Engineered transaminase peptides

Abstract

The present invention provides engineered transaminase polypeptides and compositions thereof, and polynucleotides encoding engineered transaminase polypeptides. Also provided are methods for producing transaminases. In some embodiments, the engineered transaminase polypeptides are optimized to provide improved enzyme properties in wet organic solvents under minimal water-containing conditions.

Description

[0001] This application claims priority to U.S. Patent Application Serial No. 62 / 792,518, filed on January 15, 2019, which is hereby incorporated by reference in its entirety for all purposes. Field of the Invention

[0002] The present disclosure relates to engineered transaminase polypeptides that can be used to produce pharmaceutical and fine chemical amine compounds under industrial process conditions.

[0003] Reference to a sequence listing, table or computer program

[0004] An official copy of the sequence listing is submitted with the specification as a text file in ASCII format via EFS-Web, with the file name "CX2-183WO1_ST25.txt", the creation date of January 10, 2020, and the size of 1.72 megabytes. The sequence listing submitted via EFS-Web is part of the specification and is incorporated herein by reference in its entirety.

[0005] background

[0006] Transaminases (EC 2.6.1) catalyze the transfer of an amino group, a pair of electrons, and a proton from an amino donor compound to a keto group of an amino acceptor compound. The transaminase reaction can result in the formation of a chiral amine product compound. As shown in Scheme 1, an amino acceptor compound (B) (which is a keto substrate precursor of the desired chiral amine product (D)) reacts with an amino donor compound (A) in the presence of a transaminase. The transaminase catalyzes the transfer of a primary amine group of an amino donor compound (A) to a keto group of an amino acceptor compound (B). The transaminase reaction produces a chiral amine product compound (D) (assuming R 1 With R 2 different) and a new amino acceptor byproduct (or "carbonyl byproduct") compound (C) having a keto group.

[0007] Solution 1

[0008]

[0009] Chiral amine compounds are often used as intermediates or synthons in the pharmaceutical, agrochemical and chemical industries for the preparation of a wide range of commercially desirable compounds, such as cephalosporins or pyrrolidine derivatives. Typically these industrial applications of chiral amine compounds involve the use of only one specific stereoisomeric form of the molecule, e.g. only the (R) or (S) enantiomer is physiologically active. Transaminases are highly stereoselective and have many potential industrial uses for the synthesis of optically pure chiral amine compounds.

[0010] Examples of the use of transaminases to prepare chiral amine compounds include: enantiomeric enrichment of amino acids (see, e.g., Shin et al., 2001, Biosci. Biotechnol. Biochem. 65:1782-1788; Iwasaki et al., 2003, Biotech. Lett. 25:1843-1846; Iwasaki et al., 2004, Appl. Microb. Biotech. 69:499-505, Yun et al., 2004, Appl. Environ. Microbiol. 70:2529-2534; and Hwang et al., 2004, Enzyme Microbiol. Technol. 34:429-426); preparation of intermediates and precursors of pregabalin (e.g., WO 2008 / 127646); enzymatic transamination of cyclopamine analogs (e.g., WO2011 / 017551); stereospecific synthesis and enantiomeric enrichment of β-amino acids (e.g., WO2005 / 005633); enantiomeric enrichment of amines (e.g., U.S. Pat. Nos. 4,950,606; 5,300,437; and 5,169,780); production of amino acids and derivatives (e.g., U.S. Pat. No. 5,316,943; U.S. Pat. No. 4,518,692; U.S. Pat. No. 4,826,766; U.S. Pat. No. 6,197,558; and U.S. Pat. No. 4,600,692); and in the production of the pharmaceutical compounds sitagliptin, rivastigmine, and vernakalant (see, e.g., U.S. Pat. No. 8,293,507 B2, issued Oct. 23, 2012; Savile et al., 2010, “Biocatalytic asymmetric synthesis of chiral amines from ketones applied to sitagliptin manufacture,” Science 329(5989):305-9; WO2011 / 159910, published Dec. 22, 2011; and WO2012 / 024104, published Feb. 23, 2012).

[0011] Wild-type aminotransferases having the ability to catalyze the reaction of Scheme 1 have been isolated from a variety of microorganisms, including but not limited to Alcaligenes denitrificans, Bordetella bronchiseptica, Bordetella parapertussis, Brucella melitensis, Burkholderia mallei, Burkholderia pseudomallei, Chromobacterium violaceum, Oceanicola granulosus HTCC2516, Oceanobacter species RED65, Oceanospirillum species MED92, Pseudomonas putida, Ralstonia solanacearum, Rhizobium melitensis, meliloti), Rhizobium species (strain NGR234), Bacillus thuringensis, Klebsiella pneumonia, Vibrio fluvialis (see, e.g., Shin et al., 2001, Biosci. Biotechnol, Biochem. 65: 1782-1788), and Arthrobacter species KNK168 (see, e.g., Iwasaki et al., Appl. Microbiol. Biotechnol., 2006, 69: 499-505, U.S. Pat. No. 7,169,592).Several of these wild-type aminotransferase genes and the encoded polypeptides have been sequenced, including, for example, Ralstonia solanacearum (Genbank Accession No. YP_002257813.1, GI: 207739420), Burkholderia pseudomallei 1710b (Genbank Accession No. ABA47738.1, GI: 76578263), Bordetella petrii (Genbank Accession No. AM902716.1, GI: 163258032), Vibrio fluviatilis JS17 (Genbank Accession No. AEA39183.1, GI: 327207066), and Arthrobacter sp. KNK168 (GenBank Accession No. BAK39753.1, GI: 336088341). At least two wild-type aminotransferases of the EC 2.6.1.18 and EC 2.6.1-19 classes have been crystallized and structurally characterized (see, eg, Yonaha et al., 1983, Agric. Biol. Chem. 47(10):2257-2265).

[0012] Transaminases are known to have (R)-selective or (S)-selective stereoselectivity. For example, the wild-type transaminase from Arthrobacter species KNK168 is considered to be (R)-selective and produces mainly (R)-amine compounds from certain substrates (see, e.g., Iwasaki et al., Appl. Microbiol. Biotechnol., 2006, 69:499-505, U.S. Patent No. 7,169,592), while the wild-type transaminase from Vibrio fluvialis JS17 is considered to be (S)-selective and produces mainly (S)-amine compounds from certain substrates (see, e.g., Shin et al., "Purification, characterization, and molecular cloning of a novel amine:pyruvate transaminase from Vibriofluvialis JS17," Appl. Microbiol. Biotechnol. 61 (5-6), 463-471 (2003)).

[0013] Non-naturally occurring transaminases with (R)-selectivity, increased solvent and thermal stability, and other improved properties for converting a wide range of amino acceptor substrates have been generated by mutagenesis and / or directed evolution of wild-type and other engineered transaminase backbone sequences (see, e.g., U.S. Pat. No. 8,293,507 B2, issued Oct. 23, 2012; WO2011 / 005477A1, published Jan. 13, 2011; WO2012 / 024104, published Feb. 23, 2012; and Savile et al., 2010, “Biocatalytic asymmetric synthesis of chiral amines from ketones applied to sitagliptin manufacture,” Science 329(5989):305-9).

[0014] Wild-type transaminases generally have properties that are undesirable for commercial use in the preparation of chiral amine compounds, such as instability toward industrially useful process conditions (e.g., solvents, temperature), poor recognition and stereoselectivity for commercially useful amino-acceptor and / or amino-donor substrates, and low product yields due to unfavorable reaction equilibrium.

[0015] In particular, transaminase reactions are usually carried out under aqueous conditions at high pH. Typical aqueous reaction conditions usually result in low substrate solubility, requiring organic co-solvents. Reactions under aqueous conditions are usually complicated by the instability of the product and substrate. Additional process steps are usually required, such as alkalization of the reaction mixture and extraction of the amine product with an organic solvent. SUMMARY OF THE INVENTION

[0017] The present invention provides engineered transaminase polypeptides and compositions thereof and polynucleotides encoding engineered transaminase polypeptides. A method for producing transaminase is also provided. In some embodiments, the engineered transaminase polypeptides are optimized to provide improved enzyme properties under minimal water-containing conditions in wet organic solvents. The transaminase polypeptides of the present disclosure have been engineered to have one or more residue differences compared to the engineered transaminase polypeptides before (of the amino acid sequence of SEQ ID NO: 2) and the enzyme properties of the related improvements relative to the engineered transaminase polypeptides before (see, for example, US 8,293,507 B2 authorized on October 23, 2012; PCT published WO2011005477A1 and PCT published WO2012024104 published on February 23, 2012, published on January 13, 2011). The amino acid residue differences are located at the improved residue positions that lead to a variety of enzyme properties, and the enzyme properties include, among others, activity, stereoselectivity, stability, expression, product tolerance and thermal activity in wet organic solvents.

[0018] In particular, the engineered transaminase polypeptides of the present disclosure have been engineered for the efficient conversion of the substrate 4-methyl-3-oxo-pentanoic acid methyl ester (referred to herein as "compound (2)") to its corresponding chiral amine product compound (S)-3-amino-4-methyl-pentanoic acid methyl ester (referred to herein as "compound (1)") in a wet organic solvent under minimal aqueous conditions as shown in Scheme 2.

[0019] Solution 2

[0020]

[0021] The evolved structural features of the engineered transaminase polypeptides of the present disclosure also allow for the biocatalytic conversion of a range of ketoester substrate compounds of formula (II) (including compounds other than compound (2)) into their corresponding chiral amine product compounds of formula (I) (including compounds other than compound (1)) as shown in Scheme 3,

[0022] Solution 3

[0023]

[0024] in

[0025] Z is a linear C1-6 alkyl group or a branched C1-6 alkyl group, optionally substituted with one or more hydroxyl groups, cyano groups or nitro groups; and

[0026] R1 is C1-6 alkyl, aryl-C1-2 alkyl, heteroaryl-C1-2 alkyl, or a 5- to 6-membered heterocyclic ring system optionally containing additional heteroatoms selected from O, S and N, which is unsubstituted or substituted with one to three substituents independently selected from oxo, hydroxy, halogen, C1-4 alkoxy and C1-4 alkyl.

[0027] In some embodiments, the engineered transaminase polypeptide is capable of biocatalytically converting a compound of formula (II) into a compound of formula (I) having the indicated stereochemical configuration at the stereocenter marked with * in an enantiomeric excess of at least 70% over the relative enantiomer.

[0028] The present invention provides an engineered polypeptide having transaminase activity, the engineered polypeptide comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to SEQ ID NO: 2, 6, 332, 518 and / or 656, or a functional fragment thereof, wherein the engineered polypeptide includes a polypeptide comprising at least one substitution or set of substitutions in the polypeptide sequence, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 2, 6, 332, 518 and / or 656. In some embodiments, the reference sequence is SEQ ID NO: 2. In some embodiments, the reference sequence is SEQ ID NO: 6. In some embodiments, the reference sequence is SEQ ID NO: 332. In some embodiments, the reference sequence is SEQ ID NO: 518. In some embodiments, the reference sequence is SEQ ID NO: 656.

[0029] In some embodiments, the polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:2, and wherein the engineered polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 284 and 284 / 155, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2. In some embodiments, the polypeptide sequence of the engineered polypeptide has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:2, and wherein the polypeptide sequence of the engineered polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 284A and 284A / 155W, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2. In some embodiments, the polypeptide sequence of the engineered polypeptide has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 2, and wherein the polypeptide sequence of the engineered polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: G284A and G284A / L155W, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 2. In some embodiments, the engineered polypeptide comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 2. In some embodiments, the engineered polypeptide comprises a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 2. In some embodiments, the engineered polypeptide comprises a polypeptide sequence having at least 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 2.

[0030] In some embodiments, the invention provides an engineered polypeptide having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:6, wherein the engineered polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 5 / 35, 9, 9 / 20 / 23 / 24 / 134 / 244, 9 / 20 / 23 / 101 / 244, 9 / 20 / 24 / 101 / 244 / 249, 9 / 23 / 24, 9 / 23 / 24 / 29 / 244, 9 / 23 / 24 / 101 / 262, 9 / 23 / 24 / 244 / 262, 9 / 23 / 24 / 29 / 244, 9 / 23 / 24 / 101 / 263, 9 / 23 / 24 / 24 / 29 / 244, 9 / 23 / 24 / 101 / 264, 9 / 23 / 24 / 24 / 29 / 244. 29, 9 / 24 / 244, 9 / 24 / 244 / 262, 9 / 24 / 249, 9 / 45 / 89 / 93, 9 / 79 / 101 / 244, 9 / 82 / 89, 9 / 89 / 246, 9 / 89 / 271, 9 / 93 / 142 / 246, 9 / 101 / 244, 9 / 110 / 302, 9 / 244 / 249, 9 / 244 / 262 / 264, 9 / 246, 9 / 262, 15, 15 / 16 / 18 / 20 / 89 / 93 / 160, 15 / 24 / 26 / 89 / 93 / 160, 15 / 35 / 273, 15 / 218 / 273, 15 / 249 / 273, 16, 17 / 25, 17 / 25 / 41、17 / 25 / 160、17 / 160、18、18 / 26 / 246 / 273、18 / 146、18 / 146 / 168、18 / 146 / 262、18 / 168、18 / 168 / 244、18 / 168 / 262 / 324、18 / 244、18 / 244 / 246、18 / 244 / 246 / 284、18 / 244 / 248、18 / 244 / 262 / 273、18 / 244 / 273、18 / 262、18 / 273、20、20 / 23 / 24 / 55 / 134 / 244 / 262、20 / 23 / 134 / 244、20 / 24 / 89 / 197、2 0 / 33 / 160, 20 / 324, 22, 22 / 102, 22 / 160, 23, 23 / 25 / 55 / 321, 23 / 25 / 102 / 142, 23 / 102, 23 / 321, 24, 25, 25 / 33, 25 / 104, 25 / 160, 25 / 321, 26, 26 / 273, 29, 29 / 89, 32, 33, 35, 35 / 215 / 218 / 249, 35 / 218 / 249 / 273, 35 / 221, 40, 40 / 168 / 208 / 244, 40 / 244 / 248, 41, 41 / 102, 42, 45, 46, 50, 55, 56, 71 / 102, 74,79、82、82 / 244、89、89 / 93 / 156 / 324、89 / 93 / 321 / 324、90、93、93 / 324、95、98、101、101 / 160 / 244 / 249、102、104、106、111、114、117、119、134、134 / 244、134 / 262、139、140 / 266、142、142 / 321、145、146、146 / 244、146 / 273、156、160、160 / 215 / 216 / 218、160 / 273、168、168 / 244 / 273、179、 186, 186 / 200, 197, 203, 207, 208, 208 / 246, 215, 215 / 216 / 249, 216, 216 / 273, 218, 218 / 249, 218 / 313, 234, 236, 244, 244 / 248, 244 / 248 / 284, 244 / 273, 246, 246 / 248 / 273, 248, 249, 249 / 273, 256, 262, 264, 265, 266, 271, 273, 284, 301, 316, 320, 321, 324, and 329, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 6. In some embodiments, the polypeptide sequence of the engineered polypeptide is identical to SEQ ID NO: 6. NO:6 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity, and wherein the polypeptide sequence of the engineered polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 5T / 35Y, ​​9H, 9H / 45L / 89Q / 93H, 9H / 82S / 89Q, 9H / 89Q / 246L, 9H / 89Q / 271C, 9H / 93H / 142M / 246L, 9H / 110V / 302F, 9H / 246L, 9T, 9T / 20T / 23Q / 24F / 134S / 2 44G, 9T / 20T / 23Q / 101S / 244L, 9T / 20T / 24F / 101S / 244G / 249G, 9T / 23Q / 24F, 9T / 23Q / 24F / 29M / 244G, 9T / 23Q / 24F / 101S / 262V, 9T / 23Q / 24F / 244L / 262T, 9T / 23Q / 29M, 9T / 24F / 244G, 9T / 24F / 244G / 262T, 9T / 24F / 249G, 9T / 79G / 101 S / 244G, 9T / 101S / 244G, 9T / 244G / 249G, 9T / 244G / 262T / 264N, 9T / 262T, 15I,<h2 style=";text-align:left;direction:ltr">15I / 16I / 18R / 20H / 89M / 93M / 160L,15I / 24P / 26P / 89M / 93M / 160L,15T,15T / 35G / 273K,15T / 218R / 273K,15T / 249M / 273K,16I,17I / 25R,17I / 25R / 41F,1 7I / 25R / 160V, 17I / 160V, 18G, 18G / 26H / 246V / 273T, 18G / 146L, 18G / 146L / 262V, 18G / 168P, 18G / 244G, 18G / 244G / 248M, 18G / 244L / 273V, 18G / 273T, 18G / 273V、18K、18K / 146L、18K / 146L / 168P、18K / 168P、18K / 168P / 244L、18K / 16 8P / 262V / 324W、18K / 244G / 246V、18K / 244G / 246V / 284G、18K / 244L、18K / 244 L / 246V、18K / 244T / 262V / 273T、18K / 262V、18K / 273T、18R、20H、20H / 24P / 89 M / 197T、20H / 33L / 160L、20H / 324C、20T、20T / 23Q / 24F / 55F / 134G / 244G / 262 T, 20T / 23Q / 134S / 244G, 22H, 22L, 22L / 102L, 22L / 160V, 22W, 23H, 23H / 25R / 55L / 321Y, 23H / 25R / 102L / 142L, 23H / 102L, 23H / 321Y, 23Q, 24D, 24F, 24P, 2 5R, 25R / 33M, 25R / 104V, 25R / 160V, 25R / 321Y, 26H / 273K, 26P, 29K, 29K / 89Q, 29M, 32L, 32S, 33L, 33M, 33S, 35G, 35G / 215L, 218R / 249T, 35G / 218R / 249T / 273K、35G / 221D、40M、40M / 168P / 208L / 244G、40M / 244G / 248M、41F / 102L、41 K、41Y、42T、45L、46C、50T、55F、55G、55K、55S、56R、71Y / 102P、74A、79G、79H 82P, 82S / 244D, 82T, 89L, 89M, 89M / 93M / 156L / 324C, 89M / 93M / 321V / 324C, 89Q, 89W, 90A, 90C, 90S, 90T, 93H, 93M, 93M / 324C, 93P, 95P, 95T, 98M, 101L101L / 160Y / 244E / 249M, 101S, 102L, 104I, 104S, 106S, 111L, 111T, 114T, 117D, 119A, 134G, 134G / 244L, 134G / 26 2V, 134S, 139A, 140N / 266N, 142L / 321Y, 142T, 145N, 146A, 146A / 244D, 146H, 146I, 146L, 146L / 244L, 146L / 273V , 156E, 156L, 156T, 160A, 160L, 160V, 160Y, 160Y / 215L / 216G / 218R, 160Y / 273K, 168P, 168P / 244L / 273T, 179R, 1 86R, 186R / 200R, 197L, 197T, 203P, 207Y, 208K, 208L, 208L / 246V, 208P, 208R, 215L, 215L / 216G / 249M, 216G, 216P , 216P / 273V, 216S, 218N / 313H, 218R, 218R / 249M, 218S, 234H, 236T, 244E, 244G, 244G / 248M, 244G / 248M / 284G, 2 44L, 244L / 273C, 244L / 273V, 244T, 246A, 246L, 246V, 246V / 248M / 273V, 248M, 248V, 249G, 249M, 249M / 273K, 249T , 256C, 262L, 262P, 262T, 262V, 264A, 264E, 264G, 264N, 264P, 264R, 264W, 265R, 266G, 266S, 271C, 273L, 273M, 273T, 273V, 284G, 301E, 301M, 316R, 320R, 320W, 321L, 321V, 321Y, 324C, 324V, 324W and 329R, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:6. In some embodiments, the polypeptide sequence of the engineered polypeptide has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:6, and wherein the polypeptide sequence of the engineered polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of A5T / A35Y, E9H, E9H / F45L / S89Q / S93H, E9H / D82S / S89Q, E9H / S89Q / I246L, E9H / S89Q / E271C, E9H / S93H / K142M / I246L,E9H / L110V / V302F、E9H / I246L、E9T、E9T / Y20T / Y23Q / S24F / T134S / P244G、E9T / Y20T / Y23Q / T101S / P244L、E9T / Y20T / S24F / T101S / P244G / K249G、E9T / Y23Q / S24F、E9T / Y23Q / S24F / D29M / P244G、E9T / Y23Q / S24F / T101S / A262V、E9T / Y23Q / S24F / P244L / A262T、E9T / Y23Q / D29M、E9T / S24F / P244G、E9T / S24F / P244G / A262T、E9T / S24F / K249G、E9T / R79G / T101S / P244G、E9T / T101S / P244G、E9T / P244G / K249G、E9T / P244G / A262T / L264N、E9T / A262T、D15I、D15I / T16I / L18R / Y20H / S89M / S93M / F160L、D15I / S24P / Y26P / S89M / S93M / F160L、D15T、D15T / A35G / Y273K、D15T / L218R / Y273K、D15T / K249M / Y273K、T16I、G17I / D25R、G17I / D25R / I41F、G17I / D25R / F160V、G17I / F160V、L18G、L18G / Y26H / I246V / Y273T、L18G / Q146L、L18G / Q146L / A262V、L18G / H168P、L18G / P244G、L18G / P244G / R248M、L18G / P244L / Y273V、L18G / Y273T、L18G / Y273V、L18K、L18K / Q146L、L18K / Q146L / H168P、L18K / H168P、L18K / H168P / P244L、L18K / H168P / A262V / L324W、L18K / P244G / I246V、L18K / P244G / I246V / A284G、L18K / P244L、L18K / P244L / I246V、L18K / P244T / A262V / Y273T、L18K / A262V、L18K / Y273T、L18R、Y20H、Y20H / S24P / S89M / R197T、Y20H / P33L / F160L、Y20H / L324C、Y20T、Y20T / Y23Q / S24F / I55F / T134G / P244G / A262T、Y20T / Y23Q / T134S / P244G、T22H、T22L、T22L / Q102L、T22L / F160V、T22W、Y23H、Y23H / D25R / I55L / P321Y、Y23H / D25R / Q102L / K142L、Y23H / Q102L、Y23H / P321Y、Y23Q、S24D、S24F、S24P、D25R、D25R / P33M、D25R / E104V、D25R / F160V、D25R / P321Y、Y26H / Y273K、Y26P、D29K、D29K / S89Q、D29M、N32L、N32S、P33L、P33M、P33S、A35G、A35G / G215L / L218R / K249T、A35G / L218R / K249T / Y273K、A35G / E221D、W40M、W40M / H168P / E208L / P244G、W40M / P244G / R248M、I41F / Q102L、I41K、I41Y、G42T、F45L、V46C、E50T、I55F、I55G、I55K、I55S、F56R、H71Y / Q102P、N74A、R79G、R79H、D82P、D82S / P244D、D82T、S89L、S89M、S89M / S93M / Q156L / L324C、S89M / S93M / P321V / L324C、S89Q、S89W、N90A、N90C、N90S、N90T、S93H、S93M、S93M / L324C、S93P、R95P、R95T、P98M、T101L、T101L / F160Y / P244E / K249M、T101S、Q102L、E104I、E104S、K106S、E111L、E111T、A114T、E117D、R119A、T134G、T134G / P244L、T134G / A262V、T134S、D139A、I140N / D266N、K142L / P321Y、K142T、P145N、Q146A、Q146A / P244D、Q146H、Q146I、Q146L、Q146L / P244L、Q146L / Y273V、Q156E、Q156L、Q156T、F160A、F160L、F160V、F160Y、F160Y / G215L / D216G / L218R、F160Y / Y273K、H168P、H168P / P244L / Y273T、P179R、Q186R、Q186R / Q200R、R197L、R197T、H203P、F207Y、E208K、E208L、E208L / I246V、E208P、E208R、G215L、G215L / D216G / K249M, D216G, D216P, D216P / Y273V, D216S, L218N / R313 H, L218R, L218R / K249M, L218S, V234H, R236T, P244E, P244G, P244G / R24 8M, P244G / R248M / A284G, P244L, P244L / Y273C, P244L / Y273V, P244T, I 246A, I246L, I246V, I246V / R248M / Y273V, R248M, R248V, K249G, K249M,

[0149] The invention relates to polypeptides of the invention wherein the amino acid positions of the polypeptides are numbered with reference to SEQ ID NO: 6. In some embodiments, the engineered polypeptide comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:6. In some embodiments, the engineered polypeptide comprises a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:6. In some embodiments, the engineered polypeptide comprises a polypeptide sequence having at least 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:6.

[0031] In some additional embodiments, the present invention provides engineered polypeptides having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 332, wherein the engineered polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 9 / 89 / 93 / 101 / 160, 9 / 89 / 160, 33, 35 , 44, 49, 74, 89, 89 / 93 / 160 / 249, 107, 108, 117, 140, 141, 142, 144, 156, 168, 203, 207, 234, 270, 273, 274, 292, 296, 297, 300, 302, 305, 317, 319, 321, 323, 327 and 329, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 332. In some embodiments, the polypeptide sequence of the engineered polypeptide is identical to SEQ ID NO: NO:332 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity, and wherein the polypeptide sequence of the engineered polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 9H / 89M / 93M / 101S / 160Y, 9H / 89Q / 160Y, 33N, 35H, 35M, 44T, 49P, 74K, 74R, 89L, 89M / 93M / 160V / 249M, 89V, 89W, 107M, 107R, 107V, 108L, 117D, 329T, 331A, 331S, 332T, 333R, 333V, 334H, 334Q, 334S, 335R, and 335V, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 332.In some embodiments, the polypeptide sequence of the engineered polypeptide has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 332, and wherein the polypeptide sequence of the engineered polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: E9H / S89M / S93 M / T101S / F160Y, E9H / S89Q / F160Y, P33N, A35H, A35M, A44T, S49P, N74K, N74R, S89L, S 89M / S93M / F160V / K249M, S89V, S89W, E107M, E107R, E107V, I108L, E117D, I140L, I14 0M, I140S, T141A, T141H, K142N, K142S, R144A, Q156C, Q156T, H168P, H168R, H203L, F 207G, V234I, V234T, A270R, A270S, Y273L, D274A, D274G, S292T, N296M, N296V, N296W , S297L, S297T, D300T, V302I, V302L, V302Q, P305Q, P305T, L317W, V319T, P321A, P321S, P321T, S323R, S323V, P327H, P327Q, P327S, Q329R, and Q329V, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 332. In some embodiments, the engineered polypeptide comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 332. In some embodiments, the engineered polypeptide comprises a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 332. In some embodiments, the engineered polypeptide comprises a polypeptide sequence having at least 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 332.

[0032] In some additional embodiments, the present invention provides an engineered polypeptide having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:518, wherein the engineered polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 35 / 273, 46 / 273, 111 / 159 / 273, 159, 159 / 273, 174, 199, 273 and 287, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:518. In some embodiments, the polypeptide sequence of the engineered polypeptide has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:518, and wherein the polypeptide sequence of the engineered polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 35H / 273L, 46L / 273L, 111Q / 159D / 273L, 159D, 159D / 273L, 174G, 199L, 273L, and 287P, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:518. In some embodiments, the polypeptide sequence of the engineered polypeptide has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:518, and wherein the polypeptide sequence of the engineered polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of A35H / Y273L, V46L / Y273L, E111Q / P159D / Y273L, P159D, P159D / Y273L, S174G, I199L, Y273L, and V287P, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:518. In some embodiments, the engineered polypeptide comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 518. In some embodiments, the engineered polypeptide comprises a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 518.In some embodiments, the engineered polypeptide comprises a polypeptide sequence having at least 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:518.

[0033] In some additional embodiments, the present invention provides an engineered polypeptide having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 332, wherein the engineered polypeptide has increased thermal activity in a wet organic solvent compared to the engineered polypeptide of SEQ ID NO: 332, and wherein the engineered polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 23 / 29 / 55, 34, 44, 74, 92, 108, 117, 140, 141, 144, 156, 160, 168, 215, 256, 269, 270, 274, 292, 296, 302, 319, 323 and 329, wherein the amino acid positions of the polypeptide sequence are referenced to SEQ ID NO: In some embodiments, the polypeptide sequence of the engineered polypeptide has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 332, and wherein the polypeptide sequence of the engineered polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 23Y / 29Y / 55K, 34Y, 44N, 74K, 74R, 92D, 10 8L, 117R, 140L, 141H, 144V, 156T, 160V, 160W, 160Y, 168Q, 215A, 215S, 215T, 256G, 269T, 270Q, 270S, 274A, 292T, 296M, 296V, 296Y, 302L, 302Q, 302S, 319R, 323L, 329R and 329V, wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO:332.In some embodiments, the polypeptide sequence of the engineered polypeptide has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 332, and wherein the polypeptide sequence of the engineered polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of Q23Y / D29Y / I55K, L34Y, A44N, N74K, N74R, E92D, I108L, E117R, I140L, T141H, R144V, Q156T, F160V, F160W, F160Y, H168Q, G215A, G215S, G215T, E256G, P269T, A270Q, A270S, D274A, S292T, N296M, N296V, N296Y, V302L, V302Q, V302S, V319R, S323L, Q329R and Q329V, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:332. In some embodiments, the engineered polypeptide comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 332. In some embodiments, the engineered polypeptide comprises a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 332. In some embodiments, the engineered polypeptide comprises a polypeptide sequence having at least 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 332.

[0034] In some additional embodiments, the present invention provides an engineered polypeptide having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:518, wherein the engineered polypeptide has increased thermal activity in a wet organic solvent compared to the engineered polypeptide of SEQ ID NO:518, and wherein the engineered polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 34 / 74 / 92, 35 / 273 / 323, 39, 46 / 273, 48, 55, 74 / 92, 126, 133, 138, 150, 160 / 215 / 329, 178, 199, 207, 273 and 292, wherein the amino acid positions of the polypeptide sequence are referenced to SEQ ID NO:518. NO:518. In some embodiments, the polypeptide sequence of the engineered polypeptide has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:518, and wherein the polypeptide sequence of the engineered polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 34Y / 74K / 92D, 35H / 273L / 323R, 39G, 46L / 273L, 48G, 55V, 74K / 92D, 126L, 126V, 133T, 138A, 150W, 160V / 215S / 329R, 178S, 199L, 207Y, 273L, and 292T, wherein the amino acid positions of the polypeptide sequence are referenced to SEQ ID NO:518. NO: 518. In some embodiments, the polypeptide sequence of the engineered polypeptide has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 518, and wherein the polypeptide sequence of the engineered polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: L34Y / N74K / E92D, A35 H / Y273L / S323R, A39G, V46L / Y273L, P48G, I55V, N74K / E92D, T126L, T126V, S133T, R138A, F150W, Y160V / G215S / Q329R, T178S, I199L, F207Y, Y273L and S292T, wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO:518.In some embodiments, the engineered polypeptide comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 518. In some embodiments, the engineered polypeptide comprises a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 518. In some embodiments, the engineered polypeptide comprises a polypeptide sequence having at least 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 518.

[0035] In some additional embodiments, the present invention provides an engineered polypeptide having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 656, wherein the engineered polypeptide has increased thermal activity in a wet organic solvent compared to the engineered polypeptide of SEQ ID NO: 656, and wherein the engineered polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 18, 35 / 39 / 55 / 138 / 150, 35 / 39 / 55 / 150, 35 / 39 / 126 / 150 / 273, 35 / 39 / 133 / 150 / 283. 73、35 / 39 / 150、35 / 39 / 150 / 273、35 / 133 / 178、35 / 138 / 150 / 273、35 / 150 / 273、35 / 178 / 273、35 / 273、39、39 / 55 / 150 / 178 / 273、39 / 55 / 150 / 273、39 / 126 / 150、39 / 133 / 150、39 / 133 / 178 / 273、39 / 133 / 273、39 / 150 / 178、39 / 150 / 178 / 207、39 / 150 / 178 / 207 / 273、39 / 150 / 273、39 / 178、39 / 178 / 273、39 / 207、39 / 207 / 273、39 / 273、44、55、55 / 150、88、126、126 / 133 / 178、126 / 138 / 178, 126 / 178 / 207, 132, 138 / 150 / 178, 141, 150, 150 / 178 / 273 / 323, 150 / 178 / 323, 150 / 207 / 273, 150 / 273, 168, 178 / 273, 239, 261, 263, 269, 273, 297 and 312, wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO:656.In some embodiments, the polypeptide sequence of the engineered polypeptide has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:656, and wherein the polypeptide sequence of the engineered polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 18R, 35H / 39G / 55V / 138A / 150W, 35H / 39G / 55V / 150W, 35H / 39G / 126V / 150W / 273L, 35H / 39G / 133T, 35H / 39G / 133T / 150W / 273L, 35H / 39G / 150W, 35H / 39G / 150W / 273L, 35H / 133T / 178S, 35H / 138A / 150W / 273L, 35H / 150W / 273L, 35H / 178S / 273L, 35H / 273L, 39G, 39G / 55V / 150W / 178S / 273L, 39G / 55V / 150W / 273L, 39G / 126V / 150W, 39G / 133T / 150W, 3 9G / 133T / 178S / 273L, 39G / 133T / 273L, 39G / 150W / 178S, 39G / 150W / 178S / 207Y, 39G / 150W / 178S / 207Y / 273L, 39G / 150W / 273 L, 39G / 178S, 39G / 178S / 273L, 39G / 207Y, 39G / 207Y / 273L, 39G / 273L, 44L, 44Q, 55F, 55V, 55V / 150W, 88W, 126V, 126V / 133T / 1 78S, 126V / 138A / 178S, 126V / 178S / 207Y, 132D, 132E, 138A / 150W / 178S, 141S, 150W, 150W / 178S / 273L / 323R, 150W / 178S / 323R, 150W / 207Y / 273L, 150W / 273L, 168D, 178S / 273L, 239N, 261R, 263E, 269V, 273L, 297E, 297R and 312A, wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO:656.In some embodiments, the polypeptide sequence of the engineered polypeptide has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 656, and wherein the polypeptide sequence of the engineered polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of L18R, A35H / A39G / I55V / R138A / F150W, A35H / A39G / I55V / F150W, A35H / A39G / T126V / F150W / Y273L, A35H / A39G / S133T, A35H / A39G / S134V / R135A / F150W. 3T / F150W / Y273L, A35H / A39G / F150W, A35H / A39G / F150W / Y273L, A35H / S 133T / T178S, A35H / R138A / F150W / Y273L, A35H / F150W / Y273L, A35H / T178 S / Y273L, A35H / Y273L, A39G, A39G / I55V / F150W / T178S / Y273L, A39G / I5 5V / F150W / Y273L, A39G / T126V / F150W, A39G / S133T / F150W, A39G / S133T / T178S / Y273L, A39G / S133T / Y273L, A39G / F150W / T178S, A39G / F150W / T1 78S / F207Y, A39G / F150W / T178S / F207Y / Y273L, A39G / F150W / Y273L, A39G / T178S, A39G / T178S / Y273L, A39G / F207Y, A39G / F207Y / Y273L, A39G / Y2 73L, A44L, A44Q, I55F, I55V, I55V / F150W, F88W, T126V, T126V / S133T / T1 78S, T126V / R138A / T178S, T126V / T178S / F207Y, S132D, S132E, R138A / F150W / T178S, T141S, F150W, F150W / T178S / Y273L / S323R, F150W / T178S / S323R, F150W / F207Y / Y273L, F150W / Y273L, H168D, T178S / Y273L, G239N, E261R, I263E, P269V, Y273L, S297E, S297R and R312A, wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO:656.In some embodiments, the engineered polypeptide comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 656. In some embodiments, the engineered polypeptide comprises a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 656. In some embodiments, the engineered polypeptide comprises a polypeptide sequence having at least 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 656.

[0036] In some additional embodiments, the present invention provides engineered polypeptides having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 2, wherein the engineered polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 9 / 20 / 23 / 39 / 55 / 89 / 134 / 15 0 / 155 / 160 / 178 / 199 / 244 / 273 / 284, 9 / 20 / 23 / 89 / 134 / 155 / 160 / 199 / 244 / 284, 9 / 20 / 23 / 89 / 134 / 155 / 160 / 244 / 284, 20 / 23 / 134 / 155 / 244 / 284, and 155 / 284, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 2. In some embodiments, the polypeptide sequence of the engineered polypeptide is identical to SEQ ID NO: 2. NO:2 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity, and wherein the polypeptide sequence of the engineered polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 9H / 20T / 23Q / 39G / 55V / 89Q / 134S / 150W / 155W / 160Y / 178S / 199L / 244G / 273L / 284A, 9H / 20T / 23Q / 89Q / 134S / 155W / 160Y / 199L / 244G / 284A, 9H / 20T / 23Q / 89Q / 134S / 155W / 160Y / 244G / 284A, 20T / 23Q / 134S / 155W / 244G / 284A and 155W / 284A, wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO:2.In some embodiments, the polypeptide sequence of the engineered polypeptide has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:2, and wherein the polypeptide sequence of the engineered polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: E9H / Y20T / Y23Q / A39G / I55V / S89Q / T134S / F150W / L155W / F160Y / T178S / I199 L / P244G / Y273L / G284A, E9H / Y20T / Y23Q / S89Q / T134S / L155W / F160Y / I199L / P244G / G284A, E9H / Y20T / Y23Q / S89Q / T134S / L155W / F160Y / P244G / G284A, Y20T / Y23Q / T134S / L155W / P244G / G284A and L155W / G284A, wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO:2. In some embodiments, the engineered polypeptide comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 2. In some embodiments, the engineered polypeptide comprises a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 2. In some embodiments, the engineered polypeptide comprises a polypeptide sequence having at least 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 2.

[0037] In some embodiments of the engineered polypeptides having transaminase activity of the present disclosure, the engineered polypeptide is capable of converting a substrate of compound (2) into a product of compound (1) under suitable reaction conditions. In some embodiments, the engineered polypeptide is capable of converting compound (2) into compound (1) under suitable reaction conditions with an activity at least 1, 2, 3, 5, 7, 10, 12, 15, 20 or more times greater than SEQ ID NO: 2. In some embodiments, the engineered polypeptide is capable of converting compound (2) into compound (1) with an increased activity relative to SEQ ID NO: 2, wherein suitable reaction conditions include a loading of at least 20 g / L of compound (1), wet isopropyl acetate (IPAc), 0.55 M isopropylamine (IPM) and 50°C.

[0038] In some embodiments of the present disclosure, the engineered polypeptide having transaminase activity is immobilized on a solid support, optionally wherein the solid support is selected from beads or resins comprising polymethacrylates with epoxide functional groups, polymethacrylates with aminoepoxide functional groups, styrene / DVB copolymers with octadecyl functional groups, or polymethacrylates.

[0039] The present invention also provides polynucleotide sequences encoding at least one engineered polypeptide provided herein. In some embodiments, the polynucleotide sequence encoding at least one engineered polypeptide comprises a polynucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO: 1, 5, 331, 517 and / or 655. In some embodiments, the polynucleotide sequence encoding at least one engineered polypeptide comprises a polynucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO: 1, 5, 331, 517 and / or 655, wherein the polynucleotide sequence of the engineered polypeptide comprises at least one substitution at one or more positions. In some additional embodiments, the polynucleotide sequence encoding at least one engineered polypeptide or a functional fragment thereof comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, 5, 331, 517 and / or 655. In some additional embodiments, the polynucleotide sequence includes the polynucleotide sequence listed in the odd-numbered sequence in SEQ ID NO: 3-845.

[0040] In some other embodiments, the engineered polynucleotide sequence is operably connected to a control sequence. In some embodiments, the control sequence includes a promoter. In some other embodiments, the promoter is a heterologous promoter. In some other embodiments, the control sequence includes more than one control sequence. In some embodiments, a control sequence includes a promoter, and other control sequences include different control sequences. In some embodiments, the engineered polynucleotide sequence is codon optimized. The present invention also provides an expression vector comprising at least one polynucleotide sequence provided herein. The present invention also provides a host cell comprising at least one expression vector provided herein. In some embodiments, the present invention provides a host cell comprising at least one polynucleotide sequence provided herein. In some embodiments, the host cell is a prokaryotic cell, and in some other embodiments, the host cell is a eukaryotic cell. In some other embodiments, the host cell is Escherichia coli (E.coli).

[0041] The present invention also provides a method for producing an engineered aminotransferase in a host cell, the method comprising culturing a host cell provided herein in a culture medium under suitable conditions, thereby producing at least one engineered aminotransferase provided herein. In some embodiments, the method further comprises the step of recovering at least one engineered aminotransferase from the culture medium and / or the host cell. In some other embodiments, the method further comprises the step of purifying at least one aminotransferase obtained using the method of the present invention.

[0042] The present disclosure also provides methods for preparing a wide range of chiral amine compounds using the engineered aminotransferase polypeptides disclosed herein. In some embodiments, the present disclosure provides methods for preparing compounds of structural formula (I) with an enantiomeric excess of at least 70% over the relative enantiomer:

[0043]

[0044] The compounds of formula (I) have the indicated stereochemical configuration at the stereocenters marked with *, wherein

[0045] Z is a linear C1-6 hydrocarbon group or a branched C1-6 hydrocarbon group, optionally substituted with one or more hydroxyl groups, cyano groups or nitro groups; and

[0046] R1 is C1-6 hydrocarbyl, aryl-C1-2 hydrocarbyl, heteroaryl-C1-2 hydrocarbyl, or a 5- to 6-membered heterocyclic ring system optionally containing additional heteroatoms selected from O, S and N, the heterocyclic ring being unsubstituted or substituted with one to three substituents independently selected from oxo, hydroxyl, halogen, C1-4 hydrocarbyloxy and C1-4 hydrocarbyl; the method comprising reacting a prochiral ketoester of formula (II) in the presence of an amino group donor under suitable reaction conditions:

[0047]

[0048] A step of contacting with an engineered polypeptide as disclosed herein.

[0049] In some embodiments, the present disclosure provides a method for preparing compound (1):

[0050]

[0051] The method comprises reacting a substrate of compound (2) in the presence of an amino group donor under suitable reaction conditions.

[0052]

[0053] A step of contacting with an engineered polypeptide as disclosed herein.

[0054] In some embodiments, the methods of using the engineered polypeptides disclosed herein can be performed wherein the chiral amine compound of formula (I) or compound (1) is produced in an enantiomeric excess of at least 70%, 80%, 90%, 97%, 98%, 99% or greater.

[0055] Any method disclosed herein for preparing a compound of formula (I) or compound (1) using an engineered polypeptide can be performed under a range of suitable reaction conditions, including but not limited to a range of amino group donors, temperature, wet organic solvent system, substrate loading, polypeptide loading, cofactor loading, and reaction time. In one example, in some embodiments, the preparation of a compound of formula (I) or compound (1) can be performed, wherein suitable reaction conditions include: (a) a substrate loading of about 10 g / L to 200 g / L of a substrate compound (e.g., compound (2)); (b) an engineered polypeptide of about 0.5 g / L to 25 g / L; (c) an IPM concentration of about 0.1 M to 3 M; (d) a wet organic solvent; and (e) a temperature of about 30° C. to 60° C. In some embodiments, suitable reaction conditions include: (a) about 20 g / L of substrate compound (e.g., compound (2)); (b) about 20 g / L of engineered polypeptide; (c) wet IPAc; (d) about 0.55 M IPM; and (e) about 50°C.

[0056] In some embodiments, the methods of using the engineered polypeptides disclosed herein can be performed wherein the amino donor is selected from IPM, alanine, 3-aminobutyric acid, or methylbenzylamine. In some embodiments, the amino donor is IPM.

[0057] In some embodiments, the wet organic solvent is selected from wet IPAc, wet toluene, wet methyl tert-butyl ether (MTBE), wet acetonitrile (MeCN), or wet isopropanol (IPA). In some embodiments, the wet organic solvent is wet IPAc.

[0058] In some embodiments, methods of using the engineered polypeptides disclosed herein can be performed, wherein the method further comprises the step of isolating the product compound of formula (I) or compound (1) from the reaction.

[0059] Further guidance regarding the selection of engineered polypeptides, their preparation, choice of substrates, and parameters for performing the methods is further described in the more detailed description and examples below.

[0060] Description of the invention

[0061] The present invention provides engineered transaminase polypeptides and compositions thereof, and polynucleotides encoding engineered transaminase polypeptides. Also provided are methods for producing transaminases. In some embodiments, the engineered transaminase polypeptides are optimized to provide improved enzyme properties in wet organic solvents under minimal water-containing conditions.

[0062] Unless otherwise defined, all technical terms and scientific terms used herein generally have the same meanings as those generally understood by those of ordinary skill in the art to which the present invention belongs. Generally, the nomenclature used herein and the experimental procedures in cell culture, molecular genetics, microbiology, organic chemistry, analytical chemistry and nucleic acid chemistry described below are those well known in the art and generally adopted. Such technology is well known and described in many textbooks and reference works well known to those skilled in the art. Standard techniques or their modified forms are used for chemical synthesis and chemical analysis. Therefore, the following terms are intended to have the meanings provided herein.

[0063] Although any suitable methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, some methods and materials are also described herein. It should be understood that the present invention is not limited to the specific methods, protocols and reagents described, as these can be changed according to the circumstances in which they are used by those skilled in the art. Therefore, the terms that will be defined below are more fully described by reference to the present invention as a whole.

[0064] It should be understood that the general description above and the detailed description below are only exemplary and illustrative, rather than limiting the present invention. The section titles used herein are only for organizational purposes and are not to be construed as limiting the subject matter described. Numerical ranges include numbers that limit the range. Therefore, each numerical range disclosed herein is intended to include each narrower numerical range that falls within such a wider numerical range, as such a narrower numerical range is all clearly written out herein. It is also intended that each maximum (or minimum) numerical limit disclosed herein includes each lower (or higher) numerical limit, as such a lower (or higher) numerical limit is clearly written out herein.

[0065] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes more than one polypeptide.

[0066] Similarly, "comprise," "comprises," "comprising," "include," "includes," "including," "have," and "having" are interchangeable and are not intended to be limiting. Thus, as used herein, the term "comprising" and its cognates are used in their inclusive sense (i.e., equivalent to the term "including" and its corresponding cognates).

[0067] It should also be understood that where the term "comprising" is used in the description of various embodiments, those skilled in the art will understand that in some specific instances, the embodiments may alternatively be described using the language "consisting essentially of" or "consisting of."

[0068] All patents, patent applications, articles and publications mentioned herein, both supra and infra, are hereby expressly incorporated herein by reference.

[0069] Definitions and Abbreviations

[0070] Abbreviations for the genetically encoded amino acids are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamic acid (Glu or E), glutamine (Gln or Q), glycine (Gly or G), histidine (His or H), isoleucine (Ile 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 (Val or V).

[0071] When a three-letter abbreviation is used, unless specifically preceded by "L" or "D" or clear from the context in which the abbreviation is used, the amino acid may be about the α-carbon (C α ) in the L-configuration or D-configuration of the α-carbon. For example, "Ala" represents alanine without specifying the configuration about the α-carbon, while "D-Ala" and "L-Ala" represent D-alanine and L-alanine, respectively. When single-letter abbreviations are used, capital letters represent amino acids in the L-configuration about the α-carbon, and lowercase letters represent amino acids in the D-configuration about the α-carbon. For example, "A" represents L-alanine and "a" represents D-alanine. When a polypeptide sequence is presented as a string of single-letter or three-letter abbreviations (or a mixture thereof), the sequence is presented as amino (N) to carboxyl (C) direction according to common conventions.

[0072] Abbreviations for genetically encoded nucleosides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless specifically described, abbreviated nucleotides may be ribonucleosides or 2'-deoxyribonucleosides. Nucleosides may be designated individually or collectively as ribonucleosides or 2'-deoxyribonucleosides. When a nucleic acid sequence is presented as a string of single-letter abbreviations, the sequence is presented in a 5' to 3' direction according to conventional practice, and the phosphate is not shown.

[0073] As used herein, the term "about" means an acceptable error for a particular value. In some instances, "about" means within 0.05%, 0.5%, 1.0%, or 2.0% of a given value. In some instances, "about" means within 1, 2, 3, or 4 standard deviations of a given value.

[0074] As used herein, "EC" numbers refer to the enzyme nomenclature of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). The IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reactions that they catalyze.

[0075] As used herein, "ATCC" refers to the American Type Culture Collection, whose biological deposit collections include genes and strains.

[0076] As used herein, "NCBI" refers to the National Center for Biological Information and the sequence databases provided therein.

[0077] "Protein," "polypeptide," and "peptide" are used interchangeably herein to refer to a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modifications (e.g., glycosylation, phosphorylation, lipidation, myristylation, ubiquitination, etc.). Included in this definition are D- and L-amino acids, as well as mixtures of D- and L-amino acids, and polymers comprising mixtures of D- and L-amino acids and D- and L-amino acids.

[0078] "Transaminase" or "aminotransferase" are used interchangeably herein to refer to a polypeptide having the enzymatic ability to transfer an amino group (-NH2), a pair of electrons and a proton from a primary amine of an amino group donor compound to a carbonyl group (C=O) of an amino group acceptor compound, thereby converting the amino group donor compound into its corresponding carbonyl compound and converting the carbonyl acceptor compound into its corresponding primary amine compound (see, e.g., Scheme 1). Transaminases as used herein include naturally occurring (wild-type) transaminases as well as non-naturally occurring engineered polypeptides produced by human manipulation.

[0079] "Amino group donor" or "amino donor" are used interchangeably herein to refer to a compound containing an amino group that is capable of donating an amino group to an acceptor carbonyl compound (i.e., an amino group acceptor) to become a carbonyl byproduct. The amino group donor has the general structural formula,

[0080]

[0081] Where R1 and R 2 Each of R, when used independently, is a hydrocarbyl group, a hydrocarbyl aryl group, or an aryl group, which is unsubstituted or substituted with one or more non-enzymatically inhibitory groups. 1 The structure or chirality of R 2 The same or different. 1 and R 2 Together they may form a ring which is unsubstituted, substituted, or fused to other rings. Typical amino group donors include chiral and achiral amino acids, and chiral and achiral amines.

[0082] A "chiral amine" is a amine of the general formula R 1 -CH(NH2)-R 2 Amines and used herein in their broadest sense include aliphatic and alicyclic compounds of various different and mixed functional types, characterized by the presence of a primary amino group bound to a secondary carbon atom, which carries, in addition to a hydrogen atom, (i) a divalent group forming a chiral cyclic structure, or (ii) two substituents (in addition to hydrogen) that are different from each other in structure or chirality. The divalent group forming a chiral cyclic structure includes, for example, 2-methylbutane-1,4-diyl, pentane-1,4-diyl, hexane-1,4-diyl, hexane-1,5-diyl, 2-methylpentane-1,5-diyl. The two different substituents on the secondary carbon atom (the above R 1 and R 2 ) can also vary widely and include alkyl, arylalkyl, aryl, halogen, hydroxy, lower alkyl, lower alkyloxy, lower alkylthio, cycloalkyl, carboxyl, carbalkyloxy, carbamoyl, mono- and di-(lower alkyl) substituted carbamoyl, trifluoromethyl, phenyl, nitro, amino, mono- and di-(lower alkyl) substituted amino, alkylsulfonyl, arylsulfonyl, alkylformamido, arylformamido, and the like, as well as alkyl, arylalkyl or aryl substituted with the above.

[0083] "Carbonyl byproduct" refers to a carbonyl compound formed from an amino group donor when an amino group on the amino group donor is transferred to an amino group acceptor in a transamination reaction. The carbonyl byproduct has the general structural formula,

[0084]

[0085] Where R 1 and R 2 is as defined above in relation to the amino group donor.

[0086] "Amino acceptor" and "amine acceptor", "keto substrate", "ketoester substrate" are used interchangeably herein to refer to a compound containing a carbonyl group that accepts an amino group from an amino group donor in a reaction mediated by a transaminase (see, e.g., Scheme 1). In the context of the present disclosure, amino acceptor compounds for transaminases may include, among others, compounds of formula (II) and compounds (2) as further described herein.

[0087] As used herein, "cofactor" refers to a non-protein compound that works in conjunction with an enzyme in a catalytic reaction. As used herein, "cofactor" is intended to include vitamin B6 family compounds PLP, PN, PL, PM, PNP and PMP, which are sometimes also referred to as coenzymes.

[0088] "Pyridoxal phosphate", "PLP", "pyridoxal-5'-phosphate", "PYP" and "P5P" are used interchangeably herein to refer to compounds used as cofactors in transaminase reactions. In some embodiments, pyridoxal phosphate is defined by the structure 1-(4'-formyl-3'-hydroxy-2'-methyl-5'-pyridyl)methoxyphosphonic acid, CAS number [54-47-7]. Pyridoxal-5'-phosphate can be produced in vivo by phosphorylation and oxidation of pyridoxine (also known as vitamin B6). In a transamination reaction using a transaminase, the amine group of an amino donor is transferred to a cofactor to produce a ketone byproduct, while pyridoxal-5'-phosphate is converted to pyridoxamine phosphate. Pyridoxal-5'-phosphate is regenerated by reacting with a different ketone compound (amino acceptor). The amine group is transferred from pyridoxamine phosphate to the amino acceptor to produce an amine and regenerate the cofactor. In some embodiments, pyridoxal-5'-phosphate can be replaced by other members of the vitamin B6 family, including pyridoxine (PN), pyridoxal (PL), pyridoxamine (PM), and their phosphorylated counterparts; pyridoxine phosphate (PNP), and pyridoxamine phosphate (PMP).

[0089] "Amino acids" are referred to herein by either their commonly known three letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Likewise, nucleotides may be referred to by their commonly accepted single-letter codes.

[0090] As used herein, "hydrophilic amino acids or residues" refer to amino acids or residues having side chains that exhibit a hydrophobicity less than zero according to the normalized consensus hydrophobicity scale of Eisenberg et al. (Eisenberg et al., I., 179: 125-142

[1984] ). 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).

[0091] As used herein, "acidic amino acids or residues" refer to hydrophilic amino acids or residues having side chains that exhibit a pKa value of less than about 6 when the amino acid is contained in a peptide or polypeptide. Acidic amino acids typically have negatively charged side chains at physiological pH due to loss of hydrogen ions. Genetically encoded acidic amino acids include L-Glu (E) and L-Asp (D).

[0092] As used herein, "basic amino acid or residue" refers to a hydrophilic amino acid or residue having a side chain that exhibits a pKa value greater than about 6 when the amino acid is contained in a peptide or polypeptide. Basic amino acids generally have positively charged side chains at physiological pH due to association with hydronium ions. Genetically encoded basic amino acids include L-Arg (R) and L-Lys (K).

[0093] As used herein, "polar amino acid or residue" refers to a hydrophilic amino acid or residue having a side chain that is uncharged at physiological pH but has at least one bond in which a pair of electrons shared 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).

[0094] As used herein, "hydrophobic amino acid or residue" refers to an amino acid or residue having a side chain that exhibits a hydrophobicity greater than zero according to the normalized consensus hydrophobicity scale of Eisenberg et al. (Eisenberg et al., J. Mol. Biol., 179: 125-142

[1984] ). 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).

[0095] As used herein, "aromatic amino acid or residue" refers to a hydrophilic or hydrophobic amino acid or residue having a side chain comprising at least one aromatic or heteroaromatic ring. Genetically encoded aromatic amino acids include L-Phe (F), L-Tyr (Y), and L-Trp (W). Although L-His (H) is sometimes classified as a basic residue due to the pKa of its heteroaromatic nitrogen atom, or as an aromatic residue because its side chain includes a heteroaromatic ring, histidine is classified as a hydrophilic residue or as a "constrained residue" (see below) herein.

[0096] As used herein, "constrained amino acid or residue" refers to an amino acid or residue with 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.

[0097] As used herein, "non-polar amino acid or residue" refers to a hydrophobic amino acid or residue having a side chain that is uncharged at physiological pH and has a bond in which an electron pair shared by two atoms is usually equally held 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).

[0098] As used herein, "aliphatic amino acid or residue" refers to a hydrophobic amino acid or residue with an aliphatic hydrocarbon side chain. Genetically encoded aliphatic amino acids include L-Ala (A), L-Val (V), L-Leu (L), and L-Ile (I). It is noteworthy that cysteine ​​(or "L-Cys" or "[C]") is unusual because it can form disulfide bridges with other L-Cys (C) amino acids or other sulfonyl or sulfhydryl-containing amino acids. "Cysteine-like residues" include cysteine ​​and other amino acids containing sulfhydryl moieties that can be used to form disulfide bridges. The ability of L-Cys (C) (and other amino acids with -SH side chains) to be present in a peptide in a reduced free-SH or oxidized disulfide-bridged form affects whether L-Cys (C) contributes a net hydrophobic or hydrophilic character to the peptide. Although L-Cys(C) exhibits a hydrophobicity of 0.29 according to Eisenberg's normalized consensus scale (Eisenberg et al., 1984, supra), it should be understood that for the purposes of the present disclosure, L-Cys(C) is classified into its own unique group.

[0099] As used herein, "small amino acid or residue" refers to an amino acid or residue having a side chain comprising three or less carbons and / or heteroatoms (excluding α-carbon and hydrogen). According to the above definition, small amino acids or residues can be further classified as aliphatic, non-polar, polar or acidic small amino acids or residues. 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).

[0100] As used herein, "hydroxyl-containing amino acid or 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).

[0101] As used herein, "polynucleotide" and "nucleic acid" refer to two or more nucleotides covalently linked together. A polynucleotide may completely contain ribonucleotides (i.e., RNA), completely contain 2'deoxyribonucleotides (i.e., DNA), or contain a mixture of ribonucleotides and 2'deoxyribonucleotides. Although nucleosides are usually linked together via standard phosphodiester bonds, polynucleotides may include one or more non-standard connections. A polynucleotide may be single-stranded or double-stranded, or may include both single-stranded and double-stranded regions. In addition, although a polynucleotide will usually contain naturally occurring coding nucleobases (i.e., adenine, guanine, uracil, thymine, and cytosine), it may also contain one or more modified and / or synthesized nucleobases, such as, for example, inosine, xanthine, hypoxanthine, etc. In some embodiments, such modified or synthesized nucleobases are nucleobases encoding amino acid sequences.

[0102] As used herein, "nucleoside" refers to a glycosylamine comprising a nucleoside base (i.e., a nitrogenous base) and a 5-carbon sugar (e.g., ribose or deoxyribose). Non-limiting examples of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine, and inosine. In contrast, the term "nucleotide" refers to a glycosylamine comprising a nucleoside base, a 5-carbon sugar, and one or more phosphate groups. In some embodiments, nucleosides can be phosphorylated by kinases to produce nucleotides.

[0103] As used herein, "nucleoside diphosphate" refers to a glycosylamine comprising a nucleoside base (i.e., a nitrogenous base), a 5-carbon sugar (e.g., ribose or deoxyribose) and a diphosphate (i.e., pyrophosphate) portion. In some embodiments herein, "nucleoside diphosphate" is abbreviated as "NDP". Non-limiting examples of nucleoside diphosphates include cytidine diphosphate (CDP), uridine diphosphate (UDP), adenosine diphosphate (ADP), guanosine diphosphate (GDP), thymidine diphosphate (TDP) and inosine diphosphate. In some cases, the terms "nucleoside" and "nucleotide" are used interchangeably.

[0104] "Coding sequence" refers to the portion of a nucleic acid (eg, a gene) that encodes the amino acid sequence of a protein.

[0105] As used herein, the terms "biocatalysis," "biocatalytic," "bioconversion," and "biosynthesis" refer to the use of enzymes to perform chemical reactions on organic compounds.

[0106] "Naturally occurring" or "wild-type" refers to the form found in nature. For example, a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence found in an organism that can be isolated from a source in nature and has not been intentionally modified by human manipulation.

[0107] As used herein, "recombinant," "engineered," "non-naturally occurring," and "variant" when used with respect to a cell, nucleic acid, or polypeptide refers to a material that has been modified in a manner that does not otherwise exist in nature or to a material that corresponds to a native or native form of the material. In some embodiments, the cell, nucleic acid, or polypeptide is identical to a naturally occurring cell, nucleic acid, or polypeptide, but is produced or derived from synthetic materials and / or through manipulation using recombinant techniques. Non-limiting examples include, among others, recombinant cells that express genes not found in the natural (non-recombinant) form of the cell or that express natural genes that are otherwise expressed at different levels.

[0108] The term "percentage (%) of sequence identity" is used herein to refer to the comparison between polynucleotides or polypeptides, and is determined by comparing two optimally aligned sequences in a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may include additions or deletions (i.e., gaps) compared to the reference sequence for optimal alignment of the two sequences. The percentage can be calculated as follows: determine the number of positions where the same nucleic acid base or amino acid residue appears in the two sequences to produce the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Alternatively, the percentage can be calculated as follows: determine the number of positions where the same nucleic acid base or amino acid residue appears in the two sequences or where the nucleic acid base or amino acid residue is aligned with a gap to produce the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Those skilled in the art will appreciate that there are many established algorithms that can be used to align two sequences. Optimal alignment of sequences for comparison can be performed by any suitable method, including, but not limited to, the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 2:482

[1981] ), by the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 48:443

[1970] ), by the similarity search method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444

[1988] ), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin software package), or by visual inspection, as is known in the art. Examples of algorithms suitable for determining percent sequence identity and sequence similarity include, but are not limited to, BLAST and BLAST 2.0 algorithms, described by Altschul et al. (see, respectively, Altschul et al., J. Mol. Biol., 215:403-410

[1990] ; and Altschul et al., Nucl. Acids Res., 3389-3402

[1977] ). Software for performing BLAST analysis is publicly available through the website of the National Center for Biotechnology Information. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy a certain positive threshold score T when aligned with a word of the same length in the database sequence. T is referred to as the neighborhood word score threshold (see, 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 to the point where the cumulative alignment score cannot be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for matching residue pairs; always >0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is stopped when: the cumulative alignment score drops by an amount X from its maximum achieved value; the cumulative score reaches 0 or less than 0 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 the following as defaults: word length (W) of 11, expectation (E) of 10, M=5, N=-4, and comparison of both chains. 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, Proc. Natl. Acad. Sci. USA 89:10915

[1989] ). Exemplary determinations of sequence alignments and % sequence identity can be made using the BESTFIT or GAP programs in the GCG Wisconsin software package (Accelrys, Madison WI) using the default parameters provided.

[0109] "Reference sequence" refers to a determined sequence used as the basis for sequence comparison. A reference sequence can be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Typically, a reference sequence is a length of at least 20 nucleotides or amino acid residues, a length of at least 25 residues, a length of at least 50 residues, or the full length of a nucleic acid or polypeptide. Because two polynucleotides or polypeptides can each (1) contain a similar sequence between the two sequences (i.e., a portion of a complete sequence), and (2) can also contain a different (divergent) sequence between the two sequences, a sequence comparison between two (or more) polynucleotides or polypeptides is usually performed by comparing the sequences of the two polynucleotides or polypeptides in a "comparison window" to identify and compare the sequence similarity of a local region. In some embodiments, a "reference sequence" can be based on a primary amino acid sequence, wherein a reference sequence is a sequence that can have one or more changes in a primary sequence. For example, "a reference sequence based on SEQ ID NO:2 with histidine at a residue corresponding to X9" refers to a reference sequence in which the corresponding residue (which is tyrosine) at X9 in SEQ ID NO:2 has been changed to histidine.

[0110] "Comparison window" refers to a conceptual segment of at least about 20 consecutive nucleotide positions or amino acid residues, wherein a sequence can be compared to a reference sequence of at least 20 consecutive nucleotides or amino acids, and wherein the portion of the sequence in the comparison window may comprise 20% or less additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The comparison window may be longer than 20 consecutive residues, and optionally includes windows of 30, 40, 50, 100 or more.

[0111] When used in the context of numbering a given amino acid or polynucleotide sequence, "corresponding to," "referenced to," or "relative to," means that the residues of a given reference sequence are numbered when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue numbers or residue positions of a given polymer are specified with respect to a reference sequence, rather than being specified by the actual numerical position of the residues within the given amino acid or polynucleotide sequence. For example, given an amino acid sequence, such as an engineered aminotransferase, the residue matching between the two sequences can be optimized by introducing gaps to align with the reference sequence. In these cases, the residues in a given amino acid or polynucleotide sequence are numbered with respect to the reference sequence to which it is aligned, despite the presence of gaps.

[0112] As used herein, "amino acid difference" and "residue difference" refer to the difference of the amino acid residue at one position of the polypeptide sequence relative to the amino acid residue at the corresponding position in the reference sequence. In some cases, the reference sequence has a histidine tag, but the numbering remains unchanged relative to the equivalent reference sequence without a histidine tag. The position of the amino acid difference is generally referred to as "Xn" herein, where n refers to the corresponding position in the reference sequence on which the residue difference is based. For example, "the residue difference at position X93 compared to SEQ ID NO:4" refers to the difference of the amino acid residue at the polypeptide position corresponding to position 93 of SEQ ID NO:4. Therefore, if the reference polypeptide of SEQ ID NO:4 has serine at position 93, "the residue difference at position X93 compared to SEQ ID NO:4" refers to the amino acid substitution of any residue except serine at the polypeptide position corresponding to position 93 of SEQ ID NO:4. In most examples herein, the specific amino acid residue difference at one position is indicated as "XnY", where "Xn" specifies the corresponding position as described above, and "Y" is the single letter identifier of the amino acid found in the engineered polypeptide (i.e., the residue different from the reference polypeptide). In some examples (e.g., in the tables presented in the Examples), the present invention also provides specific amino acid differences represented by the conventional symbol "AnB", where A is a single-letter identifier for a residue in a reference sequence, "n" is the number of a residue position in a reference sequence, and B is a single-letter identifier for a residue substitution in the sequence of an engineered polypeptide. In some examples, a polypeptide of the present invention may comprise one or more amino acid residue differences relative to a reference sequence, indicated by a column of designated positions where residue differences exist relative to a reference sequence. In some embodiments, when more than one amino acid can be used in a specific residue position of a polypeptide, the various amino acid residues that can be used are separated by " / " (e.g., X307H / X307P or X307H / P). Slashes can also be used to indicate more than one substitution within a given variant (i.e., there is more than one substitution in a given sequence such as in a combinatorial variant). In some embodiments, the present invention includes engineered polypeptide sequences containing one or more amino acid differences, the amino acid differences comprising conservative amino acid substitutions or non-conservative amino acid substitutions. In some additional embodiments, the present invention provides engineered polypeptide sequences comprising both conservative amino acid substitutions and non-conservative amino acid substitutions.

[0113] As used herein, "conservative amino acid substitutions" refer to the replacement of a residue with a different residue having a similar side chain, and thus generally include the replacement of an amino acid in a polypeptide with an amino acid in the same or similar amino acid defined class. For example, but not limited to, in some embodiments, an amino acid with an aliphatic side chain is replaced by another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with a hydroxyl side chain is replaced by another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acid with an aromatic side chain is replaced by another amino acid with an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain is replaced by another amino acid with a basic side chain (e.g., lysine and arginine); an amino acid with an acidic side chain is replaced by another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and / or a hydrophobic amino acid or a hydrophilic amino acid is replaced by another hydrophobic amino acid or a hydrophilic amino acid, respectively.

[0114] "Non-conservative substitutions" refer to substitutions of amino acids in a polypeptide with amino acids having significantly different side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and affect (a) the structure of the peptide backbone in the area of ​​the substitution (e.g., proline for glycine), (b) charge or hydrophobicity, or (c) side chain bulk. For example, but not limited to, exemplary non-conservative substitutions may be substitutions of acidic amino acids with basic or aliphatic amino acids; substitutions of aromatic amino acids with small amino acids; and substitutions of hydrophilic amino acids with hydrophobic amino acids.

[0115] "Deletion" refers to the modification of a polypeptide by removing one or more amino acids from a reference polypeptide. Deletion can include the 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 that make up the reference enzyme, or up to 20% of the total number of amino acids, while retaining enzymatic activity and / or retaining the improved properties of the engineered transaminase. Deletion can involve internal portions and / or terminal portions of a polypeptide. In various embodiments, deletion can include continuous segments or can be discontinuous. Deletion is generally represented by a "-" in an amino acid sequence.

[0116] "Insertion" refers to the modification of a polypeptide by adding one or more amino acids to a reference polypeptide. In some embodiments, the improved engineered transaminase comprises one or more amino acids inserted into a naturally occurring transaminase polypeptide, and one or more amino acids inserted into other improved transaminase polypeptides. The insertion may be in an internal portion of the polypeptide or may be an insertion into the carboxyl or amino terminus. Insertion as used herein includes fusion proteins as known in the art. The insertion may be a continuous stretch of amino acids or separated by one or more amino acids in a naturally occurring polypeptide.

[0117] The term "amino acid substitution set" or "substitution set" refers to a set of amino acid substitutions in a polypeptide sequence compared to a reference sequence. A substitution set can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions. In some embodiments, a substitution set refers to a collection of amino acid substitutions present in any of the variant transaminases listed in the tables provided in the Examples.

[0118] "Functional fragment" and "biologically active fragment" are used interchangeably herein to refer to polypeptides having an amino-terminal deletion and / or a carboxyl-terminal deletion and / or an internal deletion, but wherein the remaining amino acid sequence is identical to the corresponding positions in the sequence to which it is compared (e.g., a full-length engineered transaminase of the invention) and retains substantially all of the activity of the full-length polypeptide.

[0119] "Isolated polypeptide" refers to a polypeptide that is substantially separated from other contaminants (e.g., proteins, lipids, and polynucleotides) that naturally accompany it. The term includes polypeptides that have been removed or purified from their naturally occurring environment or expression system (e.g., host cells or in vitro synthesis). The improved transaminase may be present in cells, in cell culture media, or prepared in various forms (such as lysates or isolated preparations). Therefore, in some embodiments, the improved transaminase may be an isolated polypeptide.

[0120] "Substantially pure polypeptide" 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 when the target species constitutes at least about 50% of the macromolecular species present by mole or % weight, it is generally a substantially purified composition. However, in some embodiments, the composition comprising the transaminase comprises less than 50% pure (e.g., about 10%, about 20%, about 30%, about 40%, or about 50%) transaminase. Typically, a substantially pure transaminase composition will constitute 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 present in the composition by mole or % weight. In some embodiments, the target species is purified to substantially homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods), wherein the composition is substantially composed of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ionic species are not considered macromolecular species.In some embodiments, the isolated improved transaminase polypeptide is a substantially pure polypeptide composition.

[0121] "Stereoselectivity" refers to the preferential formation of one stereoisomer relative to another stereoisomer in a chemical or enzymatic reaction. Stereoselectivity can be partial, in which case the formation of one stereoisomer is preferred over the other stereoisomer, or stereoselectivity can be complete, in which case only one stereoisomer is formed. When the stereoisomers are enantiomers, stereoselectivity is referred to as enantioselectivity, i.e., the fraction (usually reported as a percentage) of one enantiomer in the sum of the two. Alternatively, it is usually reported in the art as enantiomeric excess (ee) (usually a percentage) calculated therefrom according to the following formula: [major enantiomer-minor enantiomer] / [major enantiomer+minor enantiomer]. In the case where the stereoisomers are diastereomers, stereoselectivity is referred to as diastereoselectivity, i.e., the fraction (usually reported as a percentage) of one diastereomer in a mixture of two diastereomers, usually optionally reported as diastereomeric excess (de). In cases where a mixture contains more than two diastereomers, the ratio of the diastereomers or "diastereomeric ratio" is usually reported rather than diastereomeric excess. Enantiomeric excess and diastereomeric excess are types of stereoisomeric excess. "High stereoselectivity" refers to a transaminase polypeptide that is capable of converting a substrate to the corresponding chiral amine product with a stereoisomeric excess of at least about 85%.

[0122] As used herein, "regioselectivity" and "regioselective reaction" refer to reactions in which one direction of bond formation or breaking occurs in preference to all other possible directions. A reaction can be completely (100%) regioselective if the discrimination is complete, substantially regioselective (at least 75%) if the reaction product at one site is preferred over the reaction products at other sites, or partially regioselective (x%, where the percentage is set depending on the reaction of interest).

[0123] As used herein, "chemoselectivity" refers to the preferential formation of one product over another in a chemical or enzymatic reaction.

[0124] As used herein, "improved enzyme properties" refers to at least one improved property of an enzyme. In some embodiments, the invention provides engineered transaminase polypeptides that show improvements in any enzyme properties compared to reference transaminase polypeptides and / or wild-type transaminase polypeptides and / or another engineered transaminase polypeptide. Therefore, various transaminase polypeptides can be determined and compared, including the level of "improvement" between wild-type and engineered transaminase. Improved properties include but are not limited to properties such as: increased protein expression, increased thermal activity, increased thermal stability, increased pH activity, increased stability, increased enzyme activity, increased substrate specificity or affinity, increased specific activity, increased resistance to substrate or end product inhibition, increased chemical stability, improved chemical selectivity, improved solvent stability, increased tolerance to acidic pH, increased tolerance to proteolytic activity (i.e., reduced sensitivity to proteolysis), reduced aggregation, increased solubility and changed temperature profiles (temperature profile). In other embodiments, the term is used to refer to at least one improved property of a transaminase. In some embodiments, the invention provides engineered transaminase polypeptides that exhibit improvements in any enzymatic property compared to a reference transaminase polypeptide and / or a wild-type transaminase polypeptide and / or another engineered transaminase polypeptide. Thus, the level of "improvement" between various transaminase polypeptides, including wild-type and engineered transaminases, can be determined and compared.

[0125] "Increased enzyme activity" and "enhanced catalytic activity" refer to improved properties of engineered polypeptides, which can be expressed as an increase in specific activity (e.g., product produced / time / weight protein) or an increase in the percentage of conversion of a substrate into a product (e.g., using a specified amount of enzyme in a specified time period, the percentage of conversion of a starting amount of substrate into a product) compared to a reference enzyme. In some embodiments, these terms refer to improved properties of engineered transaminase polypeptides provided herein, which can be expressed as an increase in specific activity (e.g., product produced / time / weight protein) or an increase in the percentage of conversion of a substrate into a product (e.g., using a specified amount of transaminase in a specified time period, the percentage of conversion of a starting amount of substrate into a product) compared to a reference transaminase. In some embodiments, these terms are used to refer to improved transaminases provided herein. Exemplary methods for determining the enzymatic activity of engineered transaminases of the present invention are provided in the Examples. Any property associated with enzyme activity can be affected, including typical enzyme properties. m 、V max or k cat , their changes can result in increased enzyme activity. For example, the improvement in enzyme activity can be from about 1.1 times the enzyme activity of the corresponding wild-type enzyme to up to 2 times, 5 times, 10 times, 20 times, 25 times, 50 times, 75 times, 100 times, 150 times, 200 times or more enzyme activity compared to another engineered transaminase from which the naturally occurring transaminase or transaminase polypeptide is derived.

[0126] "Increased thermal activity" or "improved thermal activity" is used herein to refer to a polypeptide having increased catalytic activity under high temperature conditions relative to a reference or wild-type protein.

[0127] "Conversion" refers to the enzymatic conversion of a substrate into a corresponding product. "Percent conversion" refers to the percentage of substrate converted into product under specified conditions within a certain period of time. Therefore, the "enzyme activity" or "activity" of a transaminase polypeptide can be expressed as the "percent conversion" of substrate into product.

[0128] By "thermostable" is meant that the transaminase polypeptide maintains similar activity (eg, more than 60% to 80%) compared to the wild-type enzyme after exposure to elevated temperature (eg, 40-80°C) for a period of time (eg, 0.5 hr-24 hr).

[0129] "Solvent stable" means that the transaminase polypeptide maintains similar activity (e.g., more than 60% to 80%) after exposure to different concentrations (e.g., 5%-99%) of solvents (ethanol, IPA, dimethyl sulfoxide (DMSO), tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butyl acetate, methyl tert-butyl ether, etc.) for a period of time (e.g., 0.5 hr-24 hr), compared to the wild-type enzyme.

[0130] As used herein, "pH stable" means that the transaminase polypeptide maintains similar activity (e.g., more than 60% to 80%) after exposure to high or low pH (e.g., 4.5-6 or 8 to 12) for a period of time (e.g., 0.5 hr-24 hr) compared to the untreated enzyme.

[0131] The term "stringent hybridization conditions" is used herein to refer to conditions under which nucleic acid hybrids are stable. As known to those skilled in the art, the stability of the hybrid is reflected in the melting temperature (T m ). In general, the stability of the hybrid varies with ionic strength, temperature, G / C content, and the presence of chaotropic agents. m Values ​​can be calculated using known methods for predicting melting temperatures (see, e.g., Baldino et al., Meth. Enzymol., 168:761-777

[1989] ; Bolton et al., Proc. Natl. Acad. Sci. USA 48:1390

[1962] ; Bresslauer et al., Proc. Natl. Acad. Sci. USA 83:8893-8897

[1986] ; Freier et al., Proc. Natl. Acad. Sci. USA 83:9373-9377

[1986] ; Kierzek et al., Biochem., 25:7840-7846

[1986] ; Rychlik et al., Nucl. Acids Res., 18:6409-6412

[1990] (erratum, Nucl. Acids Res., 26:6413 ... Res., 19:698

[1991] ); Sambrook et al., supra; Suggs et al., 1981, in Developmental Biology Using Purified Genes, Brown et al. [Eds.], pp. 683-693, Academic Press, Cambridge, MA

[1981] ; and Wetmur, Crit. Rev. Biochem. Mol. Biol. 26:227-259

[1991] ). In some embodiments, the polynucleotide encodes a polypeptide disclosed herein and hybridizes under defined conditions, such as moderately stringent or highly stringent conditions, to the complement of a sequence encoding an engineered aminotransferase of the invention.

[0132] "Hybridization stringency" refers to hybridization conditions, such as washing conditions, in nucleic acid hybridization. Typically, hybridization reactions are performed under conditions of lower stringency, followed by washes of different but higher stringency. The term "moderately stringent hybridization" refers to conditions that allow target DNA to bind to complementary nucleic acids that have about 60% identity, preferably about 75% identity, about 85% identity, and greater than about 90% identity with the target polynucleotide. Exemplary moderately stringent conditions are conditions equivalent to hybridization at 42°C in 50% formamide, 5×Denhart solution, 5×SSPE, 0.2% SDS, followed by washing at 42°C in 0.2×SSPE, 0.2% SDS. "High stringency hybridization" generally refers to conditions with a thermal melting temperature T as determined for a defined polynucleotide sequence under solution conditions. m Conditions that differ by about 10°C or less. In some embodiments, high stringency conditions refer to conditions that allow only hybridization of those nucleic acid sequences that form stable hybrids in 0.018M NaCl at 65°C (i.e., if the hybrid is unstable in 0.018M NaCl at 65°C, it is unstable under high stringency conditions as contemplated herein). High stringency conditions can be provided, for example, by hybridizing under conditions equivalent to 50% formamide, 5×Denhart solution, 5×SSPE, 0.2%SDS at 42°C, followed by washing in 0.1×SSPE and 0.1%SDS at 65°C. Another high stringency condition is hybridization under conditions equivalent to hybridization at 65°C in 5X SSC containing 0.1% (w:v) SDS and washing at 65°C in 0.1×SSC containing 0.1% SDS. Other high stringency hybridization conditions as well as moderate stringency conditions are described in the references cited above.

[0133] "Codon optimized" refers to that the codons of the polynucleotide encoding the protein are changed to those codons that are preferentially used in a specific organism, so that the encoded protein is more effectively expressed in the organism of interest. Although the genetic code is degenerate, that is, most amino acids are represented by several codons referred to as "synonyms" or "synonymous" codons, it is well known that the codon usage of a specific organism is non-random and biased for specific codon triplets. For a given gene, a gene with a common function or ancestral origin, a highly expressed protein versus a low copy number protein and an aggregated protein coding region of the genome of an organism, this codon usage bias may be higher. In some embodiments, codon optimization can be performed on the polynucleotide encoding the transaminase for optimized production in a host organism selected for expression.

[0134] As used herein, "preferred," "optimal," and "high codon usage bias" codons, when used alone or in combination, refer interchangeably to codons in a protein coding region that are used at a higher frequency than other codons encoding the same amino acid. Preferred codons can be determined based on codon usage in a single gene, a group of genes of a common function or origin, highly expressed genes, codon frequency in aggregating protein coding regions of an entire organism, codon frequency in aggregating protein coding regions of related organisms, or a combination thereof. Codons whose frequency increases with the level of gene expression are generally the optimal codons for expression. Various methods for determining codon frequency (e.g., codon usage, relative synonymous codon usage) and codon preference in a particular organism are known, including multivariate analysis, such as using cluster analysis or correlation analysis, and the effective number of codons used in a gene (see, e.g., GCG Codon Preference, Genetics Computer Group Wisconsin Package; Codon W, Peden, University of Nottingham; McInerney, Bioinform., 14:372-73

[1998] ; Stenico et al., Nucl. Acids Res., 222437-46

[1994] ; and Wright, Gene 87:23-29

[1990] ). Codon usage tables for many different organisms are available (see, e.g., Wada et al., Nucl. Acids Res., 20:2111-2118

[1992] ; Nakamura et al., Nucl. Acids Res., 28:292

[2000] ; Duret et al., supra; Henaut and Danchin, in Escherichiacoli and Salmonella, Neidhardt et al. (Eds.), ASM Press, Washington DC, pp. 2047-2066

[1996] ). The data source for obtaining codon usage can rely on any available nucleotide sequence capable of encoding a protein.These data sets include nucleic acid sequences that are actually known to encode expressed proteins (e.g., complete protein coding sequences - CDS), expressed sequence tags (ESTS), or predicted coding regions of genomic sequences (see, e.g., Mount, Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

[2001] ; Uberbacher, Meth. Enzymol., 266:259-281

[1996] ; and Tiwari et al., Comput. Appl. Biosci., 13:263-270

[1997] ).

[0135] "Control sequences" herein refer to all components that are necessary or advantageous for the expression of the polynucleotides and / or polypeptides of the present disclosure. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoters, signal peptide sequences, start sequences, and transcription terminators. At a minimum, control sequences include promoters and transcription and translation termination signals. Control sequences may be provided with linkers for the purpose of introducing specific restriction sites that facilitate the connection of the control sequences to the coding region of the nucleic acid sequence encoding the polypeptide.

[0136] "Operably linked" is defined herein as a configuration in which a control sequence is appropriately placed (i.e., in a functional relationship) at a position relative to a polynucleotide of interest such that the control sequence directs or regulates expression of the polynucleotide and / or polypeptide of interest.

[0137] "Promoter sequence" refers to a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence comprises a transcriptional control sequence that mediates the expression of the polynucleotide of interest. The promoter can be any nucleic acid sequence that exhibits transcriptional activity in a selected host cell, including mutants, truncated and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell.

[0138] "Alkyl" refers to a linear or branched group of 1 to 18 carbon atoms, particularly 1 to 8 carbon atoms, and more particularly 1 to 6 carbon atoms. Alkyl groups with a specified number of carbon atoms are indicated in parentheses, for example (C1-C4)alkyl refers to a hydrocarbon group of 1 to 4 carbon atoms.

[0139] "Aryl" refers to an unsaturated aromatic carbocyclic group of 5 to 14 carbon atoms having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthracenyl). For multiple condensed rings, at least one of the rings is aromatic. Representative aryl groups include phenyl, pyridyl, naphthyl, etc.

[0140] "Arylalkyl" refers to an alkyl group substituted with an aryl moiety. Representative arylalkyl groups include benzyl, phenethyl, and the like.

[0141] "Heteroaryl" refers to an aromatic heterocyclic group of 5 to 14 ring atoms containing 1 to 4 ring heteroatoms selected from oxygen, nitrogen and sulfur within the ring. A heteroaryl group can have a single ring (e.g., pyridyl or furanyl) or multiple fused rings (e.g., indolizinyl or benzothienyl). For multiple fused rings, at least one of the rings is aromatic.

[0142] "Heteroarylalkyl" refers to an alkyl group substituted with a heteroaryl moiety as defined herein.

[0143] "Heterocycle" and interchangeably "heterocycloalkyl" refer to a saturated or unsaturated group having from 3 to 14 ring atoms in a single ring or more than one fused ring, with 1 to 4 heteroatoms selected from nitrogen, sulfur or oxygen within the ring. A heterocyclic group can have a single ring (e.g., piperidinyl or tetrahydrofuranyl) or multiple fused rings (e.g., indolinyl, dihydrobenzofuran or quinuclidinyl). Representative heterocycles and heteroaryls include, but are not limited to, furan, thiophene, thiazole, oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, and imidazolidine.

[0144] "Alkoxy" or "Alkyloxy" refers to the group alkyl-O- wherein the alkyl group is as defined above, including optionally substituted alkyl groups also as defined above.

[0145] "Amino" refers to the group -NH2. Substituted amino refers to the groups -NHR', NR'R' and NR'R'R', where each R' is independently selected from substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkoxy, aryl, heteroaryl, arylalkyl, heteroarylalkyl, acyl, alkoxycarbonyl, thiol, sulfinyl, sulfonyl, and the like. Typical amino groups include, but are not limited to, dimethylamino, diethylamino, trimethylammonium, triethylammonium, methylsulfonylamino, furanyl-oxy-sulfonamido, and the like.

[0146] "Carboxyl" refers to -COOH.

[0147] "Carbonyl" refers to -C(O)-, which can have various substituents to form different carbonyl groups including acids, acyl halides, aldehydes, amides, esters and ketones.

[0148] "Hydroxyl" refers to -OH.

[0149] "Cyano" refers to -CN.

[0150] "Halogen" or "halo" refers to fluoro, chloro, bromo and iodo.

[0151] "Nitro" refers to -NO2.

[0152] "Fused" or "fused ring" such as in fused aryl or fused heteroaryl refers to two or more rings connected so that they have at least 2 ring atoms in common. Fused aryl refers to a fused ring in which at least one ring is aryl. Fused heteroaryl refers to a fused ring in which at least one ring is heteroaryl.

[0153] Unless otherwise specified, "substituted" means that the position occupied by hydrogen in the preceding groups is replaced with a substituent such as, but not limited to, hydroxy, oxo, nitro, methoxy, ethoxy, alkoxy, substituted alkoxy, trifluoromethoxy, haloalkoxy, fluoro, chloro, bromo, iodo, halogen, methyl, ethyl, propyl, butyl, alkyl, alkenyl, alkynyl, substituted alkyl, trifluoromethyl, haloalkyl, hydroxyalkyl, alkoxyalkyl, thio, alkylthio, acyl, carboxyl, alkoxycarbonyl, formamido, substituted formamido, alkylsulfonyl, alkylsulfinyl, alkylsulfonylamino, sulfonamido, substituted sulfonamido , cyano, amino, substituted amino, alkylamino, dialkylamino, aminoalkyl, acylamino, amidino, amidoximo, hydroxamoyl, phenyl, aryl, substituted aryl, aryloxy, arylalkyl, arylalkenyl, arylalkynyl, pyridyl, imidazolyl, heteroaryl, substituted heteroaryl, heteroaryloxy, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, substituted cycloalkyl, cycloalkyloxy, pyrrolidinyl, piperidinyl, morpholino, heterocycle, (heterocycle)oxy and (heterocycle)alkyl; and preferred heteroatoms are oxygen, nitrogen and sulfur. It is understood that where open valences exist on these substituents, they may be further substituted with hydrocarbyl, cyclohydrocarbyl, aryl, heteroaryl and / or heterocyclic groups, where such open valences exist on carbon, they may be further substituted with halogen and oxygen-, nitrogen- or sulfur-bonded substituents, and where more than one such open valence exists, these groups may be joined to form rings by direct bond formation or by bond formation with new heteroatoms (preferably oxygen, nitrogen or sulfur). It is also understood that the above substitutions may be made provided that replacement of hydrogen with a substituent does not introduce unacceptable instability into the molecules of the invention and is otherwise chemically reasonable.

[0154] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and means that the description includes instances when the event or circumstance occurs and instances in which the event or circumstance does not occur. One of ordinary skill in the art will understand that for any molecule described as containing one or more optional substituents, only sterically feasible and / or synthetically feasible compounds are intended to be included.

[0155] "Optionally substituted" refers to all subsequent modifiers in one or a series of chemical groups. For example, in the term "optionally substituted arylalkyl", the "alkyl" part and the "aryl" part of the molecule may be substituted or unsubstituted, and for a series of "optionally substituted alkyl, cycloalkyl, aryl and heteroaryl", the alkyl group, cycloalkyl group, aryl group and heteroaryl group may be substituted or unsubstituted independently of each other.

[0156] "Protective group" refers to a group of atoms that shields, reduces or prevents the reactivity of a functional group when attached to a reactive functional group in a molecule. Typically, a protective group can be selectively removed as desired during a building-up process. In Wuts and Greene, "Greene's Protective Groups in Organic Synthesis," the 4th edition, Wiley Interscience (2006), and Harrison et al., Compendium of Synthetic Organic Methods, 1-8 volumes, 1971-1996, John Wiley & Sons, NY, examples of protective groups can be found. Functional groups that may have protective groups include, but are not limited to, hydroxyl groups, amino groups, and carboxyl groups. Representative amino protecting groups include, but are not limited to, formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl ("CBZ"), tert-butoxycarbonyl ("Boc"), trimethylsilyl ("TMS"), 2-trimethylsilyl-ethanesulfonyl ("SES"), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethoxycarbonyl ("FMOC"), nitro-veratryloxycarbonyl ("NVOC"), and the like. Representative hydroxy protecting groups include, but are not limited to, those in which the hydroxy group is acylated (e.g., methyl and ethyl esters, acetate or propionate groups, or glycol esters) or those in which the hydroxy group is alkylated, such as benzyl and trityl ethers, as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers (e.g., TMS or TIPPS groups), and allyl ethers. Other protecting groups can be found in the references cited herein.

[0157] "Leaving group" generally refers to any atom or moiety that can be replaced by another atom or moiety in a chemical reaction. More specifically, a leaving group refers to an atom or moiety that is easily replaced and substituted by a nucleophile (e.g., an amine, thiol, alcohol, or cyanide). Such leaving groups are well known and include carboxylates, N-hydroxysuccinimide ("NHS"), N-hydroxybenzotriazole, halogen (fluorine, chlorine, bromine, or iodine), and oxy groups. Non-limiting characteristics and examples of leaving groups can be found, for example, in Organic Chemistry, 2nd Edition, Francis Carey (1992), pages 328-331; Introduction to Organic Chemistry, 2nd Edition, Andrew Streitwieser and Clayton Heathcock (1981), pages 169-171; and Organic Chemistry, 5th Edition, John McMurry, Brooks / Cole Publishing (2000), pages 398 and 408; all of which are incorporated herein by reference.

[0158] "Suitable reaction conditions" refer to those conditions in a biocatalytic reaction solution (e.g., ranges of enzyme loading, substrate loading, cofactor loading, temperature, pH, buffer, cosolvents, etc.) under which the aminotransferase polypeptides of the present disclosure are able to convert a substrate into a desired amino product compound, such as compound (2) into compound (1). Exemplary "suitable reaction conditions" are provided in the present disclosure and illustrated by the Examples.

[0159] "Loading," such as in "compound loading" or "enzyme loading," refers to the concentration or amount of a component in the reaction mixture at the start of the reaction.

[0160] In the context of a biocatalyst-mediated process, "substrate" refers to the compound or molecule upon which the biocatalyst acts.

[0161] In the context of a biocatalyst-mediated process, "product" refers to a compound or molecule produced by the action of the biocatalyst. For example, an exemplary product of a transaminase biocatalyst in the methods disclosed herein is compound (1).

[0162] "Wet organic solvent" is defined as an organic solvent containing up to the maximum amount of water that can be dissolved in the organic solvent. For example, those skilled in the art believe that the solubility of water in IPAc is about 1.9 wt%, and wet IPAc containing up to about 1.9 wt% water can be produced as described in the Examples below or by other suitable methods. Wet organic solvents, as used in this application, produce minimal aqueous conditions without adding any water to a chemical reaction, such as the screening assay reactions described in the Examples.

[0163] "Minimally aqueous conditions" are conditions produced by using a wet organic solvent without adding additional water. Minimally aqueous conditions use an organic solvent as a reaction condition or solvent system that contains up to the maximum amount of water that can be dissolved in the organic solvent. In contrast to aqueous conditions, minimally aqueous conditions do not include the addition of water to the reaction or solvent system. Rather, minimally aqueous conditions are produced by first dissolving up to the maximum amount of water that can be dissolved in the organic solvent, then removing any undissolved water, and then using the wet organic solvent in a solvent system that does not include any additional water that is not dissolved in the wet organic solvent. DETAILED DESCRIPTION OF THE INVENTION

[0165] The present invention provides engineered transaminase polypeptides and compositions thereof, and polynucleotides encoding engineered transaminase polypeptides. Also provided are methods for producing transaminases. In some embodiments, the engineered transaminase polypeptides are optimized to provide improved enzyme properties in wet organic solvents under minimal water-containing conditions.

[0166] The transamination of a methyl ketoester substrate (4-methyl-3-oxo-pentanoic acid methyl ester) to the corresponding S-homovaline ester product ((S)-3-amino-4-methyl-pentanoic acid methyl ester) is depicted in Scheme 2.

[0167] Solution 2

[0168]

[0169] Under typical aqueous conditions, product yields are limited by spontaneous hydrolysis of the aminoester product and spontaneous hydrolysis of the ketoester followed by decomposition. See Scheme 4 below.

[0170] Solution 4

[0171]

[0172] One method to overcome the limitations of asymmetric enzymatic synthesis in industrial processes is to use wet organic solvents. However, due to the lack of enzymes that show activity in organic solvents, this method is limited. Recently, preliminary studies have shown the activity of ω-aminotransferases from Arthrobacter species in wet organic solvents without the need for fixation (Mutti & Kroutil, Adv. Synth. Catal., 2012, 354, 3409-4313).

[0173] The polypeptides of the present invention have been engineered to be highly enantioselective and to have catalytic activity in wet organic solvents under minimally aqueous conditions. This allows the direct conversion of methylketoesters to S-homovaline esters in wet organic solvents, which greatly reduces the spontaneous hydrolysis of methylketoesters and S-homovaline esters. In addition, the engineered polypeptides of the present invention have been engineered to have increased thermal activity, allowing reactions at elevated temperatures in the presence of wet organic solvents.

[0174] Engineered transaminases with improved activity in wet organic solvents and improved activity at elevated temperatures in wet organic solvents can be used in industrial processes for preparing chiral amine compounds in optically active form.

[0175] Engineered polypeptides with transaminase activity

[0176] The present invention provides engineered transaminase polypeptides and compositions thereof, and polynucleotides encoding engineered transaminase polypeptides. Also provided are methods for producing transaminases. In some embodiments, the engineered transaminase polypeptides are optimized to provide improved enzyme properties in wet organic solvents under minimal water-containing conditions.

[0177] The engineered polypeptides of the present disclosure are non-naturally occurring transaminases that are engineered to have improved enzymatic properties (such as increased stereoselectivity) compared to the wild-type transaminase polypeptide of Arthrobacter sp. KNK168 (GenBank Accession No. BAK39753.1, GI:336088341), and also compared to the reference engineered transaminase polypeptide of SEQ ID NO:2, which is used as a starting backbone sequence for the directed evolution of the engineered polypeptides of the present disclosure. The reference engineered aminotransferase polypeptide of SEQ ID NO:2 has the following 30 amino acid differences relative to the wild-type Arthrobacter sp. KNK168 polypeptide sequence: S8P, E42G, S54P, Y60F, L61Y, H62T, V65A, V69T, D81G, M94I, I96L, F122M, S124T, S126T, G136F, Y150F, V152S, Q155L, W156Q, A169L, V199I, A209L, G217N, S223P, L269P, L273Y, T282S, A284G, P297S, and S321P.

[0178] The engineered aminotransferase polypeptides of the present disclosure are generated by directed evolution of SEQ ID NO: 2, are used to efficiently convert compound (2) to compound (1) under certain industrially relevant conditions, and have one or more residue differences compared to the reference engineered aminotransferase polypeptide of SEQ ID NO: 2. These residue differences are associated with improvements in various enzyme properties under minimally aqueous conditions in wet organic solvents, particularly increased activity, increased stereoselectivity, increased stability, and tolerance to increased substrate and / or product concentrations (e.g., reduced product inhibition). Accordingly, in some embodiments, the engineered polypeptide having transaminase activity is capable of converting substrate compound (2) into compound (1) under suitable reaction conditions with an activity that is increased by at least about 1 to 2 times, 1.5 to 2.5 times, 2 to 3 times, 2.5 to 3.5 times, 3 to 4 times, 3.5 to 4.5 times, 4 to 5 times, 4.5 to 5 times, 5 to 10 times, 10 to 15 times or more relative to the activity of the reference polypeptide of SEQ ID NO: 2, 6, 332, 518 and / or 656. In some embodiments, the engineered polypeptide having transaminase activity is capable of converting a substrate of compound (2) into compound (1) at a conversion percentage of at least about 1%, at least about 2%, at least about 3%, at least about 10%, at least about 15%, or at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40% under suitable reaction conditions within a reaction time of about 72 h, about 48 h, about 36 h, about 24 h, about 20 h, or even less. In some embodiments, the engineered polypeptide having transaminase activity is capable of converting compound (2) into compound (1) at an enantiomeric excess of at least 90%, 95%, 97%, 98%, 99% or more under suitable reaction conditions.

[0179] Suitable reaction conditions for the above-described improved properties of engineered polypeptides can be determined about the concentration or amount of polypeptide, substrate, amino group donor, cofactor and / or the conditions comprising temperature and reaction time. In some embodiments, suitable reaction conditions include the minimum water-containing conditions in a wet organic solvent. In some embodiments, suitable reaction conditions include the HTP or SFP assay conditions described below and in the examples.

[0180] In some embodiments, additional reaction components or additional techniques are utilized to supplement reaction conditions. In some embodiments, these include taking steps to stabilize the enzyme or prevent enzyme inactivation, reduce product inhibition, or shift the reaction equilibrium to desired product formation.

[0181] In some other embodiments, any of the above-described methods for converting a substrate compound into a product compound may also include one or more steps selected from the following: extraction, separation, purification, crystallization, filtration and / or lyophilization of the product compound. Methods, techniques and protocols for extracting, separating, purifying and / or crystallizing products from a biocatalytic reaction mixture produced by the methods provided herein are known to those of ordinary skill and / or are obtainable by routine experimentation. In addition, illustrative methods are provided in the examples hereinafter.

[0182] The structural and functional information of exemplary non-naturally occurring engineered aminotransferase polypeptides of the present disclosure are shown in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7 and Table 8 above. The odd sequence identifier (i.e., SEQ ID NO) refers to the nucleotide (nt) sequence encoding the amino acid (aa) sequence provided by the even SEQ ID NO, and the sequence is provided in the electronic sequence table file attached to the present disclosure, which is incorporated herein by reference. The amino acid residue differences are based on comparisons with the reference polypeptide sequences of SEQ ID NO: 2, 6, 332, 518 and / or 656. The activity of each engineered polypeptide is determined using a high throughput (HTP) assay (as a primary screen), and in some cases, a second shake flask powder (SFP) assay is determined. Additional details of HTP and SFP preparations and assays are described in the embodiments.

[0183] As will be apparent to the skilled artisan, the foregoing residue positions and the specific amino acid residues at each residue position can be used alone or in various combinations to synthesize transaminase polypeptides having desired improved properties including, among others, enzyme activity, substrate / product preference, stereoselectivity, substrate / product tolerance, and stability under various conditions, such as minimal aqueous conditions in wet organic solvents.

[0184] Given the guidance provided herein, it is also contemplated that any of the exemplary engineered polypeptides having even-numbered sequence identifiers of SEQ ID NOs: 4-846 can be used as a starting amino acid sequence for the synthesis of other engineered transaminase polypeptides, for example by adding new combinations of various amino acid differences from other polypeptides in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, and Table 8 and subsequent rounds of evolution of other residue positions described herein. Additional improvements can be generated by including amino acid differences at positions that remain unchanged through earlier rounds of evolution.

[0185] In some embodiments, the disclosure also provides engineered transaminase polypeptides comprising fragments of any engineered transaminase polypeptide described herein, wherein the fragments of the engineered transaminase polypeptides retain the functional transaminase activity and / or improved properties of the engineered transaminase polypeptides. Accordingly, in some embodiments, the disclosure provides polypeptide fragments having transaminase activity (e.g., capable of converting compound (2) to compound (1) under suitable reaction conditions), wherein the fragment comprises at least about 80%, 90%, 95%, 98% or 99% of the full-length amino acid sequence of the engineered polypeptides of the disclosure, such as the exemplary engineered polypeptides having even-numbered sequence identifiers of SEQ ID NOs: 4-846.

[0186] In some embodiments, the engineered aminotransferase polypeptides of the present disclosure may have an amino acid sequence comprising a deletion compared to any of the engineered aminotransferase polypeptide sequences described herein, such as the exemplary engineered polypeptide sequences with even sequence identifiers of SEQ ID NOs: 4-846. Thus, for each and every embodiment of the engineered aminotransferase polypeptides of the present disclosure, the amino acid sequence may comprise a deletion of one or more amino acids, 2 or more amino acids, 3 or more amino acids, 4 or more amino acids, 5 or more amino acids, 6 or more amino acids, 8 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids of the aminotransferase polypeptide, up to 20% of the total number of amino acids of the aminotransferase polypeptide, or up to 30% of the total number of amino acids of the aminotransferase polypeptide, wherein the relevant functional activity and / or improved properties of the engineered aminotransferase described herein are maintained. In some embodiments, the deletion may include 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 amino acid residues. In some embodiments, the number of deletions can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, 50, 55, or 60 amino acid residues. In some embodiments, the deletion can include the deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, 25, or 30 amino acid residues.

[0187] In some embodiments, the disclosure provides engineered transaminase polypeptides having an amino acid sequence comprising an insertion compared to any of the engineered transaminase polypeptide sequences described herein, such as the exemplary engineered polypeptide sequences with even sequence identifiers of SEQ ID NO: 4-846. Thus, for each and every embodiment of the transaminase polypeptides of the disclosure, the insertion may include one or more amino acids, 2 or more amino acids, 3 or more amino acids, 4 or more amino acids, 5 or more amino acids, 6 or more amino acids, 8 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, wherein the relevant functional activity and / or improved properties of the engineered transaminase described herein are maintained. The insertion may be at the amino terminal or carboxyl terminal or middle portion of the transaminase polypeptide.

[0188] In some embodiments, the present disclosure provides an engineered polypeptide having transaminase activity, the engineered polypeptide comprising a polypeptide having SEQ ID The sequences of the even-numbered sequence identifiers of NO:4-846 have an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical, provided that the amino acid sequence is different from (i.e., it does not include) any of the exemplary engineered transaminase polypeptide amino acid sequences disclosed in: U.S. Patent No. 8,293,507B2, issued on October 23, 2012, PCT Publication WO2011005477A1, published on January 13, 2011, PCT Publication WO2012024104, published on February 23, 2012, and PCT Application No. PCT / US12 / 54300, filed on September 7, 2012, each of which is hereby incorporated by reference into this document.

[0189] In the above embodiments, suitable reaction conditions for engineered polypeptides are those described in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7 and Table 8. Thus, in some embodiments, the preparation of a compound of formula (I) or compound (1) can be performed, wherein suitable reaction conditions include: (a) a substrate loading of about 10 g / L to 200 g / L substrate compound (e.g., compound (2)); (b) about 0.5 g / L to 25 g / L engineered polypeptide; (c) an IPM concentration of about 0.1 M to 3 M; (d) a wet organic solvent; and (e) a temperature of about 30° C. to 60° C. In some embodiments, suitable reaction conditions include: (a) about 20 g / L substrate compound (e.g., compound (2)); (b) about 20 g / L engineered polypeptide; (c) wet IPAc; (d) about 0.55 M IPM; and (e) about 55° C. Guidance for the use of these reaction conditions and transaminase polypeptides is provided in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, and Table 8, among others, and in the Examples.

[0190] In some embodiments, the polypeptides of the present disclosure may be in the form of fusion polypeptides, wherein the engineered polypeptides are fused to other polypeptides, such as by, for example, but not limited to, antibody tags (e.g., myc epitopes), purification sequences (e.g., His tags for metal binding), and cell localization signals (e.g., secretion signals). Thus, the engineered polypeptides described herein may be used with or without fusion to other polypeptides.

[0191] The engineered transaminase polypeptides described herein are not limited to genetically encoded amino acids. Thus, in addition to genetically encoded amino acids, the polypeptides described herein may include, in whole or in part, naturally occurring and / or synthetic non-encoded amino acids. Certain common non-encoded amino acids that the polypeptides described herein may contain include, but are not limited to: D-stereoisomers of genetically encoded amino acids; 2,3-diaminopropionic acid (Dpr); α-aminoisobutyric acid (Aib); ε-aminohexanoic acid (Aha); δ-aminovaleric acid (Ava); N-methylglycine or sarcosine (MeGly or Sar); ornithine (Orn); citrulline (Cit); tert-butylalanine (Bua); tert-butylglycine (Bug); N-methylisoleucine (MeIle); phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (Nle); naphthylalanine (Nal); 2-chlorophenylalanine Acid (Ocf); 3-chlorophenylalanine (Mcf); 4-chlorophenylalanine (Pcf); 2-fluorophenylalanine (Off); 3-fluorophenylalanine (Mff); 4-fluorophenylalanine (Pff); 2-bromophenylalanine (Obf); 3-bromophenylalanine (Mbf); 4-bromophenylalanine (Pbf); 2-methylphenylalanine (Omf); 3-methylphenylalanine (Mmf); 4-methylphenylalanine (Pmf); 2-nitrophenylalanine (Onf); 3-nitrophenylalanine (Mnf); 4-nitrophenylalanine (Pnf); 2-cyanophenylalanine (Ocf); 3-cyanophenylalanine (Mnf); alanine (Mcf); 4-cyanophenylalanine (Pcf); 2-trifluoromethylphenylalanine (Otf); 3-trifluoromethylphenylalanine (Mtf); 4-trifluoromethylphenylalanine (Ptf); 4-aminophenylalanine (Paf); 4-iodophenylalanine (Pif); 4-aminomethylphenylalanine (Pamf); 2,4-dichlorophenylalanine (Opef); 3,4-dichlorophenylalanine (Mpcf); 2,4-difluorophenylalanine (Opff); 3,4-difluorophenylalanine (Mpff); pyridin-2-ylalanine (2pAla); pyridin-3-ylalanine (3pAla); pyridin- 4-aminophenylalanine (4pAla); 1-naphthylalanine (1nAla); 2-naphthylalanine (2nAla); thiazolylalanine (taAla); benzothienylalanine (bAla); thienylalanine (tAla); furanylalanine (fAla); homophenylalanine (hPhe); homotyrosine (hTyr); homotryptophan (hTrp); pentafluorophenylalanine (5ff); styrylkalanine (sAla); anthrylalanine (aAla); 3,3-diphenylalanine (Dfa); 3-amino-5-phenylpentanoic acid (Afp); penicillamine (Pen);1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic); β-2-thienylalanine (Thi); methionine sulfoxide (Mso); N(w)-nitroarginine (nArg); homolysine (hLys); phosphonomethylphenylalanine (pmPhe); phosphoserine (pSer); phosphothreonine (pThr); homoaspartic acid (hAsp); homoglutamic acid (hGlu); 1-aminocyclopent-(2 or 3)-ene-4-carboxylic acid; pipecolic acid (PA); azetidine-3-carboxylic acid (ACA); 1-aminocyclopentane-3-carboxylic acid ; Allylglycine (aOly); Propargylglycine (pgGly); Homoalanine (hAla); Norvaline (nVal); Homoleucine (hLeu), Homovaline (hVal); Homoisoleucine (hlle); Homoarginine (hArg); N-acetylleucine (AcLys); 2,4-diaminobutyric acid (Dbu); 2,3-diaminobutyric acid (Dab); N-methylvaline (MeVal); Homocysteine ​​(hCys); Homoserine (hSer); Hydroxyproline (Hyp) and Homoproline (hPro). Additional non-encoded amino acids that the polypeptides described herein may contain will be apparent to those skilled in the art (see, e.g., Fasman, 1989, CRC Practical Handbook of Biochemistry and Molecular Biology, CRC Press, Boca Raton, FL, various amino acids provided in pages 3-70 and references cited therein, all of which are incorporated by reference). These amino acids can be in L- or D-configuration. ;

[0192] Those skilled in the art will recognize that amino acids or residues with side chain protecting groups may also constitute the polypeptides described herein. Non-limiting examples of such protected amino acids, which in this case belong to the aromatic class, include, but are not limited to (protecting groups are listed in parentheses): Arg (tos), Cys (methylbenzyl), Cys (nitropyridinesulfinyl), Glu (δ-benzyl ester), Gln (xanthene), Asn (N-δ-xanthene), His (bom), His (benzyl), His (tos), Lys (fmoc), Lys (tos), Ser (O-benzyl), Thr (O-benzyl) and Tyr (O-benzyl).

[0193] Non-coded amino acids that can constitute conformational constraints of the polypeptides described herein include, but are not limited to, N-methyl amino acids (L-configuration); 1-aminocyclopent-(2 or 3)-ene-4-carboxylic acid; pipecolic acid; azetidine-3-carboxylic acid; homoproline (hPro) and 1-aminocyclopentane-3-carboxylic acid.

[0194] In some embodiments, the engineered polypeptide can be provided on a solid support such as a membrane, a resin, a solid carrier or other solid phase material. The solid support can include organic polymers such as polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy and polyacrylamide and their copolymers and grafts. The solid support can also be inorganic, such as glass, silica, controlled pore glass (CPG), reversed silica or metals such as gold or platinum. The configuration of the solid support can be in the form of beads, balls, particles, granules, gels, membranes or surfaces. The surface can be planar, substantially planar or non-planar. The solid support can be porous or non-porous and can have swelling or non-swelling properties. The solid support can be configured in the form of a hole, a depression or other container, a vessel, a feature or a position.

[0195] In some embodiments, the engineered polypeptide with transaminase activity is bound or fixed on a solid support so that the engineered polypeptide retains its improved activity, enantioselectivity, stereoselectivity and / or other improved properties relative to the reference polypeptide of SEQ ID NO:2. In such embodiments, the fixed polypeptide can facilitate the biocatalytic conversion of the substrate compound of formula (II) or compound (2) into the corresponding amine product compound of formula (I) or compound (1), and is easily retained after the reaction is completed (e.g., by retaining the beads on which the polypeptide is fixed) and then reused or recycled in subsequent reactions. Such fixed enzyme methods allow further efficiency improvements and cost reductions. Accordingly, it is also contemplated that any method using the engineered transaminase polypeptide of the present disclosure can be performed using the same transaminase polypeptide bound or fixed on a solid support.

[0196] Engineered transaminase polypeptides can be non-covalently or covalently bound. A variety of methods for conjugating and fixing enzymes to solid supports (e.g., resins, membranes, beads, glass, etc.) are well known in the art. In particular, PCT publication WO2012 / 177527A1 discloses an engineered transaminase polypeptide (including a reference polypeptide of SEQ ID NO: 2) capable of converting compound (2) into a fixed compound (1), and a method for preparing a fixed polypeptide, wherein the polypeptide is physically attached to the resin by hydrophobic interactions or covalent bonds, and is stable in a solvent system comprising at least up to 100% organic solvent. Various methods for conjugating and immobilizing enzymes to solid supports (e.g., resins, membranes, beads, glass, etc.) are well known in the art and are described, for example, in Yi et al., "Covalent immobilization of ω-transaminase from Vibriofluvialis JS17 on chitosan beads," Process Biochemistry 42(5):895-898 (May 2007); Martin et al., "Characterization of free and immobilized (S)-aminotransferase for acetophenone production," Applied Microbiology and Biotechnology 76(4):843-851 (Sept. 2007); Koszelewski et al., "Immobilization of ω-transaminases by encapsulation in a sol-gel / celite matrix," Journal of Molecular Catalysis B: Enzymatic, 63:39-44 (Apr. 2010); Truppo et al., "Development of an Improved Immobilized CAL-B for the Enzymatic Resolution of a KeyIntermediate to Odanacatib,” Organic Process Research & Development, published online: dx.doi.org / 10.1021 / op200157c; Hermanson, GT, Bioconjugate Techniques, Second Edition, Academic Press (2008); Mateo et al., "Epoxy sepabeads: a novel epoxy support for stabilization of industrial enzymes via very intense multipoint covalent attachment," Biotechnology Progress 18(3):629-34 (2002); and Bioconjugation Protocols: Strategies and Methods, In Methods in Molecular Biology, ed. C. M. Niemeyer, Humana Press (2004); the disclosures of each are incorporated herein by reference. .

[0197] Solid supports that can be used to fix the engineered aminotransferase polypeptides of the present disclosure include, but are not limited to, beads or resins comprising polymethacrylates with epoxide functional groups, polymethacrylates with aminoepoxide functional groups, styrene / DVB copolymers or polymethacrylates with octadecyl functional groups. Exemplary solid supports that can be used to fix the engineered aminotransferases of the present disclosure include, but are not limited to, chitosan beads, Eupergit C, and SEPABEAD (Mitsubishi), including the following different types of SEPABEAD: EC-EP, EC-HFA / S, EXA252, EXE119, and EXE120.

[0198] In some embodiments, engineered aminotransferase polypeptides can be provided in the form of arrays, in which polypeptides are arranged in different positions. In some embodiments, the different positions are holes in a solid support such as a 96-well plate. More than one support can be configured at multiple positions on the array, and the positions can be automatically delivered by reagents or addressed by detection methods and / or instruments. Such arrays can be used to test various substrate compounds for being converted by polypeptides.

[0199] In some embodiments, the engineered polypeptide described herein can be provided in the form of a kit. The polypeptide in the kit can exist alone or as more than one polypeptide. The kit can also include reagents for enzymatic reactions, substrates for evaluating the activity of the polypeptide, and reagents for detecting the product. The kit can also include instructions for use of a reagent dispenser and the kit. In some embodiments, the kit of the present disclosure includes an array containing more than one different engineered aminotransferase polypeptides at different addressable locations, wherein different polypeptides are different variants of a reference sequence, and each polypeptide has at least one different improved enzyme property. Such arrays comprising more than one engineered polypeptide and methods of use thereof are known (see, e.g., WO2009 / 008908A2).

[0200] Polynucleotides, control sequences, expression vectors and host cells useful for preparing engineered transaminase polypeptides

[0201] In another aspect, the disclosure provides polynucleotides encoding engineered polypeptides with transaminase activity described herein. The polynucleotides can be operably linked to one or more heterologous regulatory sequences that control gene expression to produce a recombinant polynucleotide capable of expressing a polypeptide. An expression construct comprising a heterologous polynucleotide encoding an engineered transaminase can be introduced into a suitable host cell to express the corresponding engineered transaminase polypeptide.

[0202] As will be apparent to the skilled person, the availability of protein sequences and the knowledge of codons corresponding to a variety of amino acids provide a description of all polynucleotides capable of encoding a target protein sequence. The degeneracy of the genetic codons in which the same amino acid is encoded by selectable or synonymous codons allows the preparation of a very large number of nucleic acids, all of which encode the improved transaminases disclosed herein. Therefore, having identified a specific amino acid sequence, one skilled in the art can prepare any number of different nucleic acids by simply modifying one or more codons of the sequence in a manner that does not change the amino acid sequence of the protein. In this regard, the present disclosure specifically contemplates each and every possible change of a polynucleotide that can be prepared by selecting a combination based on possible codon options, and for any polypeptide disclosed herein, including the amino acid sequences of the exemplary engineered polypeptides provided in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, and Table 8, and any polypeptide disclosed in the sequence table incorporated herein by reference as SEQ ID NO: 4-846 even sequence identifiers, all such changes are considered to be specifically disclosed.

[0203] In various embodiments, codons are preferably selected to adapt to the host cell in which the protein is produced. For example, preferred codons used in bacteria are used to express genes 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. In some embodiments, not all codons need to be replaced to optimize the codon usage of the transaminase, because the native sequence will include preferred codons and may not be required for all amino acid residues due to the use of preferred codons. Therefore, the codon-optimized polynucleotide encoding the transaminase can include preferred codons at about 40%, 50%, 60%, 70%, 80% or more than 90% of the codon positions in the full-length coding region.

[0204] In some embodiments, the polynucleotide encodes a transaminase polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a reference sequence of an even-numbered sequence identifier selected from SEQ ID NOs: 4-846, wherein the polypeptide has transaminase activity and one or more of the improved properties as described herein, such as the ability to convert compound (2) into product compound (1) with increased activity compared to the polypeptide of SEQ ID NO: 2. In some embodiments, the reference sequence is selected from SEQ ID NOs: 6, 332, 518 and 656.

[0205] In some embodiments, the polynucleotide encoding the engineered transaminase polypeptide comprises a sequence of an odd sequence identifier selected from SEQ ID NO: 3-845. In some embodiments, the polynucleotide sequence is selected from SEQ ID NO: 5, 331, 517 and 655.

[0206] In some embodiments, polynucleotide can be selected from any polynucleotide sequence provided herein or its complementary sequence or the polynucleotide sequence of any variant enzyme polypeptide provided herein and hybridize under high stringency conditions.In some embodiments, the polynucleotide encoding that can hybridize under high stringency conditions comprises the enzyme polypeptide that has the amino acid sequence of one or more residue differences compared with the reference sequence.

[0207] In some embodiments, the polynucleotide encodes a polypeptide described herein, but has about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity at the nucleotide level to a reference polynucleotide encoding an engineered transaminase polypeptide. In some embodiments, the reference polynucleotide sequence is selected from sequences having odd sequence identifiers of SEQ ID NOs: 3-845.

[0208] In some embodiments, the polynucleotide of any separation in the engineered enzyme polypeptide of encoding this paper is manipulated in various ways to promote the expression of enzyme polypeptide.In some embodiments, the polynucleotide of encoding enzyme polypeptide constitutes expression vector, wherein there is one or more control sequence to regulate the expression of enzyme polynucleotide and / or polypeptide.According to the expression vector used, the manipulation of the polynucleotide of separation before the polynucleotide of separation is inserted into the vector can be desired or necessary.The technology of utilizing recombinant DNA method to modify polynucleotide and nucleotide sequence is well known in the art.In some embodiments, control sequence comprises, among others, promoter, leader sequence, polyadenylation sequence, propeptide sequence, signal peptide sequence and transcription terminator.In some embodiments, suitable promoter is selected based on the selection of host cell. 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 Escherichia coli lac operon, the Streptomyces coelicolor agarase gene (dagA), the Bacillus subtilis levansucrase gene (sacB), the Bacillus licheniformis alpha-amylase gene (amyL), the Bacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), the Bacillus licheniformis penicillinase gene (penP), the Bacillus subtilis xylA and xylB genes, and prokaryotic β-lactamase genes (see, e.g., Villa-Kamaroff et al., Proc. Natl Acad. Sci. USA 75:3727-3731

[1978] ), and 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 alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (see, e.g., WO 99 / 054585). 96 / 00787), and the NA2-tpi promoter (a hybrid of the promoters from the Aspergillus niger neutral α-amylase gene and the Aspergillus oryzae triose phosphate isomerase gene), and mutants, truncated, and hybrid promoters thereof. Exemplary yeast cell promoters can be derived from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are known in the art (see, e.g., Romanos et al., Yeast 8:423-488

[1992] ).

[0209] In some embodiments, the control sequence is also a suitable transcription terminator sequence (i.e., a sequence recognized by the host cell to terminate transcription). In some embodiments, the terminator sequence is operably connected to the 3' end of the nucleic acid sequence encoding the enzyme polypeptide. Any suitable terminator that is functional in the selected host cell can be used in the present invention. Exemplary transcription terminators for filamentous fungal host cells can be obtained from the following genes: Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger α-glucosidase, and Fusarium oxysporum trypsin-like protease. Exemplary terminators for yeast host cells can be obtained from the following genes: Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are known in the art (see, for example, Romanos et al., above).

[0210] In some embodiments, the control sequence is also a suitable leader sequence (i.e., the non-translated region of the mRNA important for translation by the host cell). In some embodiments, the leader sequence is operably linked to the 5' end of the nucleic acid sequence encoding the enzyme polypeptide. Any suitable leader sequence that is functional in the selected host cell can be used in the present invention. Exemplary leaders for filamentous fungal host cells are obtained from the following genes: Aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase. Suitable leaders for yeast host cells are obtained from the following genes: Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0211] In some embodiments, the control sequence is also a polyadenylation sequence (i.e., a sequence operably linked to the 3' end of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to the transcribed mRNA). Any suitable polyadenylation sequence that is functional in the host cell of choice may be used in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to, the following genes: 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. Bio., 15:5983-5990

[1995] ). Exemplary mammalian polyadenylation sequences can be found in Zhang et al., 2005, Nucleic Acids Res. 33:D116-D120.

[0212] In some embodiments, the control sequence is also a signal peptide (i.e., a coding region encoding an amino acid sequence that is linked to the amino terminus of a polypeptide and directs the encoded polypeptide to the secretory pathway of the cell). In some embodiments, the 5' end of the coding sequence of the nucleic acid sequence inherently contains a signal peptide coding region that is naturally linked in translation reading frame to a segment of the coding region encoding 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 to the secretory pathway of the selected host cell can be used for expression of one or more engineered polypeptides. Effective signal peptide coding regions for bacterial host cells include, but are not limited to, those obtained from the following genes: Bacillus NClB 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM), and Bacillus subtilis prsA. Additional signal peptides are known in the art (see, e.g., Simonen and Palva, Microbiol. Rev., 57: 109-137

[1993] ). In some embodiments, effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to, those 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 α-factor and Saccharomyces cerevisiae invertase.

[0213] In some embodiments, the control sequence is also a propeptide coding region encoding an amino acid sequence positioned at the amino terminus of a polypeptide. The polypeptide produced is referred to as a "proenzyme", "propolypeptide" or "zymogen". The propolypeptide can be converted into a mature active polypeptide by catalyzing or autocatalyzing the cleavage of the propeptide from the propolypeptide. The propeptide coding region can be obtained from any suitable source including but not limited to the following genes: Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae α-factor, Rhizomucor miehei aspartic protease and Myceliophthora thermophila lactase (see, e.g., WO 95 / 33836). When both the signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is positioned adjacent to the amino terminus of the polypeptide and the signal peptide region is positioned adjacent to the amino terminus of the propeptide region. In some embodiments, regulatory sequences are also utilized. These sequences promote the regulation of polypeptide expression relative to host cell growth. Examples of regulatory systems are those that cause the expression of a 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, lac, tac, and trp operator systems. In yeast host cells, suitable regulatory systems include, but are not limited to, the ADH2 system or the GAL1 system. In filamentous fungi, suitable regulatory sequences include, but are not limited to, the TAKA alpha-amylase promoter, the Aspergillus niger glucoamylase promoter, and the Aspergillus oryzae glucoamylase promoter.

[0214] On the other hand, the present invention relates to a polynucleotide comprising an engineered enzyme polypeptide, and one or more expression regulatory regions such as promoters and terminators, replication origins, etc., according to the type of host to be introduced. In some embodiments, various nucleic acids and control sequences described herein are linked together to produce a recombinant expression vector comprising one or more convenient restriction sites to allow insertion or replacement 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 a suitable vector for expression. In some embodiments relating to the generation of an expression vector, the coding sequence is located in a vector such that the coding sequence is operably linked to a suitable control sequence for expression.

[0215] The recombinant expression vector can be any suitable vector (e.g., plasmid or virus) that can be easily subjected to recombinant DNA procedures and cause expression of the enzyme polynucleotide sequence. The choice of vector generally depends on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear plasmid or a closed circular plasmid.

[0216] In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extrachromosomal entity, whose replication is independent of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome). The vector may include any means for ensuring self-replication. In some selectable embodiments, the vector is a vector that, when introduced into a host cell, is integrated into the genome and replicated with one or more chromosomes into which it is integrated. In addition, in some embodiments, a single vector or plasmid is utilized, or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, and / or a transposon.

[0217] In some embodiments, the expression vector comprises one or more selectable markers that allow easy selection of transformed cells. A "selectable marker" is a gene whose product provides antimicrobial or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, etc. Examples of bacterial selectable markers include, but are not limited to, dal genes from Bacillus subtilis or Bacillus licheniformis, or markers that 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 carbamoyltransferase), 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. oryzae), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), and equivalents thereof.

[0218] The expression vector of the present disclosure may also include elements that allow the vector to be integrated into the host cell genome or that allow the vector to replicate autonomously in the cell independently of the genome. In order to be integrated into the host cell genome, the vector may rely on the nucleic acid sequence encoding the polypeptide or any other element to enable the vector to be integrated into the genome by homologous recombination or non-homologous recombination.

[0219] Alternatively, expression vector can include other nucleic acid sequences for guiding integration into the genome of host cell by homologous recombination. Other nucleic acid sequences enable carrier to be integrated into host cell genome at the precise position in chromosome. In order to increase the possibility of integration at the precise position, integration element should preferably include sufficient number of nucleic acids, such as 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000 base pairs, which are highly homologous to the corresponding target sequence, to improve the possibility of homologous recombination. Integration element can be any sequence homologous to the target sequence in the genome of host cell. In addition, integration element can be non-coding or encoding nucleic acid sequence. On the other hand, carrier can be integrated into the genome of host cell by non-homologous recombination.

[0220] For autonomous replication, the vector can also include a replication origin so that the vector can replicate autonomously in the host cell in question. Examples of bacterial replication origins are the replication origins of P15A ori or plasmids pBR322, pUC19, pACYCl77 (the plasmid has P15A ori) or pACYC184 that allow replication in Escherichia coli, and the replication origins of pUB110, pE194, pTA1060 or pAMβ1 that allow replication in Bacillus. Examples of replication origins for use in yeast host cells are 2 μm replication origins, ARS1, ARS4, a combination of ARS1 and CEN3, and a combination of ARS4 and CEN6. The replication origin can be a replication origin with a mutation that allows a function in a temperature-sensitive manner in a host cell (see, e.g., Ehrlich, 1978, Proc Natl Acad Sci. USA 75: 1433).

[0221] More than one copy of the nucleic acid sequence of the present disclosure can be inserted into a host cell to increase the production of a gene product. The increase in the number of copies 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 selection marker gene in the nucleic acid sequence, wherein cells containing amplified copies of the selection marker gene and thus additional copies of the nucleic acid sequence can be selected by culturing the cells in the presence of a suitable selection agent.

[0222] Many expression vectors for use with embodiments of the present disclosure are commercially available. Suitable commercial expression vectors include p3xFLAG™ from Sigma-Aldrich Chemicals. TMExpression vectors including a CMV promoter and hGH polyadenylation site for expression in mammalian host cells and a pBR322 origin of replication and an ampicillin resistance marker for amplification in E. coli. Other suitable expression vectors are commercially available pBluescriptII SK(-) and pBK-CMV from Stratagene, LaJolla CA, and plasmids derived from pBR322 (Gibco BRL), pUC (GibcoBRL), pREP4, pCEP4 (Invitrogen), or pPoly (Lathe et al., 1987, Gene 57:193-201).

[0223] An exemplary expression vector can be prepared by operably linking a polynucleotide encoding an improved aminotransferase to a plasmid pCK110900I comprising a lac promoter under the control of a lacI repressor. The expression vector also comprises a P15a origin of replication and a chloramphenicol resistance gene.

[0224] In another aspect, the present disclosure provides a host cell comprising a polynucleotide encoding an improved transaminase polypeptide of the present disclosure, the polynucleotide being operably linked to one or more control sequences for expressing a transaminase in a host cell. Host cells for expressing polypeptides encoded by expression vectors of the present disclosure are well known in the art, and include, but are not limited to, bacterial cells, such as Escherichia coli, Arthrobacter species KNK168, Streptomyces, and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC accession number 201178)); insect cells, such as Drosophila S2 and Spodoptera Sf9 cells; animal cells, such as CHO, COS, BHK, 293, and Bowes melanoma cells; and plant cells. An exemplary host cell is Escherichia coli W3110 (ΔfhuA). Suitable culture media and growth conditions for the host cells described above are well known in the art.

[0225] Polynucleotides for expressing transaminases can be introduced into cells by a variety of 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.

[0226] Methods for producing engineered transaminase polypeptides

[0227] In some embodiments, in order to prepare the improved engineered polynucleotides and engineered polypeptides of the present disclosure, the naturally occurring catalytic transaminase of the catalytic transamination reaction is obtained (or derived) from Arthrobacter species KNK168. In some embodiments, the parent polynucleotide sequence is codon optimized to improve the expression of the transaminase in a specific host cell. The parent polynucleotide sequence of the wild-type polypeptide encoding Arthrobacter species KNK168 has been described (see, for example, Iwasaki et al., Appl.Microbiol.Biotechnol., 2006, 69: 499-505). The preparation of engineered transaminase based on the parent sequence is also described in U.S. Patent Publication No. 2010 / 0285541A1 and the international application WO2010 / 099501 published.

[0228] Engineered transaminases can be produced using any suitable method known in the art. The gene encoding the wild-type polypeptide of interest can be cloned into a vector such as a plasmid and expressed in a desired host such as Escherichia coli. Variants of recombinant polypeptides can be produced by various methods known in the art. In fact, there are various different mutagenesis techniques well known to those skilled in the art. In addition, mutagenesis kits can also be obtained from many commercial molecular biology suppliers. The method can be used to make specific substitutions at a determined amino acid (site-directed), specificity (region-specificity) or random mutations in a local region of a gene, or random mutagenesis (e.g., saturation mutagenesis) in the entire gene. Many suitable methods for producing enzyme variants are known to those skilled in the art, including but not limited to, using PCR to single-stranded DNA or double-stranded DNA site-directed mutagenesis, cassette mutagenesis, gene synthesis, error-prone PCR, reorganization and chemical saturation mutagenesis, or any other suitable method known in the art. Mutagenesis and directed evolution methods can be easily applied to polynucleotides encoding enzymes to produce variant libraries that can be expressed, screened and measured. Any suitable mutagenesis and directed evolution methods can be used in the present invention and are well known in the art (see, e.g., U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, 5,837,458, 5,928,905, 6,096,548, 6,117,679, 6,132,970, 6,165,793, 6,180,406, 6,251,674, 、6,265,201、6,277,638、6,287,861、6,287,862、6,291,242、6,297,053、6,303,344、6,309,883、6,319,713、6,319,714、6,323,030、6,326,204、6,335,160、6,335,198、6,344,356、6,352,85 9, 6,355,484, 6,358,740, 6,358,742, 6,365,377, 6,365,408, 6,368,861, 6,372,497, 6,337,186, 6,376,246, 6,379,964, 6,387,702, 6,391,552, 6,391,640, 6,395,547, 6,406,855, 6,406,9 10. 6,413,745, 6,413,774, 6,420,175, 6,423,542, 6,426,224, 6,436,675, 6,444,468, 6,455,253, 6,479,652, 6,482,647, 6,483,011, 6,484,105, 6,489,146, 6,500,617, 6,500,639, 6,506,602、6,506,603、6,518,065、6,519,065、6,521,453、6,528,311、6,537,746、6,573,098、6,576,467、6,579,678、6,586,182、6,602,986、6,605,430 、6,613,514、6,653,072、6,686,515、6,703,240、6,716,631、6,825,001、6,902,922、6,917,882、6,946,296、6,961,664、6,995,017、7,024,312、7,0 58,515、7,105,297、7,148,054、7,220,566、7,288,375、7,384,387、7,421,347、7,430,477、7,462,469、7,534,564、7,620,500、7,620,502、7,629, 170、7,702,464、7,747,391、7,747,393、7,751,986、7,776,598、7,783,428、7,795,030、7,853,410、7,868,138、7,783,428、7,873,477、7,873,499、 Nos. 7,904,249, 7,957,912, 7,981,614, 8,014,961, 8,029,988, 8,048,674, 8,058,001, 8,076,138, 8,108,150, 8,170,806, 8,224,580, 8,377,681, 8,383,346, 8,457,903, 8,504,498, 8,589,085, 8,762,066, 8,768,871, 9,593,326, and all related U.S. and PCT and non-U.S. counterpart applications; Ling et al., Anal. Biochem., 254(2):157- 78

[1997] ; Dale et al., Meth. Mol. Biol., 57:369-74

[1996] ; Smith, Ann. Rev. Genet., 19:423-462

[1985] ; Botstein et al., Science, 229:1193-1201

[1985] ; Carter, Biochem. J., 237:1-7

[1986] ; Kramer et al., Cell, 38:879-887

[1984] ; Wells et al., Gene, 34:315-323

[1985] ; Minshull et al., Curr. Op. Chem. Biol.,3:284-290

[1999] ; Christians et al., Nat. Biotechnol., 17:259-264

[1999] ; Crameri et al., Nature, 391:288-291

[1998] ; Crameri, et al., Nat. Biotechnol., 15:436-438

[1997] ; Zhang et al., Proc. Nat. Acad. Sci. USA, 94:4504-4509

[1997] ; Crameri et al., Nat. Biotechnol., 14:315-319

[1996] ; Stemmer, Nature, 370:389-391

[1994] ; Stemmer, Proc. Nat. Acad. Sci. USA, 91:10747-10751

[1994] ; WO 95 / 22625; WO 97 / 0078; WO 97 / 35966; WO 98 / 27230; WO 00 / 42651; WO 01 / 75767; and WO 2009 / 152336, all of which are incorporated herein by reference).

[0229] The clones obtained after the mutagenesis treatment can be screened for engineered transaminases with the desired improved enzymatic properties.Measuring enzyme activity from expression libraries can be performed using standard biochemical techniques such as HPLC analysis after OPA derivatization of the product amines.

[0230] When the desired improved enzyme property is thermostability, the enzyme activity can be measured after subjecting the enzyme preparation to a defined temperature and measuring the amount of enzyme activity remaining after the heat treatment. A clone comprising a polynucleotide encoding the transaminase is then isolated, sequenced to determine changes in the nucleotide sequence (if any), and used to express the enzyme in a host cell.

[0231] When the sequence of the engineered polypeptide is known, the polynucleotide encoding the enzyme can be prepared by standard solid phase methods according to known synthetic methods. In some embodiments, up to about 100 base fragments can be synthesized individually, then connected (for example, by enzymatic or chemical ligation method or polymerase-mediated method) to form any desired continuous sequence. For example, the polynucleotides and oligonucleotides of the present disclosure can be prepared by chemical synthesis, using, for example, by the people such as Beaucage, 1981, the classical phosphoramidite method described by Tet Lett 22:1859-69, or by the people such as Matthes, 1984, the method described by EMBO J.3:801-05, for example, as typically practiced in automated synthesis methods. According to the phosphoramidite method, oligonucleotides are synthesized, purified, annealed, connected and cloned into suitable vectors in, for example, an automatic DNA synthesizer. In addition, any nucleic acid can be obtained substantially from any one of various commercial sources.

[0232] In some embodiments, the disclosure also provides a method for preparing or manufacturing an engineered aminotransferase polypeptide, wherein the method includes culturing a host cell capable of expressing a polynucleotide encoding an engineered polypeptide under culture conditions suitable for the expression of the polypeptide. In some embodiments, the method for preparing a polypeptide also includes separating the polypeptide. The engineered polypeptide can be expressed in a suitable cell (as described above) and separated (or recovered) from the host cell and / or culture medium using any one or more of the techniques known for protein purification, including, among others, lysozyme treatment, sonication, filtration, salting out, ultracentrifugation and chromatography. Chromatographic techniques for separating polypeptides include, among others, reverse phase chromatography, high performance liquid chromatography, ion exchange chromatography, gel electrophoresis and affinity chromatography. The conditions for purifying a specific engineered polypeptide will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, and will be apparent to those skilled in the art. The separated polypeptide must be lyophilized after purification.

[0233] Methods of using engineered transaminases and compounds prepared using engineered transaminases

[0234] In another aspect, the engineered aminotransferase polypeptides disclosed herein can be used in a method for converting a substrate compound (2) or a structural analog thereof into a product of compound (1) or a corresponding structural analog. Generally, a structural analog of compound (1) is included in structural formula (I).

[0235] In some embodiments, the present disclosure provides methods of preparing compounds of structural formula (I) in an enantiomeric excess of at least 70% over the relative enantiomer:

[0236]

[0237] The compounds of formula (I) have the indicated stereochemical configuration at the stereocenters marked with *, wherein

[0238] Z is a linear C1-6 hydrocarbon group or a branched C1-6 hydrocarbon group, optionally substituted with one or more hydroxyl groups, cyano groups or nitro groups; and

[0239] R1 is C1-6 hydrocarbyl, aryl-C1-2 hydrocarbyl, heteroaryl-C1-2 hydrocarbyl, or a 5- to 6-membered heterocyclic ring system optionally containing additional heteroatoms selected from O, S and N, the heterocyclic ring being unsubstituted or substituted with one to three substituents independently selected from oxo, hydroxyl, halogen, C1-4 hydrocarbyloxy and C1-4 hydrocarbyl; the method comprising reacting a prochiral ketoester of formula (II) in the presence of an amino group donor under suitable reaction conditions:

[0240]

[0241] A step of contacting with an engineered polypeptide as disclosed herein.

[0242] In some embodiments, the present disclosure provides a method for preparing compound (1):

[0243]

[0244] The method comprises reacting a substrate of compound (2) in the presence of an amino group donor under suitable reaction conditions.

[0245]

[0246] A step of contacting with an engineered polypeptide as disclosed herein.

[0247] In some embodiments of the method for preparing compounds of structural formula (I), the compound of formula (II) specifically excludes compound (2), and the compound of formula (I) prepared by the method specifically excludes compound (1).

[0248] As described herein and illustrated in the Examples, the present disclosure contemplates a range of suitable reaction conditions that can be used in the methods herein, including, but not limited to, ranges of temperature, wet organic solvent systems, substrate loadings, mixtures of substrate compound stereoisomers, polypeptide loadings, cofactor loadings, and reaction times. Additional suitable reaction conditions for performing the methods of biocatalytically converting substrate compounds into product compounds using the engineered aminotransferase polypeptides described herein can be readily optimized by routine experimentation, including, but not limited to, contacting the engineered aminotransferase polypeptide with the substrate compound under experimental reaction conditions of concentration, temperature, wet organic solvent conditions, and detecting the product compound, for example, using the methods described in the Examples provided herein.

[0249] As described above, the engineered polypeptides having transaminase activity used in the methods of the present disclosure generally comprise an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with a reference amino acid sequence selected from any of the even-numbered sequences of SEQ ID NOs: 4-846, and the engineered transaminase polypeptide comprises an amino acid sequence having one or more amino acid residue differences compared to SEQ ID NO: 2. In some embodiments, the polynucleotide capable of hybridizing under highly stringent conditions encodes a transaminase polypeptide having the percentages of identity described above and one or more residue differences compared to SEQ ID NO: 2.

[0250] In some embodiments, suitable reaction conditions include at least about 0.5g / L to about 200g / L, 1g / L to about 200g / L, 5g / L to about 150g / L, about 10g / L to about 100g / L or about 50g / L to about 100g / L substrate compound carrying capacity. In some embodiments, suitable reaction conditions include at least about 0.5g / L, at least about 1g / L, at least about 5g / L, at least about 10g / L, at least about 15g / L, at least about 20g / L, at least about 30g / L, at least about 50g / L, at least about 75g / L, at least about 100g / L, at least about 150g / L or at least about 200g / L or even higher substrate compound carrying capacity. Although the values ​​of substrate loading provided herein are based on the molecular weight of compound (2), it is also contemplated that various hydrates and salts of compound (2) in equal molar amounts may also be used in the method. In addition, the substrate compound encompassed by formula (II) may also be used in an appropriate amount depending on the amount of compound (2) used.

[0251] In the methods described herein, the engineered transaminase polypeptide uses an amino donor to form a product compound. In some embodiments, the amino donor in the reaction conditions includes a compound selected from IPM, putrescine, L-lysine, α-phenylethylamine, D-alanine, L-alanine, D, L-alanine, D, L-ornithine, 3-aminobutyric acid or methylbenzylamine. In some embodiments, the amino donor is selected from the group consisting of isopropylamine, alanine, 3-aminobutyric acid or methylbenzylamine. In some embodiments, the amino donor is IPM. In some embodiments, suitable reaction conditions include an amino donor present at a concentration of at least about 0.1M to about 3.0M, 0.2M to about 2.5M, about 0.5M to about 2M or about 1M to about 2M, particularly IPM. In some embodiments, the amino donor is present at a concentration of about 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 1 M, 1.5 M, 2 M, 2.5 M, or 3 M.

[0252] The suitable reaction conditions of the method also generally include the presence of a cofactor. Because the engineered aminotransferase generally uses a member of the vitamin B6 family, the reaction conditions may include a cofactor selected from: pyridoxal-5'-phosphate (also referred to as pyridoxal phosphate, PLP, P5P), pyridoxine (PN), pyridoxal (PL), pyridoxamine (PM), and their phosphorylated counterparts pyridoxine phosphate (PNP), and pyridoxamine phosphate (PMP). In some embodiments, suitable reaction conditions may include a cofactor selected from PLP, PN, PL, PM, PNP, and PMP to exist at a concentration of about 0.1 g / L to about 10 g / L, about 0.2 g / L to about 5 g / L, about 0.5 g / L to about 2.5 g / L. In some embodiments, the cofactor is PLP. Therefore, in some embodiments, suitable reaction conditions may include a cofactor PLP to exist at a concentration of about 0.1 g / L to about 10 g / L, about 0.2 g / L to about 5 g / L, about 0.5 g / L to about 2.5 g / L. In some embodiments, the reaction conditions include a PLP concentration of about 10 g / L or less, about 5 g / L or less, about 2.5 g / L or less, about 1.0 g / L or less, about 0.5 g / L or less, or about 0.2 g / L or less. In some embodiments, the cofactor is added to the cell lysate instead of the reaction mixture. In some embodiments, the cofactor is added to the cell lysate before lyophilization. In some embodiments, before lyophilization, the cofactor PLP is added to the cell lysate at a concentration of about 0.1 g / L to about 10 g / L, about 0.2 g / L to about 5 g / L, about 0.5 g / L to about 2.5 g / L.

[0253] In some embodiments of the method (e.g., wherein intact cells or lysates are used), the cofactor is naturally present in the cell extract and does not need to be supplemented. In some embodiments of the method (e.g., using partially purified or purified transaminases), the method can also include the step of adding the cofactor to the enzyme reaction mixture. In some embodiments, the cofactor is added at the beginning of the reaction and / or additional cofactors are added during the reaction.

[0254] In some embodiments, the reaction mixture comprises a wet organic solvent. Therefore, in some embodiments, the pH of the reaction mixture is not monitored or maintained. In some embodiments, the wet organic solvent is wet IPAc, wet toluene, wet MTBE, wet MeCN or wet IPA.

[0255] In the embodiments of the methods herein, suitable temperatures can be used for reaction conditions, for example, taking into account the increase in reaction rate at higher temperatures, the activity of the enzyme for a sufficiently long reaction duration, and the minimal water-containing conditions used in wet organic solvents as further described herein. For example, the engineered polypeptides of the present disclosure have increased thermal activity in wet organic solvents relative to naturally occurring transaminase polypeptides and the engineered polypeptides of SEQ ID NO: 2, which allows the engineered polypeptides of the present disclosure to be used at higher temperatures to increase conversion rates. Therefore, in some embodiments, suitable reaction conditions include temperatures of about 10°C to about 70°C, about 10°C to about 65°C, about 15°C to about 60°C, about 20°C to about 60°C, about 20°C to about 55°C, about 30°C to about 55°C, or about 40°C to about 50°C. In some embodiments, suitable reaction temperatures include temperatures of about 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C. In some embodiments, the temperature during the enzymatic reaction can be maintained at a certain temperature throughout the reaction process. In some embodiments, the temperature during the enzymatic reaction can be adjusted on a temperature curve during the reaction process.

[0256] In some embodiments of the methods, the method using the engineered aminotransferase polypeptide is carried out in a wet organic solvent without an aqueous component. In some embodiments of the methods, the wet organic solvent is prepared by mixing 200 mL of water with 800 mL of anhydrous wet organic solvent above, then mixing overnight with a magnetic stir bar, and allowing to separate before removing from the organic layer.

[0257] Suitable reaction conditions can include a combination of reaction parameters that provide for the biocatalytic conversion of a substrate compound into its corresponding product compound. Thus, in some embodiments of the method, the combination of reaction parameters includes: (a) a substrate loading of about 10 g / L to 200 g / L of the substrate compound (e.g., compound (2)); (b) about 0.5 g / L to 25 g / L of the engineered polypeptide; (c) an IPM concentration of about 0.1 M to 3 M; (d) a wet organic solvent; and (e) a temperature of about 30° C. to 60° C.

[0258] In some embodiments, the combination of reaction parameters includes: (a) about 20 g / L of substrate compound (e.g., compound (2)); (b) about 20 g / L of engineered polypeptide; (c) wet IPAc; (d) about 0.55 M IPM; and (e) about 50°C.

[0259] Additional exemplary reaction conditions include the assay conditions provided in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, and Table 8, and in the Examples.

[0260] When carrying out transamination reaction described herein, engineered transaminase polypeptide can be added to reaction mixture in the form of partially purified or purified enzyme, intact cell transformed with the gene encoding the enzyme, and / or cell extract and / or lysate of such cell.Intact cell transformed with the gene encoding engineered transaminase, or its cell extract, its lysate, and separated enzyme can be used in a variety of different forms, and different forms include solid (for example, freeze-dried, spray-dried, etc.) or semisolid (for example, thick paste).Cell extract or cell lysate can be partially purified by precipitation (for example, ammonium sulfate, polyethyleneimine, heat treatment or similar treatment), and then desalting procedure (for example, ultrafiltration, dialysis and similar procedures) is carried out before freeze-drying.Any enzyme product can be stabilized by using known cross-linking agent, such as for example glutaraldehyde cross-linked or fixed to solid phase material (for example, resin, pearl such as chitosan, Eupergit C, SEPABEAD, etc.).

[0261] In some embodiments of transamination described herein, the reaction is carried out under suitable reaction conditions described herein, wherein the engineered transaminase polypeptide is fixed to a solid support. The solid support that can be used for fixing the engineered transaminase for transamination reaction includes but is not limited to pearls or resins, and the pearls or resins include polymethacrylates with epoxy functional groups, polymethacrylates with aminoepoxide functional groups, styrene / DVB copolymers or polymethacrylates with octadecyl functional groups. Exemplary solid supports include but are not limited to chitosan beads, Eupergit C and SEPABEAD (Mitsubishi), and the SEPABEAD includes the following different types of SEPABEAD: EC-EP, EC-HFA / S, EXA252, EXE119 and EXE120.

[0262] In some embodiments, solid reactants (e.g., enzymes, salts, etc.) can be provided to the reaction in a variety of different forms, including powders (e.g., freeze-dried, spray-dried, etc.). Reactants can be readily freeze-dried or spray-dried using methods and equipment known to those of ordinary skill in the art. For example, a protein solution can be frozen at -80°C in small aliquots and then added to a pre-cooled freeze-drying chamber, followed by application of a vacuum.

[0263] In some embodiments, it is also contemplated that the method comprising the biocatalytic conversion of an amine acceptor substrate compound into a chiral amine product compound using a transaminase polypeptide of the present disclosure may also include steps of forming a pharmaceutically acceptable salt or acid, a pharmaceutically acceptable formulation, examining the product, extracting, isolating, purifying and / or crystallizing, each of which may be performed under a range of conditions.

[0264] In some embodiments, a method of using an engineered polypeptide disclosed herein can be performed, wherein the method further comprises the step of isolating the compound of formula (I) or compound (1) from the reaction.

[0265] In some embodiments, the methods of using the engineered polypeptides disclosed herein can be performed, wherein the amino group donor is selected from IPM, alanine, 3-aminobutyric acid, or methylbenzylamine. In some embodiments, the amino group donor is IPM.

[0266] Methods, techniques and protocols for extracting, isolating, forming salts thereof, purifying and / or crystallizing amination product compounds or cyclized compounds from the biocatalytic reaction mixture produced by the methods disclosed above are known to those of ordinary skill and / or can be obtained by routine experimentation. In addition, illustrative methods are provided in the following examples.

[0267] Various features and embodiments of the present disclosure are illustrated in the following representative examples, which are intended to be illustrative rather than limiting. Example

[0268] Example 1: Escherichia coli expression host containing recombinant transaminase gene

[0269] The codon optimized and engineered transaminase gene (SEQ ID NO: 1) encoding the reference engineered polypeptide of SEQ ID NO: 2 was used as a starting backbone for directed evolution to generate genes encoding engineered polypeptides with transaminase activity of even-numbered sequence identifiers of SEQ ID NO: 4-846, each of which is capable of converting a substrate compound (2) into a product compound (1) with improved enzymatic properties relative to it and / or the reference polypeptide of SEQ ID NO: 2. The gene of SEQ ID NO: 1 and the polypeptide of SEQ ID NO: 2 of the present disclosure correspond to SEQ ID NO: 129 and 130 of US 9,617,573 B2. The engineered aminotransferase polypeptide of SEQ ID NO:2 has the following 30 amino acid differences relative to the wild-type Arthrobacter sp. KNK168 polypeptide sequence (GenBank Accession Nos.: BAK39753.1; GI:336088341): S8P, E42G, S54P, Y60F, L61Y, H62T, V65A, V69T, D81G, M94I, I96L, F122M, S124T, S126T, G136F, Y150F, V152S, Q155L, W156Q, A169L, V199I, A209L, G217N, S223P, L269P, L273Y, T282S, A284G, P297S, and S321P. The cloning of SEQ ID NO: 1 in the pCK110900 vector system (see, e.g., U.S. Patent Application Publication No. 2006 / 0195947A1) and the subsequent expression in E. coli W3110fhuA was as described in US 8,293,507 B2. Briefly, E. coli W3110 expresses the transaminase polypeptide as an intracellular protein under the control of the lac promoter. The polypeptide accumulates primarily as a soluble, cytoplasmic active enzyme. Standard methods of directed evolution of iterative variant libraries by gene synthesis, followed by screening and sequencing of hits were used to generate engineered derivatives of the gene sequence SEQ ID NO: 1 disclosed herein.

[0270] Example 2: Preparation of wet cell pellet containing HTP aminotransferase

[0271] The E. coli cells containing the recombinant transaminase encoding gene from the monoclonal colony are inoculated into 180 μl Luria broth (LB) containing 1% glucose and 30 μg / mL chloramphenicol (CAM) in the wells of 96-well shallow well microtiter plates. The plate is sealed with an O2 permeable seal and the culture is grown overnight at 30°C, 200rpm and 85% humidity. Then, 10 μl of each cell culture is transferred to the wells of a 96-well deep-well plate containing 390 μl Terrific broth (TB) and 30 μg / ml CAM. The deep-well plate is sealed with an O2 permeable seal and incubated at 30°C, 250rpm and 85% humidity until an OD of 0. 600 0.6-0.8. The cell culture was then induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM and incubated overnight under the same conditions used initially. The plate was centrifuged at 4,000 rpm for 10 minutes, the supernatant discarded, and the pellet frozen at -80°C before lysis, as described in Example 3. Alternatively, cells from two identical plates were pooled using wide-bore pipette tips and pelleted using centrifugation at 4000 rpm for 10 minutes. The supernatant discarded and the double pellets frozen at -80°C before lysis, as described in Example 5.

[0272] Example 3: Preparation of cell lysate containing HTP aminotransferase

[0273] First, 200 μl of lysis buffer containing 100 mM triethanolamine buffer pH 7.5, 0.25 mg / mL lysozyme, 0.2 mg / mL polymyxin b sulfate (PMBS) and 100 uM pyridoxal-5-phosphate (PLP) was added to the cell paste in each well produced as described in Example 2. Cells were lysed at room temperature for 2 hours with shaking on a bench top shaker. The plate was then centrifuged at 4,000 rpm and 4° C. for 10 minutes. The clarified supernatant was then used for biocatalytic reactions to determine their stereoselectivity as described in Example 4.

[0274] Example 4: Improved stereoselectivity relative to SEQ ID NO: 2

[0275] Select SEQ ID NO:2 as the parent enzyme. Use well-established techniques (e.g., saturation mutagenesis, and recombination of previously identified beneficial mutations) to generate a library of engineered genes. The polypeptides encoded by each gene were produced with HTP as described in Example 2, and the soluble lysate was produced as described in Example 3. Each reaction well contained 80 μL of HTP lysate prepared as explained in Example 3 and the following final conditions of 200 uL volume per well: 20 g / L substrate compound (2), DMSO 40 vol%, 0.2 M borate, and 1.5 M IPM 50 vol% and 1 g / L PLP; pH was adjusted to pH = 8.5. The plate was heat sealed and shaken at 60 ° C overnight (18-22 hours). The reaction was quenched by adding 200 uL acetonitrile per well. The plate was heat sealed and shaken at 1,000 rpm for 15 minutes, and then centrifuged at 4,000 rpm for 10 minutes. The clear supernatant of 200 uL per well was transferred to a filter plate. The filter plate was placed on top of a 96-well round bottom Costar plate and centrifuged at 4,000 rpm for 5 minutes. The 96-well round bottom Costar plate was heat sealed and analyzed using chiral LC-MS (Method 3, Example 10).

[0276] Stereoselectivity was calculated as % enantiomeric excess (%ee): desired (S) product peak area−undesired (R) product peak area / desired (S) product peak area+undesired (R) product peak area*100.

[0277] Table 1: HTP stereoselectivity

[0278]

[0279] Example 5: Preparation of Lyophilized HTP Lysate from Wet Cell Lysate Containing Transaminases

[0280] HTP Lysate and Lyophilization of HTP Lysate: The wet cell pellet containing the aminotransferase (generated as a double pellet as described in Example 2) was subjected to HTP lysis and the resulting HTP lysates were lyophilized using three conditions: A, B, and C.

[0281] Condition A:First, 300 μl lysis buffer containing 0.25 mg / mL (1 mg / mL for condition B and condition C) lysozyme; 0.2 mg / mL (0.5 mg / mL for condition B and condition C) PMBS; in 100 mM (25 mM for condition B and condition C) triethanolamine buffer pH 7.5; 10 μl of 2,000 U / mL endonuclease per plate was added to the double precipitated cell paste in each well produced as described in Example 2. With shaking on a bench top shaker, the cells were lysed at room temperature for 2 hours. The plate was then centrifuged at 4,000 rpm and 4°C for 20 minutes. 220 μl (150 μl for condition C) of the clarified lysate was transferred to a 96-well glass-coated deep-well plate. In preliminary experiments, glass-coated deep-well and polypropylene plates warped under certain reaction conditions. Therefore, conditions B and C require glass plates or glass vials (Freeslate / Chemglass glass vial plates). The plates are covered with air-hole seals and placed at -80°C with the 96-well samples in direct contact with the freezing rack for 2 hours. The plates are placed in a freeze dryer. The freeze dryer vacuum is reduced to 200 bar and the following program is run: precooling at -40°C, heat treatment at -40°C for 120 minutes, 100mTorr; drying step: 10°C, 100mTorr, 1,000-1,200 minutes. The plates are removed from the freeze dryer and the assay is performed almost immediately. For conditions B and C: the plates are frozen in the freeze dryer at -40°C for ~2 hrs, with the frost seal set at ~600-700mTorr. The freeze dryer vacuum was brought to 200 mTorr, and the following program was run: precooling: -40°C; heat treatment: -40°C, 100 min, 100 mTorr; drying steps: 1. -40°C to -15°C (1°C / min), 100 mTorr; 2. -15°C, 600 min, 100 mTorr; 3. -15°C to 0°C (1°C / min), 100 mTorr; 0°C, 900 min, 100 mTorr (the method was completed after overnight and stopped after 1,000-1,200 min in step 4).

[0282] Example 6: Preparation of lyophilized lysate from shake flask (SF) cultures

[0283] Selected HTP cultures grown as described above were plated onto LB agar plates with 1% glucose and 30 μg / ml CAM and grown overnight at 37° C. A single colony from each culture was transferred to 6 ml LB with 1% glucose and 30 μg / ml CAM. The cultures were grown at 30° C., 250 rpm for 18 hours and subcultured approximately 1:50 into 250 ml TB containing 30 μg / ml CAM to a final OD of 0.05. 600Grow the culture at 30°C, 250 rpm for approximately 195 minutes to an OD between 0.6-0.8. 600 , and induced with 1 mM IPTG. The culture was then grown at 30 ° C, 250 rpm for 20 hours. The culture was centrifuged at 4000 rpm for 20 minutes. The supernatant was discarded and the precipitate was resuspended in 30 ml of 25 mM triethanolamine pH 7.5 and cracked at 18,000 psi using a Microfluidizer system (Microfluidics). The lysate was precipitated (10,000 rpm for 60 minutes) and the supernatant was frozen and lyophilized to produce a shake flask (SF) enzyme.

[0284] Example 7: HTP Screening for Improved Activity

[0285] HTP screening, conversion assay: Wet IPAc was prepared by mixing 200mL of water with 800mL of anhydrous IPAc above, which was mixed overnight with a magnetic stir bar and allowed to separate before being removed from the organic layer. The ester substrate was premixed with 4 equivalents of IPM relative to the substrate concentration in wet IPAc. The final reaction parameters in the conversion HTP assay were: 20g / L ester substrate (2), 4 equivalents of IPM and HTP freeze-dried blocks, as prepared as explained in Conditions AC in Example 5. First, 200μl / well of 20g / L ester substrate and 4 equivalents of IPM in wet IPAc were added to a 96-well glass-coated plate. The glass-coated plate was heat-sealed and incubated at 30°C, 400rpm for 24 hours. The reaction in the 96-well glass-coated plate was quenched by adding 1000μl / well of anhydrous IPAc. The plate was heat-sealed, shaken for 1 minute, and then centrifuged at 4°C to 4,000rpm for 5 minutes. The 6-fold IPAc diluted samples were transferred to a 96-well Costar round-bottom plate for GC-FID analysis. Alternatively, 50 μl of the 6-fold IPAc diluted samples were transferred to 950 μl of water in a deep-well plate. Then, 5 μl of the 20-fold water-diluted IPAc to quench the reaction was added to 195 μl of water in a 96-well Costar round-bottom plate for RapidFire MS analysis.

[0286]

[0287] Table 2: HTP activity in organic solvents of the HTP lyophilized powder produced in Condition A of Example 5

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295] 1 Activity fold improvement Calculated as the ratio of product compound (1) produced by the specified engineered polypeptide under the activity assay reaction conditions (set forth in Example 7) / product produced by the engineered polypeptide of SEQ ID NO: 6 under the same reaction conditions. The amount of product produced is determined by GC-FID or RapidFire-MS analysis of quenched assay samples prepared as set forth in Example 10.

[0296] Table 3: HTP activity in organic solvents of HTP lyophilized powder produced in Condition A of Example 5

[0297]

[0298]

[0299]

[0300] 1 Activity fold improvement Calculated as the ratio of product compound (1) produced by the specified engineered polypeptide under the activity assay reaction conditions (set forth in Example 7) / product produced by the engineered polypeptide of SEQ ID NO: 332 under the same reaction conditions. The amount of product produced is determined by GC-FID or RapidFire-MS analysis of quenched assay samples prepared as set forth in Example 10.

[0301] Table 4: HTP activity in organic solvents of the HTP lyophilized powder produced in Condition B of Example 5

[0302]

[0303]

[0304] 1 Activity fold improvementCalculated as the ratio of product compound (1) produced by the specified engineered polypeptide under the activity assay reaction conditions (set forth in Example 7) / product produced by the engineered polypeptide of SEQ ID NO: 518 under the same reaction conditions. The amount of product produced is determined by GC-FID or RapidFire-MS analysis of quenched assay samples prepared as set forth in Example 10.

[0305] Example 8: HTP Screening for Improved Thermal Activity

[0306] HTP screening, thermal activity assay: Wet IPAc is prepared by mixing 200mL water with 800mL anhydrous IPAc above, which is mixed overnight with a magnetic stirring bar and allowed to separate before being removed from the organic layer. The ester substrate is premixed in wet IPAc with 4 equivalents of IPM relative to the substrate concentration. The final reaction parameters in the conversion HTP assay are: 20g / L ester substrate (2), 4 equivalents of IPM and HTP freeze-dried blocks, as prepared as explained in Conditions AC in Example 5. First, 200μl / well of 20g / L ester substrate and 4 equivalents of IPM in wet IPAc are added to the Freeslate glass vial plate. The Freeslate glass vial plate is evenly tightened with screws and incubated at 50°C at 400rpm for 24 hours. The reaction in the Freeslate glass vial plate is quenched by adding 400μl / well of anhydrous IPAc. Next, 100 μl / well from the Freeslate 96 glass vial plate was transferred to a 96-well Costar plate containing 100 μl / well of anhydrous IPAc and submitted for GC-FID analysis.

[0307] Table 5: HTP thermal activity in organic solvents of HTP lyophilized powder produced in Condition A of Example 5

[0308]

[0309]

[0310] 1 Activity fold improvement Calculated as the ratio of product compound (1) produced by the specified engineered polypeptide under the activity assay reaction conditions (set forth in Example 8) / product produced by the engineered polypeptide of SEQ ID NO: 332 under the same reaction conditions. The amount of product produced was determined by GC-FID analysis of quenched assay samples prepared as set forth in Example 10.

[0311] Table 6: HTP thermal activity in organic solvents of HTP lyophilized powder produced in Condition B of Example 5

[0312]

[0313]

[0314] 1 Activity fold improvement Calculated as the ratio of product compound (1) produced by the specified engineered polypeptide under the activity assay reaction conditions (set forth in Example 8) / product produced by the engineered polypeptide of SEQ ID NO: 518 under the same reaction conditions. The amount of product produced was determined by GC-FID analysis of quenched assay samples prepared as set forth in Example 10.

[0315] Table 7: HTP thermal activity in organic solvents of HTP lyophilized powder produced in Condition C of Example 5

[0316]

[0317]

[0318] 1 Activity fold improvement Calculated as the ratio of product compound (1) produced by the specified engineered polypeptide under the activity assay reaction conditions (set forth in Example 8) / product produced by the engineered polypeptide of SEQ ID NO: 656 under the same reaction conditions. The amount of product produced was determined by GC-FID analysis of quenched assay samples prepared as set forth in Example 10.

[0319] Example 9: Shake bottle powder test

[0320] Wet IPAc (or MTBE or toluene) is prepared by mixing 200mL water with 800mL anhydrous IPAc above, which is mixed overnight with a magnetic stirring bar and allowed to separate before being taken out from the organic layer. The ester substrate is pre-mixed with 4 equivalents of IPM relative to substrate concentration in wet IPAc (or MTBE or toluene). The final reaction parameters are: 20g / L ester substrate, 4 equivalents of IPM, 20mg of freeze-dried shake flask powder prepared as explained in Example 6. Next, 20mg of freeze-dried shake flask powder is weighed into 4mL glass vials. Then, 1mL of reaction mixture is added to the vial, and the vial is sealed with a Teflon seal. The vial is incubated at 50°C for 24 hours. The reaction is quenched by adding 2mL of anhydrous IPAc. Then, 500μL of quenched reaction is diluted into 500μL of dry IPAc in a GC glass vial. The vial is submitted for GC-FID analysis.

[0321] Table 8: Thermal activity of shake flask powders produced as described in Example 6 in organic solvents

[0322]

[0323] 1 Activity-fold improvement in wet IPAc Calculated as the ratio of product compound (1) produced by the specified engineered polypeptide under the activity assay reaction conditions (set forth in Example 9) / product produced by the engineered polypeptide of SEQ ID NO: 6 under the same reaction conditions. The amount of product produced was determined by GC-FID analysis of quenched assay samples prepared as set forth in Example 10.

[0324] 2 Activity fold improvement in wet MTBE Calculated as the ratio of product compound (1) produced by the specified engineered polypeptide under the activity assay reaction conditions (set forth in Example 9) / product produced by the engineered polypeptide of SEQ ID NO: 6 under the same reaction conditions. The amount of product produced was determined by GC-FID analysis of quenched assay samples prepared as set forth in Example 10.

[0325] 3 Activity fold improvement in wet toluene Calculated as the ratio of product compound (1) produced by the specified engineered polypeptide under the activity assay reaction conditions (set forth in Example 9) / product produced by the engineered polypeptide of SEQ ID NO: 6 under the same reaction conditions. The amount of product produced was determined by GC-FID analysis of quenched assay samples prepared as set forth in Example 10.

[0326] Example 10: Analytical Methods

[0327] Method 1 - Achiral GC-FID Method

[0328] The quenched reactions prepared as explained in Examples 7, 8 and 9 were subjected to GC-FID analysis under the following conditions:

[0329]

[0330] Method 2—Achiral RapidFire-MS Method

[0331] The quenched reaction prepared as explained in Example 7 was subjected to RapidFire-MS analysis under the following conditions:

[0332]

[0333]

[0334] Method 3—Chiral LC-MS Method

[0335] The quenched reaction prepared as explained in Example 4 was subjected to chiral LC-MS analysis under the following conditions:

[0336]

[0337] The present invention also provides the following items:

[0338] 1. An engineered polypeptide having transaminase activity, the engineered polypeptide comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 2, 6, 332, 518 and / or 656, or a functional fragment thereof, wherein the polypeptide sequence of the engineered polypeptide comprises at least one substitution or set of substitutions, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 2, 6, 332, 518 and / or 656.

[0339] 2. An engineered polypeptide according to item 1, wherein the polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO:2, and wherein the polypeptide sequence of the engineered polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 20, 23, 244, 284 and 284 / 155, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2.

[0340] 3. The engineered polypeptide according to item 1, wherein the polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:6, and wherein the polypeptide sequence of the engineered polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 5 / 35, 9, 9 / 20 / 23 / 24 / 134 / 244, 9 / 20 / 23 / 101 / 244, 9 / 20 / 24 / 101 / 244 / 249, 9 / 23 / 24, 9 / 23 / 24 / 29 / 244, 9 / 23 / 24 / 101 / 262, 9 / 23 / 24 / 244 / 262、9 / 23 / 29、9 / 24 / 244、9 / 24 / 244 / 262、9 / 24 / 249、9 / 45 / 89 / 93、9 / 79 / 101 / 244、9 / 82 / 89、9 / 89 / 246、9 / 89 / 271、9 / 93 / 142 / 246、9 / 101 / 244、9 / 110 / 302、9 / 244 / 249、9 / 244 / 262 / 264、9 / 246、9 / 262、15、15 / 16 / 18 / 20 / 89 / 93 / 160、15 / 24 / 26 / 89 / 93 / 160、15 / 35 / 273、15 / 218 / 273、15 / 249 / 273、16、 17 / 25、17 / 25 / 41、17 / 25 / 160、17 / 160、18、18 / 26 / 246 / 273、18 / 146、18 / 146 / 168、18 / 146 / 262、18 / 168、18 / 168 / 244、18 / 168 / 262 / 324、18 / 244、18 / 244 / 246、18 / 244 / 246 / 284、18 / 244 / 248、18 / 244 / 262 / 273、18 / 244 / 273、18 / 262、18 / 273、20、20 / 23 / 24 / 55 / 134 / 244 / 262、20 / 23 / 134 / 244、20 / 24 20 / 33 / 160, 20 / 324, 22, 22 / 102, 22 / 160, 23, 23 / 25 / 55 / 321, 23 / 25 / 102 / 142, 23 / 102, 23 / 321, 24, 25, 25 / 33, 25 / 104, 25 / 160, 25 / 321, 26, 26 / 273, 29, 29 / 89, 32, 33, 35, 35 / 215 / 218 / 249, 35 / 218 / 249 / 273, 35 / 221, 40, 40 / 168 / 208 / 244, 40 / 244 / 248, 41, 41 / 102, 42, 45, 46, 50, 55, 56,71 / 102、74、79、82、82 / 244、89、89 / 93 / 156 / 324、89 / 93 / 321 / 324、90、93、93 / 324、95、98、101、101 / 160 / 244 / 249、102、104、106、111、114、117、119、134、134 / 244、134 / 262、139、140 / 266、142、142 / 321、145、146、146 / 244、146 / 273、156、160、160 / 215 / 216 / 218、160 / 273、168、168 / 244 / 273、 179, 186, 186 / 200, 197, 203, 207, 208, 208 / 246, 215, 215 / 216 / 249, 216, 216 / 273, 218, 218 / 249, 218 / 313, 234, 236, 244, 244 / 248, 244 / 248 / 284, 244 / 273, 246, 246 / 248 / 273, 248, 249, 249 / 273, 256, 262, 264, 265, 266, 271, 273, 284, 301, 316, 320, 321, 324 and 329, wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO: 6.

[0341] 4. The engineered polypeptide according to item 1, wherein the polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 332, and wherein the engineered polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 9 / 89 / 93 / 101 / 160, 9 / 89 / 160, 33 / ... 5, 44, 49, 74, 89, 89 / 93 / 160 / 249, 107, 108, 117, 140, 141, 142, 144, 156, 168, 203, 207, 234, 270, 273, 274, 292, 296, 297, 300, 302, 305, 317, 319, 321, 323, 327 and 329, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:332.

[0342] 5. An engineered polypeptide according to item 1, wherein the polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:518, and wherein the engineered polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 35 / 273, 46 / 273, 111 / 159 / 273, 159, 159 / 273, 174, 199, 273 and 287, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:518.

[0343] 6. The engineered polypeptide according to item 1, wherein the polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 332, wherein the engineered polypeptide has increased thermal activity in a wet organic solvent compared to the engineered polypeptide of SEQ ID NO: 332, and wherein the engineered polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 23 / 29 / 55, 34, 44, 74, 92, 108, 117, 140, 141, 144, 156, 160, 168, 215, 256, 269, 270, 274, 292, 296, 302, 319, 323 and 329, wherein the amino acid positions of the polypeptide sequence refer to SEQ ID NO:332 number.

[0344] 7. An engineered polypeptide according to claim 1, wherein the polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:518, wherein the engineered polypeptide has increased thermal activity in a wet organic solvent compared to the engineered polypeptide of SEQ ID NO:518, and wherein the engineered polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 34 / 74 / 92, 35 / 273 / 323, 39, 46 / 273, 48, 55, 74 / 92, 126, 133, 138, 150, 160 / 215 / 329, 178, 199, 207, 273 and 292, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:518.

[0345] 8. The engineered polypeptide according to item 1, wherein the polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 656, wherein the engineered polypeptide has increased thermal activity in a wet organic solvent compared to the engineered polypeptide of SEQ ID NO: 656, and wherein the engineered polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 18, 35 / 39 / 55 / 138 / 150, 35 / 39 / 55 / 150, 35 / 39 / 126 / 150 / 273, 35 / 39 / 133, 35 / 39 / 133 / 150 / 273, 35 / 39 / 150, 35 / 39 / 150 / 273, 35 / 133 / 178, 35 / 138 / 150 / 273, 35 / 150 / 273, 35 / 178 / 273, 35 / 273, 39, 39 / 55 / 150 / 178 / 273, 39 / 55 / 150 / 273, 39 / 126 / 150, 39 / 133 / 150, 39 / 133 / 178 / 273、39 / 133 / 273、39 / 150 / 178、39 / 150 / 178 / 207、39 / 150 / 178 / 207 / 273、39 / 150 / 273、39 / 178、39 / 178 / 273、39 / 207、39 / 207 / 273、39 / 273、44、55、55 / 150、88、126、126 / 133 / 178、126 / 138 / 178, 126 / 178 / 207, 132, 138 / 150 / 178, 141, 150, 150 / 178 / 273 / 323, 150 / 178 / 323, 150 / 207 / 273, 150 / 273, 168, 178 / 273, 239, 261, 263, 269, 273, 297 and 312, wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO:656.

[0346] 9. An engineered polypeptide according to any one of items 6-8, wherein the engineered polypeptide has increased thermal activity in a wet organic solvent relative to the engineered polypeptide of SEQ ID NO: 6.

[0347] 10. An engineered polypeptide according to item 1, wherein the engineered polypeptide comprises a polypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of at least one engineered polypeptide variant listed in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7 and / or Table 8.

[0348] 11. An engineered polypeptide according to claim 1, wherein the engineered polypeptide comprises a polypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 2, 6, 332, 518 and / or 656.

[0349] 12. The engineered polypeptide according to item 1, wherein the engineered polypeptide comprises a variant engineered polypeptide listed in SEQ ID NO: 6, 332, 518 and / or 656.

[0350] 13. An engineered polypeptide according to item 1, wherein the engineered polypeptide comprises a polypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of at least one engineered polypeptide variant listed in the even-numbered sequences in SEQ ID NO:4-846.

[0351] 14. The engineered polypeptide according to item 1, wherein the engineered polypeptide comprises a polypeptide sequence listed in at least one of the even-numbered sequences in SEQ ID NO: 4-846.

[0352] 15. A polynucleotide sequence encoding at least one engineered polypeptide according to any one of items 1-14.

[0353] 16. A polynucleotide sequence encoding at least one engineered polypeptide, the polynucleotide sequence comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, 5, 331, 517 and / or 655, wherein the polynucleotide sequence of the engineered polypeptide comprises at least one substitution at one or more positions.

[0354] 17. A polynucleotide sequence encoding at least one engineered polypeptide or a functional fragment thereof, wherein the polynucleotide sequence comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, 5, 331, 517 and / or 655.

[0355] 18. A polynucleotide sequence according to any one of items 15-17, wherein the polynucleotide sequence is operably linked to a control sequence.

[0356] 19. The polynucleotide sequence according to any one of items 15-17, wherein the polynucleotide sequence is codon-optimized.

[0357] 20. The polynucleotide sequence according to any one of items 15-17, wherein the polynucleotide sequence comprises a polynucleotide sequence listed in the odd-numbered sequence in SEQ ID NO: 3-845.

[0358] 21. An expression vector comprising at least one polynucleotide sequence according to any one of items 15 to 20.

[0359] 22. A host cell comprising at least one expression vector according to item 21.

[0360] 23. A host cell comprising at least one polynucleotide sequence according to any one of items 15-20.

[0361] 24. A method for producing an engineered polypeptide in a host cell, the method comprising culturing the host cell according to item 22 and / or 23 under suitable conditions, thereby producing at least one engineered polypeptide.

[0362] 25. The method according to item 24, further comprising recovering at least one engineered polypeptide from the culture and / or the host cell.

[0363] 26. The method according to item 24 and / or 25, further comprising a step of purifying the at least one engineered polypeptide.

[0364] 27. A process for preparing a compound of formula (I) in an enantiomeric excess of at least 70% over the relative enantiomer:

[0365]

[0366] The compound of formula (I) has the indicated stereochemical configuration at the stereocenter marked with *,

[0367] in

[0368] Z is a linear C1-6 hydrocarbon group or a branched C1-6 hydrocarbon group, optionally substituted with one or more hydroxyl groups, cyano groups or nitro groups; and

[0369] R1 is C1-6 hydrocarbyl, aryl-C1-2 hydrocarbyl, heteroaryl-C1-2 hydrocarbyl, or a 5- to 6-membered heterocyclic ring system optionally containing additional heteroatoms selected from O, S and N, the heterocyclic ring being unsubstituted or substituted with one to three substituents independently selected from oxo, hydroxyl, halogen, C1-4 hydrocarbyl and C1-4 hydrocarbyl; the method comprising reacting a compound of formula (II) under suitable reaction conditions in the presence of an amino group donor:

[0370]

[0371] A step of contacting with an engineered polypeptide according to any one of items 1 to 14.

[0372] 28. The method according to item 27, wherein the compound of structural formula (II) does not include compound (2) and the compound of structural formula (I) does not include compound (1).

[0373] 29. A method for preparing compound (1),

[0374]

[0375] The method comprises reacting a substrate of compound (2) in the presence of an amino group donor under suitable reaction conditions.

[0376]

[0377] A step of contacting with the engineered polypeptide described in any one of items 1 to 14.

[0378] 30. The process according to any one of items 27 to 29, wherein the compound of formula (I) or the compound (1) is produced in an enantiomeric excess of at least 90%.

[0379] 31. The process according to any one of items 27 to 29, wherein the compound of formula (I) or the compound (1) is produced in an enantiomeric excess of at least 99%.

[0380] 32. The method according to any one of items 27 to 29, wherein the amino group donor is selected from isopropylamine, alanine, 3-aminobutyric acid or methylbenzylamine.

[0381] 33. The method according to any one of items 27 to 29, wherein the amino group donor is isopropylamine, optionally at a concentration of about 0.1 M to about 3.0 M, 0.2 M to about 2.5 M, about 0.5 M to about 2 M or about 1 M to about 2M.

[0382] 34. The method according to any one of items 27 to 29, wherein the suitable reaction conditions include a temperature of about 45°C to about 60°C.

[0383] 35. A method according to any one of items 27 to 29, wherein the suitable reaction conditions include a wet organic solvent.

[0384] 36. The method according to item 35, wherein the wet organic solvent is selected from wet isopropyl acetate, wet toluene, wet methyl tert-butyl ether, wet acetonitrile or wet isopropanol.

[0385] 37. The method according to item 36, wherein the wet organic solvent is isopropyl acetate.

[0386] 38. A method according to any one of items 27 to 29, wherein the suitable reaction conditions include a substrate compound loading of about 5 g / L to about 200 g / L, about 10 g / L to about 150 g / L, or about 50 g / L to about 100 g / L.

[0387] 39. A method according to any one of items 27 to 29, wherein the suitable reaction conditions include the engineered polypeptide at a concentration of about 0.5 g / L to about 5 g / L, about 0.5 g / L to about 3 g / L, about 0.5 g / L to about 2 g / L, or about 0.5 g / L to about 1 g / L.

[0388] 40. A method according to claim 29, wherein the suitable reaction conditions include: (a) a substrate loading of substrate compound (2) of about 10 g / L to 200 g / L; (b) an engineered polypeptide concentration of about 0.5 g / L to 25 g / L; (c) an isopropylamine concentration of about 0.1 M to 3 M; (d) a wet organic solvent; and (e) a temperature of about 30°C to 60°C.

[0389] 41. A method according to claim 29, wherein the suitable reaction conditions include: (a) about 20 g / L of substrate compound (2); (b) about 20 g / L of engineered polypeptide; (c) wet isopropyl acetate; (d) about 0.55 M of isopropylamine; and (e) about 50°C.

[0390] 42. The method according to any one of items 27 to 29, further comprising the step of isolating the compound of formula (I) or the compound (1) from the reaction.

Claims

1. An engineered polypeptide having transaminase activity, wherein the engineered polypeptide is a polypeptide sequence listed in the even-numbered sequence in SEQ ID NO: 6-846.

2. A polynucleotide encoding at least one engineered polypeptide according to claim 1.

3. The polynucleotide of claim 2, wherein the polynucleotide is operably linked to a control sequence, wherein the control sequence is selected from the group consisting of a promoter, a leader sequence, a polyadenylation sequence, a propeptide sequence, a signal peptide sequence, and a transcription terminator.

4. The polynucleotide of claim 2 or 3, wherein the polynucleotide is codon optimized.

5. The polynucleotide according to claim 2 or 3, wherein the polynucleotide is a polynucleotide listed in the odd-numbered sequence in SEQ ID NO: 5-845.

6. An expression vector comprising at least one polynucleotide according to any one of claims 2 to 5.

7. A host cell comprising at least one expression vector according to claim 6.

8. A host cell comprising at least one polynucleotide according to any one of claims 2 to 5.

9. A method for producing an engineered polypeptide in a host cell, the method comprising culturing the host cell according to claim 7 or 8 under suitable conditions, thereby producing at least one engineered polypeptide.

10. The method of claim 9, further comprising recovering at least one engineered polypeptide from the culture and / or the host cell.

11. The method according to claim 9 or 10, further comprising the step of purifying the at least one engineered polypeptide.

12. A process for preparing a compound of formula (I) with an enantiomeric excess of at least 70% over the relative enantiomer: (I) The compound of formula (I) has the indicated stereochemical configuration at the stereocenter marked with *, in Z is a linear C1-6 hydrocarbon group or a branched C1-6 hydrocarbon group, optionally substituted with one or more hydroxyl groups, cyano groups or nitro groups; and R1 is C1-6 hydrocarbyl, aryl-C1-2 hydrocarbyl, heteroaryl-C1-2 hydrocarbyl, or a 5- to 6-membered heterocyclic ring system optionally containing additional heteroatoms selected from O, S and N, the heterocyclic ring being unsubstituted or substituted with one to three substituents independently selected from oxo, hydroxyl, halogen, C1-4 hydrocarbyl and C1-4 hydrocarbyl; the method comprising reacting a compound of formula (II) under suitable reaction conditions in the presence of an amino group donor: (II) The step of contacting with the engineered polypeptide according to claim 1.

13. A method for preparing compound (1), (1) The method comprises reacting a substrate of compound (2) in the presence of an amino group donor under suitable reaction conditions. (2) The step of contacting with the engineered polypeptide of claim 1.

14. The process according to claim 12 or 13, wherein the compound of formula (I) or the compound (1) is produced in an enantiomeric excess of at least 90%.

15. The process according to claim 12 or 13, wherein the compound of formula (I) or the compound (1) is produced in an enantiomeric excess of at least 99%.

16. The method according to claim 12 or 13, wherein the amino group donor is selected from isopropylamine, alanine, 3-aminobutyric acid or methylbenzylamine.

17. The method of claim 12 or 13, wherein the amino group donor is isopropylamine at a concentration of 0.1 M to 3.0 M, 0.2 M to 2.5 M, 0.5 M to 2 M or 1 M to 2 M.

18. The method of claim 12 or 13, wherein the suitable reaction conditions include a temperature of 45°C to 60°C.

19. The method of claim 12 or 13, wherein the suitable reaction conditions comprise a wet organic solvent.

20. The method according to claim 19, wherein the wet organic solvent is selected from wet isopropyl acetate, wet toluene, wet methyl tert-butyl ether, wet acetonitrile or wet isopropyl alcohol.

21. The method of claim 20, wherein the wet organic solvent is isopropyl acetate.

22. The method of claim 12 or 13, wherein the suitable reaction conditions include a substrate compound at a loading of 5 g / L to 200 g / L, 10 g / L to 150 g / L, or 50 g / L to 100 g / L.

23. The method of claim 12 or 13, wherein the suitable reaction conditions comprise the engineered polypeptide at a concentration of 0.5 g / L to 5 g / L, 0.5 g / L to 3 g / L, 0.5 g / L to 2 g / L, or 0.5 g / L to 1 g / L.

24. The method of claim 13, wherein the suitable reaction conditions include: (a) substrate loading of substrate compound (2) ranging from 10 g / L to 200 g / L; (b) engineered polypeptide concentration ranging from 0.5 g / L to 25 g / L; (c) isopropylamine concentration ranging from 0.1 M to 3 M; (d) wet organic solvent; and (e) temperature ranging from 30°C to 60°C.

25. The method of claim 13, wherein the suitable reaction conditions comprise: (a) about 20 g / L of substrate compound (2); (b) about 20 g / L of engineered polypeptide; (c) wet isopropyl acetate; (d) about 0.55 M isopropylamine; and (e) about 50°C.

26. The method according to claim 12 or 13, further comprising the step of isolating the compound of formula (I) or the compound (1) from the reaction.

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