, Rhodopseudomonas, Rhodopseudomonas, Roseburia, Rhodospirillum, Rhodococcus. Scenedesmus, Streptomyces. Streptococcus, Synecoccus, Saccharomonospora. Staphylococcus. Serratia, Salmonella. Shigella. Thermoanaerobacterium, Tropheryma, Tularensis, Temecula, Thermosynechococcus, Thermococcus, Ureaplasma, Xanthomonas, Xylella, Yersinia and Zymomonas. In some embodiments, the host cell is a species26080 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 invention. In some embodiments of the present invention, the bacterial host cell is an Agrobacterium species (e.g., A. radiobacter, A. rhizogenes, and A. rubi). In some embodiments of the present invention, the bacterial host cell is wArthrobacter species (e.g., A. aurescens, A. citreus, A. globiformis, A. hydrocarboglutamicus, A. mysorens, A. nicotianae, A. parafflneus, A. protophonniae, A. roseoparqffinus,A. sulfur eus, or A. ureafaciens) . In some embodiments of the present invention, 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 a Clostridium 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. coll). In some embodiments, the host cell is Escherichia coli W3110. In some embodiments the host is Escherichia coli BL21 or BL21(DE3). In some embodiments, the bacterial host cell is an Erwinia species (e.g., E. uredovora, E. carotovora, E. ananas, E. herbicola, E. punctata, and E. terreus). In some embodiments, the bacterial host cell is Pantoea species (e.g., P. citrea or P. agglomerans). In some embodiments the bacterial host cell is a Pseudomonas species (e.g., P. putida, P. aeruginosa, P. mevalonii, and P. sp. D-01 10). In some embodiments, the bacterial host cell is a Streptococcus species (e.g., S. equisimiles, S. pyogenes, and S. uberis). In some embodiments, the bacterial host cell is a Streptomyces species (e.g., S. ambofaciens, S. achromogenes . S. avermitilis, S. coelicolor, S. aureofaciens, S. aureus, S. fungicidicus , S. griseus, or S. lividans). In some embodiments, the bacterial host cell is a Zymomonas species (e.g., Z. mobilis, or Z. lipolytica).26080

[0234] 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).

[0235] 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 macrocyclase 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 known in the art for reducing expression of protein in cells, including, but not limited to deletion of all or part of the gene encoding the protein and site-specific mutagenesis to disrupt expression or activity of the gene product, (see e.g., Chaveroche et al., Nucl. Acids Res., 28:22 e97, 2000; Cho et al., Molec. Plant Microbe Interact., 19:7-15, 2006; Maruyama and Kitamoto, Biotechnol. Lett., 30: 1811-1817, 2008;Takahashi et al., Mol. Gen. Genom, 272: 344-352, 2004; and You et al., Arch.Microbiol. ,191 :615-622, 2009, all of which are incorporated by reference herein). Random mutagenesis, followed by screening for desired mutations may also be used (see e.g., Combier et al., FEMS Microbiol. Lett., 220: 141-8, 2003; and Firon et al., Eukary. Cell 2:247-55, 2003, both of which are incorporated by reference).

[0236] Introduction of a vector or polynucleotide (e.g., DNA) 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.

[0237] In some embodiments, the engineered 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, pHand 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.

[0238] In some embodiments, cells expressing a polypeptide (e.g., a macrocyclase polypeptide) of the present disclosure are grown under batch or continuous fermentations conditions. Classical batch fermentation is a closed system, wherein the compositions of the medium are set at the beginning of the fermentation and is not subject to artificial alternations during the fermentation. A variation of the batch system is a fed-batch fermentation that also finds use in the present disclosure. In this variation, the substrate is added in increments as the fermentation progresses. Fed-batch systems are useful when catabolite repression is likely to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the medium. Batch and fed- batch fermentations are common and well known in the art. Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth. Continuous fermentation systems strive to maintain steady state growth conditions. Methods for modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are w ell known in the art of industrial microbiology.

[0239] 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 w here 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.

[0240] In some embodiments, cell-free transcription and translation systems find use in producing the polypeptide(s) (e.g., macrocyclase polypeptide(s)). Several systems are commercially available, and the methods are well-known to those skilled in the art.Methods of Evolving Macrocyclase Polypeptides

[0241] Evolution (e.g., directed evolution) may be used to identify polypeptides (e.g., macrocyclase polypeptides) of the present disclosure. For example, in some embodiments, tomake a macrocyclase polypeptide of the present disclosure, a macrocyclase polypeptide may be obtained (or derived) from any suitable bacterial species, e.g., Streptomyces sp. KCB13F003. In some embodiments, the parent polynucleotide sequence is codon optimized to enhance expression of the macrocyclase polypeptide in a specified host cell (e.g., E. coli).

[0242] The polypeptides (e.g., macrocyclase polypeptides) of the disclosure may be obtained by subjecting a polynucleotide encoding a parent sequence to mutagenesis and / or directed evolution methods. An exemplary directed evolution technique is mutagenesis and / or DNA shuffling as described in Stemmer, 1994, Proc. Natl. Acad. Sci. USA 91 : 10747-10751; WO 95 / 22625; WO 97 / 20078; WO 97 / 35966; WO 98 / 27230; WO 00 / 42651; WO 01 / 75767 and U.S. Pat. No. 6,537,746. Other directed evolution procedures that can be 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).

[0243] The clones obtained following mutagenesis treatment may be screened for macrocyclase 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. In this reaction, peptides, peptidy 1- thioesters and peptidyl -esters such as the ones described in Tables 1.1 and 1.2 can be coupled in the presence of an macrocyclase polypeptide. The reaction may be run under conditions where the macrocyclase polypeptide is the yield-limiting catalyst, such that a doubling or halving in concentration of the macrocyclase polypeptide will effect a doubling or halving of the yield of product observed at a given timepoint. Where the improved enzyme property desired is thermal stability, enzyme activity may be measured after subjecting the enzyme preparations to a defined temperature and measuring the amount of enzyme activity remaining after heat treatments.Clones containing a polynucleotide encoding macrocyclase polypeptide are then isolated, sequenced to identify the nucleotide sequence changes (if any), and used to express the enzyme in a host cell.

[0244] 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 macrocyclase may then be isolated, sequenced to identify the nucleotide sequence changes (if any), and used to express the enzy me in a host cell.26080

[0245] Where the sequence of the polypeptide is known, the polynucleotides encoding the enzyme can be prepared 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.

[0246] Polypeptides (e g., macrocyclase polypeptides) expressed in a host cell can be recovered from the cells and or the culture medium using any one or more of the well-known techniques for protein purification, including, among others, lysozyme treatment, sonication, filtration, saltingout, ultra-centrifugation, and chromatography. Suitable solutions for lysing and the high efficiency extraction of proteins from bacteria, such as E. coli, are commercially available under the trade name CELLYTIC B® from Sigma- Aldrich.

[0247] Chromatographic techniques for isolation of the polypeptide (e.g., macrocyclase 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.

[0248] In some embodiments, affinity techniques may be used to isolate the improved polypeptides (e.g., macrocyclase polypeptides). For affinity chromatography purification, the protein sequence can be tagged with a recognition sequence to enable purification. Common 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 macrocyclase polypeptide may be used.26080Methods of Using Macrocyclase Polypeptides

[0249] Also provided herein are methods of using a polypeptide (e.g., a macrocyclase polypeptide) of the disclosure. In some examples, a polypeptide (e.g., a macrocyclase polypeptide) of the disclosure is used in a method of catalyzing amide bond formation in macrocycles or 11-mer peptides (e.g., in a protecting group-free manner) from activated aminoacyl-thioesters, aminoacyl-esters and cyclic peptides. In embodiments, the polypeptide (e.g., a macrocyclase polypeptide) described herein can couple two peptides that can be further used in the synthesis of multimer peptides or can macrocyclize linear peptides. In some embodiments, the polypeptide (e.g., a macrocyclase polypeptide) may be used in a method of preparing compounds such as 3e or 2b.EXAMPLES

[0250] The following examples are meant to be illustrative and should not be construed as further limiting. The contents of the figures and all references, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference.Example 1: Synthesis, Optimization, and Assay of NRPS Macrocyclase Enzy mes with Macrocyclization Activity

[0251] This example describes methods to synthesize, optimize, and assay NRPS macrocyclase enzymes and macrocyclization activity, and the composition of optimized enzy mes.Gene Synthesis and Optimization:

[0252] The polynucleotide sequence (SEQ ID NO: 1) encoding the wild-type polypeptide (SEQ ID NO: 2) was first annotated as a possible member of macrocyclizing thioesterases in a literature report (see, e.g., Matsuda, Zhai, et al. Nat. Catal. 2020, 3, 507-515). An N-terminally hexahistidine-tagged (SEQ ID NO: 76) construct (polynucleotide SEQ ID NO: 3 and polypeptide SEQ ID NO: 4) was included in a panel of macrocyclases. SEQ ID NO: 3 was synthesized as SEQ ID NO: 5, codon-optimized for expression in E. colt, cloned into a pET30a(+) vector containing a Lad promoter, a ColEl origin of replication and a KanR selection marker. The cloned gene was propagated into the DH5a strain, sequence verified and transformed into BL21(DE3). Likewise, genes of engineered NRPS macrocyclases were cloned into pET30a vector and transformed into BL21(DE3) cells for expression.HTP Growth. Expression, and Lysate Preparation:

[0253] For panel expression, glycerol stocks of E. coli BL21(DE3) transformed with SEQ ID NO: 4 were used to inoculate 200 pL of LB media supplemented with 30 pg / mL of kanamycin26080 and 1% v / v glucose in 96-well plates. Cultures grew overnight at 200 rpm, 30 °C, 85% relative humidity. The following morning, 20 pL of the starter cultures were used to inoculate 1 mL of TB media supplemented with 30 pg / mL kanamycin in fresh 96-well plates. Cultures were shaken at 250 rpm, 37 °C, 85% RH for 4 h. The incubator temperature was adjusted to 25 °C and cultures continued to grow for 1 h. Protein expression was induced by the addition of 20 pL of 10 mM IPTG in water (final concentration 0.2 rnM) and cultures grew overnight at 250 rpm, 25 °C, 85% RH. The following day, cells were pelleted by centrifugation at 4,000 x g for 20 min, and the supernatant was discarded. Cell pellets were lysed in 200 pL of lysis buffer (50 rnM HEPES pH 7.5. 50 mM NaCl, 4 mM MgCh. 3 U / mL DNase I, 1 g / L chicken egg white lysozyme, 0.5 g / L polymyxin B sulfate) for 1 h at 1,000 rpm. Cell debris was pelleted at 4,000 x g for 20 min. Production of Shake Flask Powders (SFP):

[0254] Single colonies or glycerol stocks of BL21(DE3) E. coli transformed with macrocyclase variants were used to inoculate 20 mL of LB media supplemented with 30 pg / mL of kanamycin and 1% v / v glucose. Cultures grew overnight at 200 rpm, 30 °C. The following morning, sufficient volume of the starter cultures was used to inoculate 1 L of TB media supplemented with 30 pg / mL kanamycin to an initial ODeoo value of 0.05. Cultures were shaken at 250 rpm, 30 °C for 3.5 h. The incubator temperature was low ered to 25 °C and cultures continued growing for 45 min. Protein expression was induced by the addition of 200 pL of 1 M IPTG in water (0.2 mM final). Plates continued to grow for 18 h at 250 rpm, 30 °C, 85% RH. The following day, cells w ere pelleted by centrifugation at 4,000 x g for 15 min, and the supernatant w as discarded. Cell pellets were stored at -80 °C for at least 30 min before thawing and resuspension in lysis buffer (20 mM triethanolamine pH 7.5, 5 mL per g of wet cell weight). Cells were lysed by 2 passages through a microfluidizer, and insoluble debris was pelleted at 22,000 x g for 50 mm at 4 °C. The supernatant was frozen at -80 °C for 2 h and lyophilized over 48 h.Assay method for intermol ecular coupling activity:

[0255] A substrate master mix consisting of 20 g / L Id and 252 g / L 2d dissolved in DMSO w-as prepared. Reactions consisting of 100 pL of high-throughput cell lysate and 5 pL of substrate master mix were combined and shaken for 18 h at 25 °C, 700 rpm.Analytical method for intermolecular coupling activity (UPLC):

[0256] Column: Waters ACQUITY BEH Cl 8, 2.1 mm x 50 mm, 130 A, 1.7 pm. Mobile phase A: 5 mM ammonium formate in water pH 6.5. Mobile phase B: MeCN. Flow rate: 0.75 mL / min. Method = 15% to 68% B linear gradient over 2.0 min, followed by a linear gradient to 95% B over 0.3 min, hold at 95% B for 0.4 min, then a linear gradient to 15% B over 0. 1 min and hold 0.4 min at 15% B (total time 3.2 min). tR 3d = 2.0 min at 254 nm.26080Assay method for macrocvclization activity:

[0257] A substrate master mix consisting of 40 g / L la dissolved in DMSO was prepared. Reactions consisting of 100 pL of 40 g / L shake flask powder dissolved in 100 mM Bis-Tris pH 7.0 and 5 pL of substrate master mix were combined and shaken for 18 h at 25 °C, 700 rpm. Analytical UPLC method for macrocvclization activity:

[0258] Column: ASCENTIS® Express Cl 8, 4.6 mm x 100 mm, 2.7 pm. Mobile phase A: 5 mM ammonium formate in water pH 6.5. Mobile phase B: MeCN. Flow rate: 1.8 mL / min. Method = 10% to 95% B linear gradient over 2.5 min, followed by 1 min at 95% B (total time 3.5 min). 1R 2a = 2.2 min at 210 nm.Identification of starting point for evolution:

[0259] Activity was detected for SEQ ID NO: 4. 1.2% of la was converted to 2a in the HPLC analysis of the assay samples described above. The codons of SEQ ID NO: 3 were optimized for expression in E. coll and the polyhistidine tag was relocated to the C-terminus, cloned into a pET30a(+) vector and expressed in E. coli strain BL21(DE3) under the control of the T7 promoter as polynucleotide SEQ ID NO: 5 encoding polypeptide SEQ ID NO: 6. SEQ ID NO: 6 became the basis for evolution.Example 2 (Evolution Round (Rd) 1)Enzyme variants of SEQ ID NO: 6

[0260] In this Example, directed evolution of SEQ ID NO: 6 for improved macrocyclization activity and the resulting improved variants are described. Directed evolution was carried out byconstructing libraries of variant genes in which positions associated with 2a binding 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, and high-throughput assay described below.HTP Growth. Expression, and Lysate Preparation:

[0261] Single colonies of BL21(DE3) E. coli transformed with macrocyclase variants were used to inoculate 200 pL of LB media supplemented with 30 pg / mL of kanamycin and 1% v / v glucose in 96-well plates. Cultures grew overnight at 200 rpm, 30 °C, 85% relative humidity. The following morning, 20 pL of the starter cultures were used to inoculate 380 pL of TB media supplemented with 30 pg / mL kanamycin in fresh 96-well plates. Cultures were shaken at 250 rpm, 30 °C, 85% RH for 4 h. Protein expression was then induced by the addition of 40 pL of 2 mM IPTG in water (0.2 mM final concentration). Plates continued to grow for 18 h at 250 rpm, 30 °C, 85% RH. The following day, cells were pelleted by centrifugation at 4,000 x g for 15 min,26080 and the supernatant was discarded. Cell pellets were lysed in 200 pL of lysis buffer (50 mM MOPS pH 7.5, 4 mM MgCh, 3 U / mL DNase I, 0.5 g / L chicken egg white lysozyme, 0.5 g / L polymyxin B sulfate) for 1 h at 1,000 rpm. Cell debris was pelleted at 4,000 x g for 20 min. HTP assay for macrocvclase-catalyzed cyclization:

[0262] 55 pL reactions consisting of 50 pL of cell lysate prepared as above and 5 pL of a 10 g / L solution of la in DMSO were combined and incubated for 18 h at 25 °C, 1,000 rpm. Reactions were quenched with 150 pL MeCN, fdtered, and analyzed by liquid chromatographymass spectrometry.Analytical LC-MS method for macrocyclization activity:

[0263] Column: Waters ACQUITY BEH Cl 8, 2.1 mm x 50 mm, 130 A, 1.7 pm. Mobile phase A: 10 mM ammonium formate in water pH 8.5. Mobile phase B: MeCN. Flow rate: 0.75 mL / min. Method = 10% B for 0.2 min, followed by a linear gradient to 70% B over 0.8 min, hold at 70% B for 0.2 min, linear gradient to 50% B over 0.05 min, followed by 0.15 min at 70% B (total time 1.4 min). tR la = 0.67 min; tR 2a = 0.86 min; tR 5h = 0.35 min.

[0264] Engineered polypeptides with >2-fold conversion 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 ConversionLevels of increased conversion were determined relative to the reference polypeptide of SEQ ID NO: 6 and defined as:“+” = conversion at least 2-fold that of reference polypeptide, but less than 3-fold; and “++” = conversion at least 3-fold, as compared to the reference polypeptide.

[0265] These variants produced 2a from la, and these engineered macrocyclizing thioesterases provide new biocatalytic reagents for use in new methods for the macrocyclization of la to 2a. The variant with mutation S429A (SEQ ID NO: 8) had the highest activity. Thus, the encoding polynucleotide (SEQ ID NO: 7) was selected for further directed evolution.26080Example 3 (Evolution Rd2)Enzyme variants of SEQ ID NO: 8

[0266] In this Example, directed evolution of SEQ ID NO: 8 for improved macrocyclization and the resulting improved variants are described. Directed evolution was carried out by constructing libraries of variant genes in which positions associated with 2b binding and solvent exposure were subjected to mutagenesis. These libraries were plated to form single colonies, which were grow n and screened using the high-throughput growth and expression method, the high-throughput assay described in Example 2.HTP assay for macrocvclase-catalyzed cyclization:

[0267] Cell pellets from 400 pL cultures of sequence variants w ere lysed with 200 pL of lysis buffer (50 mM MOPS pH 7.5, 4 mM MgCE, 3 U / mL DNase I, 0.5 g / L chicken egg white lysozy me, 0.5 g / L polymyxin B sulfate) for 1 h at 1,000 rpm. Cell debris was pelleted at 4,000 x g for 20 min. 55 pL reactions consisting of 50 pL of cell lysate prepared as above and 5 pL of a 10 g / L solution of la in DMSO were combined and incubated for 18 h at 30 °C, 1,000 rpm.Reactions were quenched with 150 pL MeCN, filtered, and analyzed by liquid chromatographymass spectrometry7using the method described in Example 2.

[0268] Engineered polypeptides with >2-fold conversion from la to 2a 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 ConversionLevels of increased conversion w ere determined relative to the reference polypeptide of SEQ ID NO: 8 and defined as:"+“ = conversion at least 2-fold that of reference polypeptide, but less than 3-fold; and“++” = conversion at least 3-fold, as compared to the reference polypeptide.Additional cyclization reactions with lb:

[0269] Cell lysate prepared as above was diluted 10-fold with 50 mM MOPS buffer pH 7.5. 55 pL reactions consisting of 50 pL of diluted cell lysate prepared and 5 pL of a 10 g / L solution of26080 lb in DMSO were combined and incubated at 30 °C for 1 h, 1.000 rpm. Reactions were quenched with 150 pL MeCN, filtered, and analyzed by liquid chromatography.Analytical method for cyclization of lb (UPLC):

[0270] Column: ASCENTIS® Express C18, 2. 1 mm x 50 mm, 90 A, 2.7 pm. Mobile phase A: water with 0.1% v / v phosphoric acid. Mobile phase B: MeCN. Flow rate: 0.5 mL / min. Method = 20% to 30% B linear gradient over 2.0 min, followed by a linear gradient to 95% B over 1 min, hold at 95% B for 0.5 min, then a linear gradient to 20% B over 0. 1 min and hold 0.45 min at 15% B (total time 4.05 min). tR 6i = 1.67 min at 210 mm; tR lb = 1.97 min; tR 2b = 2.38 min.

[0271] These variants produced 2a from la, and 2b from lb, respectively, and these engineered macrocyclizing thioesterases provide new biocatalytic reagents for use in new methods for the macrocyclization of la to 2a. The variant with mutations A80M; L275F; R285V; A303C (SEQ ID NO: 10) had the highest activity7. Thus, the encoding polynucleotide (SEQ ID NO: 9) was selected for further directed evolution.Example 4 (Evolution Rd3)Enzy me variants of SEQ ID NO: 10

[0272] In this Example, directed evolution of SEQ ID NO: 10 for improved macrocyclization and the resulting improved variants are described. Directed evolution was carried out by constructing libraries of variant genes in which positions associated with solvent exposure 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 below and the high-throughput assay described below.HTP Growth and Expression:

[0273] Single colonies of BL21(DE3) E. coll transformed with macrocyclase variants were used to inoculate 200 pL of LB media supplemented with 30 pg / mL of kanamycin and 1% v / v glucose in 96-well plates. Cultures grew overnight at 200 rpm, 30 °C, 85% relative humidity. The following morning, 20 pL of the starter cultures were used to inoculate 380 pL of TB media supplemented with 30 pg / mL kanamycin in fresh 96-well plates. Cultures were shaken at 250 rpm, 30 °C, 85% RH for 4 h. Incubation temperature was lowered to 25 °C for 45 min. Protein expression was then induced by the addition of 40 pL of 2 mM IPTG in water (0.2 mM final concentration). Plates continued to grow for 18 h at 250 rpm, 30 °C, 85% RH. The following day, cells were pelleted by centrifugation at 4,000 x g for 15 min, and the supernatant was discarded.26080HTP assay for macrocyclase-catalyzed cyclization:

[0274] Cell pellets from 400 pL cultures of sequence variants were lysed with 200 pL of lysis buffer (50 mM MOPS pH 7.5, 4 mM MgCh, 3 U / mL DNase I, 0.5 g / L chicken egg white lysozyme, 0.5 g / L polymyxin B sulfate) for 1 h at 1,000 rpm. Cell debris was pelleted at 4,000 x g for 20 min. 1c was dissolved in a 1: 1 mixture of iPrOH and DMSO to a final concentration of 10 g / L. 55 pL reactions consisting of 25 pL of cell lysate, 25 pL of 50 mM MOPS pH 7.5 and 5 pL of a 10 g / L solution of 1c were combined and incubated for 18 h at 30 °C, 1,000 rpm. Reactions were quenched with 150 pL MeCN, diluted 2-fold with MeCN, filtered, and analyzed by liquid chromatography-mass spectrometry as described below.Analytical LC-MS method for macrocvclization activity:

[0275] Column: Waters ACQUITY BEH C18, 2.1 mm x 50 mm, 130 A, 1.7 pm. Mobile phase A: water with 0.1% formic acid and 0.01% trifluoroacetic acid. Mobile phase B: MeCN with 0.1% formic acid and 0.01% trifluoroacetic acid. Column temperature: 55 °C. Flow rate: 0.65 mL / min. Method: 32% B for 0.2 min, followed by a linear gradient to 38% B over 1 min, linear gradient to 95% B over 0.05 min, hold 0.25 min at 95% B, linear gradient to 32% B over 0.05 min and hold at 32% B for 0.35 min (total time 1.8 min). tR 1c = 0.83 min; tR 2b = 0.97 min; tR 6i = 0.53 min; tR 5i = 1.55 min.

[0276] Engineered polypeptides with >1. 1-fold selectivity for 2b 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 SelectivityLevels of increased conversion were determined relative to the reference polypeptide of SEQ IDNO: 10 and defined as:“+■’ = selectivity at least 1. 1-fold that of reference polypeptide, but less than 2.2-fold; and“++” = selectivity at least 2.2-fold, but less than 2.4-fold increased selectivity, as compared to the reference polypeptide.26080

[0277] These variants produced 2b from 1c and these engineered macrocyclizing thioesterases provide new biocatalytic reagents for use in new methods for the macrocyclization of 1c to 2b. The variant with mutations L82F; R94T; F204L; P281L; A354P; G407P; A418N (SEQ ID NO: 12) had the highest activity. Thus, the encoding polynucleotide (SEQ ID NO: 11) was selected for further directed evolution.Example 5 (Evolution Rd4)Enzyme variants of SEQ ID NO: 12

[0278] In this Example, directed evolution of SEQ ID NO: 12 for improved macrocyclization and the resulting improved variants are described. Directed evolution was carried out by constructing libraries of variant genes in which positions associated with product 2b binding and the lid domain 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 4 and the high-throughput assay described below.HTP assay for macrocvclase-catalvzed cyclization:

[0279] Cell pellets from 400 pL cultures of sequence variants were lysed with 200 pL of lysis buffer (50 rnM MOPS pH 7.5, 4 rnM MgCk, 3 U / mL DNase I, 0.5 g / L chicken egg white lysozyme, 0.5 g / L polymyxin B sulfate) for 1 h at 1,000 rpm. Cell debris was pelleted at 4.000 x g for 20 min. 1c was dissolved MeCN to 50 mM MOPS pH 7.5 buffer to a final concentration of 20 g / L. 50 pL reactions consisting of 15 pL of cell lysate, 30 pL of 50 mM MOPS pH 7.5 and 5 pL of a 20 g / L solution of 1c w ere combined and incubated for 18 h at 30 °C, 1,000 rpm. Reactions were quenched with 150 pL MeCN, diluted 2-fold with MeCN, filtered, and analyzed by liquid chromatography-mass spectrometry using the method described in Example 4.

[0280] Engineered polypeptides with >6-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 Conversion26080Levels of increased conversion were determined relative to the reference polypeptide of SEQ ID NO: 12 and defined as:“+” = conversion at least 8-fold that of reference polypeptide, but less than 140-fold; and “++’■ = conversion at least 140-fold, but less than 150-fold increased conversion, as compared to the reference polypeptide.

[0281] These variants produced 2b from 1c, and these engineered macrocyclizing thioesterases provide new biocatalytic reagents for use in new methods for the macrocyclization of 1c to 2b. The variant with mutations D7K; L45T; D87S; D89L; A212N; D250S; G284A; T432L (SEQ ID NO: 14) had the highest activity. Thus, the encoding polynucleotide (SEQ ID NO: 13) was selected for further directed evolution.Example 6 (Evolution Rd5)Enzyme variants of SEQ ID NO: 14

[0282] In this Example, directed evolution of SEQ ID NO: 14 for improved macrocyclization and the resulting improved variants are described. Directed evolution was carried out by constructing libraries of variant genes in which positions product 2b binding and the lid domain 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 4 and the high-throughput assay described below.HTP assay for macrocvclase-catalvzed cyclization:

[0283] Cell pellets from 400 pL cultures of sequence variants were lysed with 200 pL of lysis buffer (50 mM MOPS pH 7.5, 4 rnM MgCL, 3 U / mL DNase I, 0.5 g / L chicken egg white lysozyme, 0.5 g / L polymyxin B sulfate) for 1 h at 1,000 rpm. Cell debris was pelleted at 4,000 x g for 20 min. 1c was dissolved in MeCN to a final concentration of 20 g / L. 50 pL reactions consisting of 15 pL of cell lysate, 30 pL of 4.2% v / v iPrOH in 50 mM MOPS pH 7.5 and 5 pL of a 20 g / L solution of 1c w ere combined and incubated for 18 h at 30 °C, 1,000 rpm. Reactions were quenched with 150 pL MeCN. diluted 2-fold with MeCN. filtered, and analyzed by liquid chromatography-mass spectrometry using the method described in Example 4.

[0284] Engineered polypeptides with >1. 1-fold conversion of 1c to 2a relative to the parent polypeptide are listed in Table 6. 1 and w ere re-expressed as shake flask powders as described in Example 1.HTP assay for thioesterase-catalvzed peptide-peptide coupling:

[0285] Cell pellets were lysed as above, then diluted 2-fold in 50 mM MOPS pH 7.5. 2e and le were dissolved in a 1 : 1 mixture of MeCN and 50 mM MOPS pH 7.5 to final concentrations of 1526080 and 8 g / L, respectively. 50 pL reactions consisting of 15 pL of diluted cell lysate, 30 pL of 5% v / v iPrOH in 50 mM MOPS pH 7.5 and 5 pL of the solution of 2e and le were combined and incubated for 18 h at 30 °C, 1,000 rpm. Reactions were quenched with 150 pL MeCN, diluted 2- fold with MeCN. filtered, and analyzed by liquid chromatography-mass spectrometry' using the method below.Analytical LC-MS method for intermolecular pepti de-peptide coupling:

[0286] Column: Zorbax Eclipse Plus Cl 8, 2.1 mm x 100 mm, 95 A, 1.8 pm. Mobile phase A: water with 0.1% v / v formic acid. Mobile phase B: 7:3 MeCN / MeOH. Flow rate: 0.6 mL / min. Method = 35% B for 0.6 min, followed by a linear gradient to 50% B over 0.8 min, linear gradient to 65% B over 1 min, linear gradient to 95% B over 0.2 min, hold at 95% B for 0.3 min, linear gradient to 35% B over 0.05 min, followed by 0.4 min at 35% B (total time 3. 15min). tR 2e = 1.11 min; tR 3e = 1.52 min.Table 6.1 Variants and ConversionLevels of increased conversion to 2b were determined relative to the reference polypeptide of SEQ ID NO: 14 and defined as:”+‘‘ = conversion at least 1. 1 -fold that of reference polypeptide, but less than 1.8-fold; and “++” = conversion at least 1.8-fold, but less than 2.1 -fold increased conversion, as compared to the reference polypeptide.

[0287] These variants produced 2b from 1c, and these engineered macrocyclizing thioesterases provide new biocatalytic reagents for use in new methods for the macrocyclization of 1c to 2b. The variant with mutations A6E; T218K; L433F; E436M (SEQ ID NO: 16) had the highest activity. Thus, the encoding polynucleotide (SEQ ID NO: 15) was selected for further directed evolution.26080Example 7 (Evolution Rd6)Enzyme variants of SEQ ID NO: 16

[0288] In this Example, directed evolution of SEQ ID NO: 16 for improved conversion in the presence of a higher iPrOH concentration and the resulting improved variants are described. Directed evolution was carried out by constructing libraries of variant genes in which positions not previously targeted were subjected to mutagenesis. These libraries were plated to form single colonies, which were grown and screened using the high-throughput grow th and expression method described in Example 4 and the high-throughput assay described below-.HTP assay for macrocvclase-catalyzed cyclization:

[0289] Cell pellets from 400 pL cultures of sequence variants w ere lysed with 200 pL of lysis buffer (50 mM MOPS pH 7.5, 4 mM MgCE, 3 U / mL DNase I, 0.5 g / L chicken egg white lysozy me, 0.5 g / L polymyxin B sulfate) for 1 h at 1,000 rpm. Cell debris was pelleted at 4,000 x g for 20 min. The supernatant was diluted 2-fold with 50 mM MOPS buffer pH 7.5. 1c w as dissolved in MeCN to a final concentration of 20 g / L. 50 pL reactions consisting of 15 pL of diluted cell lysate, 30 pL of 12.5% v / v iPrOH in 50 mM MOPS pH 7.5 and 5 pL of a 20 g / L solution of 1c w ere combined and incubated for 18 h at 30 °C, 1,000 rpm. Reactions were quenched with 150 pL MeCN, diluted 2-fold with MeCN, filtered, and analyzed by liquid chromatography-mass spectrometry using the method described in Example 4.

[0290] Engineered polypeptides with >1.0-fold conversion relative to the parent polypeptide are listed in Table 7.1 and w ere re-expressed as shake flask powders as described in Example 1.Table 7.1 Variants and ConversionLevels of increased conversion were determined relative to the reference polypeptide of SEQ ID NO: 16 and defined as:“+” = conversion at least 1.0-fold that of reference polypeptide, but less than 1.7-fold; and “++” = conversion at least 1.7-fold, but less than 2.1-fold increased conversion, as compared to the reference polypeptide.26080

[0291] These variants produced 2b from 1c, and these engineered macrocyclizing thioesterases provide new biocatalytic reagents for use in new methods for the macrocyclization of 1c to 2b. The variant with K218R; L238C; A259I; L294V; L300R (SEQ ID NO: 20) had the highest activity. Thus, the encoding polynucleotide (SEQ ID NO: 19) was selected for further directed evolution.Example 8 (Evolution Rd7)Enzyme variants of SEQ ID NO: 20

[0292] In this Example, directed evolution of SEQ ID NO: 20 for improved macrocyclization activity and the resulting improved variants are described. Directed evolution was carried out by constructing libraries of variant genes in which positions associated with solvent exposure 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 4 and the high-throughput assay described below.HTP assay for macrocvclase-catalvzed cyclization:

[0293] Cell pellets from 400 pL cultures of sequence variants were lysed with 200 pL of lysis buffer (50 rnM MOPS pH 7.5, 4 rnM MgCk, 3 U / mL DNase I, 0.5 g / L chicken egg white lysozyme, 0.5 g / L polymyxin B sulfate) for 1 h at 1,000 rpm. Cell debris was pelleted at 4.000 x g for 20 min. 1c was dissolved in MeCN to a final concentration of 50 g / L. 50 pL reactions consisting of 15 pL of cell lysate, 30 pL of 20.8% v / v iPrOH in 50 rnM MOPS pH 7.5 and 5 pL of a 50 g / L solution of 1c were combined and incubated for 18 h at 25 °C, 1,000 rpm. Reactions were quenched with 200 pL MeCN, diluted 4-fold with MeCN. filtered, and analyzed by liquid chromatography-mass spectrometry using the method described in Example 4.

[0294] Engineered polypeptides with >1.5-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 ConversionLevels of increased conversion were determined relative to the reference polypeptide of SEQ IDNO: 20 and defined as:= conversion at least 1.5-fold that of reference polypeptide, but less than 2.0-fold; and26080”++“ = conversion at least 2.0-fold, but less than 2.5-fold increased conversion, as compared to the reference polypeptide.

[0295] These variants produced 2b from 1c, and these engineered macrocyclizing thioesterases provide new biocatalytic reagents for use in new methods for the macrocyclization of 1c to 2b. The variant with mutations A163R; A399E (SEQ ID NO: 22) had the highest activity. Thus, the encoding polynucleotide (SEQ ID NO: 21) was selected for further directed evolution.Example 9 (Evolution Rd8)Enzyme variants of SEQ ID NO: 22

[0296] In this Example, directed evolution of SEQ ID NO: 22 for improved tolerance to the presence of MeCN and the resulting improved variants are described. Directed evolution was carried out by constructing libraries of variant genes in which positions associated with the polypeptide surface 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 4 and the high-throughput assay described below.HTP assay for macrocvclase-catalvzed cyclization:

[0297] Cell pellets from 400 pL cultures of sequence variants were lysed with 200 pL of lysis buffer (50 mM MOPS pH 7.0. 4 mM MgCk, 3 U / mL DNase I, 0.5 g / L chicken egg white lysozyme, 0.5 g / L polymyxin B sulfate) for 1 h at 1,000 rpm. Cell debris was pelleted at 4,000 x g for 20 min. 1c was dissolved in MeCN to a final concentration of 25 g / L. 51 pL reactions consisting of 13 pL of cell lysate, 28 pL of 1.8% v / v iPrOH in 50 mM MOPS pH 7.0 and 10 pL of a 25 g / L solution of 1c in MOPS were combined and incubated for 18 h at 25 °C, 1,000 rpm. Reactions were quenched with 150 pL MeCN, diluted 2-fold with MeCN, filtered, and analyzed by liquid chromatography-mass spectrometry using the method described in Example 4.

[0298] Engineered polypeptides with >4-fold conversion relative to the parent polypeptide are listed in Table 9.1 and were re-expressed as shake flask powders as described in Example 1.Table 9.1 Variants and Conversion26080Levels of increased conversion were determined relative to the reference polypeptide of SEQ ID NO: 22 and defined as:■‘+’:= conversion at least 4-fold that of reference polypeptide, but less than 10-fold; and “++” = conversion at least 10-fold, but less than 35-fold increased conversion, as compared to the reference polypeptide.

[0299] These variants produced 2b from 1c, and these engineered macrocyclizing thioesterases provide new biocatalytic reagents for use in new methods for the macrocychzation of 1c to 2b. The variant with mutations T267L; A328S (SEQ ID NO: 24) had the highest activity. Thus, the encoding polynucleotide (SEQ ID NO: 23) was selected for further directed evolution.Example 10 (Evolution Rd9)Enzyme variants of SEQ ID NO: 24

[0300] In this Example, directed evolution of SEQ ID NO: 24 for improved macrocyclization activity and the resulting improved variants are described. Directed evolution was carried out byconstructing libraries of variant genes in which positions previously mutated and adjacent sites 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 4 and the high-throughput assay described below.HTP assay for macrocvclase-catalvzed cyclization:

[0301] Cell pellets from 400 pL cultures of sequence variants were lysed with 200 pL of lysis buffer (50 mM MOPS pH 7.5, 4 mM MgCE, 3 U / mL DNase I, 0.5 g / L chicken egg white lysozyme, 0.5 g / L polymyxin B sulfate) for 1 h at 1,000 rpm. Cell debris was pelleted at 4,000 x g for 20 min. 1c and le were dissolved in MeCN to final concentrations of 71.4 g / L and 13.1 g / L, respectively. 50 pL reactions consisting of 13 pL of cell lysate, 30 pL of 1.7% v / v iPrOH in 50 mM MOPS pH 7.5 and 5 pL of the solution of 1c and le were combined and incubated for 18 h at 25 °C, 1,000 rpm. Reactions were quenched with 150 pL MeCN, diluted 20-fold with MeCN, filtered, and analyzed by liquid chromatography-mass spectrometry .Analytical LC-MS method for macrocyclization activity:

[0302] Column: Waters ACQU1TY BEH Cl 8, 2.1 mm x 50 mm, 130 A, 1.7 pm. Mobile phase A: water with 0.1% formic acid and 0.01% trifluoroacetic acid. Mobile phase B: MeCN with 0.1% formic acid and 0.01% trifluoroacetic acid. Column temperature: 55 °C. Flow rate: 0.65 mL / min. Method: 32% B for 0.2 min, followed by a linear gradient to 38% B over 1 min, linear26080 gradient to 95% B over 0.05 min, hold 0.25 min at 95% B, linear gradient to 32% B over 0.05 min and hold at 32% B for 0.35 min (total time 1.8 min). tR 1c = 0.83 min; tR 2b = 0.97 min; tR 6i = 0.53 min; tR 5i = 1.55 min; tR 5f = 1.25 min; tR 6f = 0.74 min.

[0303] Engineered polypeptides with >1.5-fold conversion relative to the parent polypeptide are listed in Table 10.1 and were re-expressed as shake flask powders as described in Example 1.Table 10.1 Variants and ConversionLevels of increased conversion were determined relative to the reference polypeptide of SEQ IDNO: 24 and defined as:“+” = conversion at least 1.5-fold that of reference polypeptide, but less than 5-fold; and “++” = conversion at least 5-fold, but less than 9-fold increased conversion, as compared to the reference polypeptide.

[0304] These variants produced 2b from 1c, and these engineered macrocyclizing thioesterases provide new biocatalytic reagents for use in new methods for the macrocyclization of 1c to 2b in the presence of le. The variant with mutation L434V (SEQ ID NO: 28) had the highest activity. Thus, the encoding polynucleotide (SEQ ID NO: 27) was selected for further directed evolution.Example 11 (Evolution RdlO)Enzyme variants of SEQ ID NO: 28

[0305] In this Example, directed evolution of SEQ ID NO: 28 for improved macrocyclization 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 lid and previously mutated sites were subjected to mutagenesis. These libraries were plated to form single colonies, which were grown and screened using the high-throughput grow th and expression method described in Example 4 and the high-throughput assay described below.26080HTP assay for macrocvclase-catalyzed cyclization:

[0306] Cell pellets from 400 pL cultures of sequence variants were lysed with 200 pL of lysis buffer (100 mM MOPS pH 7.5, 4 mM MgCh, 3 U / mL DNase I, 0.5 g / L chicken egg white lysozyme, 0.5 g / L poly myxin B sulfate) for 1 h at 1,000 rpm. Cell debris was pelleted at 4,000 x g for 20 min. 1c and le were dissolved in a 3:2 solution of MeCN and 100 mM MOPS pH 7.5 to final concentrations of 50 g / L and 9. 1 g / L, respectively. 100 pL reactions consisting of 50 pL of cell lysate, 20 pL of 5% v / v iPrOH in 100 mM MOPS pH 7.5 and 30 pL of the solution of 1c and le were combined and incubated for 18 h at 25 °C, 1,000 rpm. Reactions were quenched with 200 pL MeCN, diluted 20-fold with MeCN, filtered, and analyzed by liquid chromatographymass spectrometry using the method described in Example 10.

[0307] Engineered polypeptides with >1.8-fold conversion relative to the parent polypeptide are listed in Table 11.1 and were re-expressed as shake flask powders as described in Example 1.Table 11.1 Variants and ConversionLevels of increased conversion were determined relative to the reference polypeptide of SEQ IDNO: 28 and defined as:“+” = conversion at least 1.8-fold that of reference polypeptide, but less than 2.5-fold; and“++” = conversion at least 2.5-fold, but less than 3-fold increased conversion, as compared to the reference polypeptide.

[0308] These variants produced 2b from 1c, and these engineered macrocyclizing thioesterases provide new biocatalytic reagents for use in new methods for the macrocyclization of 1c to 2b in the presence of le. The variant with mutations D203G; R218C; L223Q; T299F (SEQ ID NO: 30) had the highest activity. Thus, the encoding polynucleotide (SEQ ID NO: 29) was selected for further directed evolution.Example 12 (Evolution Rdl 1)Enzyme variants of SEQ ID NO: 30

[0309] In this example, directed evolution of SEQ ID NO: 30 for improved macrocyclization activity and the resulting improved variants are described. Directed evolution was carried out by26080 constructing libraries of variant genes in which positions associated with previously mutated sites and the lid domain were subjected to mutagenesis. These libraries were plated to form single colonies, which were grown and screened using the high-throughput grow th and expression method described in Example 4 and the high-throughput assay described below;HTP assay for macrocvclase-catalvzed cyclization:

[0310] Cell pellets from 400 pL cultures of sequence variants w ere lysed with 200 pL of lysis buffer (100 mM MOPS pH 7.5, 4 mM MgCh, 3 U / mL DNase I, 0.5 g / L chicken egg white lysozyme, 0.5 g / L polymyxin B sulfate) for 1 h at 1,000 rpm. Cell debris was pelleted at 4,000 x g for 20 min. 1c and le were dissolved in a 2.5: 1 solution of MeCN and 100 mM MOPS pH 7.5 to final concentrations of 57. Ig / L and 10.4 g / L, respectively. 100 pL reactions consisting of 50 pL of cell lysate, 15 pL of 6.7% v / v iPrOH in 100 mM MOPS pH 7.5 and 35 pL of the solution of 1c and le were combined and incubated for 18 h at 25 °C, 1,000 rpm. Reactions w ere quenched with 200 pL MeCN, diluted 20-fold with MeCN, filtered, and analyzed by liquid chromatography-mass spectrometry using the method described in Example 10.

[0311] Engineered polypeptides with >2-fold conversion relative to the parent polypeptide are listed in Table 12.1 and were re-expressed as shake flask powders as described in Example 1.Table 12.1 Variants and ConversionLevels of increased conversion were determined relative to the reference polypeptide of SEQ ID NO: 30 and defined as:“+” = conversion at least 2-fold that of reference polypeptide, but less than 3-fold; and“++” = conversion at least 3-fold, but less than 4-fold increased conversion, as compared to the reference polypeptide.

[0312] These variants produced 2b from 1c. and these engineered macrocyclizing thioesterases provide new biocatalytic reagents for use in new methods for the macrocyclization of 1c to 2b in the presence of le. The variant with mutations P8T; A17T; S250F (SEQ ID NO: 32) had the highest activity. Thus, the encoding polynucleotide (SEQ ID NO: 31) was selected for further directed evolution.Example 13 (Evolution Rd 12)Enzyme variants of SEQ ID NO: 32

[0313] In this Example, directed evolution of SEQ ID NO: 32 for improved selectivity for 2b formation 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 surface were subjected to mutagenesis. These libraries were plated to form single colonies, which were grow n and screened using the high-throughput growth and expression method described in Example 4 and the high-throughput assay described below.HTP assay for macrocvclase-catalyzed cyclization:

[0314] Cell pellets from 400 pL cultures of sequence variants were lysed with 200 pL of lysis buffer (100 mM MOPS pH 7.5, 4 mM MgCE, 3 U / mL DNase I, 0.5 g / L chicken egg white lysozy me, 0.5 g / L polymyxin B sulfate) for 1 h at 1,000 rpm. Cell debris was pelleted at 4,000 x g for 20 min. 1c and le were dissolved in a 4:3 solution of MeCN and 100 mM MOPS to final concentrations of 71.4 g / L and 13.1 g / L, respectively. 100 pL reactions consisting of 13 pL of cell lysate, 32 pL of 1.9% v / v iPrOH in 100 mM MOPS pH 7.5 and 35 pL of the solution of 1c and le were combined and incubated for 18 h at 25 °C, 1,000 rpm. Reactions were quenched with 150 pL MeCN, diluted 20-fold with MeCN, filtered, and analyzed by liquid chromatography-mass spectrometry using the method described in Example 10.

[0315] Engineered polypeptides with >1.4-fold selectivity improvement relative to the parent polypeptide are listed in Table 13. 1 and w ere re-expressed as shake flask powders as described in Example 1.Table 13.1 Variants and ConversionLevels of increased conversion were determined relative to the reference polypeptide of SEQ IDNO: 32 and defined as:“+” = selectivity7at least 1.4 that of reference polypeptide, but less than 1.6-fold; and“++’■ = selectivity at least 1.6-fold, but less than 1.8-fold increased selectivity, as compared to the reference polypeptide.

[0316] These variants produced 2b from 1c. and these engineered macrocyclizing thioesterases provide new biocatalytic reagents for use in new methods for the macrocyclization of 1c to 2b in the presence of le. The variant with mutations CUP; G14I; T325R (SEQ ID NO: 34) had the highest activity. Thus, the encoding polynucleotide (SEQ ID NO: 33) was selected for further directed evolution.Example 14 (Evolution Rdl3)Enzyme variants of SEQ ID NO: 34

[0317] In this Example, directed evolution of SEQ ID NO: 34 for improved selectivity for 2b formation and the resulting improved variants are described. Directed evolution was carried out by constructing libraries of variant genes in which positions associated with the enzy me core 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 4 and the high-throughput assay described below.HTP assay for macrocvclase-catalvzed cyclization:

[0318] Cell pellets from 400 pL cultures of sequence variants were lysed with 200 pL of lysis buffer (100 mM triethanolamine pH 7.5. 4 mM MgCh, 3 U / rnL DNase I, 0.5 g / L chicken egg white lysozyme, 0.5 g / L polymyxin B sulfate) for 1 h at 1,000 rpm. Cell debris was pelleted at 4,000 x g for 20 min. The supernatant was diluted 4-fold with 100 mM triethanolamine buffer pH 7.5. 1c and le were dissolved in a 2:1 solution of MeCN to 100 mM triethanolamine pH 7.5 to final concentrations of 50.0 g / L and 9.1 g / L, respectively. 100 pL reactions consisting of 10 pL of diluted cell lysate. 30 pL of 3.3% v / v iPrOH in 100 mM MOPS pH 7.5 and 60 pL of the solution of 1c and le were combined and incubated for 18 h at 25 °C, 1,000 rpm. Reactions were quenched by diluting 100-fold with MeCN, filtered, and analyzed by liquid chromatography - mass spectrometry7using the method described in Example 10.

[0319] Engineered polypeptides with >l-fold selectivity improvement relative to the parent polypeptide are listed in Table 14. 1 and were re-expressed as shake flask powders as described in Example 1.Table 14.1 Variants and Conversion26080Levels of increased selectivity were determined relative to the reference polypeptide of SEQ IDNO: 34 and defined as:“+” = selectivity at least l.O-fold that of reference polypeptide, but less than 1. 1 -fold; and“++” = selectivity at least 1. 1 -fold, but less than 1.2-fold increased selectivity, as compared to the reference polypeptide.

[0320] These variants produced 2b from 1c, and these engineered macrocyclizing thioesterases provide new biocatalytic reagents for use in new methods for the macrocyclization of 1c to 2b in the presence of le. The variant with mutations A9K; V19L; G20V; G269T; S405V (SEQ ID NO: 36) had the highest activity. Thus, the encoding polynucleotide (SEQ ID NO: 35) was selected for further directed evolution.Example 15 (Evolution Rdl4)Enzyme variants of SEQ ID NO: 36

[0321] In this Example, directed evolution of SEQ ID NO: 36 for increased selectivity against side product 5f and the resulting improved variants are described. Directed evolution was carried out by constructing libraries of variant genes in which positions associated with 5f binding 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 4 and the high-throughput assay described below.HTP assay for macrocvclase-catalyzed cyclization:

[0322] Cell pellets from 400 pL cultures of sequence variants were lysed with 200 pL of lysis buffer (100 mM MOPS pH 7.5, 4 mM MgCh. 3 U / mL DNase I, 0.5 g / L chicken egg white lysozyme, 0.5 g / L polymyxin B sulfate) for 1 h at 1 ,000 rpm. Cell debris was pelleted at 4,000 x g for 20 min. The supernatant was diluted 4-fold with 100 mM MOPS buffer pH 7.5. 1c and le were dissolved in a 2: 1 solution of 100 mM MOPS pH 7.5 to MeCN to final concentrations of 100 g / L and 18.3 g / L, respectively. 50 pL reactions consisting of 10 pL of diluted cell lysate, 10 pL of 5% v / v iPrOH in 100 mM MOPS pH 7.5 and 30 pL of the solution of le and le were combined and incubated for 18 h at 25 °C, 1,000 rpm. Reactions were quenched by diluting 10- fold with MeCN and diluting an additional 10-fold with 1: 1 MOPS / MeCN, filtered, and analyzed by liquid chromatography-mass spectrometry using the method described in Example 10.26080

[0323] Engineered polypeptides with >1.2-fold selectivity relative to the parent polypeptide are listed in Table 15.1 and were re-expressed as shake flask powders as described in Example 1.Table 15.1 Variants and SelectivityLevels of increased selectivity were determined relative to the reference polypeptide of SEQ ID NO: 36 and defined as:“+” = selectivity at least 1.2-fold that of reference polypeptide, but less than 2.4-fold; and “++” = selectivity at least 2.4-fold, but less than 3-fold increased selectivity, as compared to the reference polypeptide.

[0324] These variants produced 2b from 1c, and these engineered macrocyclizing thioesterases provide new biocatalytic reagents for use in new methods for the macrocyclization of 1c to 2b in the presence of le. The variant with mutations G203D; G389N; R406K; A408F; F410V (SEQ ID NO: 38) had the highest activity7. Thus, the encoding polynucleotide (SEQ ID NO: 37) was selected for further directed evolution.Example 16 (Evolution Rdl5)Enzy me variants of SEQ ID NO: 38

[0325] In this Example, directed evolution of SEQ ID NO: 38 for improved hydrolysis selectivity against 6i and the resulting improved variants are described. Directed evolution was carried out by constructing libraries of variant genes in which positions associated with 6i binding were subjected to mutagenesis. These libraries were plated to form single colonies, which were grown and screened using the high-throughput grow th and expression method described in Example 4 and the high-throughput assay described below.HTP assay for macrocvclase-catalyzed cyclization:

[0326] Cell pellets from 400 pL cultures of sequence variants w ere lysed with 200 pL of lysis buffer (100 mM MOPS pH 7.5, 4 mM MgCh, 3 U / mL DNase I, 0.5 g / L chicken egg white26080 lysozyme, 0.5 g / L polymyxin B sulfate) for 1 h at 1,000 rpm. Cell debris was pelleted at 4,000 x g for 20 min. The supernatant was diluted 4-fold with 100 mM MOPS buffer pH 7.5. 1c and le were dissolved in a 2: 1 solution of 100 mM MOPS pH 7.5 to MeCN to final concentrations of 100 g / L and 18.3 g / L, respectively. 100 pL reactions consisting of 10 pL of diluted cell lysate, 30 pL of 3.3% v / v iPrOH in 100 mM MOPS pH 7.5 and 60 pL of the solution of 1c and le were combined and incubated for 18 h at 25 °C, 1,000 rpm. Reactions were quenched by diluting 10- fold with MeCN, then diluting again 10-fold with 1 : 1 MOPS / MeCN, filtered, and analyzed by liquid chromatography-mass spectrometry using the method described in Example 10.

[0327] Engineered polypeptides with >1.6-fold hydrolysis selectivity relative to the parent polypeptide are listed in Table 16. 1 and were re-expressed as shake flask powders as described in Example 1.Table 16.1 Variants and ConversionLevels of increased selectivity were determined relative to the reference polypeptide of SEQ ID NO: 38 and defined as:“+” = selectivity at least 1.6-fold that of reference polypeptide, but less than 2-fold; and“++” = selectivity at least 2-fold, but less than 2.5-fold increased selectivity, as compared to the reference polypeptide.

[0328] These variants produced 2b from 1c, and these engineered macrocyclizing thioesterases provide new biocatalytic reagents for use in new methods for the macrocyclization of 1c to 2b in the presence of le. The variant with mutations G12K; D15P; D31G; Q223M; Q327G; L387M (SEQ ID NO: 41) had the highest activity. Thus, the encoding polynucleotide (SEQ ID NO: 39) was selected for further directed evolution. An optimized polynucleotide sequence of SEQ ID NO: 40 that also encodes SEQ ID NO: 41 was generated.26080Example 17 (Evolution Rd 16)Enzyme variants of SEQ ID NO: 41

[0329] In this Example, directed evolution of SEQ ID NO: 41 for improved selection against undesired side product 6i and the resulting improved variants are described. Directed evolution was carried out by constructing libraries of variant genes in which positions associated with 6i binding were subjected to mutagenesis. These libraries were plated to form single colonies, which were grown and screened using the high-throughput grow th and expression method described in Example 4 and the high-throughput assay described below.HTP assay for macrocvclase-catalyzed cyclization:

[0330] Cell pellets from 400 pL cultures of sequence variants w ere lysed with 200 pL of lysis buffer (100 mM MOPS pH 7.5, 4 mM MgCE, 3 U / mL DNase I, 0.5 g / L chicken egg white lysozy me, 0.5 g / L polymyxin B sulfate) for 1 h at 1,000 rpm. Cell debris was pelleted at 4,000 x g for 20 min. The supernatant was diluted 8-fold with 100 m MOPS buffer pH 7.5. 1c and le were dissolved in a 2: 1 solution of 100 mM MOPS pH 7.5 to MeCN to final concentrations of 100 g / L and 18.3 g / L, respectively. 50 pL reactions consisting of 10 pL of diluted cell lysate, 10 pL of 5% v / v iPrOH in 100 mM MOPS pH 7.5 and 30 pL of the solution of 1c and le w ere combined and incubated for 18 h at 25 °C, 1,000 rpm. Reactions were quenched by diluting 10- fold with MeCN. then diluting again 10-fold with 1 : 1 MOPS / MeCN, filtered, and analyzed by liquid chromatography-mass spectrometry using the method described in Example 10.

[0331] Engineered polypeptides with >l-fold selectivity improvement relative to the parent polypeptide are listed in Table 17.1 and were re-expressed as shake flask pow ders as described in Example 1.Table 17.1 Variants and ConversionNO: 41 and defined as:“+” = selectivity at least 1-fold that of reference polypeptide, but less than 15-fold; and“++” = selectivity at least 15-fold, but less than 25-fold increased conversion, as compared to the reference polypeptide.26080

[0332] These variants produced 2b from 1c, and these engineered macrocyclizing thioesterases provide new biocatalytic reagents for use in new methods for the macrocyclization of 1c to 2b in the presence of le. The variant with mutations R59G; SI 15L; Al 19L; F299S; A391L (SEQ ID NO: 44) had the highest activity. Thus, the encoding polynucleotide (SEQ ID NO: 42) was used to generate an optimized polynucleotide sequence (SEQ ID NO: 43), and this optimized sequence was selected for further directed evolution.Example 18 (Evolution Rdl7)Enzyme variants of SEQ ID NO: 44

[0333] In this Example, directed evolution of SEQ ID NO: 44 for improved macrocyclization activity and the resulting improved variants are described. Directed evolution was carried out by constructing libraries of variant genes in which positions previously mutated or not targeted in at least 4 rounds 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 4 and the high-throughput assay described below7.HTP assay for macrocvclase-catalvzed cyclization:

[0334] Cell pellets from 400 pL cultures of sequence variants were lysed with 200 pL of lysis buffer (100 mM MOPS pH 7.5, 4 mM MgCh. 3 U / mL DNase I, 0.5 g / L chicken egg white lysozyme, 0.5 g / L polymyxin B sulfate) for 1 h at 1,000 rpm. Cell debris was pelleted at 4,000 x g for 20 min. The supernatant w7as diluted 10-fold with 100 mM MOPS buffer pH 7.5. 1c and le were dissolved in a 5: 1 solution of 100 mM MOPS pH 7.5 to MeCN to final concentrations of 100 g / L and 34.9 g / L, respectively. 50 pL reactions consisting of 10 pL of diluted cell lysate, 10 pL of 5% v / v iPrOH in 100 mM MOPS pH 7.5 and 30 pL of the solution of 1c and le were combined and incubated for 18 h at 25 °C, 1,000 rpm. 1,000 rpm. Reactions were quenched by diluting 10-fold with MeCN, then diluting again 10-fold w ith 1: 1 MOPS / MeCN, filtered, and analyzed by liquid chromatography-mass spectrometry using the method described in Example 10.

[0335] Engineered polypeptides with >l-fold conversion relative to the parent polypeptide are listed in Table 18.1 and w7ere re-expressed as shake flask powders as described in Example 1.Table 18.1 Variants and Conversion26080Levels of increased conversion were determined relative to the reference polypeptide of SEQ ID NO: 44 and defined as:“+” = conversion at least 1-fold that of reference polypeptide, but less than 1.1 -fold; and“++” = conversion at least 1.1 -fold, but less than 1.5-fold increased conversion, as compared to the reference polypeptide.

[0336] These variants produced 2b from 1c, and these engineered macrocyclizing thioesterases provide new biocatalytic reagents for use in new methods for the macrocyclization of 1c to 2b in the presence of le. The variant with mutations K9G; L391Q (SEQ ID NO: 46) had the highest activity. Thus, the encoding polynucleotide (SEQ ID NO: 45) was selected for further directed evolution.Example 19 (Evolution Rdl8)Enzyme variants of SEO ID NO: 46

[0337] In this Example, directed evolution of SEQ ID NO: 46 for improved selectivity for 2b formation and the resulting improved variants are described. Directed evolution was carried out by constructing libraries of variant genes in which positions associated with 2b-binding loops 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 4 and the high-throughput assay described below.HTP assay for macrocvclase-catalvzed cyclization:

[0338] Cell pellets from 400 pL cultures of sequence variants were lysed with 200 pL of lysis buffer (100 mM MOPS pH 7.5, 4 mM MgCh. 3 U / mL DNase I, 0.5 g / L chicken egg white lysozyme, 0.5 g / L polymyxin B sulfate) for 1 h at 1,000 rpm. Cell debris was pelleted at 4,000 x g for 20 min. The supernatant was diluted 5-fold with 100 mM MOPS buffer pH 7.5. 1c and le were dissolved in a 5: 1 solution of 100 mM MOPS pH 7.5 to MeCN to final concentrations of 100 g / L and 37.4 g / L, respectively. 50 pL reactions consisting of 10 pL of diluted cell lysate, 10 pL of 5% v / v iPrOH in 100 mM MOPS pH 7.5 and 30 pL of the solution of le and le were combined and incubated for 18 h at 25 °C, 1,000 rpm. Reactions were quenched by diluting 10- fold with MeCN, then diluting again 10-fold with 1 : 1 MOPS / MeCN, filtered, and analyzed by liquid chromatography-mass spectrometry using the method described in Example 10.26080

[0339] Engineered polypeptides with >1.9-fold selectivity ratio relative to the parent polypeptide are listed in Table 19. 1 and were re-expressed as shake flask powders as described in Example 1.Table 19.1 Variants and ConversionIn Table 19. 1, -391N and -392G indicate that a residue was inserted at the positions that were originally 391 or 392 (asparagine and glycine insertions, respectively) with reference to SEQ ID NO: 46. Amino acid positions downstream of the insertion use numbering accounting for the inserted positions in this Example and all later Examples.Levels of increased selectivity were determined relative to the reference polypeptide of SEQ ID NO: 46 and defined as:“+” = selectivity at least 1.9-fold that of reference polypeptide, but less than 3.5-fold; and“++” = selectivity at least 3.5-fold, but less than 5.5-fold increased selectivity, as compared to the reference polypeptide.

[0340] These variants produced 2b from 1c, and these engineered macrocyclizing thioesterases provide new biocatalytic reagents for use in new methods for the macrocyclization of 1c to 2b in the presence of le. The variant with mutations D258H; C303K; G327Y;-391N;-392G; F435W (SEQ ID NO: 48) had the highest activity. Thus, the encoding polynucleotide (SEQ ID NO: 47) was selected for further directed evolution.26080Example 20 (Evolution Rdl9)Enzy me variants of SEQ ID NO: 48

[0341] In this Example, directed evolution of SEQ ID NO: 48 for improved macrocyclization activity and the resulting improved variants are described. Directed evolution was carried out by constructing libraries of variant genes in which positions associated with 1c binding and positions spatially proximal to the -391N;-392G insertion mutations of the three-dimensional structure of SEQ ID NO: 48 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 4 and the high-throughput assay described below.HTP assay for macrocvclase-catalvzed cyclization:

[0342] Cell pellets from 400 pL cultures of sequence variants were lysed with 200 pL of lysis buffer (100 mM MOPS pH 7.5, 4 mM MgCh. 3 U / mL DNase I, 0.5 g / L chicken egg white lysozyme, 0.5 g / L polymyxin B sulfate) for 1 h at 1,000 rpm. Cell debris was pelleted at 4,000 x g for 20 min. The supernatant was diluted 5-fold with 100 mM MOPS buffer pH 7.5. The pH of 50 mM 1c coupling stream with 20 mM 1g dissolved in 100 mM MOPS and 12.5% v / v MeCN was adjusted to pH 7.5. 50 pL reactions consisting of 10 pL of diluted cell lysate and 5 pL of the solution of 1c and 1g coupling stream were combined and incubated for 18 h at 25 °C, 1.000 rpm. Reactions were quenched by diluting 10-fold with MeCN, then diluting again 10-fold with 1 : 1 MOPS / MeCN, filtered, and analyzed by liquid chromatography -mass spectrometry using the method described in Example 10.

[0343] Engineered polypeptides with >l-fold conversion relative to the parent polypeptide are listed in Table 20.1 and were re-expressed as shake flask powders as described in Example 1.Table 20.1 Variants and ConversionLevels of increased conversion were determined relative to the reference polypeptide of SEQ ID NO: 48 and defined as:“+” = conversion at least 1-fold that of reference polypeptide, but less than 1.15-fold; and “++” = conversion at least 1.15-fold, but less than 1.3-fold increased conversion, as compared to the reference polypeptide.26080

[0344] These variants produced 2b from 1c. and these engineered macrocyclizing thioesterases provide new biocatalytic reagents for use in new methods for the macrocyclization of 1c to 2b in the presence of le. The variant with mutations D301E; A431G (SEQ ID NO: 51) had the highest activity. Thus, the encoding polynucleotide (SEQ ID NO: 49) was used to generate an optimized polynucleotide sequence (SEQ ID NO: 50), and this optimized sequence was selected for further directed evolution.Example 21 (Evolution Rd20)Enzyme variants of SEQ ID NO: 51

[0345] In this Example, directed evolution of SEQ ID NO: 51 for improved tolerance to the presence of MeCN and the resulting improved variants are described. Directed evolution was carried out by constructing libraries of variant genes in which positions associated with the polypeptide surface 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 4 and the high-throughput assay described below.HTP assay for macrocvclase-catalvzed cyclization:

[0346] Cell pellets from 400 pL cultures of sequence variants were lysed with 200 pL of lysis buffer (100 mM MOPS pH 7.5, 4 mM MgCh. 3 U / mL DNase I, 0.5 g / L chicken egg white lysozyme, 0.5 g / L polymyxin B sulfate) for 1 h at 1,000 rpm. Cell debris was pelleted at 4,000 x g for 20 min. The supernatant was diluted 10-fold with 100 mM MOPS buffer pH 7.5. The pH of 48.9 mM 1c coupling stream with 19.6 mM 1g dissolved in 100 mM MOPS and 16.7% v / v MeCN was adjusted to pH 7.5. 50 pL reactions consisting of 5 pL of diluted cell lysate and 45 pL of the solution of 1c and 1g coupling stream were combined and incubated for 18 h at 25 °C, 1,000 rpm. Reactions were quenched by diluting 10-fold with MeCN, then diluting again 10-fold with 1: 1 MOPS / MeCN, filtered, and analyzed by liquid chromatography-mass spectrometry using the method described in Example 10.

[0347] Engineered polypeptides with >1. 1-fold conversion relative to the parent polypeptide are listed in Table 21.1 and were re-expressed as shake flask powders as described in Example 1.Table 21.1 Variants and Conversion26080Levels of increased conversion were determined relative to the reference polypeptide of SEQ ID NO: 51 and defined as:“+” = conversion at least 1.1 -fold that of reference polypeptide, but less than 1.6-fold; and “++” = conversion at least 1.6-fold, but less than 2.2-fold increased conversion, as compared to the reference polypeptide.

[0348] These variants produced 2b from 1c, and these engineered macrocyclizing thioesterases provide new biocatalytic reagents for use in new methods for the macrocyclization of 1c to 2b in the presence of 1g. The variant with mutations D122G; E245D; H258S (SEQ ID NO: 54) had the highest activity7. Thus, the encoding polynucleotide (SEQ ID NO: 52) was used to generate an optimized polynucleotide sequence (SEQ ID NO: 53), and this optimized sequence was selected for further directed evolution.Example 22 (Evolution Rd21)Enzyme variants of SEQ ID NO: 54

[0349] In this Example, directed evolution of SEQ ID NO: 54 for improved selectivity for the formation of 2b and the resulting improved variants are described. Directed evolution was earned out by constructing libraries of variant genes in which positions associated with 2b binding 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 4 and the high-throughput assay described below.HTP assay for macrocv clase-catalvzed cyclization:

[0350] Cell pellets from 400 pL cultures of sequence variants were lysed with 200 pL of lysis buffer (100 mM MOPS pH 7.5, 4 mM MgCh, 3 U / mL DNase I, 0.5 g / L chicken egg white lysozyme, 0.5 g / L polymyxin B sulfate) for 1 h at 1,000 rpm. Cell debris was pelleted at 4.000 x g for 20 min. The supernatant was diluted 20-fold with 100 mM MOPS buffer pH 7.5. The pH of 48.9 mM 1c coupling stream with 19.6 mM 1g dissolved in 100 mM MOPS and 16.7% v / v MeCN was adjusted to pH 7.5. 50 pL reactions consisting of 5 pL of diluted cell lysate and 45 pL of the solution of 1c and 1g coupling stream were combined and incubated for 18 h at 25 °C, 1,000 rpm. Reactions were quenched by diluting 10-fold with MeCN, then diluting again 10-fold with 1: 1 MOPS / MeCN, filtered, and analyzed by liquid chromatography -mass spectrometry using the method described in Example 10.26080

[0351] Engineered polypeptides with >1.4-fold selectivity 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 ConversionNO: 54 and defined as:“+” = selectivity at least 1.4-fold that of reference polypeptide, but less than 1.7-fold; and“++” = selectivity at least 1.7-fold, but less than 2.1 -fold increased selectivity’, as compared to the reference polypeptide.

[0352] These variants produced 2b from 1c, and these engineered macrocyclizing thioesterases provide new biocatalytic reagents for use in new methods for the macrocyclization of 1c to 2b in the presence of 1g. The variant with mutations S87R; P172K; D244H; F250T; P257N; R313T; R325K; G390Q (SEQ ID NO: 57) had the highest activity’. Thus, the encoding polynucleotide (SEQ ID NO: 55) was selected for further directed evolution. An optimized polynucleotide sequence of SEQ ID NO: 56 that also encodes SEQ ID NO: 57 yvas generated.Example 23 (Evolution Rd22)Enzyme variants of SEQ ID NO: 57

[0353] In this Example, directed evolution of SEQ ID NO: 57 for improved enzyme expression and selectivity, and the resulting improved variants are described. Directed evolution was carried26080 out by constructing libraries of variant genes in which positions associated with the enzyme surface were subjected to mutagenesis. These libraries were plated to form single colonies, which were grown and screened using the high-throughput grow th and expression method described in Example 2 and the high-throughput assay described below.HTP assay for macrocvclase-catalvzed cyclization:

[0354] Cell pellets from 400 pL cultures of sequence variants w ere lysed with 200 pL of lysis buffer (100 mM MOPS pH 7.5, 4 mM MgCh, 3 U / rnL DNase I, 0.5 g / L chicken egg white lysozyme, 0.5 g / L polymyxin B sulfate) for 1 h at 1,000 rpm. Cell debris was pelleted at 4,000 x g for 20 min. The supernatant was diluted 5-fold with 100 m MOPS buffer pH 7.5. The pH of 51.6 mM 1c coupling stream with 20.6 mM 1g dissolved in 100 mM MOPS and 10.4% v / v MeCN w as adjusted to pH 7.5. 50 pL reactions consisting of 4 pL of diluted cell lysate and 46 pL of the solution of 1c and 1g coupling stream were combined and incubated for 18 h at 25 °C, 1,000 rpm. Reactions were quenched by diluting 10-fold with MeCN, then diluting again 10-fold with 1: 1 MOPS / MeCN, filtered, and analyzed by liquid chromatography-mass spectrometry using the method described in Example 10.

[0355] Engineered polypeptides with >1. 1-fold selectivity relative to the parent polypeptide are listed in Table 23. 1 and were re-expressed as shake flask powders as described in Example 1.Table 23.1 Variants and ConversionNO: 57 and defined as:■‘+’?= selectivity at least 1.1-fold that of reference polypeptide, but less than 1.2-fold; and“++” = selectivity at least 1.2-fold, but less than 1.4-fold increased selectivity, as compared to the reference polypeptide.

[0356] These variants produced 2b from 1c, and these engineered macrocyclizing thioesterases provide new biocatalytic reagents for use in new methods for the macrocyclization of 1c to 2b in the presence of 1g. The variant with mutations L164T; S450H (SEQ ID NO: 61) had the highest activity7. Thus, the encoding polynucleotide (SEQ ID NO: 58) w as used to generate two optimized polynucleotide sequences (SEQ ID NO: 59 and SEQ ID NO: 60). SEQ ID NO: 59, which also encodes SEQ ID NO: 61, was selected for further directed evolution26080Example 24 (Evolution Rd23)Enzy me variants of SEQ ID NO: 61

[0357] In this Example, directed evolution of SEQ ID NO: 61 for improved enzyme expression and selectivity, 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 surface 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 2 and the high-throughput assay described below.HTP assay for macrocvclase-catalvzed cyclization:

[0358] Cell pellets from 400 pL cultures of sequence variants were lysed with 200 pL of lysis buffer (100 mM EPPS pH 7.6, 4 mM MgCh, 3 U / mL DNase I, 0.5 g / L chicken egg white lysozyme, 0.5 g / L polymyxin B sulfate) for 1 h at 1,000 rpm. Cell debris was pelleted at 4.000 x g for 20 min. The supernatant was diluted 10-fold with 100 mM EPPS buffer pH 7.6. The pH of 58.9 mM 1c coupling stream with 23.6 mM 1g dissolved in 100 mM EPPS pH 7.6, 12.2% v / v MeCN, 0.6% v / v iPrOH, 2.8% v / v methyl -tert-butyl ester and 0.6% v / v heptane was adjusted to pH 7.6. 100 pL reactions consisting of 10 pL of diluted cell lysate and 90 pL of the solution of 1c and 1g coupling stream were combined and incubated for 18 h at 25 °C, 1,000 rpm. Reactions were quenched by diluting 10-fold with MeCN, then diluting again 10-fold with 1 : 1 EPPS / MeCN, filtered, and analyzed by liquid chromatography-mass spectrometry using the method described in Example 10.

[0359] Engineered polypeptides with >1. 1-fold selectivity relative to the parent polypeptide are listed in Table 24.1 and were re-expressed as shake flask powders as described in Example 1.Table 24.1 Variants and ConversionNO: 61 and defined as:“+” = selectivity at least 1.1 -fold that of reference polypeptide, but less than 1.4-fold; and■‘++” = selectivity at least 1.4-fold, but less than 1.7-fold increased selectivity, as compared to the reference polypeptide.26080

[0360] These variants produced 2b from 1c. and these engineered macrocyclizing thioesterases provide new biocatalytic reagents for use in new methods for the macrocyclization of 1c to 2b in the presence of 1g. The variant with mutations G9D; C218G; P351V (SEQ ID NO: 64) had the highest activity. Thus, the encoding polynucleotide (SEQ ID NO: 62) was selected for further directed evolution. An optimized polynucleotide sequence of SEQ ID NO: 63 that also encodes SEQ ID NO: 64 was generated.Example 25 (Evolution Rd24)Enzyme variants of SEQ ID NO: 64

[0361] In this Example, directed evolution of SEQ ID NO: 64 for improved enzyme expression and selectivity, 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 core 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 2 and the high-throughput assay described below.HTP assay for macrocvclase-catalvzed cyclization:

[0362] Cell pellets from 400 pL cultures of sequence variants were lysed with 200 pL of lysis buffer (100 mM MOPS pH 7.5, 4 mM MgCh. 3 U / mL DNase I, 0.5 g / L chicken egg white lysozyme, 0.5 g / L polymyxin B sulfate) for 1 h at 1,000 rpm. Cell debris was pelleted at 4,000 x g for 20 min. The supernatant was diluted 10-fold with 100 mM MOPS buffer pH 7.5. The pH of 62.2 mM 1c coupling stream with 24.9 mM 1g dissolved in 100 mM MOPS, 13.3% v / v MeCN and 1.1% v / v heptane was adjusted to pH 7.5. 50 pL reactions consisting of 5 pL of diluted cell lysate and 45 pL of the solution of 1c and 1g coupling stream were combined and incubated for 18 h at 25 °C, 1,000 rpm. Reactions were quenched by diluting 10-fold with MeCN, then diluting again 10-fold with 1 :1 MOPS / MeCN, filtered, and analyzed by liquid chromatography -mass spectrometry using the method described in Example 10.

[0363] Engineered polypeptides with > 1.0-fold selectivity relative to the parent polypeptide are listed in Table 25.1 and were re-expressed as shake flask powders as described in Example 1.Table 25.1 Variants and Conversion26080Levels of increased selectivity were determined relative to the reference polypeptide of SEQ ID NO: 64 and defined as:“+” = selectivity at least l.O-fold that of reference polypeptide, but less than 1.2-fold; and“++” = selectivity at least 1.2-fold, but less than 1.4-fold increased selectivity, as compared to the reference polypeptide.

[0364] These variants produced 2b from 1c, and these engineered macrocyclizing thioesterases provide new7biocatalytic reagents for use in new methods for the macrocyclization of 1c to 2b in the presence of 1g. The variant with mutations P18D; V351I; N391A; G392S (SEQ ID NO: 67) had the highest activity. Thus, the encoding polynucleotide (SEQ ID NO: 65) was selected for further directed evolution. An optimized polynucleotide sequence of SEQ ID NO: 66 that also encodes SEQ ID NO: 67 was generated.Example 26 (Evolution Rd25)Enzyme variants of SEQ ID NO: 67

[0365] In this Example, directed evolution of SEQ ID NO: 67 for improved organic solvent tolerance 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 surface and 2b binding 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 2 and the high-throughput assay described below7.HTP assay for macrocvclase-catalyzed cyclization:

[0366] Cell pellets from 400 pL cultures of sequence variants were lysed with 200 pL of lysis buffer (100 mM MOPS pH 7.5, 4 mM MgCk, 3 U / mL DNase I, 0.5 g / L chicken egg white lysozyme, 0.5 g / L polymyxin B sulfate) for 1 h at 1,000 rpm. Cell debris was pelleted at 4,000 x g for 20 min. The supernatant was diluted 10-fold with 100 mM MOPS buffer pH 7.5. The pH of 69.4 mM 1c coupling stream with 27.8 mM 1g dissolved in 100 mM MOPS, 13.3% v / v MeCN and 1.1% v / v heptane was adjusted to pH 7.5. 50 pL reactions consisting of 5 pL of diluted cell lysate and 45 pL of the solution of 1c and 1g coupling stream w ere combined and incubated for 18 h at 25 °C, 1,000 rpm. Reactions were quenched by diluting 10-fold with MeCN, then diluting again 10-fold with 1 :1 MOPS / MeCN, filtered, and analyzed by liquid chromatography -mass spectrometry using the method described in Example 10.26080

[0367] Engineered polypeptides with >1.0-fold selectivity relative to the parent polypeptide are listed in Table 26.1 and were re-expressed as shake flask powders as described in Example 1.Table 26.1 Variants and ConversionNO: 67 and defined as:“+” = selectivity at least 1.0-fold that of reference polypeptide, but less than 1.2-fold; and“++” = selectivity at least 1.2-fold, but less than 1.7-fold increased selectivity’, as compared to the reference polypeptide.

[0368] These variants produced 2b from 1c, and these engineered macrocyclizing thioesterases provide new biocatalytic reagents for use in new methods for the macrocyclization of 1c to 2b in the presence of 1g. The variant with mutations Y5I; A151S; P220Q; A391G (SEQ ID NO: 73) had the highest activity’ and selectivity. Thus, the encoding polynucleotide (SEQ ID NO: 71) was selected for production. An optimized polynucleotide sequence of SEQ ID NO: 72 that also encodes SEQ ID NO: 73 yvas generated and can be used for production.

[0369] Table 27 provides a summary of exemplary' sequences provided herein.

[0370] All references (e.g.. publications or patents or patent applications) cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) w as specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Other embodiments are within the following claims.26080260802608026080260802608026080260802608026080260802608026080260802608026080260802608026080260802608026080260802608026080260802608026080260802608026080260802608026080

Claims

WHAT IS CLAIMED IS:

1. An engineered polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 6, wherein the polypeptide comprises an amino acid substitution at one or more amino acid positions selected from 5, 6. 7, 8, 9. 11, 12, 14, 15, 17, 18, 19, 20, 31, 45, 59, 80, 82, 87, 89, 94, 115, 1 19, 122, 151, 163, 164, 172, 204, 212, 218, 220, 223, 238, 244, 245, 250, 257, 258, 259, 267, 269, 275, 281, 284, 285, 294, 299, 300, 301, 303, 313, 325, 327, 328, 351, 354, 387, 389, 390, 391, 399, 405, 406, 407, 408, 410, 418, 429, 432, 433, 434, 436. and 448, 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 74 amino acid substitutions, each amino acid substitution at an amino acid position selected from 5, 6, 7, 8, 9, 11, 12, 14, 15, 17, 18, 19, 20, 31, 45, 59, 80, 82, 87, 89, 94, 115, 119, 122, 151, 163, 164, 172, 204, 212, 218, 220, 223, 238, 244, 245, 250, 257, 258, 259, 267, 269, 275, 281, 284, 285, 294, 299, 300, 301, 303, 313, 325, 327, 328, 351, 354, 387, 389, 390, 391, 399, 405, 406, 407, 408, 410, 418. 429, 432, 433, 434, 436. and 448.

3. The polypeptide of claim 1 or 2, wherein the polypeptide comprises at least one amino acid substitution selected from Y5I, A6E, D7K, P8T, A9D, CUP, G12K, G14I, D15P, A17T, P18D, V19L, G20V, D31G, L45T, R59G. A80M. L82F, D87R, D89L, R94T, S115L, A119L, D122G, A151S, A163R. L164T. P172K, F204L, A212N. T218G, P220Q, L223M. L238C, D244H, E245D, D250T, P257N, D258S, A259I, T267L, G269T, L275F, P281L, G284A, R285V, L294V, T299S, L300R, D301E, A303K, R313T, T325K, Q327Y, A328S, P351I, A354P, L387M, G389N. G390Q, A391Q, A399E, S405V, R406K, G407P, A408F, F410V, A418N, S429G, T432L, L433W, L434V, E436M, and S448H.

4. The polypeptide of 3, wherein the polypeptide comprises from 4 to 74 amino acid substitutions selected from Y5I, A6E, D7K, P8T, A9D, CUP, G12K, G14I, D15P, A17T, P18D, V19L, G20V, D31G, L45T, R59G, A80M, L82F, D87R, D89L, R94T, S115L, A119L, D122G, A151S, A163R, L164T, P172K, F204L, A212N, T218G, P220Q, L223M, L238C, D244H, E245D, D250T, P257N, D258S, A259I, T267L, G269T, L275F, P281L, G284A, R285V, L294V, T299S, L300R, D301E, A303K, R313T, T325K, Q327Y, A328S, P351I, A354P, L387M,G389N, G390Q. A391Q, A399E, S405V. R406K, G407P, A408F, F410V. A418N, S429G, T432L, L433W, L434V, E436M, and S448H.

5. The polypeptide of 4, wherein the polypeptide comprises all of the following amino acid substitutions: Y5I, A6E, D7K. P8T, A9D. Cl IP, G12K, G14I, D15P. A17T, P18D, V19L.G20V, D31G, L45T, R59G, A80M, L82F, D87R, D89L, R94T, SI 15L, A119L, D122G, A151S, A163R, L164T, P172K, F204L, A212N, T218G, P220Q, L223M, L238C, D244H, E245D, D250T, P257N, D258S, A259I, T267L, G269T, L275F, P281L, G284A, R285V, L294V, T299S, L300R, D301E, A303K, R313T. T325K, Q327Y, A328S, P351I, A354P. L387M, G389N. G390Q, A391Q, A399E, S405V, R406K, G407P, A408F, F410V, A418N, S429G, T432L, L433W, L434V, E436M, and S448H.

6. The polypeptide of any one of claims 1-5, further comprising an amino acid insertion between amino acid positions 390 and 391, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 6.

7. The polypeptide of claim 6, wherein the amino acid insertion comprises the amino acid sequence of GS.

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 Tables 1.4, 1.5, 2.1, 3.1, 4.1, 5.1, 6.1, 7.1,8.1, 9.1, 10.1, 11.1, 12.1, 13.

1. 14.1, 15.1, 16.1, 17.1, 18.1, 19.1, 20.1, 21.1, 22.1, 23.

1. 24.1.25.

1. or 26.1.

9. The polypeptide of any one of claims 1-8, wherein the amino acid sequence comprises any one of SEQ ID NO: 73, 70, 8, 10, 12, 14, 16, 18. 20, 22, 24, 26, 28, 30, 32. 34, 36, 38, 41, 44. 46, 48, 51, 54, 57, 61, 64, or 67.

10. The polypeptide of any one of claims 1-9, wherein the amino acid sequence comprises SEQ ID NO: 73.

11. The polypeptide of any one of claims 1-9, wherein the amino acid sequence comprises SEQ ID NO: 70.

12. An engineered polypeptide comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 73.

13. The polypeptide of claim 12. wherein the amino acid sequence has at least 99% sequence identity to SEQ ID NO: 73.

14. The polypeptide of claim 12 or 13, comprising the amino acid sequence of SEQ ID NO: 73.

15. An engineered polypeptide comprising an amino acid sequence having at least 85% sequence identity to amino acid residues 1-449 of SEQ ID NO: 73.

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 or an ester 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; and / or f) increased thermostability.

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.2608021. The polynucleotide of claim 19 or 20, wherein the polynucleotide comprises any one of SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 40, 42, 43, 45, 47, 49, 50, 52, 53, 55, 56, 58, 59, 60, 62, 63, 65, 66, 68, 69, 71, or 72.

22. A polynucleotide comprising at least 80% sequence identity to any one of SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 40, 42, 43, 45, 47, 49, 50, 52, 53, 55, 56, 58, 59, 60, 62, 63, 65, 66, 68, 69, 71, or 72, wherein the polynucleotide does not comprise SEQ ID NO: 1.

23. The polynucleotide of claim 22, comprising the polynucleotide sequence of SEQ ID NO: 71 or 72.

24. The polynucleotide of claim 22, comprising the polynucleotide sequence of SEQ ID NO: 68 or 69.

25. An expression vector comprising at least one polynucleotide sequence of any one of claims 19-24.

26. The expression vector of claim 25, wherein the polynucleotide sequence is operably linked to a control sequence.

27. The expression vector of claim 26. wherein the control sequence is a promoter.

28. The expression vector of claim 27, wherein the promoter is a heterologous promoter.

29. A host cell comprising the polynucleotide of any one of claims 19-23 or the expression vector of any one of claims 25-28.

30. The host cell of claim 29, wherein the host cell is prokaryotic or eukaryotic.

31. A method of producing a polypeptide, the method comprising culturing the host cell of claim 29 or 30 under conditions such that the polypeptide encoded by the polynucleotide is produced.2608032. The method of claim 31. further comprising the step of recovering the polypeptide.

33. The method of claim 32, further comprising the step of purifying the polypeptide.

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

35. The method of claim 34, wherein the method produces a cyclic peptide.

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