Biocatalyst for synthesizing ubrogepant intermediate and method for synthesizing same

Abstract

The present application provides an engineered aminotransferase polypeptide for synthesizing ubrogepant intermediates with high stereoselectivity, high catalytic activity, and excellent stability. The present application also provides a reaction process for the asymmetric synthesis of ubrogepant intermediates ((5S,6R)-6-methyl-2-oxo-5-phenyl-3-carbamic acid tert-butyl ester)piperidine using the aminotransferase polypeptide, and a purification process for the product.

Description

[Technical field]

[0001] The present invention relates to the technical field of biotechnology, and in particular to the use of engineering aminotransferase polypeptides in catalyzing the synthesis of Ubrogepant intermediates. [Background technology]

[0002] Migraine is a common primary headache condition, characterized by a predominantly unilateral, moderate to severe, throbbing headache that may be accompanied by nausea, vomiting, photophobia, and other symptoms. Currently, more than 10% of the world's population suffers from migraine, with the number of women being approximately twice that of men. The development of migraine has a complex genetic cause, but it is still unclear which genes are involved in the development of migraine, and the pathogenesis of migraine has not yet been clearly elucidated. Traditionally, triptan drugs, such as sumatriptan, zolmitriptan, and almotriptan, which act mainly on the 5HT1B / 1D receptor, have been used to treat migraine. Due to their inherent vasoconstrictive effect, they are not suitable for use in migraine patients with cardiovascular disease. Calcitonin gene-related peptide (CGRP) is a neuropeptide containing 37 amino acids, which has vasodilatory properties, can act on multiple sites, and can be involved in nociception and sensitization of peripheral and central neurons of the trigeminovascular system, which are related to the pathophysiology of migraine. Currently, CGRP receptor antagonism has proven to be an effective way to relieve migraines. Ubrogepant was approved for marketing by the FDA in 2019 and is the first oral calcitonin gene-related peptide (CGRP) receptor antagonist approved by the FDA for the treatment of migraines. Ubrogepant relieves migraine symptoms by blocking the binding of CGRP to its receptor. Its mechanism of action is completely different from conventional triptan drugs and exerts its effect in a new way without constricting blood vessels, a problem with many existing migraine treatments.

[0003] In the paper "Practical Asymmetric Synthesis of a Calcitonin Gene-Related Peptide (CGRP) Receptor Antagonist Ubrogepant" (Org. Process Res. Dev. 2017, 21, 1851-1858), Nobuyoshi Yasuda et al. disclosed a method for synthesizing ubrogepant (see Figure 1). Ubrogepant contains two core structural fragments, one of which is a lactam (shown in structural formula L2) containing three chiral centers, and the other fragment is a spiro acid (shown in structural formula A1), and the lactam synthesis is the most difficult. To synthesize the key intermediate L2, Nobuyoshi Yasuda and his colleagues first synthesized the compound represented by the structural formula S1 (isopropyl 4-phenyl-2-(tert-butoxycarbonylamino)-5-oxohexanoate), and then carried out the dynamic kinetic catalysis of aminotransferase in 50% dimethyl sulfoxide (DMSO) using S1 (theoretically containing four different isomers: ST1, ST2, SD1, and SD2) as a substrate to obtain lactam L1 with two immobilized chiral centers. The reaction catalyzed by this aminotransferase can prepare L1 with high chiral purity in one go by utilizing the epimerization of the substrate. If the aminotransferase is only active against the isomers ST1 and ST2 of the S1 substrate but not against SD1 or SD2, ST1 and ST2 can be converted by the aminotransferase to IT1 and IT2, respectively (the ester bonds in the structures of IT1 and IT2 can be spontaneously broken and then cyclically formed to form L1). Therefore, ID1 or ID2 does not exist in the product. In addition, under appropriate reaction conditions, when ST1 and ST2 are consumed, the isomers SD1 and SD2 that could not participate in the aminotransferase reaction are spontaneously converted in situ to ST1 and ST2, and the resulting ST1 and ST2 are converted to IT1 and IT2 by the aminotransferase. The key to this reaction is the need to develop an aminotransferase with extremely high selectivity for the isomers ST1 and ST2 of the substrate S1.That is, it is only active against ST1 and ST2, but not against SD1 and SD2, and when the target carbonyl is converted to amino, only the amino of the R-configuration is produced. This results in lactam L1 with extremely high chiral purity. In the aminotransferase reaction disclosed by Nobuyoshi Yasuda et al., the ratio of the sum of the concentrations of IT1 and IT2 to the sum of the concentrations of ID1 and ID2 in the product (i.e., diastereomeric ratio, abbreviated as dr) is up to 61:1. Nobuyoshi Yasuda et al. obtained intermediate L1 by aminotransferase reaction, then carried out alkylation reaction and induced diastereomeric conversion using crystallization to prepare L2.

[0004] JPEG2025501365000002.jpg60170

[0005] JPEG2025501365000003.jpg83170JPEG2025501365000004.jpg52170

[0006] The present invention discloses an engineered aminotransferase with better performance suitable for the dynamic kinetic catalytic reaction of aminotransferase synthesizing L1 and its analogs. The engineered aminotransferase provided by the present invention has better resistance to the solvent used in the reaction, better activity, and better thermal stability. In addition, the present invention optimizes the aminotransferase reaction system and post-treatment system using a mixed solvent of dimethyl sulfoxide (DMSO) and acetonitrile (ACN) as the reaction solvent, greatly improving the defect of low solubility of the substrate S1 when DMSO solvent alone is used, and also avoids the influence on enzyme activity caused by too high concentration of acetonitrile, a single solvent with higher substrate solubility, and allows part of the solvent to be recycled in the post-treatment, which is economical and environmentally friendly. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention provides an engineered aminotransferase polypeptide capable of asymmetric synthesis of chiral amine compounds, particularly ubrogepant intermediate L1, with high stereoselectivity, high catalytic activity and excellent stability. The present invention also provides a gene for the engineered aminotransferase polypeptide, a recombinant expression vector containing the gene, an engineered strain, an efficient preparation method thereof, and an asymmetric synthesis reaction process for L1 using the engineered aminotransferase polypeptide, as well as a product purification process. [Means for solving the problem]

[0008] A first aspect of the present invention provides an improved engineered aminotransferase polypeptide, which has been modified by an artificial directed evolution process to generate mutations, such as substitutions, insertions, or deletions of a specific number of amino acid residues, to obtain an aminotransferase active in the reaction shown in FIG. JPEG2025501365000005.jpg6170 spherase enzyme library was screened, and finally, an aminotransferase having the sequence shown in SEQ ID NO:2, which is active in the reaction shown in FIG. 2, was determined. SEQ ID NO:2 was developed from a wild-type aminotransferase derived from Aspergillus fumigatus. However, SEQ ID NO:2 has low activity and stereoselectivity for the substrate S1, and low solvent resistance. As a result of detection by the present inventors, when the reaction shown in FIG. 2 was performed using SEQ ID NO:2, the reaction yield was 35% in the case of a 24-hour reaction under the conditions of a substrate S1 loading amount of 5 g / L and an aminotransferase loading amount of 10 g / L. In addition, high-concentration solvents such as methanol and DMSO have an inhibitory effect on SEQ ID NO:2. For example, if the activity of SEQ ID NO:2 under 20% methanol is defined as 100%, the relative activities of SEQ ID NO:2 under 35% and 50% methanol conditions were 59% and 43%, respectively, and the relative activities of the aminotransferase under 20% DMSO, 35% DMSO, and 50% DMSO conditions were 74%, 23%, and 8%, respectively. In the reaction system using methanol as a solvent, the product catalyzed by SEQ ID NO:2 had a dr value of 1.7, and in the DMSO system, the dr value was 0.3. For this aminotransferase to achieve industrial production of L1, it is necessary to engineer this aminotransferase to improve its activity and selectivity.

[0009] The present invention uses computational biology techniques to construct models and perform virtual screening of mutants of aminotransferase SEQ ID NO:2. First, 112 stable mutants are obtained, and then activity virtual screening techniques are used to select 40 mutants from the 112 stable mutants that potentially contribute to improving the activity of catalyzing the reaction shown in FIG. 2. Then, the inventors carry out gene construction and recombinant expression of these 40 mutants predicted by virtual screening in the laboratory, and experimentally verify their performance of catalyzing the reaction shown in FIG. 2 by setting appropriate reaction conditions, and finally, 15 mutants with improved catalytic activity and / or selectivity for the production of S1 to L1 are identified. Among them, the performance of SEQ ID NO:24 is better. Compared with SEQ ID NO:2, SEQ ID NO:24 contains the mutation W183A. Based on these 15 experimentally verified mutants, the inventors carried out a new round of virtual screening of the combinatorial mutation library and identified 7 beneficial mutants suitable for combination. Then, we constructed a combinatorial mutation library containing these seven beneficial mutations, screened the library using experimental methods, and finally obtained the optimal mutant SEQ ID NO: 130. Compared with SEQ ID NO: 2, the amino acid mutations contained in SEQ ID NO: 130 are T52Y, Q53T, W183A, and N190I.

[0010] The engineered aminotransferase polypeptides provided herein are active for the reaction shown in FIG. 2 and comprise an amino acid sequence having one or more residue differences compared to the SEQ ID NO:2 sequence at amino acid residue positions corresponding to X52, X53, X115, X126, X146, X183, and X190.

[0011] Additionally, compared to the sequence of SEQ ID NO:2, the engineered aminotransferase polypeptides provided herein include amino acid sequences that include at least one of the following characteristics: T52Y, Q53TKFEH, N115GE, R126L, I146Q, W183AST, N190LI, and may include insertions or deletions 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 more amino acid residues based on these differences.

[0012] More specifically, in some embodiments, the improved engineered aminotransferase polypeptides based on SEQ ID NO:2 are selected from the group consisting of the corresponding aminotransferase polypeptides of SEQ ID NO:4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 100, 102, 104, 106, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 120, 122, 124, 126, 128, 129, 130, 131, 132, 133, 134, 135, 136, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 1 , 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, and 158.

[0013] In some embodiments, the improved engineered aminotransferase polypeptide is selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158.

[0014] The identity between two amino acid sequences or two nucleotide sequences can be obtained by algorithms commonly used in the field, and can be calculated using NCBI Blastp and Blastn software according to default parameters, or the Clustal W algorithm (Nucleic Acid Research, 22 (22): 4673-4680, 1994) can be employed. For example, using the Clustal W algorithm, the amino acid sequence identity between SEQ ID NO:2 and SEQ ID NO:130 is 98.7%.

[0015] In another aspect, the invention provides polynucleotide sequences encoding engineered aminotransferase polypeptides. In some embodiments, the polynucleotide may be part of an expression vector having one or more regulatory sequences for expressing the engineered aminotransferase polypeptide. In some embodiments, the polynucleotide may be selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 220, 221, 222, 223, 225, 227, 229, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 9, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157.

[0016] As known to those of skill in the art, due to the degeneracy of nucleotide codons, the sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 100, 102, Polynucleotide sequences encoding the amino acid sequences of 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, and 158 are set forth in SEQ ID NO: NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 , 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93 , 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157. Nucleic acid sequences encoding engineered aminotransferases of the present invention are represented by SEQ ID NOs. NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 100, 102, 104 , 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158.

[0017] In another aspect, the disclosure provides polynucleotides encoding engineered aminotransferases or expression vectors capable of expressing the engineered aminotransferases, and host cells. In some embodiments, the host cells may be bacterial host cells, such as E. coli. The host cells may be used to express and isolate the engineered aminotransferases described herein, or may be used directly in reactions to convert substrates to products.

[0018] In some embodiments, the engineered aminotransferase in the form of a whole cell, a crude extract, an isolated polypeptide, or a purified polypeptide can be used, either alone or in immobilized form (e.g., immobilized on a resin).

[0019] The engineered aminotransferase polypeptides disclosed in the present invention can catalyze the conversion of a ketone substrate, represented by structure XI, to an amine product, represented by structure I.

[0020] JPEG2025501365000006.jpg73170 (where R 1 , R 2 , R 3 , R 4 , and R 5 may be optionally substituted -H, C1-C6 hydrocarbyl, halogen (e.g., -F, -Cl, -Br, -I), -NO:2, -NO, -SOR' or -SOR', -SR', -NR'R', -OR', -COR' or -COR', -C(O)NR', ​​-SONH2 or -SONH2, -CN, CF3, and R 6 may be a C1-C6 hydrocarbyl, a C1-C6 halogenated hydrocarbon, or a C1-C6 hydroxy-substituted hydrocarbon; R 7 may be a C1-C6 hydrocarbyl, a C1-C6 halogenated hydrocarbon, or a C1-C6 hydroxy-substituted hydrocarbon; R 8may be a CBZ protecting group, a BOC protecting group, a Fomc protecting group, a Bn protecting group, or a methoxy(ethoxy)carbonyl protecting group. Each R' is independently selected from H or a C1-C4 hydrocarbyl.

[0021] The amine product of formula I is a mixture of one or more of the chiral amine products of formulas II-V.

[0022] JPEG2025501365000007.jpg60170

[0023] Due to the activity of the ester group in the compound I structure, under appropriate reaction conditions, the enzyme-catalyzed amine product of formula I can spontaneously undergo ring formation to form a lactam of formula VI.

[0024] JPEG2025501365000008.jpg61153

[0025] The chiral amine product of formula VI is a mixture of one or more of the chiral amine products of formulas VII-X below.

[0026] JPEG2025501365000009.jpg57153

[0027] The substrate, structural formula XI, which corresponds to the chiral amine product, structural formula IX, that can be produced catalyzed by the engineered aminotransferase polypeptides disclosed herein, is shown below:

[0028] JPEG2025501365000010.jpg61153

[0029] Preferably, the engineered aminotransferase polypeptides disclosed in the present invention have significant catalytic activity towards a substrate S1, the structural formula of which is shown below:

[0030] JPEG2025501365000011.jpg64170

[0031] S1 may include the following four different isomers ST1, ST2, SD1 or SD2.

[0032] JPEG2025501365000012.jpg40170

[0033] The engineered aminotransferase polypeptides disclosed in the present invention may convert S1 to I1.

[0034] JPEG2025501365000013.jpg67170

[0035] I1 may include the following four different isomers IT1, IT2, ID1 or ID2.

[0036] JPEG2025501365000014.jpg30131

[0037] A compound of formula IT represents IT1 and / or IT2.

[0038] JPEG2025501365000015.jpg53131

[0039] The ester bond on the I1 structure may spontaneously break to form a ring, forming the corresponding compounds denoted T1, T2, D1, and D2.

[0040] JPEG2025501365000016.jpg41131

[0041] In the products produced by the engineered aminotransferase polypeptides disclosed herein catalyzing substrate S1, IT1 and IT2, and T1 and T2 are excess products. T1 and T2 are represented by the structural formula L1.

[0042] JPEG2025501365000017.jpg47131

[0043] In the present invention, the "diastereomeric ratio" (i.e., diastereomeric ratio, abbreviated as dr) is the ratio of the sum of the concentrations of diastereomeric compounds T1 and T2 in the product to the sum of the concentrations of diastereomeric compounds D1 and D2, and is calculated by dr = [T1 + T2] / [D1 + D2].

[0044] In some embodiments, the engineered aminotransferase polypeptide has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity compared to SEQ ID NO:2 and is capable of converting compound S1 to one or more of the amine products of compounds T1, T2, D1, and D2.

[0045] In some embodiments, the dr value of the product (i.e., [T1+T2] / [D1+D2]) is at least 1, 2, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more.

[0046] Specific embodiments of engineered aminotransferase polypeptides for use in the methods are further described in the detailed description of the invention. Improved engineered aminotransferase polypeptides that may be used in the above methods include those of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 66 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158. Any of the methods of preparing a compound of Formula I, Formula VI, Formula II or Formula L1 using engineered polypeptides disclosed herein may be carried out under a range of suitable reaction conditions including, but not limited to, ranges of amino donors, pH, temperature, buffers, solvent systems, substrate loading, polypeptide loading, cofactor loading, pressure, and reaction time. For example, in some embodiments, preparation of compounds of formula T1 and T2 may be performed, where suitable reaction conditions include (a) loading of substrate S1 from about 10 g / L to 100 g / L, (b) loading of engineering polypeptide from about 1 g / L to 50 g / L, (c) loading of isopropylamine from about 0.1 M to 4.0 M, (d) pH from about 7.0 to 11.5, (e) temperature from about 10° C. to 65° C., and (f) 0% to 70% solvent. Organic solvents described herein include, but are not limited to, methanol, dimethyl sulfoxide (DMSO), acetonitrile (ACN), dimethylformamide (DMF), methyl tert-butyl ether (MTBE), isopropyl acetate, ethanol, propanol, isopropanol (IPA), or mixtures of two or more thereof.

[0047] detail definition For the purposes of this disclosure, unless expressly defined otherwise, technical and scientific terms used herein have the meanings that are commonly understood by those of ordinary skill in the art.

[0048] "Protein," "polypeptide," and "peptide" are used interchangeably herein to mean a polymer of at least two amino acids covalently joined by amide bonds, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristoylation, ubiquitination, etc.). Included within this definition are D- and L-amino acids, and mixtures of D- and L-amino acids.

[0049] The terms "engineered aminotransferase," "engineered aminotransferase polypeptide," "improved aminotransferase polypeptide," and "engineered polypeptide" are used interchangeably herein.

[0050] "Bacteria" or "wet bacteria" refers to host cells expressing a polypeptide or an engineered polypeptide, including wet bacteria obtained by the preparation process shown in Example 2.

[0051] "Polynucleotide" and "nucleic acid" are used interchangeably herein.

[0052] As used herein, "cofactor" refers to a non-protein compound that acts in conjunction with an enzyme in a catalytic reaction. As used herein, "cofactor" is intended to include the vitamin B6 family of compounds pyridoxal-5'-phosphate (or PLP), pyridoxine (pyridoxol, PN), pyridoxal (PL), pyridoxamine (PM), pyridoxinephosphate (PNP), and pyridoxaminephosphate (PMP), sometimes referred to as coenzymes. "PLP", "pyridoxal-5'-phosphate", "pyridoxal-5'-phosphate", "PYP", and "P5P" are used interchangeably herein to refer to compounds that function as cofactors in enzyme-catalyzed reactions.

[0053] "Coding sequence" refers to a nucleic acid portion (eg, a gene) that codes for the amino acid sequence of a protein.

[0054] "Naturally-occurring" or "wild-type" refers to a form found in nature. For example, a naturally-occurring or wild-type polypeptide or polynucleotide sequence is a sequence that occurs in an organism, isolatable from a source in nature, and has not been intentionally modified by human manipulation.

[0055] "Recombinant" or "engineered" or "non-naturally occurring," when used to refer to, for example, a cell, nucleic acid, or polypeptide, refers to material that is altered in a way that does not occur in nature, or that is the same but produced or obtained from synthetic material and / or by manipulation using recombinant techniques, or material that corresponds to the native or native form of such material.

[0056] "Sequence identity" and "homology" are used interchangeably herein to refer to a comparison between polynucleotides or polypeptides ("sequence identity" and "homology" are usually expressed as a percentage) and are determined by comparing two best aligned sequences in a comparison window, where a portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) for best alignment of the two sequences compared to the reference sequence. The percentage is obtained by determining the number of positions in the two sequences where the same nucleic acid base or amino acid residue occurs to obtain the number of matching positions, and dividing this number of matching positions by the total number of positions in the comparison window, multiplying by 100 to obtain the percentage of sequence identity. Alternatively, the percentage may be obtained by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences, or the number of positions where the nucleic acid base or amino acid residue aligns with a gap to obtain the number of matching positions, and dividing this number of matching positions by the total number of positions in the comparison window, multiplied by 100 to obtain the percentage of sequence identity. Those skilled in the art will recognize that there are many established algorithms that can be used to align two sequences. Sequences for comparison can be best aligned using, for example, the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the similarity search method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, computer implementations of these algorithms (GAP, BESTFIT, FASTA, or TFASTA in the GCCG Wisconsin package), or by visual inspection (see generally, Current Protocols in Molecular Biology, edited by F. M. Ausubel et al., Current Protocols, a joint venture of Greene Publishing Associates Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).Examples of suitable algorithms for determining percentage of sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms described in Altschul et al., 1990, J. Mol. Biol. 215:403-410, and Altschul et al., 1977, Nucleic Acids Res. 3389-3402, respectively. The software used to perform BLAST analysis is publicly available from the National Center for Biotechnology Information website. This algorithm involves identifying high-scoring sequence pairs (HSPs) by first identifying short words of length W in the query sequence that match or meet some positive threshold score T when compared to words of the same length in the database sequences. T is called the neighborhood word score threshold (Altschuletal et al., supra). These initial neighborhood word hits act as seeds to initiate searches to find longer HSPs that contain 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 mismatching residues, always <0). For amino acid sequences, a score matrix is ​​used to calculate the cumulative score. Extension of the word hit string in each direction is terminated when the accumulation of one or more negative scoring residue alignments that would reduce the cumulative alignment score by an amount X from the maximum achieved causes the cumulative score to fall below 0 or when either end of the 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 default values ​​of a word length (W) of 11, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.For amino acid sequences, the BLASTP program uses as default values ​​a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989, Proc NatlAcad Sci USA 89:10915). Exemplary sequence alignments and determination of percent sequence identity can be performed using the BESTFIT or GAP programs of the GCG Wisconsin software package (Accelrys, Madison WI) using the default parameters provided. A "reference sequence" refers to a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of a larger sequence, such as a fragment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotides or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, or the full length of a nucleic acid or polypeptide. Because two polynucleotides or polypeptides may each (1) contain sequences that are similar between the two sequences (i.e., a portion of the complete sequence) and (2) may further contain sequences that differ between the two sequences, comparison of sequences between two (or more) polynucleotides or polypeptides is typically performed by comparing the sequences of the two polynucleotides or polypeptides within a "comparison window" to identify and compare local regions of sequence similarity. In some embodiments, the "reference sequence" is not intended to be limited to a wild-type sequence, but may include engineered or modified sequences. A "comparison window" refers to a conceptual fragment of at least about 20 contiguous nucleotide positions or amino acid residues in which a sequence may be compared to a reference sequence of at least 20 contiguous nucleotides or amino acid residues, where the portion of the sequence within the comparison window may contain no more than 20% additions or deletions (i.e., gaps) compared to the reference sequence (not including additions or deletions) for optimal alignment of the two sequences. The comparison window may be longer than 20 contiguous residues, and optionally includes a window of 30, 40, 50, 100, or more.In the case of numbers used for a specified amino acid sequence or polynucleotide sequence, "corresponding to," "referring to," or "against" refers to the number of the specified reference sequence residue when the specified amino acid sequence or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given sequence is assigned based on the reference sequence, not the actual numerical position of the residue in the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as an amino acid sequence of an engineered polypeptide, may be aligned with a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, gaps exist, but the residue number of a given amino acid or polynucleotide sequence is formulated relative to the reference sequence to which it is aligned. An "amino acid difference" or "residue difference" refers to the difference in an amino acid residue at a position in a polypeptide sequence relative to the amino acid residue at the corresponding position in the reference sequence. The position of the amino acid difference is generally referred to herein as "Xn," where n refers to the corresponding position in the reference sequence that is the basis for the residue difference. For example, "residue difference at position X183 compared to SEQ ID NO:2" refers to an amino acid residue difference at a polypeptide position corresponding to position 183 of SEQ ID NO:2. Thus, if a reference polypeptide of SEQ ID NO:2 has tryptophan at position 183, then "residue difference at position X183 compared to SEQ ID NO:2" refers to an amino acid substitution of any residue other than tryptophan at a polypeptide position corresponding to position 183 of SEQ ID NO:2. In most examples herein, a specific amino acid residue difference at a position is indicated as "XnY," where "Xn" refers to the corresponding position above, and "Y" is the one-letter code for the amino acid that appears in the engineered polypeptide (i.e., a residue that differs from that of the reference polypeptide). In some examples (e.g., Table 2), the disclosure also provides a specific amino acid difference represented by the conventional code "AnB," where A is the one-letter code for the residue in the reference sequence, "n" is the number of the residue position in the reference sequence, and B is the one-letter code for the residue substitution in the sequence of the engineered polypeptide.In some examples, the polypeptides of the disclosure may include one or more amino acid residue differences relative to a reference sequence, represented by a list of specific positions where residue differences exist compared to the reference sequence. "Deletion" refers to the modification of a polypeptide by removing one or more amino acids from a reference polypeptide. Deletions may include the removal of one or more, two or more, five or more, ten or more, fifteen or more, or twenty 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 that make up the reference enzyme, while retaining activity of the engineered aminotransferase for the reactions shown in FIG. 1. Deletions may include internal and / or terminal portions of the polypeptide. In various embodiments, deletions may include contiguous segments or may be discontinuous. "Insertion" refers to the modification of a polypeptide by adding one or more amino acids from a reference polypeptide. In some embodiments, the engineered polypeptides of the disclosure include one or more amino acid insertions into naturally occurring aminotransferase polypeptides and one or more amino acid insertions into other engineered polypeptides. Insertions may be made internally in the polypeptide, or at the carboxy or amino terminus. As used herein, an insertion includes fusion proteins as known in the art. An insertion may be a contiguous segment of amino acids or may be separated by one or more amino acids in a naturally occurring polypeptide. As used herein, a "fragment" refers to a polypeptide having an amino-terminal and / or carboxy-terminal deletion, but in which the amino acid sequence retained is the same as the corresponding positions in the sequence. A fragment may be at least 10 amino acids long, at least 20 amino acids long, at least 50 amino acids long or more, and up to 70%, 80%, 90%, 95%, 98%, and 99% of the full-length engineered polypeptide.

[0057] An "isolated polypeptide" or "purified polypeptide" refers to a polypeptide that has been substantially isolated from other materials that are naturally associated with it, such as proteins, lipids, and polynucleotides. The term includes polypeptides that have been removed or purified from the naturally occurring environment or expression system (e.g., a host cell or in vitro synthesis). An engineered polypeptide may be present intracellularly, in cell culture medium, or prepared in various forms, such as a lysate or isolated preparation. Thus, in some embodiments, an engineered polypeptide may be an isolated polypeptide.

[0058] A "chiral center" refers to a carbon atom that connects four different groups.

[0059] "Stereoselectivity" refers to the preferential formation of one stereoisomer over another or isomers in a chemical or enzymatic reaction. Stereoselectivity may be partial, with one stereoisomer being formed in preference to the other, or complete, with only one stereoisomer being formed. Stereoselectivity is referred to as diastereomer selectivity when the stereoisomers are diastereoisomers, and the ratio of one diastereomer to the other is usually reported as the "diastereomer ratio" (dr for short). This ratio is usually alternatively reported in the art as the "diastereomer ratio" (dr for short), calculated from the formula {major diastereomer concentration} / {minor diastereomer concentration}.

[0060] "Stereoisomers," "stereoisomeric forms," ​​and similar expressions are used interchangeably herein and refer to all isomers whose molecules differ only in the orientation of their atoms in space. This includes enantiomers and isomers of compounds with multiple chiral centers that are not mirror images of one another (i.e., "diastereomers").

[0061] "Improved enzyme properties" refers to any improved enzyme properties that an engineered polypeptide exhibits compared to a reference sequence, the evolution of which begins with aminotransferase SEQ ID NO: 22. Enzyme properties for which improvement is desirable include, but are not limited to, enzyme activity (which can be expressed as a percentage of substrate conversion), thermal stability, solution stability (e.g., stability to alcohol-based compounds), pH activity profile, cofactor requirements, resistance to inhibitors (e.g., sediment or product inhibition), stereospecificity, and stereoselectivity.

[0062] "Reaction yield" refers to the molar percentage of the product produced in a reaction system relative to the initial input substrate under specified reaction conditions and within a specified reaction time. Therefore, the "enzyme activity" or "activity" of an aminotransferase or engineered polypeptide can be expressed as a "reaction yield". The reaction is generally calculated as {molar concentration of product} / {molar concentration of starting substrate} by taking samples and measuring the molar concentration of the product and the molar concentration of the starting substrate in the reaction system.

[0063] "Thermostable" refers to an engineered polypeptide that retains similar activity as the starting template after exposure to elevated temperatures (eg, 65°C or higher) for a period of time (eg, 0.5 hours or more).

[0064] "Solvent stability" or "solvent resistance" refers to an engineered polypeptide that retains activity similar to the starting template after exposure to various concentrations (e.g., 5-99%) of solvents (e.g., methanol, ethanol, isopropanol, dimethyl sulfoxide (DMSO), tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butyl acetate, methyl tert-butyl ether, etc.) for a period of time (e.g., 0.5-24 hours).

[0065] "Suitable reaction conditions" refers to conditions in a biocatalytic reaction solution (e.g., ranges of enzyme loading, substrate loading, amino donor loading, cofactor loading, temperature, pH, buffers, co-solvents, etc.) that allow an engineered polypeptide according to the present disclosure to convert a substrate into a desired product compound. Exemplary "suitable reaction conditions" are provided in the present disclosure and verified by the examples. A compound may be referred to by its chemical structure and / or chemical name. When the chemical structure conflicts with the chemical name, the chemical structure defines the compound.

[0066] Directed evolution process and developed engineered aminotransferases The engineered aminotransferase polypeptide disclosed in the present invention is modified through an innovative directed evolution process and undergoes mutations such as substitution, insertion or deletion of a certain number of amino acid residues. The inventors have detected that the aminotransferase corresponding to SEQ ID NO:2 is active on S1, but has low activity, low chiral selectivity and poor solvent tolerance. In order to develop an engineered aminotransferase with excellent performance suitable for the reaction shown in FIG. 2, the present invention has designed and carried out three research and development stages as shown in Table 1. The research and development focus in each stage is different, and different screening reaction conditions are adopted in a targeted manner. The optimal engineered aminotransferase polypeptides obtained in each stage are shown in Table 2.

[0067] JPEG2025501365000018.jpg73137

[0068] The main objective of Phase I is to screen the developed engineering aminotransferase enzyme library to find an aminotransferase catalyst that is catalytically active for the production of product L1 from substrate S1 and can be directly applied industrially or used as a starting enzyme for directed evolution. As a result of screening by the inventors, SEQ ID NO:2 is the most suitable evolution starting enzyme developed from wild-type aminotransferase from Aspergillus fumigatus (NCBI: XP_748821.1). Table 2 shows the residue differences of SEQ ID NO:2 compared to the wild-type enzyme and the sequence identity of SEQ ID NO:2 compared to the wild-type enzyme. The amino acid sequence identity was calculated using the Clustal W algorithm (Nucleic Acid Research, 22(22):4673-4680, 1994). SEQ ID NO:2 needs to be modified through directed evolution techniques to further improve its activity, stability, selectivity and other properties to be suitable for industrial applications.

[0069] JPEG2025501365000019.jpg94137

[0070] The main objective of Phase II is to discover amino acid mutations that have significant effects on enzyme activity, stability and selectivity, and provide data support for subsequent directed evolution library design. The present invention uses bioinformatics and computational biology techniques to perform virtual screening of mutants of aminotransferase SEQ ID NO:2, and the general process of this virtual screening method is as follows:

[0071] The process of virtual screening is as follows.

[0072] Step 1: Homology modeling: For SEQ ID NO:2, homology modeling was performed through Yasara software using PDBID 4UUG as template, and the modeling parameters are shown in Table 3.

[0073] JPEG2025501365000020.jpg81137

[0074] Step 2: Autodock docking: Through the Autodock method of Yasara software, the four chiral substrates ST1, ST2, SD1, and SD2 were docked with the target enzyme to obtain the enzyme-substrate complex shown in Figure 3. Then, the amino acids within 5 Å of the substrate were selected as candidate mutation sites (T52, Q53, T60, L113, N115, R126, L141, L143, I146, L148, W183, N190, G215, S273, T274, A2755).

[0075] Step 3: Rosetta stability virtual screening: Using the Cartesian_ddg algorithm of Rosetta software, single-point saturation mutation stability virtual screening was performed on the candidate sites obtained in the previous step. The number of virtual screenings is 16*19=304 single-site mutations. As a result of the screening, 112 single-site mutations beneficial for stability were found. The results are shown in Table 4.

[0076] JPEG2025501365000021.jpg94170

[0077] The evaluation criteria for the calculation results of the stability of mutants are as follows: if ΔΔG≦-1 kcal / mol, it is a stable mutant, if ΔΔG≧1 kcal / mol, it is a non-stable mutant, if -1 kcal / mol<ΔΔG<1 kcal / mol, it is an invalid mutant. This criterion also applies to the evaluation of stability results obtained by other calculation methods.

[0078] Step 4: Activity Virtual Screening: The reaction energy barrier is the minimum energy required for a reactant molecule to become an activated molecule. The magnitude of the energy barrier reflects the difficulty of the reaction. Therefore, the present invention employs a process based on empirical valence bond theory to realize batch calculation of reaction energy barriers, and obtains mutants with improved activity by comparing the difference in the calculated reaction energy barriers between SEQ ID NO:2 and mutants. This screening step resulted in a total of 40 mutations that improve the activity of the target substrate T1 or T2. The results are shown in Table 5.

[0079] JPEG2025501365000022.jpg68170

[0080] The final experimental screening results showed that 15 of the 40 virtual screening mutations showed similar or significantly improved activity in improving the dr value of producing T1 or T2. Among them, the better mutant enzyme is SEQ ID NO:24, whose mutation is W183A. For a specific screening example, see Example 5. And the 15 advantageous mutations and their corresponding activities and selectivities are shown in Table 6.

[0081] JPEG2025501365000023.jpg108170

[0082] The method for constructing a mutation library can be site-specific mutation PCR or multi-site mutation PCR, which is common in the art (see "Mutagenesis and Synthesis of Novel Recombinant Genes Using PCR", Chapter 32, in PCR Primer, 2nd edition (eds. Dieffenbach and Dveksler). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003).

[0083] The main objective of stage III is to obtain an enzyme with significantly improved activity, selectivity, and solvent tolerance (stability). In the previous two stages, we found that when methanol or DMSO alone is used as a co-solvent for substrate S1, each has its own advantages and disadvantages. When methanol is used as a co-solvent, the solubility of the substrate in the co-solvent is high, but the substrate is easily hydrolyzed, and when DMSO is used as a co-solvent, the solubility of substrate S1 in the system is low, which affects the in situ racemization of the substrate (i.e., the conversion of ST1, ST2 to SD1, SD2). To solve the disadvantages caused by using a single solvent for the reaction, we ingeniously used a mixed solvent of DMSO / ACN as the reaction solvent instead of a single solvent. The solubility of substrate S1 in ACN is much higher than that in DMSO, and the substrate is not easily hydrolyzed in both DMSO and ACN systems, so the advantage of using a mixed solvent is to improve the solubility of substrate S1 in the reaction system, promote the in situ racemization of the substrate, greatly reduce the hydrolysis of the substrate caused by the use of alcohol-based solvents, and to some extent avoid the inhibition of enzyme activity caused by high concentrations of single acetonitrile solvents. In order to obtain enzymes with improved performance, when designing the library, the 15 favorable mutations screened in stage II were combined to obtain a combinatorial mutation library containing 1728 protein sequences, and Rosetta stability virtual screening and activity virtual screening of the combinatorial mutations were also performed, and the first 20% of the combinations of mutations with improved activity were used to obtain the probability of occurrence of the dominant amino acid, and finally the optimal amino acid combination mutations for each site were obtained, and the results are shown in Table 7.

[0084] JPEG2025501365000024.jpg42170

[0085] Finally, based on SEQ ID NO:24, the optimal mutations obtained at each site were combined into an experimental screening library, and screened using the screening reaction conditions of stage III in Table 1. For a specific screening example, see Example 7. Finally, an aminotransferase mutant SEQ ID NO:130 was obtained, which has significantly improved chiral selectivity and activity. Compared with SEQ ID NO:2, it contains four amino acid mutations, SEQ ID NO:T52Y, Q53T, W183A, and N190I. Table 8 shows the engineered aminotransferase polypeptides corresponding to each combination of mutations, the improved activity compared with SEQ ID NO:2, and the dr value of the product produced by the catalyst thereby.

[0086] JPEG2025501365000025.jpg184170JPEG2025501365000026.jpg243170JPEG2025501365000027.jpg239170

[0087] JPEG2025501365000028.jpg48170

[0088] The document "Practical Asymmetric Synthesis of a Calcitonin Gene-Related Peptide (CGRP) Receptor Antagonist Ubrogepant" discloses that the dr value of the product of aminotransferase ATA-412 and ATA-426 at a substrate concentration of 50 g / L is 61. The engineered aminotransferase polypeptide developed by the present invention has better activity, selectivity, stability (including thermal stability), solvent resistance, substrate resistance, etc. In addition, the present invention optimizes the solvent used in the reaction, making the solubility of substrate S1 in an aqueous ACN solution much higher than that in an aqueous DMSO solution, avoiding damage to enzyme activity caused by a single high-concentration ACN solvent, and improving the solubility of substrate S1 in an aqueous phase system, the present invention innovatively uses a mixed solvent of ACN and DMSO as the reaction solvent. It has also been proven that the ester hydrolysis of substrate S1 in the mixed solvent system is very effectively controlled, and therefore the engineered aminotransferase polypeptide disclosed in the present invention is more suitable for industrial production scenarios.

[0089] Polynucleotides, control sequences, expression vectors and host cells useful for preparing engineered aminotransferase polypeptides In another aspect, the disclosure provides polynucleotides that encode engineered polypeptides having aminotransferase activity as described herein. The polynucleotides can be operably linked to one or more heterologous regulatory sequences that control gene expression to generate recombinant polynucleotides capable of expressing the polypeptide. An expression construct containing a heterologous polynucleotide encoding an engineered aminotransferase can be introduced into a suitable host cell to express the corresponding engineered aminotransferase polypeptide. As will be apparent to one of skill in the art, the availability of protein sequences and knowledge of the codons that correspond to various amino acids provides a description of all polynucleotides that can encode a target protein sequence. The degeneracy of the genetic code, in which the same amino acid is encoded by alternative or synonymous codons, allows for the generation of a very large number of nucleic acids, all of which encode the improved aminotransferase polypeptides disclosed herein. Thus, after determining a particular amino acid sequence, one of skill in the art can generate any number of different nucleic acids by modifying the sequence of only one or more codons, without altering the amino acid sequence of the protein.In this regard, the present disclosure specifically contemplates all possible modifications of polynucleotides that may be prepared by selecting combinations based on possible codon choices, and for any polypeptide herein, the amino acid sequences of exemplary engineered polypeptides provided in Tables 6 and 8, and SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 16 This includes any of the polypeptides disclosed as having the sequences 0, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, all of which modifications are considered to be specifically disclosed.

[0090] In various embodiments, codons are preferably selected to be compatible with the host cell in which the protein is produced, for example, bacterially preferred codons are used to express genes in bacteria, yeast preferred codons are used to express genes in yeast, and mammalian preferred codons are used to express genes in mammalian cells.

[0091] In some embodiments, the polynucleotide is selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 170, 172, 174, 176, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188 and / or a polypeptide having one or more of the improved properties described herein, such as an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence selected from among: 42, 144, 146, 148, 150, 152, 154, 156, 158, wherein the polypeptide has one or more of the improved properties described herein, such as aminotransferase activity, increased activity compared to the polypeptide of SEQ ID NO:2, or the ability to convert compound S1 to product T2 or T2.

[0092] In some embodiments, the polynucleotide encodes an engineered aminotransferase polypeptide comprising an amino acid sequence having one or more amino acid residue differences compared to SEQ ID NO:2 and having the above percentage identity. In some embodiments, the disclosure provides engineered polypeptides with aminotransferase activity that comprise at least 80% sequence identity to the reference sequence of SEQ ID NO:2 and have a combination of residue differences at positions selected from X52, X53, X115, X126, X146, X183, X190.

[0093] In some embodiments, a polynucleotide encoding an engineered aminotransferase polypeptide is selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157.

[0094] In some embodiments, the polynucleotides encode a polypeptide as described herein but have about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity at the nucleotide level to a reference polynucleotide encoding an engineered aminotransferase.

[0095] In some embodiments, the reference polynucleotide sequence is selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93 , 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157.

[0096] An isolated polynucleotide encoding an engineered aminotransferase polypeptide can be manipulated in a variety of ways to provide for expression of the polypeptide, including further sequence modification by codon optimization to improve expression, insertion into an appropriate expression element with or without additional control sequences, and transformation into a host cell suitable for expression and production of the polypeptide. Depending on the expression vector, manipulation of the isolated polynucleotide may be desirable or necessary before it is inserted into a vector. Techniques for modifying polynucleotides and nucleic acid sequences using recombinant DNA methods are well known in the art. Guidance is provided in Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, and Current Protocols in Molecular Biology, Ausubel. F., ed., Greene Pub. Associates, 1998, updated in 2010.

[0097] In another aspect, the present disclosure also relates to a recombinant expression vector comprising a polynucleotide encoding an engineered aminotransferase polypeptide or variant thereof, and one or more expression control regions, such as a promoter terminator and an origin of replication, depending on the type of host into which it is introduced. Alternatively, the nucleic acid sequence of the present disclosure may be expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the sequence into an appropriate expression vector. In producing an expression vector, the coding sequence is placed in the vector such that the coding sequence is operably linked to appropriate control sequences for expression. The recombinant expression vector may be any vector (e.g., a plasmid or a virus) that can be conveniently used in recombinant DNA steps and that can result in the expression of a polynucleotide sequence. The choice of vector usually depends on the compatibility of the vector with the host cell into which it is introduced. The vector may be a linear plasmid or a closed circular plasmid. The expression vector may be 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. Alternatively, the vector may be one that is integrated into the genome when introduced into a host cell and replicates with the chromosome into which it is integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids that together comprise the total DNA to be introduced into the host cell genome, may be used. Many expression vectors useful for embodiments of the present disclosure are commercially available. An exemplary expression vector may be prepared by operably linking a polynucleotide encoding an improved aminotransferase polypeptide to the plasmid pACYC-Duet-1 (Novagen).

[0098] In another aspect, the disclosure provides a host cell comprising a polynucleotide encoding an improved aminotransferase polypeptide of the disclosure, said polynucleotide being operably linked to one or more control sequences for expression of the aminotransferase in the host cell. Host cells for expressing the polypeptides encoded by the expression vectors of the disclosure are well known in the art and include, but are not limited to, bacterial cells such as E. coli, Arthrobacter sp. KNK168, Streptomyces sp., and Salmonella typhimurium cells; fungal cells such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris); 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 E. coli BL21(DE3). The host cells may be wild-type or engineered cells that have been subjected to genome editing, such as knocking out the wild-type aminotransferase gene contained in the genome of the host cell. Suitable culture media and growth conditions for the host cells described above are well known in the art.

[0099] Polynucleotides for expressing aminotransferases can be introduced into cells by a variety of methods known in the art, including electroporation, bioparticle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion. The various methods of introducing polynucleotides into cells will be apparent to those skilled in the art.

[0100] Methods for Producing Engineered Aminotransferase Polypeptides If the sequence of an engineered polypeptide is known, a polynucleotide encoding the polypeptide can be prepared by standard solid-phase methods according to known synthesis methods. In some embodiments, fragments of up to about 100 bases can be synthesized separately and then ligated (e.g., by enzymatic or chemical ligation or polymerase-mediated methods) to form any desired contiguous sequence. For example, the polynucleotides and oligonucleotides of the present disclosure are typically implemented in the classical phosphoramidite method, e.g., as described in Beaucage et al., 1981, TetLett 22:1859-69, or the method described in Matthes et al., 1984, EMBOJ.3:801-05, e.g., automated synthesis methods. According to the phosphoramidite method, oligonucleotides are synthesized, e.g., in an automated DNA synthesizer, purified, annealed, ligated, and cloned into an appropriate vector. Essentially any nucleic acid can also be obtained from any of a variety of commercial sources.

[0101] In some embodiments, the disclosure also provides a method for preparing or making an engineered aminotransferase polypeptide, the method comprising culturing a host cell capable of expressing a polynucleotide encoding the engineered polypeptide under culture conditions suitable for expression of the polypeptide.

[0102] In some embodiments, the method of preparing a polypeptide further comprises isolating the polypeptide. The engineered polypeptide can be expressed in a suitable cell and isolated (or recovered) from the host cell and / or culture medium using any one or more of the well-known techniques for protein purification. The techniques for protein purification include, among others, lysozyme treatment, sonication, filtration, salting out, heat treatment, ultracentrifugation, and chromatography.

[0103] Methods for using engineered aminotransferases and preparation of compounds using the same In another aspect, the improved engineered aminotransferase polypeptides described herein can convert a prochiral acceptor ketone compound to a chiral amine compound in the presence of an amino donor. The present disclosure also provides methods for preparing a wide range of compound I or structural analogs thereof using the engineered aminotransferase polypeptides disclosed herein. In some embodiments, the engineered aminotransferase polypeptides can be used in methods for preparing compounds of structural formula I.

[0104] JPEG2025501365000029.jpg59170 (where R 1 , R 2 , R 3 , R 4 , and R 5 may be optionally substituted -H, C1-C6 hydrocarbyl, halogen (e.g., -F, -Cl, -Br, -I), -NO:2, -NO, -SOR' or -SOR', -SR', -NR'R', -OR', -COR' or -COR', -C(O)NR', ​​-SONH2 or -SONH2, -CN, CF3, and R 6 may be a C1-C6 hydrocarbyl, a C1-C6 halogenated hydrocarbon, or a C1-C6 hydroxy-substituted hydrocarbon; R 7 may be a C1-C6 hydrocarbyl, a C1-C6 halogenated hydrocarbon, or a C1-C6 hydroxy-substituted hydrocarbon; R 8 may be a CBZ protecting group, a BOC protecting group, a Fomc protecting group, a Bn protecting group, or a methoxy(ethoxy)carbonyl protecting group. Each R' is independently selected from H or a C1-C4 hydrocarbyl. The amine product of formula I is a mixture of one or more of the chiral amine isomers of formulas II-VI.

[0105] JPEG2025501365000030.jpg59170

[0106] Depending on the activity of the ester group in the compound I structure, under appropriate reaction conditions, e.g., appropriate temperature, pH and solvent conditions, a portion of the amine products of formula I may spontaneously cyclize to form lactams of formula VI.

[0107] JPEG2025501365000031.jpg63170

[0108] The amine product of formula VI is a mixture of one or more of the chiral amine isomers of formulas VII-X below.

[0109] JPEG2025501365000032.jpg67170

[0110] A substrate, structural formula XI, that can be catalyzed by an aminotransferase to produce a chiral amine product, structural formula IX, is shown below.

[0111] JPEG2025501365000033.jpg63170

[0112] Preferably, the engineered aminotransferase polypeptides disclosed in the present invention have significant catalytic activity towards a substrate S1, the structural formula of which is shown below:

[0113] JPEG2025501365000034.jpg60170

[0114] S1 may include four different isomers of ST1, ST2, SD1 or SD2.

[0115] JPEG2025501365000035.jpg40170

[0116] The engineered aminotransferase polypeptides disclosed in the present invention can convert S1 to I1.

[0117] JPEG2025501365000036.jpg65170

[0118] I1 may include four different isomers: IT1, IT2, ID1 or ID2.

[0119] JPEG2025501365000037.jpg38170

[0120] A compound of formula IT represents IT1 and / or IT2.

[0121] JPEG2025501365000038.jpg67170

[0122] The ester bond in the structure of I1 may spontaneously break to form a ring, forming the corresponding compounds denoted T1, T2, D1, and D2.

[0123] JPEG2025501365000039.jpg55170

[0124] Among the products produced by catalysis of substrate S1 by the engineered aminotransferase polypeptide disclosed in the present invention, IT1 and IT2, and T1 and T2 are the excess products. The structural formulas of T1 and T2, denoted by L1, are shown below.

[0125] JPEG2025501365000040.jpg59170

[0126] The improved engineered aminotransferase polypeptides described herein are capable of converting S1 to one or more of T1, T2, D1, D2 in the presence of an amino donor. In some embodiments, the dr value of the product (i.e., [T1+T2] / [D1+D2]) is at least 1, 2, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more.

[0127] In some embodiments, improved engineered aminotransferase polypeptides that may be used in the above methods include those of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 700, 710, 720, 730 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158; NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 6 2, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 1 Also included are amino acid sequences that have at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of the reference amino acid sequences selected from the following sequences: 24, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158.

[0128] As described herein and verified in the Examples, the present disclosure contemplates suitable reaction conditions that can be used in the methods herein, including, but not limited to, ranges of pH, temperature, buffers, solvent systems, substrate loading, polypeptide loading, and reaction times. Additional suitable reaction conditions for carrying out the methods of biocatalytically converting substrate compounds to product compounds using the 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, pH, temperature, and solvent conditions, and detecting the product compound using, for example, the methods described in the Examples provided herein.

[0129] As described above, the engineered polypeptides having aminotransferase activity used in the disclosed methods are generally selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, , 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158.

[0130] The substrate compound in the reaction mixture may vary, taking into account, for example, the amount of the desired product compound, the effect of substrate concentration on enzyme activity, the stability of the enzyme under reaction conditions, and the percentage of substrate to product conversion. In some embodiments of the method, suitable reaction conditions include a loading of substrate S1 of at least about 0.5 g / L, at least about 1 g / L, at least about 5 g / L, at least about 10 g / L, at least about 15 g / L, at least about 20 g / L, at least about 30 g / L, at least about 50 g / L, at least about 75 g / L, at least about 100 g / L or more. The substrate loading values ​​provided herein are based on the molecular weight of compound A1, although it is also contemplated that equimolar amounts of various hydrates and salts of the compound may be used in the method.

[0131] In the methods described herein, the engineered aminotransferase polypeptide catalyzes the formation of a chiral amine product from a ketone substrate and an amino donor. In some embodiments, the amino donor in the reaction conditions includes any suitable amino acid selected from alanine, isopropylamine (also known as 2-aminopropane), phenylalanine, glutamine, leucine, or 3-aminobutyric acid, or any suitable chiral or achiral amine selected from methylbenzylamine. The amino donor may also be used in the form of a salt, in embodiments (e.g., alanine hydrochloride, alanine acetate, isopropylamine hydrochloride, isopropylamine acetate, etc.). In some embodiments, the amino donor is isopropylamine. In some embodiments, the suitable reaction conditions include the amino donor, particularly isopropylamine, being present at a loading amount of at least about 1 times the molar loading amount of substrate S1. In some embodiments, isopropylamine is present at a loading amount of 0.1 M to about 4.0 M.

[0132] In reaction embodiments, reaction conditions may include a suitable pH. As noted above, the desired pH or desired pH range can be maintained by using an acid or base, a suitable buffer, or a combination of a buffer and the addition of an acid or base. The pH of the reaction mixture can be controlled before and / or during the reaction process. In some embodiments, suitable reaction conditions include a solution pH of about 7 to about 11.5. In some embodiments, reaction conditions include a solution pH of about 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5.

[0133] In embodiments of the methods herein, a suitable temperature can be used for the reaction conditions, for example, taking into account the increased reaction rate at higher temperatures, enzyme activity over a sufficiently long reaction time. Thus, in some embodiments, suitable reaction conditions include temperatures of about 10°C to about 65°C, about 25°C to about 50°C, about 25°C to about 40°C, or about 25°C to about 30°C. In some embodiments, suitable reaction temperatures include temperatures of about 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, or 60°C. In some embodiments, the temperature during the enzyme reaction can be maintained at a specific temperature throughout the reaction. In some embodiments, the temperature during the enzyme reaction can be adjusted to the temperature profile during the reaction.

[0134] Methods using engineered aminotransferases are typically carried out in water or solvents. Suitable solvents include aqueous buffers, organic solvents, and / or co-solvent systems, where the co-solvent systems typically include aqueous and organic solvents. The aqueous solutions (water or aqueous co-solvent systems) may be pH buffered or unbuffered. In some embodiments, methods using engineered aminotransferase polypeptides are typically carried out in aqueous co-solvent systems that include organic solvents (e.g., methanol, ethanol, propanol, isopropanol (IPA)), dimethylsulfoxide (DMSO), dimethylformamide (DMF), isopropyl acetate, ethyl acetate, butyl acetate, 1-octanol, heptane, octane, methyl tert-butyl ether (MTBE), toluene, etc.), ionic liquids (e.g., 1-ethyl 4-methylimidazole tetrafluoroborate, 1-butyl-3-methylimidazole tetrafluoroborate, 1-butyl-3-methylimidazole hexafluorophosphate, etc.). The organic solvent component in the aqueous co-solvent system can be miscible with the aqueous component to provide a single liquid phase, or partially miscible or immiscible with the aqueous component to provide two liquid phases. Carbon dioxide generated during the hydrolysis reaction can cause foam formation, so an antifoaming agent can be added as appropriate. Exemplary aqueous co-solvent systems include water and one or more organic solvents. Typically, the organic solvent component of the aqueous co-solvent system is selected so as not to completely inactivate the aminotransferase. Suitable co-solvent systems can be readily identified by measuring the enzymatic activity of a particular engineered aminotransferase using an enzymatic activity assay as described herein with a defined substrate of interest in a candidate solvent system. In some embodiments of the method, the suitable reaction conditions include an aqueous co-solvent comprising a mixture of DMSO and ACN at a concentration of about 1% to about 100% (v / v), about 1% to about 60% (v / v), about 2% to about 60% (v / v), about 5% to about 60% (v / v), about 10% to about 60% (v / v), about 10% to about 50% (v / v), or about 10% to about 40% (v / v).In some embodiments of the method, the suitable reaction conditions include a solvent mixture of DMSO and ACN at a concentration of at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%.

[0135] Suitable reaction conditions may include a combination of reaction parameters that provide for the biocatalytic conversion of the substrate compounds to the corresponding product compounds. Thus, in some embodiments of the method, the combination of reaction parameters includes: (a) a loading of about 10 g / L to 100 g / L substrate S1, (b) about 1 g / L to 50 g / L of the engineering polypeptide, (c) a loading of about 0.1 M to 4.0 M isopropylamine, (d) a pH of about 7.0 to 11.5, (e) a temperature of about 10° C. to 65° C., and (f) a solvent mixture of 1% to 70% DMSO and ACN.

[0136] In carrying out the enzymatic reactions described herein, the engineering polypeptide may be added to the reaction mixture in the form of partially purified or purified enzyme, heat-treated enzyme solution, whole cells transformed with a gene encoding the enzyme, and / or cell extracts of such cells, and / or lysates. Whole cells transformed with a gene encoding the engineering polypeptide, or cell extracts thereof, lysates thereof, and isolated enzymes may be used in various forms, such as solid (e.g., freeze-dried, spray-dried, etc.) or semi-solid (e.g., crude pastes such as wet cells). Cell extracts or cell lysates may be partially purified by precipitation (e.g., treatments such as ammonium sulfate, polyethyleneimine, heat treatment, etc.), followed by a desalting procedure (e.g., ultrafiltration, dialysis, etc.) before lyophilization. Any enzyme product may be stabilized by cross-linking or immobilization to a solid phase material (e.g., a resin) using known cross-linking agents, such as glutaraldehyde.

[0137] In some embodiments of the enzymatic reactions described herein, the reactions are carried out under suitable reaction conditions as described herein, and the engineering polypeptide is immobilized on a solid support. Solid supports that can be used to immobilize the engineering polypeptide for carrying out the enzymatic reaction include, but are not limited to, microspheres or resins that include epoxy-functionalized polymethacrylate, aminoepoxy-functionalized polymethacrylate, octadecyl-functionalized styrene / DVB copolymer, or octadecyl-functionalized polymethacrylate. Exemplary solid supports include, but are not limited to, chitosan beads, EupergitC, and SEPABEAD (Mitsubishi), including different types of SEPABEAD:EC-EP, EC-HFA / S, EXA252, EXE119, and EXE120.

[0138] In some embodiments, where the engineered polypeptide can be expressed as a secreted polypeptide, medium containing the secreted polypeptide can be used in the methods herein. In some embodiments, solid reactants (e.g., enzymes, salts, etc.) may be applied to the reaction in a variety of forms, such as powders (e.g., lyophilized, spray dried, etc.), solutions, emulsions, suspensions, etc. Reactants can be readily lyophilized or spray dried using methods and equipment well known to those of skill in the art. For example, a small volume of protein solution is frozen at -80°C and added to a pre-cooled lyophilization chamber, followed by vacuum.

[0139] In some embodiments, there are several options for the order or manner in which the reactants are added: The reactants can be added simultaneously to the solvent (e.g., a single-phase solvent, a two-phase aqueous co-solvent system, etc.), or some reactants can be added first and other reactants added in a flow stream or in interval batches. Various features and embodiments of the present disclosure are illustrated in the following representative examples, which are intended to be illustrative and not limiting. [Brief description of the drawings]

[0140] [Figure 1] This is the synthetic route for ubrogepant. [Diagram 2] The aminotransferase-catalyzed reactions of the present invention. [Diagram 3] This is the basic process of virtual screening. [Figure 4] Distribution of amino acid sites near substrate ST1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0141] Example 1: Aminotransferase screening in Phase I A cell lysate (1 g / L lysozyme, 0.5 g / L PMBS, 0.5 g / L nuclease, dissolved in sodium tetraborate buffer, pH 10.5) was added to a well plate containing wet cells containing various aminotransferases at 200 μL per well, and shaken for 1 h to disrupt the cells, obtaining a cell lysate. The lysate was centrifuged, and the supernatant was transferred to a new deep well plate to obtain an enzyme solution that could be used in the reaction. 40μL of 20g / L substrate mother solution dissolved in DMSO and 50μL of reaction mixture (containing 4M isopropylamine and 2g / L PLP, dissolved in sodium tetraborate buffer, adjusted to pH 10.5 (40℃) with concentrated hydrochloric acid) were added to a 96-well deep well plate, and the final concentrations of each component in the reaction system were [5g / L substrate, 55% enzyme solution (v / v), 20% DMSO, 0.5g / L PLP, 1M isopropylamine, 0.025M sodium tetraborate buffer, pH 10.5, and the well plate was placed in a thermostatic shaker at 45℃ for 24h. After the reaction was completed, the well plate was removed and placed on a water bath shaker at 70℃ for 1h, then inactivated by adding pure acetonitrile in a 1:1 ratio, and the sample was diluted to 2.5g / L and injected for detection. HPLC detection showed that SEQ ID NO:2 showed the best activity and selectivity, with a reaction yield of 29% and a dr value of 0.3 in 24 hours.

[0142] Example 2: Expression of Aminotransferase Polypeptides A single microbial colony containing E. coli BL21(DE3) carrying the aminotransferase polypeptide expression plasmid was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of LB medium (containing 30 μg / mL chloramphenicol) and grown overnight on a shaker at 30°C with shaking. 600 When the OD of the medium reached 2, 5% (v / v) of the inoculum was added to 250 mL of TB medium in a 1000 mL Erlenmeyer flask, and cultured with shaking in a shaker at 30°C. 600 When the β-aminotransferase activity reached 0.6, IPTG was added to a final concentration of 1 mM to induce the expression of aminotransferase. After culturing for 20 h, the culture was centrifuged (8000 rpm, 10 min), and the supernatant was discarded and the cells were collected to obtain wet cells. The wet cells can be used directly to prepare the enzyme solution or frozen and stored at -20°C until use.

[0143] Example 3: Testing the Solvent Tolerance of EQ ID NO:2 0.5 g of the wet bacterial cells of SEQ ID NO:2 prepared in Example 2 was weighed out, and 5 mL of cell lysis solution (containing 1 g / L lysozyme, 0.5 g / L PMBS, and 0.5 g / L nuclease, dissolved in sodium tetraborate buffer, pH 10.5) was added and the cells were disrupted by shaking for 1 hour to obtain a cell lysis solution. The cell lysis solution was centrifuged, and the supernatant was collected to obtain an enzyme solution. The reactor was opened, and S1 dissolved in DMSO, methanol, and IPA, which had been previously heated to 45°C, was added to the reaction flask, followed by the reaction mixture (containing 4M isopropylamine and 2g / L PLP, dissolved in sodium tetraborate buffer, and adjusted to pH 10.5 (40°C) with concentrated hydrochloric acid), sodium tetraborate buffer (pH 10.5), and SEQ ID NO:2 enzyme solution, so that the final concentration of the reaction system was 5g / L substrate, 20% enzyme solution (v / v), 20% solvent (v / v), 0.5g / L PLP, 1M isopropylamine, 0.025M sodium tetraborate buffer, pH 10.5. The reaction was then carried out for 24 hours with magnetic stirring at 400 rpm. After the reaction was completed, the reactor was heated to 70°C and continued to be heated for 1 hour. Then, 5mL of pure acetonitrile was added to inactivate the enzyme, and a sample was taken for HPLC detection. As a result of the detection, the yields after 24 hours of reaction in methanol, DMSO, and isopropanol systems are as follows. JPEG2025501365000041.jpg23170

[0144] Example 4: Expression of mutant enzyme library and preparation of enzyme solution for screening Colonies of the mutant enzyme library were selected from the agar plate, inoculated into a 96-well plate containing LB medium containing chloramphenicol, and cultured overnight at 30°C on a shaker. 600 When the OD of the culture reached 2-3, 20 μL was taken from the 96-well plate and inoculated into a 96-well deep well plate (400 μL TB medium per well) containing TB medium containing chloramphenicol, placed on a shaker, and cultured at 30°C. 600When the pH reached 0.6-0.8, IPTG was added as an inducer to a final concentration of 1 mM, and the mixture was placed on a shaker and expressed overnight at 30°C (18-20h). After expression was completed, the deep well plate containing the bacterial solution was centrifuged, the supernatant of the bacterial solution was removed, and wet bacterial cells were obtained. The wet bacterial cells were stored in a refrigerator at -20°C for at least 24 hours, and then 200μL of cell lysis solution (containing 1g / L lysozyme, 0.5g / L PMBS, 0.5g / L nuclease, dissolved in 0.05M sodium tetraborate buffer, pH 10.5) was added per well to the well plate containing the wet bacterial cells, and the well plate was shaken for 1h to disrupt the cells and obtain a lysis solution. The lysis solution was centrifuged, and the supernatant was transferred to a new deep well plate to obtain an enzyme solution that can be used for screening reactions.

[0145] Example 5: High-throughput screening of enzymes in Phase II 70 μL of the substrate mother solution dissolved in methanol, 40 μL of sodium tetraborate buffer (pH 10.5), and 50 μL of an isopropylamine mixture (containing 4 M isopropylamine and 2 g / L PLP, dissolved in sodium tetraborate buffer, adjusted to pH 10.5 (40 ° C) with concentrated hydrochloric acid) were added in sequence to a deep well plate, and 60 μL of the enzyme solution prepared in Example 4 was added in sequence, and the final concentrations of each component in the reaction system were 5 g / L substrate, 30% (v / v) of the enzyme solution, 35% methanol, 0.5 g / L PLP, 1 M isopropylamine, 0.025 M sodium tetraborate buffer, pH 10.5, and the well plate was placed in a thermostatic shaker at 45 ° C and reacted for 24 h. After the reaction was completed, the well plate was removed and placed in a water bath shaker at 70 ° C for 1 h, and then inactivated by adding acetonitrile at a ratio of 1:1. The sample was diluted to 2.5 g / L and injected for detection.

[0146] Example 6: DMSO:ACN mixed solvent reaction 0.5 g of the wet bacterial cells of SEQ ID NO:24 prepared in Example 2 was weighed out, and 5 mL of a cell lysis solution (containing 1 g / L lysozyme, 0.5 g / L MBS, and 0.5 g / L nuclease, dissolved in sodium tetraborate buffer, pH 10.5) was added. The cells were disrupted by shaking for 1 hour to obtain a cell lysis solution. The cell lysis solution was centrifuged, and the supernatant was collected to obtain an enzyme solution. The reactor was opened and heated to 45°C in advance. S1 dissolved in methanol, DMSO, 80% DMSO:20% ACN, 70% DMSO:30% ACN, 60% DMSO:40% ACN, and 50% DMSO:50% ACN, a reaction mixture (containing 4 M isopropylamine and 2 g / L PLP, dissolved in sodium tetraborate buffer, and adjusted to pH 10.5 (40°C) with concentrated hydrochloric acid), and the SEQ ID NO:24 enzyme solution were added to the reaction flask, resulting in a final reaction system of 50 g / L substrate, 25% (v / v) SEQ ID NO:24 enzyme solution, 50% (v / v) solvent, 0.5 g / L PLP, 1 M isopropylamine, 0.025 M sodium tetraborate buffer, pH 10.5. The reaction was then carried out for 24 hours with magnetic stirring at 400 rpm. After the reaction was completed, the reactor was heated to 70°C and continued to be heated for 1 hour. After that, 5 mL of pure acetonitrile was added to inactivate the enzyme, and samples were taken for HPLC detection. The results of detection showed that the yields after 24 h reaction in various solvent systems are as shown in the table below. JPEG2025501365000042.jpg35170

[0147] Example 7: High-throughput screening of enzymes in Phase III 70 μL of substrate mother solution dissolved in a 70% DMSO:30% ACN mixed solvent, 40 μL of sodium tetraborate buffer (pH 10.5), 50 μL of an isopropylamine mixed solution (4 M isopropylamine, containing 2 g / L PLP, dissolved in sodium tetraborate buffer, pH adjusted to 10.5 (40° C.) with concentrated hydrochloric acid), and 60 μL of the enzyme solution prepared in Example 4 were added in that order to a deep well plate, and the final concentrations of each component in the reaction system were 30 g / L substrate, 30% (v / v) of the enzyme solution, 35% DMSO:15% ACN, 0.5 g / L PLP, 1 M isopropylamine, 0.025 M sodium tetraborate buffer, pH 10.5, and the well plate was placed in a 55° C. thermostatic shaker and reacted for 24 hours. After the reaction was completed, the well plate was taken out and heated in a water bath shaker at 70°C for 1 h, then acetonitrile was added in a 1:1 ratio to inactivate the sample, and the sample was diluted to 5 g / L and injected for detection.

[0148] Example 8: Enzyme thermostability testing Preparation of enzyme mother liquor: 1.5 g of SEQ ID NO:130 cells were weighed and dissolved in 30 mL of cell lysis solution (1 g / L lysozyme, 0.5 g / L MBS, 0.5 g / L nuclease, dissolved in sodium tetraborate buffer, pH 10.5), shaken at room temperature for 1 hour, centrifuged, and the supernatant was collected for use.

[0149] Heat treatment of enzyme solution: 3 mL of the supernatant of the prepared enzyme solution was collected and heat-treated in water baths at 45°C, 55°C, and 65°C for 2 hours and 24 hours, respectively.

[0150] Detection of activity of heat-treated enzyme solution: The reactor was opened in advance, heated to 45°C, and the substrate dissolved in a mixed solvent of 70% DMSO:30% ACN, the reaction mixture [containing 4 M isopropylamine and 2 g / L PLP, dissolved in sodium tetraborate buffer, and adjusted to pH 10.5 (40°C) with concentrated hydrochloric acid], and the enzyme solution of SEQ ID NO:130 heat-treated under each condition were added to the reaction flask in order, so that the final concentration of the reaction system was 50 g / L substrate, 35% DMSO:15% ACN, 1 M isopropylamine, 0.5 g / L PLP, 0.025 M sodium tetraborate buffer, pH 10.5, and 25% (v / v) enzyme solution of SEQ ID NO:130. The reaction was carried out for 24 hours with magnetic stirring at 400 rpm. After the reaction was completed, the reactor was heated to 70°C, and the reaction flask was heated for 1 hour, and then 5 mL of pure acetonitrile was added to inactivate it. HPLC detection was performed and the enzyme activity under different heat treatment conditions is shown in the table below. JPEG2025501365000043.jpg41170

[0151] Example 9: Testing the pH tolerance of enzymes The reactor was opened in advance, and the temperature was raised to 55°C. The substrate dissolved in a mixed solvent of 70% DMSO:30% ACN, the reaction mixture (containing 4 M isopropylamine and 2 g / L PLP, dissolved in sodium tetraborate buffer, and adjusted to pH 10.5 (40°C) with concentrated hydrochloric acid), and the enzyme solution of SEQ ID NO:130 were added to the reaction flask in that order, and the final reaction concentrations were 50 g / L substrate, 35% DMSO:15% ACN, 1 M isopropylamine (pH 9.5, 10.5, 11, 11.5), 0.5 g / L PLP, 0.025 M sodium tetraborate buffer (pH 9.5, 10.5, 11, 11.5), and 25% (v / v) SEQ ID NO:130. The enzyme solution was made into NO:130 and reacted for 24 hours with magnetic stirring at 400 rpm. After the reaction was completed, the reactor was heated to 70°C and the reaction flask was heated for 1 hour, after which 5 mL of pure acetonitrile was added to inactivate it. Samples were taken and subjected to HPLC detection. The yields at pH 9.5, pH 10.5, pH 11, and pH 11.5 for 24 hours are shown in the table below. JPEG2025501365000044.jpg27170

[0152] Example 10: Optimization of reaction temperature The reactor was opened in advance, and the temperature was raised to 30°C, 45°C, 55°C, and 65°C, respectively. The substrate dissolved in a mixed solvent of 70% DMSO:30% ACN, the reaction mixture (containing 4 M isopropylamine and 2 g / L PLP, dissolved in sodium tetraborate buffer, and adjusted to pH 10.5 (40°C) with concentrated hydrochloric acid), and the enzyme solution of SEQ ID NO:130 were added to the reaction flask in that order, so that the final reaction concentrations were 50 g / L substrate, 35% DMSO:15% ACN mixed solvent, 1 M isopropylamine (pH 10.5), 0.5 g / L PLP, 0.025 M sodium tetraborate buffer (pH 10.5), and 25% (v / v) enzyme solution of SEQ ID NO:130. The reaction was carried out for 24 hours with magnetic stirring at 400 rpm. After the reaction was completed, the temperature of the reactor was raised to 70°C, and the reaction flask was heated for 1 hour, and then inactivated by adding 5 mL of pure acetonitrile. Samples were taken and subjected to HPLC detection. The yield results at 45°C, 55°C and 65°C for 24 hours are shown in the table below. JPEG2025501365000045.jpg28170

[0153] Example 11: Testing the Solvent Tolerance of Engineered Aminotransferases The reactor was opened in advance, and the temperature was raised to 55°C. The substrate dissolved in methanol, DMSO, isopropanol, and ACN, the reaction mixture (containing 4M isopropylamine and 2g / L PLP, dissolved in sodium tetraborate buffer, adjusted to pH 10.5 (40°C) with concentrated hydrochloric acid), and the SEQ ID NO:130 enzyme solution were added to the reaction flask in that order, resulting in a final reaction concentration of 50g / L substrate, 20%, 35%, 50%, and 60% methanol, DMSO, isopropanol, ACN, 1M isopropylamine (pH 10.5), 0.5g / L PLP, 0.025M sodium tetraborate buffer (pH 10.5), and 25% (v / v) SEQ ID NO:130 enzyme solution. The reaction was carried out for 24 hours with magnetic stirring at 400 rpm. After the reaction was completed, the temperature of the reactor was raised to 70°C. The reaction flask was heated for 1 hour, and then inactivated by adding 5mL of pure acetonitrile. Samples were taken and subjected to HPLC detection, and the results of the 24 hour yields under various solvent concentration conditions are shown in the table below. JPEG2025501365000046.jpg53170

[0154] Example 12: All-organic phase reactions The reactor was opened in advance, heated to 55°C, and the substrate S1 dissolved in methanol, DMSO, isopropanol, ACN, ethyl acetate, isopropyl acetate, and toluene, respectively, the isopropylamine mixture (containing 0.25mL pure water and 3.5g / L PLP), and SEQ ID NO:130 whole cells were added to the reaction flask in order, and the final reaction concentrations were 50g / L substrate, 86% methanol, DMSO, isopropanol, ACN, ethyl acetate, isopropyl acetate, toluene, 1M isopropylamine, 0.5g / L PLP, and 50g / L SEQ ID NO:130 whole cells, and the reaction was carried out for 24 hours with magnetic stirring at 400 rpm. After the reaction was completed, the reactor was heated to 70°C, and the reaction flask was heated for 1 hour, and then 5mL of pure acetonitrile was added to inactivate it. Samples were taken and subjected to HPLC detection, and the results of the yields for 24 hours under various solvent concentration conditions are shown in the table below. JPEG2025501365000047.jpg45170

[0155] Example 13: Fermentation and post-treatment A single colony of E. coli BL21(DE3) containing the desired engineered aminotransferase polypeptide expression plasmid was inoculated into 50 mL of LB broth containing 30 μg / mL chloramphenicol (5.0 g / L Yeast Extract LP0021, 10 g / L Tryptone LP0042, 10 g / L sodium chloride) and cultured at 30°C in a shaker at 250 rpm for 16 h. The OD 600 When the OD reached 3.5-4.5, the culture was removed from the shaker and immediately inoculated into a fermenter. A 1.0 L fermenter containing 0.4 L of growth medium was sterilized in a high-pressure steam sterilizer at 121 °C for 30 min. The fermenter was inoculated with the culture in the shake flask. The temperature of the fermenter was kept at 37 °C by a jacket, the fermenter was stirred at a speed of 200-800 rpm, and air was supplied into the fermenter at 0.4-0.8 L / min to maintain the dissolved oxygen concentration at 30% or higher. The pH of the culture was maintained at 7.0 by adding 25-28% v / v ammonium hydroxide. The growth of the bacteria was maintained by feeding a feed solution containing 500 g / L food grade glucose dextrose monohydrate, 12 g / L ammonium chloride, and 5 g / L magnesium sulfate heptahydrate. The OD of the culture was 0.01 g / L. 600When the pH reached 25±5, the culture temperature was lowered and maintained at 30°C, and isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM to induce the expression of aminotransferase. After fermentation for approximately 16 h, the culture was removed from the tank. The wet cells were collected by centrifugation at 8000 rpm for 10 min at 4°C in a Thermo Multifuge X3R centrifuge. The collected wet cells were either used directly in the next downstream recovery process or frozen and stored at -20°C until use. 6 g of wet cells were resuspended in 30 mL of 100 mM potassium phosphate buffer (pH 7.5) containing 250 μM pyridoxal 5'-phosphate (PLP) at 4°C. The cells were homogenized and triturated twice using a homogenizer at 800 bar to release the aminotransferase from the cells. The resulting lysate was clarified by centrifugation at 8000 rpm for 10 min at 4°C using a Thermo Multifuge X3R centrifuge. The clarified supernatant was dispensed into shallow containers, frozen at -20°C, and lyophilized to enzyme powder using a freeze dryer. The aminotransferase enzyme powder was stored frozen at -20°C.

[0156] Example 14: Aminotransferase-catalyzed reactions The water bath was started and heated to 55°C. Two 500 mL reaction flasks were fixed on iron stands and placed in the water bath to preheat. 20 g of S1, 70 mL of DMSO, and 30 mL of ACN were added to the two 500 mL reaction flasks in this order and stirred to completely dissolve S1. Then, 50mL of reaction mixture (containing 4M isopropylamine, 2g / L PLP, dissolved in sodium tetraborate buffer, adjusted to pH 10.5 (40℃) with concentrated hydrochloric acid) was added with the enzyme powder of SEQ ID NO:24 and SEQ ID NO:130 prepared in Example 13, respectively, to obtain a final reaction system of 100g / L S1, 35% DMSO:15% ACN, 1M isopropylamine (pH 10.5), 0.5g / L PLP, 0.025M sodium tetraborate buffer (pH 10.5), 20g / L SEQ ID NO:24 or SEQ ID NO:130 (numbered as reaction 1 and reaction 2, respectively). During the reaction process, the pH of the reaction system was adjusted with 6M isopropylamine aqueous solution using a real-time pH regulator, and the pH of the reaction system was maintained at pH 10.3 to pH 10.5. At 24h, 48h, 72h, and 96h, 200μL samples were taken and heated at 70℃ for 1h. Then, 200μL of pure acetonitrile was added to inactivate the reaction and HPLC detection was performed. The yields of the reactions are shown in the table below. JPEG2025501365000048.jpg39170

[0157] Example 15: Post-reaction treatment The reaction solution of Example 14 was rotary evaporated under water pump conditions of 40°C and -0.095MPa to remove isopropylamine and acetonitrile, and 2M sodium hydroxide was added to adjust the reaction system to pH 10. The reaction solution was then extracted with 100mL of ethyl acetate, the supernatant was separated, the lower aqueous phase was extracted again with 50mL of ethyl acetate, the ethyl acetate layers were combined, washed twice with 50mL of saturated saline, and separated. Next, the ethyl acetate was removed by rotary evaporation under water pump conditions of 40°C and -0.095MPa, and the ethyl acetate was recovered using a condenser. The crude product was then crystallized using a mixed solvent of ethyl acetate / n-heptane (1:2 (v / v)). In the final reaction 1, 13.1g of pure product was obtained, and the product had a total yield of 77.4%, a dr value of 79, and a purity of 98.5%. Reaction 2 gave 14.3 g of pure product with an overall yield of 84.5%, dr value >100 and purity >99%.

Claims

1. An engineered aminotransferase polypeptide, the sequence of which is the amino acid sequence set forth in SEQ ID NO:2, which, under suitable reaction conditions, is capable of converting compound S1 to compound I1.

2. The aminotransferase polypeptide of claim 1, wherein the suitable reaction conditions include a loading of 5 g / L of compound S1, a loading of 10 g / L of engineered aminotransferase polypeptide, 1.0 M isopropylamine, 0.5 g / L PLP, methanol / water or DMSO / acetonitrile / water mixed solvent, pH 10.5, and 45° C.

3. An engineered aminotransferase polypeptide, the amino acid sequence of which comprises amino acid residue differences at one or more residue positions selected from X52Y, X53T, X53K, X53F, X53E, X53H, X115G, X115E, X126L, X146Q, X183A, X183S, X183T, X190L, X190I, compared to the sequence set forth in SEQ ID NO:2, and which converts S1 to IT or L1 with superior catalytic activity, stability and / or dr value than the SEQ ID NO:2 polypeptide.

4. Corresponding SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 100, 102, 104, 106, 108, 11 4. The engineered aminotransferase polypeptide of claim 3, comprising an amino acid sequence selected from: 0, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158.

5. A polypeptide immobilized on a solid material by methods of chemical bonding or physical adsorption, the polypeptide being selected from the aminotransferase polypeptides of any one of claims 1 to 4.

6. A polynucleotide encoding the polypeptide according to any one of claims 1 to 4.

7. The polynucleotide sequence is selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 7. The polynucleotide of claim 6, which is a sequence corresponding to: 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157.

8. An expression vector comprising the polynucleotide according to claims 6 to 7.

9. The expression vector of claim 8 which is a plasmid, cosmid, phage or viral vector.

10. A host cell comprising the expression vector according to any one of claims 8 to 9, said host cell being preferably Escherichia coli.

11. 11. A method for preparing an aminotransferase polypeptide comprising culturing the host cell of claim 10 and obtaining the aminotransferase polypeptide from the culture.

12. 12. An aminotransferase catalyst selected from the culture of claim 11, an aminotransferase polypeptide-containing host cell or culture medium obtained from the culture, or a processed product using the same, wherein the product is an extract obtained from a transformed cell, an isolated product obtained by isolating or purifying the aminotransferase in the extract, or an immobilized product obtained by immobilizing an immobilized transformant cell and the extract or an isolated product of the extract.

13. A method for preparing a compound of formula I, comprising contacting a substrate of formula XI with an engineered polypeptide according to any one of claims 1 to 5 under suitable reaction conditions. (Here, R １ , R ２ , R ３ , R ４ , and R ５ The group is an optionally substituted -H, １ ~C ６ Hydrocarbyl, halogen (e.g., -F, -Cl, -Br, -I), -NO:2, -NO, -SO ２ R' or -SOR', -SR', -NR'R', -OR', -CO ２ R' or -COR', -C(O)NR', ​​-SO ２ N.H. ２ Or -SO NH ２ , -CN, CF ３ R may be ６ is C １ ~C ６ Hydrocarbyl, C １ ~C ６ Halogenated hydrocarbons, C １ ~C ６ may be a hydroxy-substituted hydrocarbon; R ７ is C １ ~C ６ Hydrocarbyl, C １ ~C ６ Halogenated hydrocarbons, C １ ~C ６ may be a hydroxy-substituted hydrocarbon; R ８ may be a CBZ protecting group, a BOC protecting group, a Fomc protecting group, a Bn protecting group, or a methoxy(ethoxy)carbonyl protecting group; each R' is independently H or C １ C ４ is selected from hydrocarbyl.

14. the product of formula I is a mixture of one or more of the chiral amine products represented by formulas II-VI; Due to the activity of the ester group of Compound I, under suitable reaction conditions, e.g., suitable temperature, pH and solvent conditions, a portion of the amine product of formula I will spontaneously undergo cyclization to form a lactam of formula VI, 14. The method of claim 13, wherein the chiral amine product of formula VI is a mixture of one or more compounds of the chiral amine products of formulas VII-X below:

15. A method for preparing a compound of formula I1, comprising contacting a substrate of formula S1 with an engineering polypeptide according to any one of claims 1 to 5 under suitable reaction conditions.

16. 。 A method for preparing a compound of formula L1, comprising contacting a substrate of formula S1 with an engineering polypeptide according to any one of claims 1 to 5 under suitable reaction conditions.

17. 17. The method of claim 16, wherein the dr value of the product (i.e., [T1+T2] / [D1+D2]) is at least 1, 2, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more.

18. The method according to any one of claims 14 to 17, wherein the reaction solvent comprises methanol, dimethylsulfoxide (DMSO), acetonitrile (ACN), dimethylformamide (DMF), methyl tert-butyl ether (MTBE), isopropyl acetate, ethanol, propanol, isopropanol (IPA), or a mixture of two or more of these solvents.

19. 18. The method of any one of claims 14 to 17, wherein the reaction conditions comprise a temperature of from 10°C to 65°C.

20. The method of any one of claims 14 to 17, wherein the reaction conditions comprise pH 7.0 to pH 11.

5.

21. The method of any one of claims 14 to 17, wherein the substrate is present at a loading of from 10 g / L to 100 g / L.

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