D-threonine aldolase mutants and uses thereof

Abstract

The application belongs to the technical field of enzyme engineering, and particularly relates to a D-threonine aldolase mutant and application thereof. Compared with wild-type D-threonine aldolase, the substrate conversion rate of the mutant or salt thereof is significantly improved, and the enzyme activity is significantly improved.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, specifically relating to D-threonine aldolase mutants and their applications. Background Technology

[0002] D-Serine has a wide range of applications. As a chiral substance used in the synthesis of pharmaceutical intermediates, it is the main raw material for the synthesis of D-cyclic serine, and also a raw material for the synthesis of dipeptides, short peptides, and polypeptides. In the development of novel antibiotics, D-serine has been favored by many scientists. Furthermore, D-serine is an important intermediate in the synthesis of some chiral drugs such as lacosamide. Simultaneously, D-serine plays a crucial role in the biological regulation of the human body. Research has found that D-serine participates in the regulation of NMDA (N-methyl-D-aspartate) in the human brain, exhibiting anti-anxiety effects. In the treatment of Alzheimer's disease and schizophrenia, D-serine can effectively improve cognitive impairment and positive symptoms in patients with mental illness. D-serine is also involved in the pathogenesis of Parkinson's disease and epilepsy. In recent years, research on D-serine in the field of neuroscience has become increasingly popular, attracting widespread attention. Currently, the demand for D-serine in domestic and international markets is increasing year by year, but the existing production technology cannot meet the large domestic and international demand.

[0003] The microbial enzymatic conversion method for preparing D-serine can be carried out under normal pressure, with mild reaction conditions, which is conducive to large-scale industrial production. It does not produce by-products, and the subsequent extraction and purification are simple and convenient, with high yields and high optical purity of the obtained D-serine product. Therefore, the process does not generate large amounts of wastewater, waste gas, or solid waste, making it an energy-saving and healthy production process that is conducive to promoting green and environmentally friendly industries. It is an industry type that the country actively encourages and focuses on supporting.

[0004] Currently reported in the literature is that the wild-type D-threonine aldolase catalyzes the preparation of D-serine with low efficiency. Therefore, it is necessary to develop a highly active D-threonine aldolase mutant to meet the ever-expanding market demand for D-serine. Summary of the Invention

[0005] The first aspect of the present invention is to provide a D-threonine aldolase mutant or a salt thereof.

[0006] A second aspect of the present invention aims to provide a nucleic acid molecule encoding a D-threonine aldolase mutant of the first aspect of the present invention.

[0007] A third aspect of the present invention is to provide an expression cassette comprising a nucleic acid molecule of the second aspect of the present invention.

[0008] A fourth aspect of the present invention is to provide a carrier comprising a nucleic acid molecule of the second aspect or an expression cassette of the third aspect of the present invention.

[0009] The fifth aspect of the present invention aims to provide a cell comprising a D-threonine aldolase mutant or a salt thereof of the first aspect of the present invention, a nucleic acid molecule of the second aspect, an expression cassette of the third aspect, or a vector of the fourth aspect.

[0010] The sixth aspect of this invention aims to provide a product.

[0011] The seventh aspect of this invention aims to provide the application of the D-threonine aldolase mutant of the first aspect of this invention or its salt, the nucleic acid molecule of the second aspect, the expression cassette of the third aspect, the vector of the fourth aspect, the cell of the fifth aspect, or the product of the sixth aspect.

[0012] The object of the eighth aspect of the present invention is to provide a method for preparing D-serine.

[0013] The object of the ninth aspect of the present invention is to provide a method for preparing the D-threonine aldolase mutant or its salt as described in the first aspect of the present invention.

[0014] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a D-threonine aldolase mutant or a salt thereof, said D-threonine aldolase mutant having the following mutations compared to wild-type D-threonine aldolase: D152T, S242G and / or A250S.

[0015] In some embodiments, the amino acid sequence of the wild-type D-threonine aldolase is shown in SEQ ID NO: 1.

[0016] In some embodiments, the amino acid sequence of the D-threonine aldolase mutant is shown in SEQ ID NO: 6.

[0017] In some embodiments, the salt comprises at least one of a metal salt, an ammonium salt, a salt formed with an organic base, a salt formed with an inorganic acid, a salt formed with an organic acid, a salt formed with a basic amino acid, and a salt formed with an acidic amino acid.

[0018] In some embodiments, the metal salt comprises at least one of alkali metal salts (e.g., sodium salts, potassium salts, etc.), alkaline earth metal salts (e.g., calcium salts, magnesium salts, barium salts, etc.), and aluminum salts.

[0019] In some embodiments, the salt formed with an organic base comprises a salt formed with one or more of the following organic bases: trimethylamine, triethylamine, pyridine, methylpyridine, 2,6-dimethylpyridine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine.

[0020] In some embodiments, the salt formed with the inorganic acid comprises a salt formed with one or more of the following inorganic acids: hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid.

[0021] In some embodiments, the salt formed with the organic acid comprises a salt formed with one or more of the following organic acids: formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.

[0022] In some embodiments, the salt formed with the basic amino acid comprises a salt formed with one or more of the following basic amino acids: arginine, lysine, ornithine.

[0023] In some embodiments, the salt formed with the acidic amino acid comprises a salt formed with one or more of the following acidic amino acids: aspartic acid, glutamic acid.

[0024] A second aspect of the invention provides a nucleic acid molecule encoding a D-threonine aldolase mutant of the first aspect of the invention.

[0025] In some embodiments, the nucleotide sequence of the nucleic acid molecule includes: a1) The nucleotide sequence shown in SEQ ID NO:5; or a2) A nucleotide sequence of SEQ ID NO:5 that has undergone substitution and / or deletion and / or addition of one or more nucleotides, and has the same function as the nucleic acid molecule shown in SEQ ID NO:5; or a3) A nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 70%, 60%, 50%, 40%, or 30% homology with SEQ ID NO:5 and having the same function as the nucleic acid molecule shown in SEQ ID NO:5.

[0026] A third aspect of the present invention provides an expression cassette comprising a nucleic acid molecule of the second aspect of the present invention.

[0027] A fourth aspect of the present invention provides a carrier comprising a nucleic acid molecule of the second aspect of the present invention or an expression cassette of the third aspect of the present invention.

[0028] In some embodiments, the vector is selected from prokaryotic expression vectors or eukaryotic expression vectors.

[0029] In some embodiments, the carrier is selected from pGEX, pMAL, or pET.

[0030] In some embodiments, the carrier is pET-42a.

[0031] A fifth aspect of the invention provides a cell comprising a D-threonine aldolase mutant or a salt thereof of the first aspect of the invention, a nucleic acid molecule of the second aspect, an expression cassette of the third aspect, or a vector of the fourth aspect.

[0032] In some embodiments, the cells do not involve reproductive material.

[0033] In some embodiments, the cells are selected from prokaryotic cells or eukaryotic cells.

[0034] In some embodiments, the cells are selected from fungal cells, bacterial cells, plant cells, insect cells, or mammalian cells.

[0035] In some embodiments, the cells are selected from Escherichia coli; more specifically, Escherichia coli BL21(DE3).

[0036] A sixth aspect of the present invention provides a product comprising a D-threonine aldolase mutant or a salt thereof from the first aspect of the present invention, a nucleic acid molecule from the second aspect, an expression cassette from the third aspect, a vector from the fourth aspect, or a cell from the fifth aspect.

[0037] In some embodiments, the product comprises a D-threonine aldolase mutant or a salt thereof from the first aspect of the present invention; And optionally, glycine, formaldehyde and / or pyridoxal phosphate.

[0038] In some embodiments, the product comprises a D-threonine aldolase mutant of the first aspect of the invention or a salt thereof, glycine, formaldehyde, and pyridoxal phosphate.

[0039] In some embodiments, the product is used to synthesize D-serine.

[0040] In some embodiments, the product is a reagent or a kit.

[0041] A seventh aspect of the invention provides the use of the D-threonine aldolase mutant of the first aspect or a salt thereof, the nucleic acid molecule of the second aspect, the expression cassette of the third aspect, the vector of the fourth aspect, the cell of the fifth aspect, or the product of the sixth aspect in any of a1)-a2): a1) Synthesizes D-serine; a2) Prepare products that synthesize D-serine.

[0042] In some implementations, the product is a reagent combination, a reagent kit, or a reagent package.

[0043] An eighth aspect of the present invention provides a method for preparing D-serine, comprising the steps of using a D-threonine aldolase mutant or a salt thereof from the first aspect of the present invention, a nucleic acid molecule from the second aspect, an expression cassette from the third aspect, a vector from the fourth aspect, a cell from the fifth aspect, or a product from the sixth aspect.

[0044] In some embodiments, the method includes the following steps: mixing a D-threonine aldolase mutant of the first aspect of the present invention or its salt, glycine, formaldehyde and pyridoxal phosphate, and reacting to obtain the desired product.

[0045] In some embodiments, the concentration of the D-threonine aldolase mutant or its salt in the reaction system is 0.005%-0.015% by mass-volume ratio.

[0046] In some implementations, the reaction time is 1-3 hours.

[0047] In some embodiments, the reaction temperature is 32-39°C; more specifically, it is 36-38°C.

[0048] A ninth aspect of the present invention provides a method for preparing a D-threonine aldolase mutant or a salt thereof from the first aspect of the present invention, obtained by culturing the cells described in the fifth aspect of the present invention.

[0049] The beneficial effects of this invention are: This invention provides a D-threonine aldolase mutant or its salt, which, compared with wild-type D-threonine aldolase, significantly improves substrate conversion and enzyme activity. Detailed Implementation

[0050] definition Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0051] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0052] The present invention will be further described in detail below through specific embodiments.

[0053] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0054] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these examples are commercially available. For reagents whose manufacturers are listed, similar products from other manufacturers are substituted.

[0055] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. For reference, please refer to "Molecular Cloning: A Laboratory Manual (3rd Edition)" (Science Press), "Microbiology Experiments (4th Edition)" (Higher Education Press), and the manufacturer's instructions for the corresponding instruments and reagents.

[0056] Example 1: Obtaining the D-threonine aldolase gene tha from Arthrobacter DK-38 strain tha-F:5'-C CATATG AGCCAGGAAGTGATCCGTGG-3' (SEQ ID NO: 3), Reverse primer sequence tha-R: 5'-C CTCGAG TTAACGGCTAAAACCACGCG-3' (SEQ ID NO: 4); The italicized letters represent the restriction enzyme sites Nde I and Xho I, respectively. The PCR reaction was carried out in a 50 µL system under the following conditions: denaturation at 95°C for 3 min, denaturation at 95°C for 50 s, annealing at 58°C for 1 min, extension at 72°C for 3 min, for a total of 35 cycles; extension at 72°C for 10 min; 3 μL of the PCR product was then used for agarose gel electrophoresis for verification; 100 μL of the PCR product was then used for agarose gel electrophoresis, and the target fragment was recovered according to the instructions of the gel recovery kit.

[0057] Example 2: Construction of the expression vector for the D-threonine aldolase gene tha from Arthrobacter DK-38 strain The PCR product from Example 1 was digested with restriction endonucleases Nde I and Xho I, and then ligated with the pET-42a plasmid (purchased from Novagen) digested with Nde I and Xho I. The constructed vector was named pET42a-tha. The ligation product pET42a-tha was then used to transform Escherichia coli DH5-α (purchased from Promega), and the plasmid of the transformed bacteria was extracted by sequencing. The plasmid was then used to transform Escherichia coli BL21(DE3) strain (purchased from Promega) to obtain the wild-type strain.

[0058] Example 3: Error-prone PCR amplification of the tha gene Taking advantage of the fact that Taq DNA polymerase lacks 3′–5′ proofreading function, the frequency of random mutations was controlled by using high magnesium ion concentration (8 mmol / L) and different concentrations of dNTPs (dATP and dGTP at 1.5 mmol / L, and dTTP and dCTP at 3.0 mmol / L). Random mutations were introduced into the target gene to construct a mutation library. The template concentration A260 value was 1000 ng / mL, the enzyme concentration was 5 U / µL, and the primer concentration was 100 µM. The optimal mutation rate in the experiment was approximately 0.6%. The error-prone PCR reaction system (100 µL) is as follows: 10×PCR reaction buffer 10 µL; dATP 2 µL; dTTP 4 µL; dCTP 4 µL; dGTP 2 µL; MgCl2 6 µL; Primer tha-F 4 µL; Primer tha-R 4 µL; DNA template (PCR product from Example 1) 4 µL; Taq 1 µL of DNA polymerase; ddH2O 59 µL; Total volume 100 µL.

[0059] The PCR program was as follows: 95℃ pre-denaturation for 3 min; 94℃ denaturation for 1 min, 54℃ annealing for 1 min, 75℃ extension for 3 min, 45 cycles; and a final extension at 75℃ for 15 min. The PCR product was recovered by gel extraction, and 5 µL of the product was examined by 1% agarose gel electrophoresis and stored at -20℃ for later use.

[0060] Example 4: Construction of a library of *Arthrobacter* DK-38 strain D-threonine aldolase gene *tha* mutants Error-prone PCR products were digested with restriction endonucleases Nde I and Xho I, and then ligated with pET-42a plasmid digested with Nde I and Xho I to construct the vector library pET42a-thaM. pET42a-thaM was then transformed into Escherichia coli BL21(DE3) strain to obtain mutant strains, and an expression mutant library was constructed.

[0061] Example 5: Construction of expression mutant library and screening of mutants The mutant strain obtained in Example 4 was randomly selected and placed into a 6-well plate of LB medium containing 60 μg / mL kanamycin. The plate was incubated at 37°C and 150 rpm until the OD600 value reached 0.6-0.8. IPTG (final concentration 0.1 mmol / L) was then added, and the plate was incubated at 30°C for another 12 h. The bacterial cells were collected by centrifugation and resuspended in 50 mmol / L, pH 8.0 Tris-HCl buffer (containing 1 mmol / L imidazole) (wet cell to buffer ratio: 1 g wet cell: 5 mL buffer). The cells were then sonicated in an ice bath, centrifuged, and the supernatant was collected for protein purification and enzyme activity assay. The specific methods are as follows: Take Ni 2+5 mL of NAT-0 agarose was packed into the chromatography column and washed twice with 25 mL of water. The column was then equilibrated with 25 mL of NAT-0 buffer (20 mM Tris-HCl pH 7.9, 0.5 M NaCl). The disrupted bacterial cells were collected, and the supernatant obtained after centrifugation was added to Ni... 2+ In the NTA chromatography column, after adding the solution 2-3 times, the medium was washed with 25 mL of NAT-1 buffer (i.e., NAT-0 buffer containing 80 mmol / L imidazole) to remove impurities. Finally, the target protein was eluted with the above buffer containing 300 mmol / L imidazole. SDS-PAGE analysis yielded a specific protein band of the expected size, and the protein concentration was determined using the Brandford method. Enzyme activity assay: Add 0.01% (w / v) purified protein to a pH 7.5 mixed reaction solution containing 0.1M glycine, 0.1M formaldehyde, and 0.01mM pyridoxal phosphate, mix well, and react at 37℃ for 2 h. Detect the glycine and D-serine content using high-performance liquid chromatography (HPLC), and calculate the conversion rate (D-serine molar conversion rate = (C...). V M1) / (m M2); where C is the final concentration of D-serine in the reaction solution (g / L), V is the final volume of the reaction solution (L), M1 is the molar mass of glycine (g / mol), M2 is the molar mass of D-serine (g / mol), and m is the mass of glycine (g) to determine its enzyme activity. The liquid chromatography conditions include: a C18 reversed-phase column (4.6... The column was 150 mm long and 5 μm wide. The mobile phase was methanol:water (20:80), the flow rate was 1 mL / min, the column temperature was 35 ℃, the detection wavelength was 210 nm, and the injection volume was 20 μL.

[0062] Calculations showed that the substrate conversion rate of the wild-type strain in Example 2 was 3.25%, while the substrate conversion rate of the mutant strain M1 (THAM1) was the highest, reaching 95.09% (Table 1).

[0063] Table 1 Glycine Conversion Rate

[0064]

[0065] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A D-threonine aldolase mutant or a salt thereof, wherein the D-threonine aldolase mutant has the following mutations compared with wild-type D-threonine aldolase: D152T, S242G and / or A250S.

2. The mutant according to claim 1, characterized in that, The amino acid sequence of the wild-type D-threonine aldolase is shown in SEQ ID NO: 1; Preferably, the amino acid sequence of the D-threonine aldolase mutant is shown in SEQ ID NO:

6.

3. A nucleic acid molecule encoding the D-threonine aldolase mutant according to any one of claims 1-2.

4. The nucleic acid molecule according to claim 3, characterized in that, The nucleotide sequence of the nucleic acid molecule includes: a1) The nucleotide sequence shown in SEQ ID NO:5; or a2) A nucleotide sequence of SEQ ID NO:5 that has undergone substitution and / or deletion and / or addition of one or more nucleotides, and has the same function as the nucleic acid molecule shown in SEQ ID NO:5; or a3) A nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 70%, 60%, 50%, 40%, or 30% homology with SEQ ID NO:5 and having the same function as the nucleic acid molecule shown in SEQ ID NO:

5.

5. An expression cassette comprising the nucleic acid molecule according to any one of claims 3-4.

6. A vector comprising the nucleic acid molecule of any one of claims 3-4 or the expression cassette of claim 5.

7. A cell comprising the D-threonine aldolase mutant or its salt as described in any one of claims 1-2, the nucleic acid molecule as described in any one of claims 3-4, the expression cassette as described in claim 5, or the vector as described in claim 6.

8. A product comprising the D-threonine aldolase mutant or a salt thereof as described in any one of claims 1-2, the nucleic acid molecule as described in any one of claims 3-4, the expression cassette as described in claim 5, the vector as described in claim 6, or the cell as described in claim 7; Preferably, the product comprises the D-threonine aldolase mutant or a salt thereof as described in any one of claims 1-2; And optionally, glycine, formaldehyde and / or pyridoxal phosphate.

9. The use of the D-threonine aldolase mutant or its salt according to any one of claims 1-2, the nucleic acid molecule according to any one of claims 3-4, the expression cassette according to claim 5, the vector according to claim 6, the cell according to claim 7, or the product according to claim 8 in any one of a1)-a2): a1) Synthesizes D-serine; a2) Prepare products that synthesize D-serine.

10. A method for preparing D-serine, comprising the steps of using the D-threonine aldolase mutant or its salt as described in any one of claims 1-2, the nucleic acid molecule as described in any one of claims 3-4, the expression cassette as described in claim 5, the vector as described in claim 6, or the cell as described in claim 7, or the product as described in claim 8.

11. The method according to claim 10, characterized in that, The method comprises the following steps: mixing the D-threonine aldolase mutant or its salt as described in any one of claims 1-2, glycine, formaldehyde and pyridoxal phosphate, and reacting to obtain the desired product.

12. The method for preparing the D-threonine aldolase mutant or its salt according to any one of claims 1-2, obtained by culturing the cells according to claim 7.

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