A transaminase mutant for synthesizing sitagliptin

Abstract

The present invention discloses a transaminase mutant SEQ ID NO: 3, which can efficiently convert (2Z)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazine-7-(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-one into sitagliptin.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme catalysis, and in particular relates to a transaminase mutant and use thereof in producing sitagliptin by an enzymatic catalytic method. Background Art

[0002] Sitagliptin, whose chemical name is 7-[(3R)-3-amino-1-oxo-4-(2,4,5-trifluorophenyl)butyl]-5,6,7,8-tetrahydro-3-trifluoromethyl-1,2,4-triazolo[4,3-a]pyrazine, was developed by Merck & Co., Inc. (Merck) in the United States. Its phosphate monohydrate is the first dipeptidyl peptidase-4 (DPP-4) inhibitor drug used for the treatment of type II diabetes, and has the advantages of good safety and low incidence of adverse reactions.

[0003] Since Sitagliptin was made public, the development and research of the synthetic route and process of the product has not been interrupted for nearly 20 years. The original research of Merck & Co., Ltd. in the United States disclosed the first synthetic route of Sitagliptin (see patent document CN1524082A), which uses a chiral source to induce chiral α-amino acids, and then produces β-amino acids through multi-step reactions to construct the chiral center of Sitagliptin. However, the reaction requires n-butyl lithium and diazomethane, and the reaction conditions are harsh, making it difficult to apply on a large scale. The synthetic routes reported in patent documents CN10219102A, CN102030683A, WO2011049344A2 and CN102485718A are also all about introducing chiral sources to synthesize Sitagliptin, but these methods have many by-products, low product yields, long preparation routes, unstable intermediates, the need for highly toxic substances, and poor atom economy, and have not been able to achieve industrial application. Genericus (UK) Ltd. published patent WO20100131025A1 in 2009, reporting a chiral induction combined with kinetic resolution method for synthesizing sitagliptin. The method uses cheap and readily available materials, but the product yield is low, resulting in its lack of economic feasibility.

[0004] At present, there are two methods for industrial production of sitagliptin, which are developed by the original Merck and its partner Codex Heath. Patent document WO2004085378A1 discloses a method for synthesizing sitagliptin by asymmetric reduction, and its key step is to use a chiral rhodium catalyst to asymmetric hydrogenate enamine to construct a chiral center, which has the advantages of simple route, fewer steps, high conversion rate and optical purity. Another industrialization route is to use Arthrobacter sp. (Arthrobacter) (R)-ω-transaminase as the research object, combined with protein structure computer-aided design, site-directed saturation mutation and full gene random mutation strategies, and finally obtain a new type of transaminase that can efficiently catalyze the synthesis of sitagliptin from sitagliptin precursor ketone (Christopher K.Savile et al.Science 329,305 (2010), patent document CN102405281A). Compared with chemical synthesis, the enzymatic conversion strategy can increase the product yield by 10-13%, increase production efficiency by 53%, reduce total waste emissions by 19%, and no heavy metals need to be added during the reaction. Currently, the bioenzymatic method has become the preferred option for synthesizing chiral pharmaceutical chemicals or chemical intermediates.

[0005] The sitagliptin phosphate patent protected by CN1832949A will expire in 2024. Currently, there is an urgent need to develop a domestic bio-enzymatic route for synthesizing sitagliptin. Summary of the invention

[0006] The inventors have focused their research on the enzymatic synthesis of sitagliptin on the improvement of transaminase. The economic efficiency of the enzymatic synthesis of sitagliptin is improved by increasing the enzyme activity of transaminase in catalyzing the synthesis of sitagliptin (compound IV) from sitagliptin precursor ketone (compound III) and / or the stereoselectivity of the enzyme and / or the tolerance of the enzyme to substrate products.

[0007]

[0008] To this end, the inventors have extensively screened the microbial sources of transaminases, and used the transaminases from Arthrobacter sp. KNK168 as the object of investigation and transformation, and constructed a computer-simulated protein 3D model using bioinformatics methods. After screening many sites, a transaminase with significantly improved enzyme activity was constructed, which can efficiently catalyze the transamination reaction of the sitagliptin precursor ketone ((2Z)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazine-7-(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-one) to obtain sitagliptin with high optical purity. Specifically, the present invention includes the following technical solutions:

[0009] A transaminase mutant, which is a mutant formed by mutations in five or more, preferably ten or more, up to forty-six of the following sites in the wild-type transaminase amino acid sequence SEQ ID NO:1 derived from Arthrobacter sp. KNK168: F3, S8, E9, I10, V11, T22, Y23, D25, L28, D29, P30, A31, P33, I55, Y60, L61, H62, V65, V69, H71, N74, F88, I96, F122, S124, S126, I127, Y131, G136, K142, Y150, W156, D165, V199, H203, A209, L211, G215, S223, T268, E271, L273, T282, A284, S321, S323, the aminotransferase mutant can be used to convert sitagliptin proketone into sitagliptin.

[0010] The above term "mutation" includes but is not limited to substitution, deletion or addition of amino acids.

[0011] Preferably, the above mutation is selected from the group consisting of F3K, S8P, E9S, I10N, V11M, T22K, Y23F, D25E, L28G, D29S, P30A, A31D, P33N, I55V, Y60F, L61Y, H62T, V65A, V69T, H71S, N74H, F88Y, I96L, F122M, S124T, S126T, I127F, Y131L, G136F, K142N, Y150T, W156T, D165N, V199I, H203N, A209L, L211V, G215C, S223P, T268S, E271D, L273Y, T282S, A284G, S321P, S323Q.

[0012] In a preferred embodiment, the aminotransferase mutant is SEQ ID NO: 1 (F3K, S8P, E9S, I10N, V11M, T22K, Y23F, D25E, L28G, D29S, P30A, A31D, P33N, I55V, Y60F, L61Y, H62T, V65A, V69T, H71S, N74H, F88Y, I96L, F122M, S124T, S12 6T, I127F, Y131L, G136F, K142N, Y150T, W156T, D165N, V199I, H203N, A209L, L211V, G215C, S223P, T268S, E271D, L273Y, T282S, A284G, S321P, S323Q) mutant, relative to SEQ ID NO: 1, 46 amino acid substitutions occurred, and its amino acid sequence is SEQ ID NO: 3:

[0013] MAKSADTPSNMYTHDTGLDYIKFSEYEGSADNNLAGGAAWIEGAFVPPSEARISVFDQGFYTSDATYTTFSVWHGNAFRLDDHIERLYSNAESMRLIPPLTQDEVKEIALELVAKTELREAMVTVTFTRGLSSTPFERDITNHRPQVYMTAVPYQTIVPFDRIRNGV HAMVAQSVRRTPRSSIDPQVKNFQWGDLIRAIQETNDRGFELPVLLDCDGLLAEGPGFNVVVIKDGVVRSPGRAALPGITRKTVLEIAESLGHEAILADISLADLYDADEVLGCSTGGGVWPFVSVDGNPISDGVPGPVTQSIIRRYWELNVEPSQLLTPVQY(SEQ ID NO:3).

[0014] The second aspect of the present invention provides a gene encoding the above-mentioned aminotransferase mutant.

[0015] Preferably, the nucleotide sequence of the gene encoding the transaminase mutant SEQ ID NO:3 is SEQ ID NO:4.

[0016] The third aspect of the present invention provides a plasmid comprising the above gene, such as SEQ ID NO: 4. The plasmid comprises a vector for expressing the above gene, such as SEQ ID NO: 4, preferably a PET series vector, such as pET22b, pET24a, pET28a, etc., but is not limited thereto.

[0017] The fourth aspect of the present invention provides a microorganism for expressing the above aminotransferase mutant. For example, the microorganism is a transformant transformed with the above plasmid.

[0018] The above-mentioned microorganism can be selected from Escherichia coli, Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, preferably Escherichia coli, more preferably Escherichia coli BL21 (DE3).

[0019] The fifth aspect of the present invention provides use of the above-mentioned transaminase mutant or the above-mentioned microorganism in the production of sitagliptin.

[0020] Specifically, in the production of sitagliptin, the sitagliptin precursor ketone ((2Z)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazine-7-(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-one) is used as a substrate raw material, and the above-mentioned aminotransferase mutant or microorganism is used as a catalyst to catalyze the 2-position amination reaction to obtain sitagliptin.

[0021] Preferably, the reaction system comprises pyridoxal phosphate as a coenzyme.

[0022] The above reaction system may further comprise o-phenylenediamine dihydrochloride, or isopropylamine and / or triethanolamine as an amino group donor.

[0023] Since the above transaminase mutant has high thermal stability, the enzyme reaction temperature during the catalytic production of sitagliptin can be 40-50°C, preferably around 45°C.

[0024] The pH value of the reaction system is 8.0-9.0, preferably 8.5.

[0025] In order to speed up the reaction, an organic solvent may be added to the reaction system as a co-solvent for the substrate, such as DMSO. The concentration of DMSO in the reaction system may be 10%-60%, preferably 60%.

[0026] Furthermore, the substrate concentration may be 20-120 g / L, preferably about 100 g / L.

[0027] The transaminase mutant SEQ ID NO: 3 screened out through 56 rounds of mutations in the present invention has high stereoselectivity and significantly improved enzyme activity compared with the wild-type transaminase SEQ ID NO: 1. The ee value of the sitagliptin (compound IV) product synthesized by catalysis is as high as 99.95% or more, and it has feasibility for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is an HPLC chart of the retention time of sitagliptin and sitagliptin proketone detected by HPLC in the examples. DETAILED DESCRIPTION

[0029] In order to screen for a transaminase with high enzyme activity and the ability to catalyze the production of sitagliptin from the vitamin sitagliptin precursor ketone ((2Z)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7-(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-one) with high stereoselectivity, the inventors screened a large number of microbial sources through literature retrieval and database retrieval analysis, and selected Arthrobacter sp. KNK168 (see Microbial synthesis of chiral amines by (R)-specific transamination with Arthrobacter sp. KNK168. A. Iwasaki, et al., Applied microbiology and biotechnology.2006,v.69,no.5,p499-505.DOI:10.1007 / s00253-005-0002-1.A novel transaminase,(R)-amine:pyruvate aminotransferase,from Arthrobacter sp.KNK168(FERM BP-5228):purification,characterization,and gene cloning.A.Iwasaki,et al.,Appliedmicrobiology and biotechnology.2012,v.93,no.4,p1563-1573.) derived wild-type transaminase, the amino acid sequence of which is SEQ ID NO:1:

[0030] MAFSADTSEIVYTHDTGLDYITYSDYELDPANPLAGGAAWIEGAFVPPSEARISIFDQGYLHSDVTYTVFHVWNGNAFRLDDHIERLFSNAESMRIIPPLTQDEVKEIALELVAKTELREAFVSVSITRGYSSTPGERDITKHRPQVYMYAVPYQWIVPFDRIRDGV HAMVAQSVRRTPRSSIDPQVKNFQWGDLIRAVQETHDRGFEAPLLLDGDGLLAEGSGFNVVVIKDGVVRSPGRAALPGITRKTVLEIAESLGHEAILADITLAELLDADEVLGCTTAGGVWPFVSVDGNPISDGVPGPVTQSIIRRYWELNVESSSLLTPVQY(SEQ ID NO:1).

[0031] In this article, the terms "wild type", "wild enzyme", and "wild-type enzyme" have the same meaning, and all refer to the transaminase with the amino acid sequence of SEQ ID NO: 1. Sometimes for the sake of convenience, the wild-type transaminase and its mutants such as SEQ ID NO: 3 may be collectively referred to as "transaminase" in this article.

[0032] The amino acid number of the aminotransferase mutant SEQ ID NO: 3 of the present invention is 330, and the structure is clear, so those skilled in the art can easily obtain its encoding gene, expression cassettes and plasmids containing these genes, and transformants containing the plasmids. These genes, expression cassettes, plasmids, and transformants can be obtained by genetic engineering construction methods well known to those skilled in the art.

[0033] In order to optimally express transaminase in the most commonly used Escherichia coli in genetic engineering, the expression genes of these enzymes can be codon optimized. Codon optimization is a technology that can be used to maximize protein expression in organisms by increasing the translation efficiency of the gene of interest. Different organisms usually show the special preference for one of some codons of the same amino acid being encoded due to mutation tendency and natural selection. For example, in fast-growing microorganisms such as Escherichia coli, the optimization codon reflects the composition of its respective genome tRNA library. Therefore, in fast-growing microorganisms, the low-frequency codons of amino acids can be replaced with codons for the same amino acids but with high frequency. Therefore, the expression of the optimized DNA sequence is improved in fast-growing microorganisms.

[0034] For example, to express the transaminase in E. coli, the gene encoding the codon-optimized wild transaminase SEQ ID NO: 1 can be SEQ ID NO: 2; the gene encoding the transaminase mutant SEQ ID NO: 3 can be SEQ ID NO: 4.

[0035] The transformant host can be any microorganism suitable for expressing transaminase, including bacteria and fungi. Preferably, the microorganism is Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, or Escherichia coli, preferably Escherichia coli, more preferably Escherichia coli BL21 (DE3).

[0036] When used as a biocatalyst for the production of sitagliptin, the transaminase of the present invention can be in the form of an enzyme or a bacterial cell. The enzyme forms include free enzymes, immobilized enzymes, including purified enzymes, crude enzymes, fermentation broths, enzymes fixed on carriers, etc.; the bacterial cells include living bacterial cells, dead bacterial cells, immobilized bacterial cells, etc.

[0037] When microorganisms such as Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae or Escherichia coli are no longer fermented and proliferated, but used for enzyme catalysis, they are themselves a natural immobilized enzyme, and do not need to be crushed or even extracted and purified, and can be used as an enzyme preparation for catalytic reactions. Since both the reaction substrate and the reaction product are small molecules, they can easily pass through the biological barrier of the bacteria - the cell membrane, so there is no need to crush the bacteria, which is economically beneficial.

[0038] The present invention is further described in detail below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0039] Example

[0040] This article involves the addition amount, content and concentration of various substances, and the percentages mentioned here refer to the mass percentages unless otherwise specified.

[0041] Materials and methods

[0042] The whole gene synthesis, primer synthesis and sequencing in the examples were all completed by Suzhou Genewise Biotechnology Co., Ltd.

[0043] The molecular biology experiments in the embodiments include plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, culture medium preparation, etc., and are mainly carried out with reference to Molecular Cloning Experiment Guide (3rd edition), edited by J. Sambrook and DW Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002). If necessary, the specific experimental conditions can be determined by simple experiments.

[0044] PCR amplification experiments were performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. If necessary, adjustments could be made through simple experiments.

[0045] LB medium: 10g / L tryptone, 5g / L yeast extract, 10g / L sodium chloride, pH 7.2. (LB solid medium plus 20g / L agar powder.)

[0046] TB medium: 24g / L yeast extract, 12g / L tryptone, 16.43g / L K2HPO4.3H2O, 2.31g / L KH2PO4, 5g / L glycerol, pH 7.0-7.5. (TB solid medium plus 20g / L agar powder.)

[0047] HPLC detection of substrate and product chromatographic conditions: chromatographic column C18 (4.6×250mm, 5μm); mobile phase A is 50mM potassium dihydrogen phosphate solution (pH3.00), mobile phase B is acetonitrile, mobile phase A: mobile phase B = 1:1; flow rate is 1.0mL / min; detection wavelength is 254nm.

[0048] HPLC chiral detection chromatographic conditions: chromatographic column CHIRALPAK AD-H (4.6×250 mm, 5 μm); mobile phase n-hexane:ethanol:triethylamine (40:60:0.1); flow rate 0.7 mL / min, detection wavelength 268 nm.

[0049] It should be noted that, for the sake of convenience of description, in the embodiments, the strain number, plasmid number, enzyme number, and enzyme encoding gene number may share one number, which is easily understood by those skilled in the art, that is, the same number can refer to different biological forms in different environments.

[0050] Example 1: Construction of wild-type transaminase gene recombinant Escherichia coli

[0051] For the wild enzyme SEQ ID NO:1, codon optimization was performed according to the codon preference of Escherichia coli to obtain the coding gene SEQ ID NO:2. Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to synthesize SEQ ID NO:2 and cloned into the NcoI and BamHI sites of plasmid pET28a to obtain plasmid pET-ATA1.

[0052] The recombinant plasmid pET-ATA1 was transformed into the expression host Escherichia coli BL21 (DE3) by electroporation to obtain the recombinant Escherichia coli EcATA1 expressing the wild transaminase.

[0053] Example 2: Construction of random mutation point libraries in the first and second rounds and high-throughput screening

[0054] 2.1 Construction of random mutation point library by error-prone PCR

[0055] The random mutant library was constructed using error-prone PCR technology with plasmid pET-ATA1 as template.

[0056] The following primer pair ATA-5 / ATA-3 was designed:

[0057] Forward primer ATA-5: 5'-CTTTAAGAAGGAGATATACCATG-3',

[0058] Reverse primer ATA-3: 5′-GAGCTCGAATTCGGATCCTTA-3′.

[0059] Using plasmid pET-ATA1 as a template, PCR amplification was performed to obtain a transaminase mutant DNA sequence of approximately 1.0 kb.

[0060] The 50 μL error-prone PCR reaction system included: 10 ng plasmid (pET-ATA1) template, 50 pmol of a pair of primers ATA-5 and ATA-3, 1× Taq buffer, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP, 1 mM dTTP, 7 mM MgCl2, (0 mM, 0.05 mM, 0.1 mM, 0.15 mM, 0.2 mM) MnCl2, and 2.5 units of Taq enzyme (Takara).

[0061] The PCR reaction conditions were: 95°C for 5 min; 94°C for 30 s, 55°C for 30 s, 72°C for 2 min / kbp, 30 cycles; 72°C for 10 min.

[0062] The PCR product was electrophoresed and recovered by gel (Axygen DNA gel recovery kit AP-GX-50). Using plasmid pET-ATA1 as template and the above-mentioned recovered product (random mutation fragment) of about 1.0 kb as large primer, MegaPrimer PCR was performed with KOD-plus DNA polymerase: 94℃5min; 98℃10s, 60℃30s, 68℃2min / kb, 25 cycles; 68℃10min. The plasmid template was digested with DpnI restriction endonuclease (Thermo Company) and electroporated into E. coli BL21 (DE3), and more than 10 4 A random mutation library of 10 clones.

[0063] 2.2 High-throughput screening of mutant libraries

[0064] Pick a single colony from the random mutation library to a 96-well plate (each well contains 110 μL of liquid LB-Kan medium), incubate at 37°C and 400 rpm for 5 hours, then take out 60 μL of bacterial solution from each well to a 96-well deep-well plate (each well contains 240 μL of liquid TB-Kan-0.2 mM IPTG), and incubate at 25°C and 400 rpm for 12 to 16 hours. Centrifuge at 4°C and 4000 rpm for 10 minutes to collect the bacteria, and remove the supernatant culture solution. Subsequently, wash the bacteria with precooled saline, centrifuge at 4°C and 4000 rpm for 10 minutes to collect the bacteria, and remove the supernatant. Add 200 μL of enzyme reaction solution (70 mM triethanolamine, 70 mM o-phenylenediamine dihydrochloride, 0.5 g / L pyridoxal phosphate, 5 g / L compound III, 10% dimethyl sulfoxide (DMSO)) to each well, and adjust the pH to 8.5 for reaction. Resuspend the cells, incubate at 45°C and 250 rpm for 1 to 5 h, and detect the value at a wavelength of 475 nm.

[0065] Select strains with significantly improved activity for nucleic acid sequencing to determine the amino acid mutation sites. At the same time, HPLC is used to detect the ee values ​​of the reaction products of these strains. The strains with product ee values ​​greater than 99.95% and the highest improvement in enzyme activity are selected as the starting strains for the next round of random mutant library construction. Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to perform genome sequencing and comparison on the strains with the highest improvement in enzyme activity to determine the changes in their amino acid sequences. Repeat the establishment of the random mutation library and high-throughput reaction screening with compound III as the substrate. The screening results are shown in Table 1.

[0066] Table 1. Results of high-throughput screening of random mutation libraries from round 1 to round 2

[0067]

[0068] Note: “-” means no reaction occurs; “+” means the difference of the absorbance value of the reaction relative to the starting strain is greater than 0 and less than 0.05; “++” means the difference of the absorbance value of the reaction relative to the starting strain is greater than 0.05 and less than 0.1; “+++” means the difference of the absorbance value of the reaction relative to the starting strain is greater than 0.1 and less than 0.2; “++++” means the difference of the absorbance value of the reaction relative to the starting strain is greater than 0.2.

[0069] Example 3: Construction of random mutation point library from round 3 to round 14 and high-throughput screening

[0070] 3.1 Construction of random mutation point library by error-prone PCR

[0071] Referring to the method of Example 2, a random mutation point library of transaminase was constructed to obtain about 10 4 A clonal strain.

[0072] 3.2 High-throughput screening of EcATA5-based mutant library

[0073] Pick a single colony from the mutant library to a 96-well plate (each well contains 110 μL of liquid LB-Kan medium), incubate at 37°C and 400 rpm for 5 hours, then take out 60 μL of bacterial solution from each well to a 96-well deep-well plate (each well contains 240 μL of liquid TB-Kan-0.2 mM IPTG), and incubate at 25°C and 400 rpm for 12 to 16 hours. Centrifuge at 4°C and 4000 rpm for 10 minutes to collect the cells, and remove the supernatant culture medium. Subsequently, wash the cells with pre-cooled saline, centrifuge at 4°C and 4000 rpm for 10 minutes to collect the cells, and remove the supernatant. Add 200 μL of enzyme reaction solution (70 mM triethanolamine, 70 mM o-phenylenediamine dihydrochloride, 0.5 g / L pyridoxal phosphate, 10 g / L compound III, 10% dimethyl sulfoxide (DMSO) to each well, adjust the pH to 8.5 for reaction. Resuspend the cells, incubate at 45°C, 250 rpm for 5 hours, and detect the wavelength value of 475 nm.

[0074] 3.3 Select strains with significantly improved vitality for nucleic acid sequencing to determine the amino acid mutation sites, and detect the ee values ​​of the reaction products of these strains by HPLC. Select strains with product ee values ​​greater than 99.95% and the highest vitality improvement as the starting strains for the next round of random mutant library construction, and repeat the random mutation library construction and high-throughput reaction screening with compound III as the substrate. The screening results are shown in Table 2.

[0075] Table 2. Results of high-throughput screening of random mutation libraries from rounds 3 to 14

[0076]

[0077]

[0078] Note: “-” means no reaction occurs; “+” means that the percentage of enzyme activity increased relative to the respective starting strain is greater than 0% and less than or equal to 50%; “++” means that the percentage of enzyme activity increased relative to the respective starting strain is greater than 50% and less than or equal to 100%; “+++” means that the percentage of enzyme activity increased relative to the respective starting strain is greater than 100% and less than or equal to 200%; “++++” means that the percentage of enzyme activity increased relative to the respective starting strain is greater than 200%.

[0079] Example 4: Construction of random mutation point library from rounds 15 to 17 and high-throughput screening

[0080] 1. Construction of random mutation point library by error-prone PCR

[0081] The random mutation point library of transaminase was constructed according to the method of Example 2.

[0082] 2. High-throughput screening of mutant libraries based on EcATA30

[0083] Pick a single colony to a 96-well plate (each well contains 110 μL of liquid LB-Kan medium), incubate at 37°C and 400 rpm for 5 hours, then take out 60 μL of bacterial solution from each well to a 96-well deep-well plate (each well contains 240 μL of liquid TB-Kan-0.2 mM IPTG), and incubate at 25°C and 400 rpm for 12 to 16 hours. Centrifuge at 4°C and 4000 rpm for 10 minutes to collect the bacteria, and remove the supernatant culture medium. Subsequently, wash the bacteria with pre-cooled saline, centrifuge at 4°C and 4000 rpm for 10 minutes to collect the bacteria, and remove the supernatant. Add 200 μL of enzyme reaction solution (70 mM triethanolamine, 70 mM o-phenylenediamine dihydrochloride, 0.5 g / L pyridoxal phosphate, 20 g / L compound III, 50% dimethyl sulfoxide (DMSO) to each well, adjust the pH to 8.5 for reaction. Resuspend the cells, incubate at 45°C, 250 rpm for 5 hours, and detect the wavelength value of 475 nm.

[0084] 3. Select strains with significantly improved vitality for nucleic acid sequencing to determine the amino acid mutation sites, and detect the ee values ​​of the reaction products of these strains by HPLC. Select strains with product ee values ​​greater than 99.95% and the highest vitality improvement as the starting strains for the next round of random mutant library construction, and repeat the random mutation library construction and high-throughput reaction screening with compound III as the substrate. The screening results are shown in Table 3.

[0085] Table 3. Results of high-throughput screening of random mutation libraries from rounds 15 to 17

[0086]

[0087] Note: “-” means no reaction occurs; “+” means that the percentage of enzyme activity increased relative to the respective starting strain is greater than 0% and less than or equal to 50%; “++” means that the percentage of enzyme activity increased relative to the respective starting strain is greater than 50% and less than or equal to 100%; “+++” means that the percentage of enzyme activity increased relative to the respective starting strain is greater than 100% and less than or equal to 200%; “++++” means that the percentage of enzyme activity increased relative to the respective starting strain is greater than 200%.

[0088] Example 5: Construction and screening of transaminase site-directed saturation mutation library based on EcATA35

[0089] 5.1 Construction of site-directed saturation mutation library of amino acids 3, 6, 7, 9, 29, 32, 151, 156, 157, 158, 159, 283, 285 of mutant ATA35

[0090] Using plasmid pET-ATA35 as a template, the primer pairs shown in Table 4 were designed for circular PCR amplification.

[0091] The 50 μL PCR reaction system includes: 10 ng template, 10 pmol primer pair, 1xKODplus buffer, 0.2 mM dNTP, 1.5 mM MgSO4, and 1 unit of KOD-plus DNA polymerase.

[0092] The PCR reaction conditions were: 95°C for 3 min; 98°C for 10 s, 57°C for 30 s, 68°C for 2 min / kbp, 30 cycles; 68°C for 10 min.

[0093] 50 μl of amplified PCR was directly added with 1 μl of DpnI restriction endonuclease (Thermo Company) to digest the plasmid template, and then electroporated into E. coli BL21 (DE3) to obtain more than 10 3 A random mutation library of 10 clones.

[0094] Table 4. List of primers related to site-directed saturation mutagenesis library

[0095]

[0096]

[0097] Note: N stands for A / T / C / G.

[0098] 5.2 High-throughput screening of saturation mutant libraries

[0099] Pick a single colony to a 96-well plate (each well contains 110 μL of liquid LB-Kan medium), incubate at 37°C and 400 rpm for 5 hours, then take out 60 μL of bacterial solution from each well to a 96-well deep-well plate (each well contains 240 μL of liquid TB-Kan-0.2 mM IPTG), and incubate at 25°C and 400 rpm for 12 to 16 hours. Centrifuge at 4°C and 4000 rpm for 10 minutes to collect the bacteria, and remove the supernatant culture medium. Subsequently, wash the bacteria with pre-cooled saline, centrifuge at 4°C and 4000 rpm for 10 minutes to collect the bacteria, and remove the supernatant. 200 μL of enzyme reaction solution (100 mM triethanolamine, 1 M isopropylamine, 0.5 g / L pyridoxal phosphate, 50 g / L compound III, 50% dimethyl sulfoxide (DMSO), adjusted to pH 8.5) was added to each well to resuspend the cells, and the mixture was incubated at 45°C and 250 rpm for 3 hours to react. Samples were taken and the product amount was determined by HPLC. The peak times of compound III and compound IV were as shown in Figure 2. Figure 1 The enzyme activity of the mutant and the R-type optical purity (ee value) of the product are shown in Table 5.

[0100] Table 5. Results of high-throughput screening of single-site saturation mutant library based on EcATA35

[0101]

[0102]

[0103] Note: Enzyme activity is defined as the amount of enzyme required to catalyze the substrate to produce 1 μmol of R-type product per hour at 45°C and pH 8.5, which is defined as one enzyme activity unit (U).

[0104] As can be seen from Table 5, the enzyme activity of strain EcATA41 is relatively the highest, and further mutations were made to it.

[0105] Example 6: Construction of double-site mutants based on EcATA35

[0106] Using plasmid pET-ATA41 as a template, the primer pair W156T-5 / W156T-3 was designed for PCR amplification to obtain the W156T mutant, and the PCR product was a plasmid of about 6.3 kb loaded with the transaminase mutant ATA48 (ATA41-W156T); the primer pair G285V-5 / G285V-3 was designed for PCR amplification to obtain the G285V mutant, and the PCR product was a plasmid of about 6.3 kb loaded with the transaminase mutant ATA49 (ATA41-G285V). The primer sequences are shown in Table 6.

[0107] The 50 μL PCR reaction system includes: 10 ng template, 10 pmol primer pair, 1xKOD plus buffer, 0.2 mM dNTP, 1.5 mM MgSO4, and 1 unit of KOD-plus DNA polymerase.

[0108] The PCR reaction conditions were: 95°C for 3 min; 98°C for 10 s, 57°C for 30 s, 68°C for 2 min / kbp, 30 cycles; 68°C for 10 min.

[0109] The plasmid template was digested with DpnI restriction endonuclease (Thermo Company) and transformed into E. coli BL21 (DE3) by electroporation to obtain the strain.

[0110] Table 6. List of primers related to targeted mutagenesis

[0111]

[0112] Example 7: Synthesis of sitagliptin based on single-site and double-site mutants of EcATA35

[0113] 7.1 Expression of recombinant protein and preparation of enzyme reaction system

[0114] Pick a single colony of genetically engineered bacteria into 5 mL of liquid LB medium containing Kan and culture it overnight at 37°C and 220 rpm. The next day, transfer the inoculum to a shake flask containing 100 mL of liquid TB medium at a volume concentration of 1 v / v%, and culture it at 37°C and 220 rpm until the OD 600nm When the pH value is 0.6-0.8, the inducer IPTG (final concentration 0.2mM) is added, and then the induction culture is carried out at 25°C and 220rpm for 15h. The bacterial cells are collected by centrifugation at 4°C and 4000rpm, the supernatant culture solution is removed, and the cells are washed with physiological saline, and the cells are collected by centrifugation at 4°C and 4000rpm, and the supernatant is removed. Subsequently, the cells are resuspended with a triethanolamine-hydrochloric acid solution with a pH value of 8.5. The shake flask is horizontally 50mL whole-cell catalytic reaction, and the system includes: 100mM triethanolamine, 1M isopropylamine, 0.5g / L pyridoxal phosphate, 50g / L compound III (dissolved in DMSO before the enzymatic reaction and added to the reaction system in batches), 60% dimethyl sulfoxide (DMSO), 2g cell mass, pH value 8.5 (pH value adjusted with concentrated hydrochloric acid or isopropylamine), and incubated at 45°C and 300rpm for 15h.

[0115] 7.2 Results of enzymatic asymmetric synthesis of sitagliptin

[0116] After the enzyme reaction is completed, centrifuge at 6000rpm for 10min and take the supernatant. Adjust the pH to 2-3 with concentrated hydrochloric acid supernatant, take another 50mL of 1M HCL and add it to the precipitate in the previous step, stir for 5min, collect the supernatant by centrifugation, combine the two supernatants, maintain 1h of stirring and circulating heating at 45℃, and then cool to room temperature. Add 25ml of isopropanol (IPA), stir evenly, add 25mL of isopropyl acetate (IPAc), stir evenly, adjust the pH to 11 with 10M NaOH, and then stir for another 5min. Add 50mL of IPAc / IPA mixed solution with a volume ratio of 80:20 to the solution, stir at room temperature for 5min, centrifuge at 6000rpm for 10min, and take the upper layer of liquid. Add 30mL of saturated saline to the upper layer of liquid, stir at room temperature for 5min, centrifuge at 6000rpm for 10min, dilute to a suitable concentration and detect by HPLC, the results are shown in Table 7.

[0117] Table 7. Enzyme activities of single-site and double-site mutants of EcATA35

[0118]

[0119]

[0120] As can be seen from Table 7, the enzyme activity of strain EcATA48 is relatively the highest, and further mutations were made to it.

[0121] Example 8: Construction of single-site, double-site, and triple-site mutants based on EcATA48

[0122] Using plasmid pET-ATA48 as a template, a primer pair F3K-5 / F3K-3 was designed for PCR amplification to obtain the F3K mutant, and the PCR product was a plasmid of approximately 6.3 kb loaded with the transaminase mutant ATA50 (ATA48-F3K); a primer pair E9S-5 / E9S-3 was designed for PCR amplification to obtain the E9S mutant, and the PCR product was a plasmid of approximately 6.3 kb loaded with the transaminase mutant ATA51 (ATA48-E9S); a primer pair G285V-5 / G285V-3 was designed for PCR amplification to obtain the G285V mutant, and the PCR product was a plasmid of approximately 6.3 kb loaded with the transaminase mutant ATA52 (ATA48-G285V). Using plasmid pET-ATA52 as a template, primer pair F3K-5 / F3K-3 was designed for PCR amplification to obtain the F3K mutant. The PCR product was a plasmid of approximately 6.3 kb loaded with the transaminase mutant ATA53 (ATA52-F3K); primer pair E9S-5 / E9S-3 was designed for PCR amplification to obtain the E9S mutant. The PCR product was a plasmid of approximately 6.3 kb loaded with the transaminase mutant ATA54 (ATA52-E9S). Using plasmid pET-ATA50 as a template, the primer pair E9S-52 / E9S-32 was designed for PCR amplification to obtain the E9S mutant, and the PCR product was a plasmid of about 6.3 kb loaded with the transaminase mutant ATA55 (ATA50-E9S). Using plasmid pET-ATA55 as a template, the primer pair G285V-5 / G285V-3 was designed for PCR amplification to obtain the G285V mutant, and the PCR product was a plasmid of about 6.3 kb loaded with the transaminase mutant ATA56 (ATA55-G285V). The primer sequences are shown in Table 8.

[0123] The 50 μL PCR reaction system includes: 10 ng template, 10 pmol primer pair, 1xKOD plus buffer, 0.2 mM dNTP, 1.5 mM MgSO4, and 1 unit of KOD-plus DNA polymerase.

[0124] The PCR reaction conditions were: 95°C for 3 min; 98°C for 10 s, 57°C for 30 s, 68°C for 2 min / kbp, 30 cycles; 68°C for 10 min.

[0125] The plasmid template was digested with DpnI restriction endonuclease (Thermo Company) and transformed into E. coli BL21 (DE3) by electroporation to obtain the strain.

[0126] Table 8. List of primers related to targeted mutagenesis

[0127]

[0128]

[0129] Example 9: Synthesis of sitagliptin based on single-site, double-site, and triple-site mutants of EcATA48

[0130] The expression of the recombinant protein and the preparation of the enzyme reaction system were performed with reference to Example 7, and the reaction was carried out. The results are shown in Table 9.

[0131] Table 9. Enzyme activities of single-site and double-site mutants of EcATA48

[0132] Strain No. Transaminase number Mutation site Enzyme activity (U) ee value EcATA48 ATA48 / 191.29 >99.95％ EcATA50 ATA50 F3K 204.68 >99.95％ EcATA51 ATA51 E9S 200.85 >99.95％ EcATA52 ATA52 G285V 187.46 >99.95％ EcATA53 ATA53 G285V、F3K 195.12 >99.95％ EcATA54 ATA54 G285V、E9S 198.94 >99.95％ EcATA55 ATA55 F3K、E9S 216.16 >99.95％ EcATA56 ATA56 F3K, E9S, G285V 202.77 >99.95％

[0133] As can be seen from Table 7, the enzyme activity of strain EcATA55 is relatively the highest. The amino acid sequence of the transaminase mutant ATA55 is SEQ ID NO:3, and the corresponding codon-optimized nucleic acid sequence of the encoding gene is SEQ ID NO:4.

[0134] Example 10: Use of transaminase mutant ATA55 in the synthesis of sitagliptin

[0135] In a 1L reaction system, EcATA55 whole cells expressing the aminotransferase mutant SEQ ID NO: 3 were used to catalyze the asymmetric synthesis of sitagliptin from the precursor ketone. The total reaction system included 100mM triethanolamine, 1M isopropylamine, 0.5g / L pyridoxal phosphate, 100g / L compound III, 25g cells (wet weight), and the volume ratio of the aqueous phase to DMSO was 4:6. The initial reaction system contained 300mL DMSO, 100g compound III was dissolved in 300mL DMSO, and added to the reaction system at a flow rate of 1mL / min. The pH value of the reaction system was controlled at about 8.5 using hydrochloric acid or isopropylamine. The reaction was completed after 8-12h.

[0136] HPLC test results showed that after 12 hours of reaction, the molar yield of the product exceeded 95% and the ee value of the product was greater than 99.95%.

[0137] In summary, the present invention uses o-phenylenediamine dihydrochloride and isopropylamine as amino donors for multiple rounds of mutation of the wild-type transaminase SEQ ID NO: 1, and the screened transaminase mutant SEQ ID NO: 3 has been greatly improved compared with the wild enzyme. The conversion rate of the catalytic reaction of the sitagliptin precursor ketone is high, the synthesized sitagliptin has high optical activity, and the ee value is as high as 99.95% or more, which has the feasibility of industrial application. Sequence Listing <110> Shanghai Banglin Biotechnology Co., Ltd. <120> A transaminase mutant for synthesizing sitagliptin <130> SHPI2110376 <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 330 <212> PRT <213> Arthrobacter sp. KNK168 <400> 1 Met Ala Phe Ser Ala Asp Thr Ser Glu Ile Val Tyr Thr His Asp Thr 1 5 10 15 Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn 20 25 30 Pro Leu Ala Gly Gly Ala Ala Trp Ile Glu Gly Ala Phe Val Pro Pro 35 40 45 Ser Glu Ala Arg Ile Ser Ile Phe Asp Gln Gly Tyr Leu His Ser Asp 50 55 60 Val Thr Tyr Thr Val Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu 65 70 75 80 Asp Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Met Arg Ile 85 90 95 Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu 100 105 110 Val Ala Lys Thr Glu Leu Arg Glu Ala Phe Val Ser Val Ser Ile Thr 115 120 125 Arg Gly Tyr Ser Ser Thr Pro Gly Glu Arg Asp Ile Thr Lys His Arg 130 135 140 Pro Gln Val Tyr Met Tyr Ala Val Pro Tyr Gln Trp Ile Val Pro Phe 145 150 155 160 Asp Arg Ile Arg Asp Gly Val His Ala Met Val Ala Gln Ser Val Arg 165 170 175 Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp 180 185 190 Gly Asp Leu Ile Arg Ala Val Gln Glu Thr His Asp Arg Gly Phe Glu 195 200 205 Ala Pro Leu Leu Leu Asp Gly Asp Gly Leu Leu Ala Glu Gly Ser Gly 210 215 220 Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg 225 230 235 240 Ala Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu 245 250 255 Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Leu Ala Glu Leu 260 265 270 Leu Asp Ala Asp Glu Val Leu Gly Cys Thr Thr Ala Gly Gly Val Trp 275 280 285 Pro Phe Val Ser Val Asp Gly Asn Pro Ile Ser Asp Gly Val Pro Gly 290 295 300 Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu 305 310 315 320 Thr Pro Val Gln Tyr Free Mp3 Download 325 330 <210> 2 <211> 993 <212> DNA <213> Container() <400> 2 atggcgttta gcgcggatac gtctgaaatt gtgtataccc atgataccgg cctggattat attack gcgattatga attackccg gccaaccctc tggcgggcgg cgcggcgtgg 120 attgaaggcg cgtttgtgcc gccgagcgaa gcgcgcatta gcattttga tcaaggctat 180 ctgcatagcg atgtgaccta taccgtgttt catgtgtgga acggcaacgc gtttcgcctg 240 300. gatgatcata ttgaacgcct gtttagcaac gcggaagta tgcgcattat tccgccgctg acccaagatg aagtgaaaga aattgcgctg gaactggtgg cgaaaaccga actgcgcgaa gcgtttgtga gtgtgagtat cacccgcggc tatagcagca ccccggggcga acgcgatatt 420 accaaacatc gcccgcaagt gtatatgtat gcggtgccgt atcagtggat tgtgccgttt 480 gatcgcattc gcgatggcgt tcatgcgatg gtggcgcaga gcgtgcgccg caccccgcgc 540 agcagcattg atccgcaagt gaaaaacttt cagtggggcg atctgattcg cgcggtacaa 600 gaaacccatg atcgcggctt tgaagcgccg ctgttactgg atggcgatgg cctgctggcg 660 gaaggctcag gctttaacgt ggttgtgatt aaagatggcg tggtgcgcag cccgggccgc 720 gcggcgctgc cgggcattac ccgcaaaacc gtgctggaaa ttgcggaaag cctgggccat 780 gaagcgattc tggcggatat taccctggcg gaactgctgg atgcggatga agtgctgggc 840 tgcaccaccg cgggcggcgt gtggccattc gtgagcgtgg atggtaaccc gattagcgat 900 ggtgtgccgg gtccggtgac gcagagcatt attcgccgct attgggaact gaacgtggaa 960 tcaagtagcc tgctgacccc ggtgcagtat taa 993 <210> 3 <211> 330 <212> PRT <213> Artificial Sequence () <400> 3 Met Ala Lys Ser Ala Asp Thr Pro Ser Asn Met Tyr Thr His Asp Thr 1 5 10 15 Gly Leu Asp Tyr Ile Lys Phe Ser Glu Tyr Glu Gly Ser Ala Asp Asn 20 25 30 Asn Leu Ala Gly Gly Ala Ala Trp Ile Glu Gly Ala Phe Val Pro Pro 35 40 45 Ser Glu Ala Arg Ile Ser Val Phe Asp Gln Gly Phe Tyr Thr Ser Asp 50 55 60 Ala Thr Tyr Thr Thr Phe Ser Val Trp His Gly Asn Ala Phe Arg Leu 65 70 75 80 Asp Asp His Ile Glu Arg Leu Tyr Ser Asn Ala Glu Ser Met Arg Leu 85 90 95 Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu 100 105 110 Val Ala Lys Thr Glu Leu Arg Glu Ala Met Val Thr Val Thr Phe Thr 115 120 125 Arg Gly Leu Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Asn His Arg 130 135 140 Pro Gln Val Tyr Met Thr Ala Val Pro Tyr Gln Thr Ile Val Pro Phe 145 150 155 160 Asp Arg Ile Arg Asn Gly Val His Ala Met Val Ala Gln Ser Val Arg 165 170 175 Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp 180 185 190 Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr Asn Asp Arg Gly Phe Glu 195 200 205 Leu Pro Val Leu Leu Asp Cys Asp Gly Leu Leu Ala Glu Gly Pro Gly 210 215 220 Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg 225 230 235 240 Ala Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu 245 250 255 Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Ser Leu Ala Asp Leu 260 265 270 Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp 275 280 285 Pro Phe Val Ser Val Asp Gly Asn Pro Ile Ser Asp Gly Val Pro Gly 290 295 300 Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu 305 310 315 320 Pro Ser Gln Leu Leu Thr Pro Val Gln Tyr 325 330 <210> 4 <211> 993 <212> DNA <213> Container() <400> 4 atggcgaaga gcgcggatac gccttcgaat atgtataccc atgataccgg cctggattat attaatta gcgaatatga aggatccgcg gacaacaatc tggcgggcgg cgcggcgtgg attgaaggcg cgtttgtgcc gccgagcgaa gcgcgcatta gcgtttttga tcaaggcttt 180 tatactagcg atgcgaccta taccacgttt tctgtgtggc acggcaacgc gtttcgcctg 240 300. gatgatcata ttgaacgcct gtatagcaac gcggaagta tgcgccttat tccgccgctg acccaagatg aagtgaaaga aattgcgctg gaactggtgg cgaaaaccga actgcgcgaa gcgatggtga ctgtgacttt cacccgcggc cttagcagca ccccgttcga acgcgatatt 420 accaaccatc gcccgcaagt gtatatgact gcggtgccgt atcagacgat tgtgccgttt 480 gatcgcattc gcaatggcgt tcatgcgatg gtggcgcaga gcgtgcgccg caccccgcgc 540 agcagcattg atccgcaagt gaaaaacttt cagtggggcg atctgattcg cgcgatacaa gaaaccaatg atcgcggctt tgaactgccg gtgttactgg attgcgatgg cctgctggcg gaaggcccag gctttaacgt ggttgtgatt aaagatggcg tggtgcgcag cccgggccgc 720 gcggcgctgc cgggcattac ccgcaaaacc gtgctggaaa ttgcggaaag cctgggccat 780 gaagcgattc tggcggatat ttccctggcg gatctgtacg atgcggatga agtgctgggc 840 tgctccaccg ggggcggcgt gtggccattc gtgagcgtgg atggtaaccc gattagcgat 900 ggtgtgccgg gtccggtgac gcagagcatt attcgccgct attgggaact gaacgtggaa 960 ccaagtcaac tgctgacccc ggtgcagtat taa 993

Claims

1. A transaminase mutant, characterized in that: The amino acid sequence is SEQ ID NO:

3.

2. A gene encoding the transaminase mutant according to claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene is SEQ ID NO:

4.

4. A microorganism expressing the transaminase mutant according to claim 1.

5. Use of the transaminase mutant according to claim 1 or the microorganism according to claim 4 in producing sitagliptin.

6. The use according to claim 5, characterized in that Sitagliptin is obtained by using (2Z)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazine-7-(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-one as a reaction substrate and using the aminotransferase mutant according to claim 1 or the microorganism according to claim 4 to catalyze the amino transfer reaction.

7. The use according to claim 6, characterized in that The reaction system contains pyridoxal phosphate as a coenzyme.

8. The use according to claim 7, characterized in that The reaction system also contains o-phenylenediamine dihydrochloride, isopropylamine or triethanolamine as an amino donor.

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