Transaminase mutants and uses thereof

Abstract

The application discloses a transaminase mutant and application thereof, and belongs to the technical field of genetic engineering and enzyme engineering. Arthrobacter sp. The transaminase mutant is obtained by mutating an amino acid sequence of wild-type Arthrobacter omega-transaminase. The application screens and modifies key residues of an active pocket substrate binding region, and obtains the transaminase mutant with significantly improved catalytic activity, so that the transaminase mutant has wide industrial application prospects in the green synthesis of chiral amine medical intermediates.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and enzyme engineering technology, and more specifically, to a transaminase mutant, its preparation method, and its application. Background Technology

[0002] ω-transaminase (ω-TA, EC 2.6.1.X) belongs to the transaminase family and catalyzes asymmetric reductive amination reactions of carbonyl compounds to generate chiral amines. It has significant applications in the green synthesis of chiral amine pharmaceutical intermediates and non-natural amino acids. Compared with traditional chemical synthesis methods, ω-transaminase-catalyzed asymmetric reductive amination reactions offer significant advantages such as mild reaction conditions, excellent stereoselectivity, and environmental friendliness, and have been widely applied in the industrial production of key intermediates for various chiral drugs, including sitagliptin and cinacalcet.

[0003] However, wild-type ω-transaminases from natural sources still face many technical bottlenecks in practical industrial applications. Firstly, the limited size of the active pocket in wild-type ω-transaminases and the unique spatial structure of their substrate-binding regions significantly restrict their catalytic ability towards sterically hindered non-natural ketone substrates, resulting in low catalytic efficiency and conversion rates. To address these issues, it is urgent to employ protein engineering techniques to target and modify ω-transaminases, thereby improving their overall performance in industrial applications. Summary of the Invention

[0004] The purpose of this invention is to provide a transaminase mutant with significantly improved catalytic activity and broadened substrate spectrum, so as to solve the technical problems of low catalytic efficiency and insufficient reaction conversion of transaminases for sterically hindered ketone substrates in the prior art.

[0005] This invention uses materials derived from the genus Arthrobacter ( Arthrobacter sp. Using ω-transaminase (SEQ ID NO: 1) as the parent material, a semi-rational design strategy was employed. Through homology modeling and molecular docking analysis, key amino acid residues in the substrate-binding region of the active pocket were identified for site-directed saturation mutagenesis screening. After high-throughput screening and multiple rounds of iterative mutagenesis combinations, several mutants with significantly better catalytic performance than the wild type were finally obtained.

[0006] Specifically, the present invention provides a transaminase mutant, which is obtained by mutation based on the wild-type transaminase with an amino acid sequence as shown in SEQ ID NO.1, and the mutation includes R442M.

[0007] Furthermore, the transaminase mutant has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO.1.

[0008] Furthermore, the transaminase mutant has at least 85%, 90%, 93%, 95%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO.1.

[0009] Sequence identity can be determined using sequence alignment algorithms known in the field (such as BLASTP, FASTA, CLUSTALW, etc.), calculated using default parameters.

[0010] Further, the mutation is one of the following single point mutations or combination mutations: R442M, R442M+C60Y, R442M+C60N, R442M+A436H, R442M+A436S, R442M+A436M, R442M+C60F+A436V, R442M+C60N+A436I, R442M+C60Y+A436P, R442M+C60F+A436M, R442M+C60N+A436V, R442M+C60V+A436H, R442M+C60Y+A436V, R442M+C60F+A436K, R442M+C60F+A436I.

[0011] Based on this invention, those skilled in the art will understand that, without altering the core catalytic function and stereoselectivity of transaminase, one or more amino acids can be conservatively substituted, added, or deleted from the transaminase mutant sequence to obtain a functionally equivalent protein variant.

[0012] In the amino acid mutation codes of this invention, the letters before and after the numbers are amino acid abbreviations known in the art. For example, V represents Valine, A represents Alanine, M represents Methionine, K represents Lysine, Q represents Glutamine, R represents Arginine, I represents Isoleucine, S represents Serine, H represents Histidine, E represents Glutamic acid, P represents Proline, F represents Phenylalanine, D represents Aspartic acid, Y represents Tyrosine, G represents Glycine, and N represents Asparagine.

[0013] In the amino acid mutation code of this invention, the numbers are the mutation site numbers, the letters before the numbers represent the amino acids before the mutation, and the letters after the numbers represent the amino acids after the mutation. For example, C60Y means that the 60th amino acid is mutated from C to Y, R442M means that the 442nd amino acid is mutated from R to M, and so on.

[0014] In the amino acid combination mutation codes of this invention, "+" indicates mutations performed before and after the "+" sign. For example, R442M+ C60Y indicates a combination of R442M and C60Y mutations, and so on.

[0015] The present invention also provides a DNA molecule encoding the above-mentioned transaminase mutant. In the nucleotide combination mutation code of this invention, ";" indicates mutations performed before and after ";". For example, "CGT1324-1326ATG;TGT178-180TAT" indicates a combination of mutations of CGT1324-1326ATG and TGT178-180TAT, and so on.

[0016] The present invention also provides a recombinant expression vector containing the aforementioned DNA molecule. The recombinant expression vector is constructed by operably linking the DNA molecule to an expression regulatory sequence, the expression regulatory sequence including a promoter, a ribosome binding site, and a terminator.

[0017] The expression vector may be selected from any vector known in the art suitable for expressing exogenous genes in host cells, including but not limited to at least one of the pET series vectors, pGEX series vectors, pUC series vectors, pBAD series vectors, and pMAL series vectors. Preferably, the recombinant expression vector is pET28a, pET21a, pBV220, or pGEX6P.

[0018] The present invention also provides a recombinant host cell containing the recombinant expression vector, or having the DNA molecule integrated into its genome.

[0019] Further, the recombinant host cell is selected from at least one of Escherichia coli, Bacillus subtilis, Pichia pastoris, or Saccharomyces cerevisiae. Preferably, the host cell is Escherichia coli BL21(DE3).

[0020] The present invention also provides a method for preparing the above-mentioned transaminase mutant, characterized by comprising the following steps: culturing the above-mentioned recombinant host cells, inducing expression, and collecting and purifying the transaminase mutant.

[0021] The present invention also provides an enzyme preparation of the above-mentioned transaminase mutant, wherein the enzyme preparation is selected from at least one of free enzyme, immobilized enzyme, crude enzyme solution, cell lysate or whole cell.

[0022] The present invention further provides a method for preparing chiral amine compounds by catalytic asymmetric reductive amination reaction, comprising: in the presence of the above-mentioned transaminase mutant or the above-mentioned recombinant host cell, using ketone substrate and amino donor as raw materials to carry out a transamination reaction to generate chiral amine compounds.

[0023] Furthermore, the structure of the ketone substrate is as follows: R1 and R2 are each independently selected from C 1-6 Alkyl groups, unsubstituted or substituted groups of the following: phenyl, 5-6 membered aromatic heterocycles, or R1 and R2 linked to form an unsubstituted or substituted 6-7 membered carbon ring benzo[a]benzene ring; the substituents on the phenyl, 5-6 membered aromatic heterocycle, 6-7 membered carbon ring benzo[a]benzene ring are each independently selected from C[a]. 1-6 Alkyl, C 1-6 alkoxy, halogen, hydroxyl, halogen-substituted C 1-6 Alkyl, halogen-substituted C 1-6 Alkoxy; The amino donor is a primary amine amino donor; The transamination reaction is carried out with the participation of a coenzyme.

[0024] Furthermore, the structure of the ketone substrate is selected from: The amino donor is isopropylamine or alanine; The coenzyme is pyridoxal phosphate.

[0025] Furthermore, the transamination reaction is carried out at a temperature of 25°C to 55°C and at a pH of 6.0 to 9.0.

[0026] The beneficial effects achieved by this invention are as follows: The transaminase mutant provided by this invention has significantly higher enzyme activity than the wild type. Compared with representative transaminase mutants in the prior art, the mutant of this invention exhibits highly efficient catalytic activity for aromatic ketone substrates such as 2'-methoxyacetophenone, 5-fluoro-2-hydroxyacetophenone, o-chloroacetophenone, and 1-benzocycloheptanone, as well as sterically hindered ketone substrates, providing a new option for the green, efficient, and industrialized production of chiral amine pharmaceutical intermediates. Attached Figure Description

[0027] Figure 1 A schematic diagram of the reaction route for the production of amines from substrate ketones using transaminase catalysis.

[0028] Figure 2 The structural formulas of the products after reduction of substrates S1 to S4 in Example 4.

[0029] Figure 3 HPLC chromatograms of the reaction solution after conversion of substrates S1 to S4 in Example 4. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and not for limiting the scope of protection of this invention. Any modifications, equivalent substitutions, and improvements made based on the principles of this invention should be included within the scope of protection of this invention.

[0031] Unless otherwise specified, the molecular biology experimental procedures (such as PCR amplification, site-directed mutagenesis, plasmid extraction, enzyme digestion and ligation, transformation, and protein expression and purification) used in the following examples were performed in accordance with the standard procedures in *Molecular Cloning: A Laboratory Manual*. All reagents used were commercially available analytical grade or biochemical reagents.

[0032] DNA fragments and protein sequences of recombinant proteins: The following examples use the transaminase with the amino acid sequence shown in SEQ ID NO:1 (nucleotide sequence SEQ ID NO:2) as the parent material for mutation experiments to obtain various variants. The amino acid sequence shown in SEQ ID NO:1 is derived from... Arthrobacter sp. The amino acid sequence of wild-type transaminase (asATA).

[0033] The amino acid sequence shown in SEQ ID NO: 1 is as follows: MGLTVQKINWEQVKEWDRKYLMRTFSTQNEYQPVPIESTEGDYLITPGGTRLLDFFNQLCCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSFRSGLVGENSESFSAQIPGSSCSSAVLMAPSSNTFQDSNGNYLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGVGSTMPPYEYVPQIRKMTKELGVLWISDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVVVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENLVEQAKNSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFRHGMNPNQIPTQIIMEKALEKGVLIGGAMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQQSA The nucleotide sequence shown in SEQ ID NO.2 is as follows: Example 1: Construction of a Mutant Library Using recombinant plasmid pGEX6p-asATA-WT as a template, degenerate mutant primers for C60, T88, F164, V242, L272, M423, A436, and R442 were designed using a saturation mutagenesis method. The saturation mutagenesis PCR conditions were: 10 μL 5×PS Buffer, 1 μL each of primers (10 μM), 4 μL dNTPs (2.5 mM), 0.5 μL PrimeSTAR HS enzyme (2.5 U / mL), 50 ng plasmid, and ultrapure water to a final volume of 50 μL. The conditions were: 98 ℃ pre-denaturation for 10 s, 98 ℃ denaturation for 10 s, 65 ℃ annealing for 15 s, and 72 ℃ extension for 4 min, for a total of 20 cycles. The PCR product was treated with 1 μL DpnI enzyme at 37 ℃ for 1 h. 0.5 μL of the PCR product was chemically transformed into an E. coli expression strain, and the resulting mutant library had a capacity of more than 100 mutants.

[0034] The construction of the combined mutant was carried out using the pGEX6p-asATA-R442M mutant plasmid as a template and a second round of saturation mutagenesis was performed using mutation primers.

[0035] Example 2: Screening of mutant libraries A single colony of the mutant strain constructed in Example 1 was picked and inoculated into a cell culture plate containing 150 μL of LB medium (100 μg / ml ampicillin). After overnight growth with shaking at 37°C and 750 rpm, 40 μL of seed culture was inoculated into 1 mL of LB medium (100 μg / ml ampicillin) and cultured with shaking at 37°C and 750 rpm. Cells were cultured until OD600 reached 0.6-0.8, then 0.2 mM IPTG was added, and protein expression was induced overnight at 25°C. After centrifugation, the supernatant was discarded (4000 rpm, 4°C, 10 min), and the resulting solution was added for enzyme activity assay. The specific reaction system was: 10 mM substrate, 5% DMSO, 0.1 M isopropylamine, 0.2 mM pyridoxal phosphate (PLP), 0.1 mol / L Tris buffer (pH 8.0), and incubated overnight at 37°C. After the reaction was complete, add 2 times the volume of acetonitrile, vortex to mix evenly and centrifuge, take the supernatant, filter and perform HPLC analysis.

[0036] By comparing the enzyme activity with that of wild-type asATA (SEQ ID NO.1), positive mutants with higher activity were screened. The gene sequences of the mutants were obtained after secondary screening and gene sequencing. Specific amino acid and nucleotide mutation sites are shown in Table 1.

[0037] Table 1. Mutation sites of asATA mutants Example 3: Expression and preparation of mutants Single colonies of the mutant strain constructed in Example 1 were inoculated into LB medium (containing 100 μg / mL ampicillin) and cultured overnight at 37°C. Then, 1% of the colonies were transferred to TB medium (containing 100 μg / mL ampicillin) and cultured at 37°C for 5 h. 0.5 mM IPTG was added for induction, and the culture was continued at 25°C for 16 h. The bacterial cells were collected by centrifugation at 4°C and 4000 rpm. The cells were resuspended in 1×PBS, sonicated, and then centrifuged at 15000 rpm and 4°C for 30 min. A portion of the supernatant was collected for protein expression level identification by electrophoresis. The remaining supernatant was flash-frozen in liquid nitrogen and then lyophilized to obtain the crude enzyme powder of the mutant.

[0038] Example 4: Enzyme activity assay of mutants The catalytic activity of the mutant obtained in Example 3 was tested according to the following method: 0.1 mol / L, pH 8.0 sodium phosphate buffer, 1 M isopropylamine, 1 mM PLP, 5 mg / mL crude enzyme solution, and substrate (one of the following: 15.01 mg / mL substrate S1, 1.61 mg / mL substrate S2, 1.54 mg / mL substrate S3, or 7.73 mg / mL substrate S4). The reaction was carried out at 37°C for 18 h. After the reaction, 2 volumes of acetonitrile were added to the reaction flask, vortexed to mix, and filtered through a 0.2 μm filter membrane. The product yield was detected by HPLC, and the conversion rate was calculated by comparing the product peak area with the standard curve of the product standard. A schematic diagram of the reaction route for the conversion of a substrate ketone to an amine using transaminase catalysis is shown below. Figure 1 As shown, the structural formulas of the products after reduction of substrates S1~S4 are as follows: Figure 2 As shown, the HPLC chromatograms of the reaction solution after the conversion of substrates S1 to S4 are as follows. Figure 3 As shown in the figure. Specific results are shown in Table 2.

[0039] Table 2 Comparison of activities between wild-type and mutant enzymes Activity results showed that the wild-type transaminase exhibited no or low activity in catalyzing the above-mentioned ketone substrates. The mutants modified through saturation mutagenesis and iterative combined mutagenesis demonstrated significantly superior catalytic activity against all tested substrates compared to the wild-type and control mutant C60Y. The mutant conversion rate could be increased to over 95%, clearly demonstrating that combined mutagenesis effectively broadened the binding capacity of the active pocket, enabling the efficient conversion of aromatic ring substrates that were previously difficult to access the catalytic center. The mutants provided by this invention significantly improve catalytic activity.

[0040] Example 5: Comparison of enzyme activity between the mutant of the present invention and the mutant of the prior art To further demonstrate the advantages of the transaminase mutant of the present invention, the gene sequences of the preferred mutant M2 of the present invention and representative transaminase mutants in the prior art (see Table 3) were cloned into the same pET28a vector, and crude enzyme lyophilized powder was prepared according to the method of Example 3. Since the expression levels of the three mutants differed, to ensure the comparability of their enzyme activities, the enzyme content was confirmed using SDS-PAGE gel analysis with two representative transaminase mutants in the prior art as standards. After adjusting to ensure that the target enzyme protein content was consistent, the enzyme catalytic activity was tested according to the method of Example 4, and the results are shown in Table 3.

[0041] Table 3 Comparison of catalytic activity of transaminase mutants in this invention and existing patents As shown in Table 3, the mutant M2 (C60Y+R442M) with two mutation sites provided by this invention exhibits superior substrate conversion. In particular, for the sterically hindered substrate S2, the multi-site mutant including C60Y / R442L reported in US12152263B2 has a substrate conversion rate of only 27.82%, and the multi-site mutant including C60Y / R442T reported in CN114277011B shows no catalytic activity. In contrast, the mutant M2 of this invention achieves a substrate conversion rate as high as 61.46%, demonstrating significantly improved catalytic activity.

[0042] Example 6: Large-scale fermentation of recombinant host cells Escherichia coli BL21(DE3) containing the mutant M2 encoding gene was inoculated into 100 mL LB medium (containing 100 μg / mL ampicillin) at an inoculation ratio of 1‰, and cultured overnight at 37°C with shaking at 220 rpm. A 5% inoculum was then transferred to a 5 L fermenter containing 2 L of fermentation medium (containing anhydrous glucose, yeast extract, peptone, KH2PO4, (NH4)2HPO4, MgSO4·7H2O, and trace metal ion solution). A fed-batch culture mode was used during fermentation. When the OD600 reached 30-40, the temperature was lowered to 25°C, and 0.5 mM IPTG was added to induce expression. Culture was continued for 14-18 h. OD600 was measured after fermentation. 600 The bacterial cells were collected by centrifugation. In a 5L fermenter for scale-up cultivation, OD... 600 The concentration reached 143.7, and the wet cell weight reached 260 g / L. After resuspending the cells in PBS, lyophilizing and freezing them, the ratio of wet cells to lyophilized powder was 4:1. The enzyme activity conversion rates of the mutant to the four substrates S1-S4 were consistent with the low-level expression data.

[0043] The results showed that the strain had a high expression level per unit volume, a high yield from the freeze-drying process, and a high substrate conversion rate, demonstrating its good potential for industrial scale-up.

[0044] In summary, the transaminase mutant provided by this invention achieves significant improvements in both catalytic activity and tolerance to sterically hindered substrates. The acquisition of this high-performance mutant offers a new option for the green, efficient, and industrialized production of chiral amine pharmaceutical intermediates.

Claims

1. A transaminase mutant, characterized in that, The transaminase mutant was obtained by mutating the wild-type transaminase with the amino acid sequence shown in SEQ ID NO.1, and the mutation includes R442M.

2. The transaminase mutant according to claim 1, characterized in that, The mutation is one of the following single point mutations or combinations of mutations: R442M, R442M+C60Y, R442M+C60N, R442M+A436H, R442M+A436S, R442M+A436M, R442M+C60F+A436V, R442M+C60N+A436I, R442M+C60Y+A436P, R442M+C60F+A436M, R442M+C60N+A436V, R442M+C60V+A436H, R442M+C60Y+A436V, R442M+C60F+A436K, R442M+C60F+A436I.

3. A DNA molecule, characterized in that, The DNA molecule encodes the transaminase mutant as described in claim 1 or 2.

4. A recombinant expression vector, characterized in that, The recombinant expression vector contains the DNA molecule as described in claim 3.

5. A recombinant host cell, characterized in that, The recombinant host cell contains the DNA molecule of claim 3 or the recombinant expression vector of claim 4.

6. The recombinant host cell according to claim 5, characterized in that, The host cells are selected from Escherichia coli, Bacillus subtilis, Pichia pastoris, or Saccharomyces cerevisiae.

7. A method for preparing the transaminase mutant according to claim 1 or 2, characterized in that, The process includes the following steps: culturing the recombinant host cell as described in claim 5, inducing expression, and collecting and purifying the transaminase mutant.

8. A method for preparing chiral amine compounds, characterized in that, In the presence of the transaminase mutant of claim 1 or 2 or the recombinant host cell of claim 5, a transamination reaction is carried out using ketone substrates and amino donors as raw materials to generate chiral amine compounds.

9. The method according to claim 8, characterized in that, The structure of the ketone substrate is as follows: R1 and R2 are each independently selected from C 1-6 Alkyl groups, unsubstituted or substituted groups of the following: phenyl, 5-6 membered aromatic heterocycles, or R1 and R2 linked to form an unsubstituted or substituted 6-7 membered carbon ring benzo[a]benzene ring; the substituents on the phenyl, 5-6 membered aromatic heterocycle, 6-7 membered carbon ring benzo[a]benzene ring are each independently selected from C[a]. 1-6 Alkyl, C 1-6 alkoxy, halogen, hydroxyl, halogen-substituted C 1-6 Alkyl, halogen-substituted C 1-6 Alkoxy; The amino donor is a primary amine amino donor; The transamination reaction is carried out with the participation of a coenzyme.

10. The method according to claim 9, characterized in that, The structures of the ketone substrates are selected from: The amino donor is isopropylamine or alanine; The coenzyme is pyridoxal phosphate.

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