WO2024098269A1 - 一种转氨酶突变体及其在西他列汀合成中的应用 - Google Patents

一种转氨酶突变体及其在西他列汀合成中的应用 Download PDF

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WO2024098269A1
WO2024098269A1 PCT/CN2022/130745 CN2022130745W WO2024098269A1 WO 2024098269 A1 WO2024098269 A1 WO 2024098269A1 CN 2022130745 W CN2022130745 W CN 2022130745W WO 2024098269 A1 WO2024098269 A1 WO 2024098269A1
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transaminase
seq
sitagliptin
microorganism
enzyme
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French (fr)
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王金刚
韦炎龙
梁岩
任亮
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上海邦林生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
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    • C12P19/00—Preparation of compounds containing saccharide radicals
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    • C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
    • C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin

Definitions

  • the invention belongs to the technical field of enzyme catalysis, and in particular relates to a transaminase and an application thereof in the synthesis of sitagliptin.
  • sitagliptin (compound II) 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.
  • Its phosphate monohydrate is a best-selling dipeptidyl peptidase 4 (DPP-4) inhibitor drug that can improve blood sugar control in patients with type 2 diabetes by increasing the level of active incretin hormones. It has the advantages of good safety and low incidence of adverse reactions.
  • transaminase biocatalysts to synthesize sitagliptin, which has the advantages of simple route, high product optical purity and high substrate conversion rate.
  • pyridoxal phosphate (PLP) is added as a coenzyme to catalyze the reversible transfer of the amino group from the donor to the hydroxyl acceptor.
  • Patent document CN102405281A discloses that based on the (R)- ⁇ -transaminase from Arthrobacter sp., computer-aided design is used to simulate the 3D structure of the protein, and strategies such as amino acid site combination mutation, site-directed saturation mutation, and random mutation of the entire gene sequence are combined to finally construct a new transaminase mutant that can efficiently catalyze (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 (Compound I) to synthesize sitagliptin using a mixed system of 50% aqueous phase
  • the transaminase mutant ATA64 (SEQ ID NO: 1 in this article) disclosed in CN2021115167214 in a methanol-containing reaction system
  • the inventors continued to modify the mutant enzyme ATA64, hoping to obtain a mutant enzyme that tolerates methanol environment and has higher catalytic efficiency, and further reduce the production cost of sitagliptin.
  • many sites were screened to screen out a mutant that can efficiently catalyze compound I to synthesize sitagliptin in a methanol-containing reaction system.
  • the present invention includes the following technical solutions:
  • transaminase which is a polypeptide selected from the following:
  • polypeptide that has more than 95%, preferably more than 96%, preferably more than 97%, preferably more than 98%, and more preferably more than 99% homology with SEQ ID NO: 3, and whose enzyme activity in a methanol solution reaction system is improved compared to SEQ ID NO: 3.
  • ATA84 which is a mutant in which the asparagine at position 35 in SEQ ID NO:1 (i.e., ATA64 disclosed in CN2021115167214) mutates to alanine (N35A), the aspartic acid at position 120 mutates to glutamate (D120E), the methionine at position 122 mutates to valine (M122V), the phenylalanine at position 127 mutates to isoleucine (F127I), the leucine at position 131 mutates to tyrosine (L131Y), the asparagine at position 165 mutates to aspartic acid (N165D), the alanine at position 169 mutates to leucine (A169L), and the leucine at position 213 mutates to arginine (L213R).
  • ATA84 is a mutant in which the asparagine at position 35 in SEQ ID NO:1 (i.e., ATA64 disclosed in CN2021115167214
  • the above enzyme activity refers to the enzyme activity when catalyzing the conversion of (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 into sitagliptin.
  • the present invention also provides a gene encoding the above transaminase.
  • the gene encoding the transaminase SEQ ID NO:3 can be a polynucleotide shown in the nucleotide sequence SEQ ID NO:4, or a polynucleotide having more than 90%, preferably more than 92%, preferably more than 95%, preferably more than 97%, preferably more than 98%, and more preferably more than 99% homology with SEQ ID NO:4.
  • the present invention also provides a plasmid comprising the above coding gene.
  • the above plasmid can be a pET vector such as pET22b, pET24a, pET28a, or other commonly used vectors such as pSH plasmid.
  • Another aspect of the present invention provides a microorganism for expressing the above-mentioned transaminase, such as SEQ ID NO: 3, into whose genome the above-mentioned coding gene, such as SEQ ID NO: 4, is integrated, or a microorganism transformed with the above-mentioned plasmid.
  • the transformation of the above plasmid can be carried out by conventional chemical transformation or electroporation into the competent cell.
  • the above gene editing technology is selected from the following groups: homologous double exchange, TALEN system, CRISPR-Cas9 system, CRISPR-Cpf1 system, CRISPR-Cas12 system, CRISPR-BEST system, MuGENT (multiplex genome editing by natural transformation), etc.
  • the microorganism is a microorganism with a fast growth rate and suitable for expressing exogenous recombinant proteins, for example, selected from Bacillus subtilis, Lactobacillus brevis, Escherichia coli, Candida magnolii, Pichia pastoris, Saccharomyces cerevisiae.
  • the microorganism is Escherichia coli, more preferably Escherichia coli BL21 (DE3).
  • the above transaminase or the above microorganism can be used to produce sitagliptin.
  • a reaction system containing an organic solvent preferably an alcohol such as methanol or ethanol as a cosolvent
  • (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 reaction substrate
  • the above transaminase or the above microorganism is used to catalyze the transamination reaction to obtain sitagliptin.
  • organic solvent is added to the above reaction system as a co-solvent for the substrate, and the organic solvent includes but is not limited to methanol, ethanol, propanol, isopropanol, DMSO, or a mixture of two or more thereof.
  • the concentration of methanol in the reaction system is 10%-60%, preferably 50%.
  • the reaction system contains pyridoxal phosphate as a coenzyme.
  • the above reaction system may further comprise o-phenylenediamine dihydrochloride and/or isopropylamine as an amino group donor.
  • reaction temperature can be 35-50° C., preferably 38-49° C., preferably 39-48° C., preferably 40-46° C., preferably about 45° C.
  • the reaction pH can be 7.0-9.5, preferably pH 7.5-9.2, preferably pH 7.8-9.0, preferably pH 8.0-8.8, preferably pH 8.3-8.6, for example, about pH 8.5.
  • the methanol concentration in the reaction system is 40%-60%, preferably 50%.
  • the concentration of substrate I is 20-200 g/L, preferably 150 g/L.
  • the present invention uses solvent methanol as the environmental screening pressure, combined with genetic engineering mutation technology, and continues to perform multiple rounds of mutations on the transaminase mutant ATA64 reported in patent document CN2021115167214 (SEQ ID NO: 1 in this article). After screening, a mutant SEQ ID NO: 3 that is tolerant to methanol environment and has higher enzyme activity is obtained.
  • the conversion rate of the catalytic reaction of compound I is high, and the product II has high stereoselectivity, which improves the economy of the enzymatic synthesis of sitagliptin process route.
  • FIG1 is an HPLC spectrum of the synthesis of sitagliptin (Compound II) catalyzed by mutant strain EcATA84.
  • FIG. 2 is an HPLC spectrum of sitagliptin catalyzed by the starting strain EcATA64 under the same reaction conditions.
  • the transaminase of the present invention is a mutant obtained by further mutation of ATA64 (i.e., SEQ ID NO: 1) disclosed in patent document CN2021115167214, and has significantly improved tolerance to alcohols (especially methanol) and improved enzyme activity. It can be applied to the sitagliptin enzyme catalytic reaction system with alcohols such as methanol/ethanol as co-solvents for substrate I.
  • ATA64 with the amino acid sequence SEQ ID NO:1 is used as the initial enzyme for mutation.
  • starting (type) enzyme As in this article, the terms "starting (type) enzyme”, “initial (type) enzyme” and “starting enzyme” have the same meaning, all referring to the transaminase ATA64 with the amino acid sequence of SEQ ID NO: 1.
  • starting enzyme the starting enzyme and its mutants such as SEQ ID NO: 3 can be collectively referred to as “transaminase” in this article.
  • (enzyme activity) increase or “increase” as used above means an increase of at least 100% compared to a reference level, for example, an increase of at least about 1 times, at least about 2 times, or at least about 3 times, or at least about 5 times, or at least about 10 times, or at least about 20 times compared to a reference level.
  • the “mutation” includes but is not limited to replacement, deletion, insertion, chemical modification of amino acid residues, preferably a forward mutation, i.e. a mutation that improves enzyme activity.
  • the substitution can be a non-conservative substitution, a conservative substitution, or a combination of a non-conservative substitution and a conservative substitution.
  • Constant amino acid substitution or mutation refers to the interchangeability of residues with similar side chains, and therefore generally includes replacing the amino acids in the polypeptide with the same or similar amino acid definition categories.
  • a conservative mutation can be replaced by aliphatic to aliphatic, non-polar to non-polar, polar to polar, acidic to acidic, alkaline to alkaline, aromatic to aromatic, or restricted residue to restricted residue substitution, then a conservative mutation does not include a hydrophilic to hydrophilic, hydrophobic to hydrophobic, hydroxyl-containing to hydroxyl-containing or small residue to small residue substitution.
  • conservative substitutions include: mutual substitutions between aromatic amino acids F, W, and Y; mutual substitutions between hydrophobic amino acids L, I, and V; mutual substitutions between polar amino acids Q and N; mutual substitutions between basic amino acids K, R, and H; mutual substitutions between acidic amino acids D and E; and mutual substitutions between hydroxyl amino acids S and T.
  • A, V, L, or I can be conservatively mutated to another aliphatic residue or another non-polar residue.
  • Exemplary conservative substitutions are, for example:
  • Non-conservative substitution refers to the substitution or mutation of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups listed above.
  • a non-conservative mutation affects (a) the structure of the peptide backbone in the area of the substitution (e.g., proline for glycine), (b) charge or hydrophobicity, or (c) side chain bulk.
  • “Deletion” refers to the modification of a polypeptide by removing one or more amino acids from a reference polypeptide. Deletion can include the removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids constituting the reference enzyme, while retaining enzymatic activity and/or retaining the improved properties of the engineered aldolase. Deletion can be directed to the interior and/or ends of the polypeptide. In various embodiments, deletion can comprise a continuous segment or can be discontinuous.
  • Insertion refers to the modification of a polypeptide by adding one or more amino acids from a reference polypeptide.
  • the improved engineered aldolase comprises one or more amino acids inserted into a naturally occurring aldolase and one or more amino acids inserted into other improved aldolase polypeptides.
  • the insertion can be inside the polypeptide, or at the carboxyl or amino termini. Insertion as used herein includes fusion proteins as known in the art.
  • the insertion can be a continuous amino acid segment or separated by one or more amino acids in a naturally occurring polypeptide.
  • the specific changes are asparagine at position 35 mutated to alanine (N35A), aspartic acid at position 120 mutated to glutamic acid (D120E), methionine at position 122 mutated to valine (M122V), phenylalanine at position 127 mutated to isoleucine (F127I), leucine at position 131 mutated to tyrosine (L131Y), asparagine at position 165 mutated to aspartic acid (N165D), alanine at position 169 mutated to leucine (A169L), and leucine at position 213 mutated to arginine (L213R).
  • the amino acid number of the aminotransferase mutant SEQ ID NO: 3 of the present invention is 330, and the structure is clear, so it is easy for those skilled in the art to 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.
  • 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 genes 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.
  • the coding gene of the codon-optimized starting transaminase SEQ ID NO:1 can be SEQ ID NO:2; the coding gene of the transaminase mutant SEQ ID NO:3 can be SEQ ID NO:4.
  • the above-mentioned transformant host can be any microorganism suitable for expressing transaminase, including bacteria and fungi.
  • the microorganism is Bacillus subtilis, Corynebacterium glutamicum, Pichia pastoris, Saccharomyces cerevisiae, or Escherichia coli, preferably Escherichia coli, more preferably Escherichia coli BL21 (DE3).
  • 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.
  • microorganisms such as Bacillus subtilis, Corynebacterium glutamicum, 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 advantageous.
  • 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 D.W. Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002). If necessary, the specific experimental conditions can be determined by simple experiments.
  • 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.
  • LB medium 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, pH 7.2. (LB solid medium plus 20g/L agar powder.)
  • TB medium 24g/L yeast extract, 12g/L tryptone, 16.43g/L K 2 HPO 4 .3H 2 O, 2.31g/L KH 2 PO 4 , 5g/L glycerol, pH 7.0-7.5.
  • TB solid medium is supplemented with 20g/L agar powder.
  • Chiral detection chromatographic conditions chromatographic column CHIRALPAK AD-H (4.6 ⁇ 250mm, 5 ⁇ m); mobile phase: n-hexane:ethanol:triethylamine (40:60:0.1); flow rate: 0.7mL/min, detection wavelength: 268nm.
  • 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.
  • Example 1 Construction of initial transaminase gene recombinant Escherichia coli
  • Example 1 based on the amino acid sequence SEQ ID NO: 1 of the initial enzyme ATA64, codon optimization was performed according to the codon preference of Escherichia coli, and Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to synthesize its coding gene SEQ ID NO: 2, and cloned into the NcoI and BamHI sites of plasmid pET28a to obtain plasmid pET-ATA64.
  • the recombinant plasmid pET-ATA64 was transformed into the expression host Escherichia coli BL21 (DE3) by electroporation to obtain the recombinant Escherichia coli EcATA64 expressing the initial aminotransferase.
  • Example 2 Construction of random mutation point library and high-throughput screening from round 1 to round 2
  • the random mutant library was constructed using error-prone PCR technology with plasmid pET-ATA64 as template.
  • Reverse primer ATA-3 5’-GAGCTCGAATTCGGATCCTTA-3’.
  • PCR amplification was performed to obtain a transaminase mutant DNA sequence of approximately 1.0 kb.
  • the 50 ⁇ L error-prone PCR reaction system included: 10 ng plasmid (pET-ATA64) 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 MgCl 2 , (0 mM, 0.05 mM, 0.1 mM, 0.15 mM, 0.2 mM) MnCl 2 , and 2.5 units of Taq enzyme (Takara).
  • 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.
  • PCR products were electrophoresed and recovered by gel (Axygen DNA gel recovery kit AP-GX-50).
  • KOD-plus DNA polymerase was used for MegaPrimer PCR: 94°C5min; 98°C10s, 60°C30s, 68°C2min/kb, 25 cycles; 68°C10min.
  • the plasmid template was digested with DpnI restriction endonuclease (Thermo Company) and electroporated into E. coli BL21 (DE3) to obtain a random mutation library of more than 10 4 clones.
  • strain EcATA73 with high enzyme activity was selected for the next round of mutation.
  • Example 3 Construction of random mutation point library from round 3 to round 5 and high-throughput screening
  • the selected bacterial plasmid was used as a template to construct a random mutation library.
  • the construction method was based on the method for constructing a transaminase random mutation point library in Example 2.
  • the starting strain EcATA64 and the mutant strain EcATA84 were cultured in a fermenter, respectively.
  • a single colony was picked from the LB plate (containing Kan) of the strain, inoculated into 5 mL of liquid LB medium containing Kan, and cultured overnight at 37°C and 220 rpm.
  • the next day the inoculation volume was transferred to a shake flask containing 100 mL of liquid TB medium at a volume concentration of 5% v/v, and cultured at 37°C and 220 rpm until OD 600nm reached 6, and then transferred to a 5L fermenter as a seed liquid.
  • the culture was carried out at 400-800 rpm/min and 37°C, and the dissolved oxygen was controlled within the range of 20-30%.
  • IPTG was added to induce transaminase expression
  • the final concentration of IPTG was 0.2 mM
  • the culture was continued at 28-30°C for 16-24 hours, and the bacterial cells were collected by centrifugation for catalysis of the synthesis of sitagliptin in a 1L reaction system.
  • Ammonia water was used to control the pH value of the whole fermentation process to 6.8-7.2.
  • HPLC test results show that after 20 hours of reaction, the molar yield of the catalytic product involved in the mutant strain EcATA84 exceeds 95% (as shown in Figure 1), and the product e.e. value is greater than 99.95%.
  • the starting strain EcATA64 whole cell catalyzes the asymmetric synthesis of sitagliptin from the precursor ketone of sitagliptin, and the product molar yield is only about 77% (as shown in Figure 2), and the product e.e. value is greater than 99.95%.

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Abstract

本发明提供了一种转氨酶，其氨基酸序列如SEQ ID NO.3所示，该转氨酶能够在甲醇溶液反应体系中催化(2Z)-4-氧代-4-[3-(三氟甲基)-5,6-二氢[1,2,4]***并[4,3-a]吡嗪-7-(8H)-基]-1-(2,4,5-三氟苯基)丁-2-酮转化为西格列汀。

Description

一种转氨酶突变体及其在西他列汀合成中的应用 技术领域
本发明属于酶催化技术领域，具体地说，涉及一种转氨酶及其在西他列汀合成中的应用。
背景技术
西他列汀(化合物II)化学名称为7-[(3R)-3-氨基-1-氧代-4-(2,4,5-三氟苯基)丁基]-5,6,7,8-四氢-3-三氟甲基-1,2,4-***并[4,3-a]吡嗪，其磷酸盐一水合物是畅销的二肽基肽酶4(DPP-4)抑制剂药物，可通过增加活性肠促胰岛激素的水平而改善2型糖尿病患者的血糖控制，具有安全性好、不良反应发生率低等优势。
目前工业上主要利用转氨酶生物催化剂合成西他列汀，具有路线简洁、产物光学纯度高、底物转化率高等优势。
Figure PCTCN2022130745-appb-000001
反应过程中添加磷酸吡哆醛(PLP)作为辅酶，催化氨基从供体到羟基受体的可逆转移。专利文献CN102405281A公开了在Arthrobacter sp.来源(R)-ω-转氨酶基础上，利用计算机辅助设计模拟蛋白质3D结构，联用氨基酸位点组合突变、定点饱和突变、全基因序列随机突变等策略，最终构建了能以50％水相-50％DMSO有机相的混合体系作为反应溶剂，高效催化(2Z)-4-氧代-4-[3-(三氟甲基)-5,6-二氢-[1,2,4]***并[4,3-a]吡嗪-7-(8H)-基]-1-(2,4,5-三氟苯基)丁-2-酮(化合物I)合成西他列汀的新型转氨酶突变体。但是酶反应体系中使用了大量价格昂贵的DMSO，造成后处理复杂，增加了产品生产成本。相比于DMSO，醇类比如甲醇或乙醇成本低廉，且易于后处理，因此利用甲醇或乙醇替代酶反应体系中的助溶剂DMSO可显著降低产品生产成本。发明人在专利文献 CN2021115167214中公开了一种Arthrobacter sp.来源R型转氨酶突变体ATA64，能够以50％水相-50％甲醇有机相为反应溶剂，催化化合物I合成化合物II。
发明内容
为了进一步提高CN2021115167214中公开的转氨酶突变体ATA64(本文中SEQ ID NO:1)在含甲醇反应体系中的酶活力，发明人继续在突变酶ATA64的基础上进行改造，期望获得耐受甲醇环境、催化效率更高的突变酶，进一步降低西他列汀的生产成本。基础上，对众多位点进行筛选，筛选出一种能在含甲醇反应体系中高效催化化合物I合成西他列汀的突变体。具体地，本发明包含如下技术方案：
一种转氨酶，其为选自下述的多肽：
(a)氨基酸序列为SEQ ID NO:3的多肽；
(b)与SEQ ID NO:3有95％以上、优选96％以上、优选97％以上、优选98％以上、更优选99％以上同源性、且在甲醇溶液反应体系中的酶活力相比SEQ ID NO:3提高的多肽。
Figure PCTCN2022130745-appb-000002
本文中，氨基酸序列为SEQ ID NO:3的转氨酶命名为ATA84，其为SEQ ID NO:1(即CN2021115167214公开的ATA64)中第35位的天冬酰胺突变为丙氨酸(N35A)、第120位的天冬氨酸突变为谷氨酸(D120E)、第122位的甲硫氨酸突变为缬氨酸(M122V)、第127位的苯丙氨酸突变为异亮氨酸(F127I)、第131位的亮氨酸突变为酪氨酸(L131Y)、第165位的天冬酰胺突变为天冬氨酸(N165D)、第169位的丙氨酸突变为亮氨酸(A169L)、第213位的亮氨酸突变为精氨酸(L213R)的突变体。
上述酶活力是指催化(2Z)-4-氧代-4-[3-(三氟甲基)-5,6-二氢-[1,2,4]***并[4,3-a]吡嗪-7-(8H)-基]-1-(2,4,5-三氟苯基)丁-2-酮转化为西格列汀时的酶活力。
本发明还提供了编码上述转氨酶的基因。
例如，编码转氨酶SEQ ID NO:3的基因可以是核苷酸序列SEQ ID NO:4所示的多核苷酸、或者与SEQ ID NO:4有90％以上、优选92％以上、优选95％以上、 优选97％以上、优选98％以上、更优选99％以上同源性的多核苷酸。
本发明还提供了包含上述编码基因的质粒。例如，上述质粒可以是pET载体例如pET22b、pET24a、pET28a，也可以是pSH质粒等其他常用载体。
本发明的另一方面提供了一种用于表达上述转氨酶比如SEQ ID NO:3的微生物，其基因组中整合了上述的编码基因比如SEQ ID NO:4，或者是转化了上述质粒的微生物。
上述质粒的转化可以通过常规的化学转化法或者电转化方法转入细胞感受态中。上述基因编辑技术例如选自下组：同源双交换，TALEN***，CRISPR-Cas9***，CRISPR-Cpf1***，CRISPR-Cas12***，CRISPR-BEST***，MuGENT(multiplex genome editing by natural transformation，通过自然转化进行多重基因组编辑)等。
优选地，上述微生物是增殖速度快、适合于表达外源重组蛋白的微生物，例如选自枯草芽孢杆菌、短乳杆菌、大肠杆菌、木兰假丝酵母、毕赤酵母、酿酒酵母。优选微生物是大肠杆菌，更优选是大肠杆菌BL21(DE3)。
显然，上述转氨酶或者上述微生物可以用于生产西格列汀。例如，在含有有机溶剂、优选醇类比如甲醇或乙醇作为助溶剂的反应体系中，以(2Z)-4-氧代-4-[3-(三氟甲基)-5,6-二氢-[1,2,4]***并[4,3-a]吡嗪-7-(8H)-基]-1-(2,4,5-三氟苯基)丁-2-酮为反应底物，采用上述转氨酶或者上述微生物催化氨基转移反应，得到西格列汀。
上述反应体系中加入有机溶剂作为底物的助溶剂，所述有机溶剂包括但不限于甲醇、乙醇、丙醇、异丙醇、DMSO、或者它们两种以上的混合物。
当助溶剂是甲醇时，反应体系中甲醇浓度为10％-60％，优选50％。
在一种实施方式中，反应体系中包含磷酸吡哆醛作为辅酶。
优选地，上述反应体系中还可以包含邻苯二甲胺二盐酸和/或异丙胺作为氨基供体。
进一步地，反应温度可以为35-50℃、优选38-49℃、优选39-48℃、优选40-46℃，优选45℃左右。反应pH值可以为7.0-9.5、优选pH 7.5-9.2、优选pH 7.8-9.0、优选pH8.0-8.8、优选pH8.3-8.6，例如pH 8.5左右。
进一步地，反应体系中甲醇浓度为40％-60％，优选50％。底物I浓度为20-200g/L，优选150g/L。
本发明采用溶剂甲醇作为环境筛选压力，结合基因工程突变技术，继续对专利文献CN2021115167214中报道的转氨酶突变体ATA64(本文中SEQ ID NO:1)进行多轮突变 后，筛选得到一种耐受甲醇环境、且酶活力更高的突变体SEQ ID NO:3，催化化合物I反应的转化率高，产物II立体选择性高，提高了酶法合成西格列汀工艺路线的经济性。
附图说明
图1是突变菌株EcATA84催化合成西格列汀(化合物II)的HPLC谱图。
图2是出发菌株EcATA64相同反应条件下催化合成西格列汀的HPLC谱图。
具体实施方式
本发明的转氨酶是对专利文献CN2021115167214公开的ATA64(即SEQ ID NO:1)作进一步突变后筛选得到的醇类(尤其是甲醇)耐受性明显提高、且酶活力提高的突变体，可以适用于甲醇/乙醇等醇类作为底物I助溶剂的西格列汀酶催化反应体系。
该技术方案中，氨基酸序列SEQ ID NO:1的ATA64作为突变的初始酶。
Figure PCTCN2022130745-appb-000003
在本文中，术语“起始(型)酶”、“初始(型)酶”、“出发酶”表示相同的意义，都是指氨基酸序列为SEQ ID NO:1的转氨酶ATA64。有时为了表述方便起见，在本文中可以将起始酶与其突变体比如SEQ ID NO:3等统称为“转氨酶”。
本文中，上述所用术语“(酶活力)提高”或“增加”表示相较于参考水平提高至少100％，例如相较于参考水平的至少约1倍、至少约2倍、或至少约3倍、或至少约5倍、或至少约10倍、或至少约20倍的提高。
所述的“突变”包括但不限于氨基酸残基的替换、删除、***、化学修饰，优选是正向突变即酶活力提高的突变。所述取代可以是非保守取代、保守取代或非保守取代和保守取代的组合。“保守的”氨基酸取代或突变是指具有相似侧链的残基的可互换性，并且因此通常包括用相同或相似的氨基酸定义类别中的氨基酸取代多肽中的氨基酸。然而，如本文所用，如果保守的突变可以代替地为脂肪族至脂肪族、非极性至非极性、极性至极性、酸性至酸性、碱性至碱性、芳族至芳族、或限制残基至限制残基的取代，则保守的突变不包括亲水至亲水、疏水至疏水、含羟基至含羟基或小残基至小残基的取代。 本技术领域公知，保守性置换的常见情况包括：芳香族氨基酸F、W、Y之间的相互置换；疏水性氨基酸L、I、V之间的相互置换，极性氨基酸Q、N之间的相互置换，碱性氨基酸K、R、H之间的相互置换，酸性氨基酸D、E之间的相互置换，羟基的氨基酸S、T之间的相互置换。此外，A、V、L或I可以保守地突变为另一脂肪族残基或另一非极性残基。示例性的保守取代例如为：
Figure PCTCN2022130745-appb-000004
“非保守取代”是指用具有显著不同的侧链特性的氨基酸进行的多肽中氨基酸的取代或突变。非保守取代可以使用上面所列的定义组之间而不是之内的氨基酸。在一个实施方案中，非保守突变影响(a)取代区域中肽主链的结构(例如，脯氨酸取代甘氨酸)、(b)电荷或疏水性、或(c)侧链体积。
“缺失”是指通过从参考多肽移除一个或多个氨基酸而对多肽进行的修饰。缺失可以包括移除1个或多个氨基酸、2个或更多个氨基酸、5个或更多个氨基酸、10个或更多个氨基酸、15个或更多个氨基酸、或20个或更多个氨基酸、多达构成参考酶的氨基酸总数的10％，同时保留酶活性和/或保留工程化醛缩酶的改良特性。缺失可以针对多肽的内部和/或端部。在多个实施方案中，缺失可以包含连续的区段或者可以是不连续的。
“***”是指通过从参考多肽添加一个或多个氨基酸而对多肽进行的修饰。在一些实施方案中，改良的工程化醛缩酶包括将一个或多个氨基酸***天然存在的醛缩酶中以及将一个或多个氨基酸***其他改良的醛缩酶多肽中。***可以是在多肽的内部，或羧基端或氨基端。如本文所用的***包括如本领域中已知的融合蛋白。***可以是连续氨 基酸区段或者被天然存在的多肽中的一个或多个氨基酸分隔开。
通过甲醇环境加压，从易错PCR法构建的随机突变点库中筛选出的突变酶SEQ ID NO:3是初始酶SEQ ID NO:1的个别氨基酸发生替换后的突变体，具体变化是第35位的天冬酰胺突变为丙氨酸(N35A)、第120位的天冬氨酸突变为谷氨酸(D120E)、第122位的甲硫氨酸突变为缬氨酸(M122V)、第127位的苯丙氨酸突变为异亮氨酸(F127I)、第131位的亮氨酸突变为酪氨酸(L131Y)、第165位的天冬酰胺突变为天冬氨酸(N165D)、第169位的丙氨酸突变为亮氨酸(A169L)、第213位的亮氨酸突变为精氨酸(L213R)。
本发明的转氨酶突变体SEQ ID NO:3的氨基酸数量有330个，且结构明确，因此本领域技术人员很容易获得其编码基因、包含这些基因的表达盒和质粒、以及包含该质粒的转化体。这些基因、表达盒、质粒、转化体可以通过本领域技术人员所熟知的基因工程构建方式获得。
为了在基因工程中最常用的大肠杆菌中最佳地表达转氨酶，可以对这些酶的表达基因进行了密码子优化。密码子优化是可用于通过增加感兴趣基因的翻译效率使生物体中蛋白质表达最大化的一种技术。不同的生物体由于突变倾向和天然选择而通常示出对于编码相同氨基酸的一些密码子之一的特殊偏好性。例如，在生长快速的微生物如大肠杆菌中，优化密码子反映出其各自的基因组tRNA库的组成。因此，在生长快速的微生物中，氨基酸的低频率密码子可以用用于相同氨基酸的但高频率的密码子置换。因此，优化的DNA序列的表达在快速生长的微生物中得以改良。
例如，为了在大肠杆菌中表达转氨酶，经密码子优化的起始转氨酶SEQ ID NO:1的编码基因可以是SEQ ID NO:2；转氨酶突变体SEQ ID NO:3的编码基因可以是SEQ ID NO:4。
上述转化体宿主可以使任何适合表达转氨酶的微生物，包括细菌和真菌。优选微生物是枯草芽孢杆菌、谷氨酸棒杆菌、毕赤酵母、酿酒酵母、或者大肠杆菌，优选大肠杆菌，更优选大肠杆菌BL21(DE3)。
当作为生物催化剂用于生产西格列汀时，本发明的转氨酶可以呈现酶的形式或者菌体的形式。所述酶的形式包括游离酶、固定化酶，包括纯化酶、粗酶、发酵液、载体固定的酶等；所述菌体的形式包括存活菌体、死亡菌体、固定化菌体等。
当微生物比如枯草芽孢杆菌、谷氨酸棒杆菌、毕赤酵母、酿酒酵母或者大肠杆菌不再进行发酵增殖、而是用于酶催化反应时，本身就是一种天然的固定化酶，而且不需要 进行破碎处理、甚至提取纯化处理，就可以作为一种酶制剂用于催化反应。由于反应底物和反应产物都是小分子化合物，可以很方便地穿过菌体的生物屏障--细胞膜，因此不需要对菌体进行破碎处理，这在经济方面是有利的。
以下结合具体实施例对本发明做进一步详细说明。应理解，以下实施例仅用于说明本发明而非用于限定本发明的范围。
实施例
本文中涉及到多种物质的添加量、含量及浓度，其中所述的百分含量，除特别说明外，皆指质量百分含量。
材料和方法
实施例中的全基因合成、引物合成及测序皆由苏州金唯智生物科技有限公司完成。
实施例中的分子生物学实验包括质粒构建、酶切、连接、感受态细胞制备、转化、培养基配制等等，主要参照《分子克隆实验指南》(第三版)，J.萨姆布鲁克，D.W.拉塞尔(美)编著，黄培堂等译，科学出版社，北京，2002)进行。必要时可以通过简单试验确定具体实验条件。
PCR扩增实验根据质粒或DNA模板供应商提供的反应条件或试剂盒说明书进行。必要时可以通过简单试验予以调整。
LB培养基：10g/L胰蛋白胨，5g/L酵母提取物，10g/L氯化钠，pH7.2。(LB固体培养基另加20g/L琼脂粉。)
TB培养基：24g/L酵母提取物、12g/L胰蛋白胨、16.43g/L K 2HPO 4.3H 2O、2.31g/L KH 2PO 4、5g/L甘油，pH7.0-7.5。(TB固体培养基另加20g/L琼脂粉。)
20X电转母液：80g/L甘氨酸，2％吐温80。
HPLC检测条件：色谱柱C18(4.6×250mm，5μm)；流动相A：流动相B＝7：3(流动相A：10mM K 2HPO 4,0.1％三乙胺，pH 3.5；流动相B：乙腈)；流速1.0mL/min；检测波长为210nm。
手性检测色谱条件：色谱柱CHIRALPAK AD-H(4.6×250mm，5μm)；流动相为正己烷：乙醇：三乙胺(40：60：0.1)；流速为0.7mL/min，检测波长为268nm。
需说明的是，为描述方便起见，在实施例中，可将菌株编号、质粒编号、酶编号、酶编码基因编号共用一个编号，这是本领域技术人员容易理解的，即同一个编号在不同环境中可以指代不同的生物形式。
实施例1：初始型转氨酶基因重组大肠杆菌的构建
1.1参照专利文献CN2021115167214中实施例1的方法，以初始酶ATA64的氨基酸序列SEQ ID NO:1为基础，根据大肠杆菌密码子偏好性进行密码子优化，委托苏州金唯智生物科技有限公司合成其编码基因SEQ ID NO:2，并克隆到质粒pET28a的NcoI、BamHI位点，获得质粒pET-ATA64。
1.2通过电转化将重组质粒pET-ATA64转化表达宿主大肠杆菌BL21(DE3)，得到表达初始转氨酶的重组大肠杆菌EcATA64。
实施例2：第1轮到第2轮随机突变点文库建立及高通量筛选
2.1易错PCR法构建随机突变点库
以质粒pET-ATA64为模板，利用易错PCR技术构建随机突变体文库。
设计如下引物对ATA-5/ATA-3：
正向引物ATA-5：5’-CTTTAAGAAGGAGATATACCATG-3’，
反向引物ATA-3：5’-GAGCTCGAATTCGGATCCTTA-3’。
以质粒pET-ATA64为模板，进行PCR扩增，获得约1.0kb的转氨酶突变体DNA序列。
50μL易错PCR反应体系包括：10ng质粒(pET-ATA64)模板，50pmol一对引物ATA-5和ATA-3，1×Taq buffer，0.2mM dGTP，0.2mM dATP，1mM dCTP，1mM dTTP，7mM MgCl 2，(0mM、0.05mM、0.1mM、0.15mM、0.2mM)MnCl 2，2.5个单位的Taq酶(Takara)。
PCR反应条件为：95℃5min；94℃30s，55℃30s，72℃2min/kbp，30个循环；72℃10min。
对PCR产物电泳、胶回收(Axygen DNA凝胶回收试剂盒AP-GX-50)。以质粒pET-ATA64为模板，以约1.0kb的回收产物(随机突变片段)作为大引物，用KOD-plus DNA聚合酶做MegaPrimer PCR：94℃5min，；98℃10s，60℃30s，68℃2min/kb，25个循环；68℃10min。DpnI限制性内切酶(Thermo公司)消化质粒模板，电转化大肠杆菌E.coli BL21(DE3)，得到超过10 4个克隆的随机突变库。
2.2突变体库的高通量筛选
从备选菌株的LB平板(含Kan)上挑取单菌落，接种至96孔板(每孔含有110μL的液体LB-Kan培养基)，在37℃、400rpm条件下孵育5h后，从每孔中取出60μL的菌液到96孔深孔板(每孔含有240μL的液体TB-Kan-0.2mM IPTG)，在25℃、400rpm条件下孵育12～16h。在4℃、4000rpm条件下离心10min收集菌体，去除上清培养液。 随后，用预冷的生理盐水洗涤菌体，在4℃、4000rpm条件下离心10min收集菌体，去除上清液。每孔加200μL酶反应液(100mM三乙醇胺、70mM邻苯二甲胺二盐酸、0.5g/L磷酸吡哆醛、20g/L化合物I、55％甲醇，调pH值为8.5进行反应。重悬菌体，在45℃、250rpm条件下孵育1～5h，检测475nm波长数值，数值越大，酶活性越高。
2.3选择活力明显提升的菌株进行核酸测序，确定氨基酸突变位点，同时HPLC检测这些菌种反应产物的ee值，选择产物ee值大于99.95％，且酶活力改善最高的菌株作为下一轮随机突变体库建库的出发菌株。委托苏州金唯智生物科技有限公司对酶活力最高的菌株进行基因组测序比对，确定其氨基酸序列改变情况。重复随机突变库建立和以化合物I为底物、甲醇助溶的反应体系中的高通量筛选。筛选结果参见表1。
表1、第1轮和第2轮随机突变库高通量筛选结果
Figure PCTCN2022130745-appb-000005
备注：“+”代表活力百分比相对各自出发菌株大于0％小于等于50％；“++”代表活力百分比相对各自出发菌株大于50％小于等于100％；“+++”代表活力百分比相 对各自出发菌株大于100％小于等于200％；“++++”代表活力百分比相对各自出发菌株大于200％。
经过比较，筛选酶活力高的菌株EcATA73用于下一轮突变。
实施例3：第3轮到第5轮随机突变点文库建立及高通量筛选
3.1易错PCR法构建随机突变点库
以筛选出的菌种质粒为模板，进行随机突变库的构建，构建方法参照实施例2中构建转氨酶随机突变点文库的方法。
3.2突变体库的高通量筛选
从备选菌株的LB平板(含Kan)上挑取单菌落，接种至96孔板(每孔含有110μL的液体LB-Kan培养基)，在37℃、400rpm条件下孵育5h后，从每孔中取出60μL的菌液到96孔深孔板(每孔含有240μL的液体TB-Kan-0.2mM IPTG)，在25℃、400rpm条件下孵育12～16h。在4℃、4000rpm条件下离心10min收集菌体，去除上清培养液。随后，用预冷的生理盐水洗涤菌体，在4℃、4000rpm条件下离心10min收集菌体，去除上清液。向每个孔加入100μL 50％水-50％甲醇溶液重悬细胞，在45℃，800rpm下孵育1h。随后向每个孔加入酶反应液补至200μL，最终含70mM三乙醇胺、70mM邻苯二甲胺二盐酸、0.5g/L磷酸吡哆醛、30g/L化合物I、55％甲醇，挑pH值为8.5进行反应。重悬菌体，在45℃、250rpm条件下孵育1～5小时，检测475nm波长数值，数值越大，酶活性越高。
3、选择活力明显提升的菌株进行核酸测序，确定氨基酸突变位点，同时HPLC检测这些菌种反应产物的ee值，选择产物ee值大于99.95％，且酶活力最高的菌株作为下一轮随机突变体库建库的出发菌株，重复随机突变库建立及以化合物I为底物、甲醇助溶反应体系的高通量筛选。筛选结果参见表2。
表2、第3轮到第5轮随机突变库高通量筛选结果
Figure PCTCN2022130745-appb-000006
Figure PCTCN2022130745-appb-000007
备注：“+”代表活力百分比相对各自出发菌株大于0％小于等于50％；“++”代表活力百分比相对各自出发菌株大于50％小于等于100％；“+++”代表活力百分比相对各自出发菌株大于100％小于等于200％；“++++”代表活力百分比相对各自出发菌株大于200％。
经过上述多轮突变和筛选，得到较为理想的突变酶ATA84，其氨基酸序列为SEQ ID NO:3。
实施例4：出发菌株EcATA64和突变菌株EcATA84的发酵培养
分别对出发菌株EcATA64和突变菌株EcATA84进行发酵罐培养。从菌株的LB平板(含Kan)上挑取单菌落，接种至5mL含有Kan的液体LB培养基中，在37℃、220rpm过夜培养。次日，按照体积浓度为5％v/v的接种量转接至含有100mL液体TB培养基的摇瓶中，37℃、220rpm条件下培养至OD 600nm达到6后，作为种子液转接到5L发酵罐。接种后400～800rpm/min、37℃条件下培养，控制溶氧在20～30％范围内。当菌体OD 600nm达到20后，加入IPTG诱导转氨酶表达，IPTG的终浓度为0.2mM，28-30℃继续培养培养16-24h，离心收集菌体用于1L反应体系中催化西他列汀的合成。全发酵过程利用氨水控制pH值为6.8～7.2。
实施例5：转氨酶突变体合成西他列汀的应用
在1L反应体系中，分别用菌株EcATA64和EcATA84的全细胞催化西格列汀前体 酮(化合物I)不对称合成西格列汀(化合物II)。总反应体系包括：100mM三乙醇胺、1M异丙胺、0.5g/L磷酸吡哆醛、150g/L化合物I、50g/L细胞(湿重)，50％甲醇。150g化合物I溶于甲醇中，以1mL/min的流速流加到反应体系中，利用盐酸或异丙胺控制反应体系的pH值在8.5左右。
HPLC检测结果显示，反应20h后，突变菌株EcATA84参与的催化产物摩尔生成率超过95％(如图1所示)，产物e.e.值大于99.95％。而在相同反应条件下，出发菌株EcATA64全细胞催化西格列汀前体酮不对称合成西格列汀，产物摩尔生成率只有大约77％(如图2所示)，产物e.e.值大于99.95％。该实验证明转氨酶突变体ATA84有较好的甲溶剂耐受性，且ATA84的酶活力明显高于初始酶ATA64，具有较好的应用前景，为实现酶催化法生产西格列汀的工业化奠定了基础。

Claims (10)

  1. 一种转氨酶，其为选自下述的多肽：
    (a)氨基酸序列为SEQ ID NO:3的多肽；
    (b)与SEQ ID NO:3有95％以上同源性、且在甲醇溶液反应体系中的酶活力相比SEQ ID NO:3提高的多肽。
  2. 如权利要求1所述的转氨酶，其特征在于，所述酶活力是指催化(2Z)-4-氧代-4-[3-(三氟甲基)-5,6-二氢-[1,2,4]***并[4,3-a]吡嗪-7-(8H)-基]-1-(2,4,5-三氟苯基)丁-2-酮转化为西格列汀时的酶活力。
  3. 编码如权利要求1或2所述转氨酶的基因。
  4. 如权利要求3所述的基因，其特征在于，编码转氨酶SEQ ID NO:3的基因是核苷酸序列SEQ ID NO:4所示的多核苷酸、或者与SEQ ID NO:4有90％以上同源性的多核苷酸。
  5. 包含如权利要求4所述基因的质粒。
  6. 一种用于表达如权利要求1所述转氨酶的微生物，其特征在于，基因组中整合了如权利要求4所述的基因，或者转化了如权利要求5所述质粒的微生物。
  7. 如权利要求6所述的微生物，其特征在于，所述微生物是大肠杆菌。
  8. 如权利要求1所述转氨酶或者如权利要求6所述微生物在生产西格列汀中的用途。
  9. 如权利要求7所述的用途，其特征在于，在含有有机溶剂、优选醇类作为助溶剂的反应体系中，以(2Z)-4-氧代-4-[3-(三氟甲基)-5,6-二氢-[1,2,4]***并[4,3-a]吡嗪-7-(8H)-基]-1-(2,4,5-三氟苯基)丁-2-酮为反应底物，采用如权利要求1所述转氨酶或者如权利要求6所述微生物催化氨基转移反应，得到西格列汀。
  10. 如权利要求8所述的用途，其特征在于，反应体系中包含磷酸吡哆醛作为辅酶。
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CN107384887A (zh) * 2017-07-05 2017-11-24 浙江工业大学 一种氨基转移酶、突变体及其制备西他列汀的应用
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