4-aminobutyric acid transaminase and its encoding gene and application

Abstract

The application discloses 4-aminobutyric acid transaminase and an encoding gene and application thereof. The 4-aminobutyric acid transaminase has an amino acid sequence shown in SEQ ID NO:1, and a mutant thereof has an amino acid sequence shown in SEQ ID NO:3. The 4-aminobutyric acid transaminase provided by the application can efficiently catalyze synthesis of glutaric acid.

Description

Technical Field

[0001] This invention belongs to the field of functional enzyme screening technology, and relates to a 4-aminobutyric acid transaminase, its encoding gene, and its applications. Background Technology

[0002] Glutaric acid is a widely used chemical, including in the production of polyamides, polyurethanes, glutaric anhydride, 1,5-pentanediol, and 5-hydroxyvalerate. A C5 dicarboxylic acid, glutaric acid is widely used in the manufacture of industrial polymers such as aliphatic polyesters, polyurethanes, and polyethylene, often as a corrosion inhibitor and plasticizer. Furthermore, dimethyl glutaric acid functions as a green solvent and can be used in cleaning products, paints, and coating components. Glutaric acid is typically produced through various petroleum-based chemical methods, including the nitric acid-catalyzed oxidation of 2-cyanopentanone and the condensation of acrylonitrile with ethyl malonate. However, these processes rely on non-renewable and toxic starting materials. Therefore, the production of glutaric acid using whole-cell systems through fermentation and biotransformation has been adopted.

[0003] 4-Aminobutyric acid transaminase can catalyze the conversion of 5-aminovaleric acid to glutaric acid. This catalytic process requires cofactors such as β-nicotinamide adenine dinucleotide (NAD+) and α-ketoglutaric acid (α-KG). Currently, large-scale production of glutaric acid has not been achieved through fermentation and biotransformation, therefore there remains a need to find a genetically engineered bacterium that can produce high levels of glutaric acid. Summary of the Invention

[0004] The purpose of this invention is to provide a 4-aminobutyric acid transaminase, its mutants, its encoding gene, and its applications, thereby solving the problems of low enzyme activity, conversion rate, and stability in existing 4-aminobutyric acid transaminase-catalyzed synthesis of glutaric acid.

[0005] Specifically, this invention obtains 4-aminobutyric acid transaminase from *Schizosaccharomyces japonicus* yFS275, the amino acid sequence of which is shown in SEQ ID NO:1 and the nucleotide sequence of which is shown in SEQ ID NO:2. Further mutations are performed to obtain a 4-aminobutyric acid transaminase mutant with significantly improved enzyme activity, conversion rate, and stability, the amino acid sequence of which is shown in SEQ ID NO:3 and the nucleotide sequence of which is shown in SEQ ID NO:4.

[0006] In a first aspect, the present invention provides a 4-aminobutyric acid transaminase having the amino acid sequence shown in SEQ ID NO:1.

[0007] The 4-aminobutyric acid transaminase provided by this invention can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically, for example, by using recombinant technology to express it from a prokaryotic host (e.g., Escherichia coli) or a eukaryotic host (e.g., yeast, higher plants).

[0008] In some embodiments, the above-mentioned 4-aminobutyric acid transaminase is obtained by introducing a recombinant vector containing its encoding gene into Escherichia coli (e.g., E.coli BL21(DE3)) to obtain a recombinant genetically engineered bacterium, and then inducing the expression of the recombinant genetically engineered bacterium to obtain 4-aminobutyric acid transaminase.

[0009] In a second aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned 4-aminobutyric acid transaminase, having the nucleotide sequence shown in SEQ ID NO:2.

[0010] The nucleic acid molecules provided by this invention can usually be obtained by PCR amplification or artificial synthesis.

[0011] In a third aspect, the present invention provides a mutant of the above-mentioned 4-aminobutyric acid transaminase, which has the amino acid sequence shown in SEQ ID NO:3, and whose catalytic activity, stability and conversion rate are further improved compared with the above-mentioned 4-aminobutyric acid transaminase.

[0012] The mutant of 4-aminobutyric acid transaminase provided by the present invention can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically, for example, by using recombinant technology to express it from a prokaryotic host (e.g., Escherichia coli) or a eukaryotic host (e.g., yeast, higher plants).

[0013] In some embodiments, the above-mentioned 4-aminobutyric acid transaminase is obtained by introducing a recombinant vector containing its encoding gene into Escherichia coli (e.g., E.coli BL21(DE3)) to obtain a recombinant genetically engineered bacterium, and then inducing expression of the recombinant genetically engineered bacterium to obtain a mutant of 4-aminobutyric acid transaminase.

[0014] In a fourth aspect, the present invention provides a nucleic acid molecule encoding the mutant of the above-mentioned 4-aminobutyric acid transaminase, having the nucleotide sequence shown in SEQ ID NO:4.

[0015] The nucleic acid molecules provided by this invention can usually be obtained by PCR amplification or artificial synthesis.

[0016] In a fifth aspect, the present invention provides a recombinant vector comprising any of the nucleic acid molecules described above.

[0017] The recombinant vector includes a cloning vector and an expression vector, wherein the cloning vector is used to replicate the relevant sequence and the expression vector is used to express the relevant gene.

[0018] In some embodiments, the recombinant vector is pET-SJAG or pET-ΔSJAG42, which is obtained by replacing the sequence between the XbaI and BamHI restriction sites of pET-28a(+) with the nucleic acid molecule encoding the above-mentioned 4-aminobutyric acid transaminase or the nucleic acid molecule of the above-mentioned mutant 4-aminobutyric acid transaminase, while keeping the rest of the sequence unchanged.

[0019] In a sixth aspect, the present invention provides a recombinant cell comprising any of the recombinant vectors described above.

[0020] In some embodiments, the recombinant cells induce the production of the above-mentioned 4-aminobutyric acid transaminase or a mutant of the above-mentioned 4-aminobutyric acid transaminase.

[0021] In some implementations, the method for constructing the recombinant cells includes the following:

[0022] The recombinant vector was transformed into host cells and induced to obtain mutants expressing 4-aminobutyric acid transaminase or the above-mentioned 4-aminobutyric acid transaminase.

[0023] Furthermore, the recombinant vector is any of the recombinant vectors described above, and the host cell is a prokaryotic cell or a eukaryotic cell, such as Escherichia coli, yeast, etc., preferably Escherichia coli BL21(DE3).

[0024] In some embodiments, the recombinant cells are recombinant bacteria W and recombinant bacteria 42, and the recombinant cells can be recombinant genetically engineered bacteria. The culture medium used when the recombinant genetically engineered bacteria express 4-aminobutyric acid transaminase or its mutants can be any culture medium in the art that enables the recombinant genetically engineered bacteria to grow and produce the 4-aminobutyric acid transaminase or its mutants of the present invention, preferably LB medium.

[0025] There are no special requirements for the culture method and culture conditions, as long as the genetically engineered bacteria can grow normally and express 4-aminobutyric acid transaminase or its mutant.

[0026] More specifically, the above-mentioned method for constructing recombinant cells includes the following steps:

[0027] (1) Amplification of the 4-aminobutyric acid transaminase gene SJAG;

[0028] (2) Obtaining the mutant ΔSJAG of the 4-aminobutyric acid transaminase gene;

[0029] (3) Construction of recombinant expression plasmids pET-SJAG and pET-ΔSJAG;

[0030] (4) Transform the recombinant expression plasmids pET-SJAG and pET-ΔSJAG into host cells;

[0031] (5) Positive clones were obtained by screening with resistant culture medium.

[0032] In a seventh aspect, the present invention provides a method for preparing 4-aminobutyric acid transaminase or a mutant thereof, comprising:

[0033] The recombinant cells described above were induced to undergo culture to obtain a culture.

[0034] Isolate the above-mentioned 4-aminobutyric acid transaminase or a mutant of the above-mentioned 4-aminobutyric acid transaminase from the culture;

[0035] The methods for inducing and culturing recombinant cells and for isolating 4-aminobutyric acid transaminase or its mutants from the culture are conventional methods in this field.

[0036] In an eighth aspect, the present invention provides the use of the above-described 4-aminobutyric acid transaminase, any of the above-described nucleic acid molecules, mutants of the above-described 4-aminobutyric acid transaminase, the above-described recombinant vector, the above-described recombinant cells, and / or the 4-aminobutyric acid transaminase or mutants thereof prepared by the above-described methods in the preparation of glutaric acid.

[0037] In a ninth aspect, the present invention provides a method for preparing glutaric acid, comprising: using the above-mentioned 4-aminobutyric acid transaminase, the above-mentioned mutant of 4-aminobutyric acid transaminase, the above-mentioned recombinant cells and / or the 4-aminobutyric acid transaminase or its mutant prepared by the above method as a catalyst to catalyze the reaction of 5-aminovaleric acid to obtain glutaric acid.

[0038] In some embodiments, the temperature in the above catalytic reaction is 20-40°C, for example 20°C, 30°C, 40°C, or any value or range between these values, preferably 30°C; the initial pH is 7-9, which can be adjusted using a Tris base (e.g., 500 mM Tris), for example, to pH 8.5.

[0039] The catalytic reaction comprises 5-aminovaleric acid, α-ketoglutaric acid, polysorbate 80, and NAD+;

[0040] The concentration of 5-aminovaleric acid is 200-500 mM, for example 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, or any value or range between these values, preferably 300 mM.

[0041] The concentration of α-ketoglutaric acid is 100-300 mM, for example 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, or any value or range between these values, preferably 150 mM.

[0042] The volume percentage of polysorbate 80 is 0.01-1%, for example 0.01%, 0.02%, 0.04%, 0.06%, 0.08%, 0.1%, or any value or range between these values, preferably 0.06%;

[0043] The concentration of NAD+ is 5-20 mM, such as 5 mM, 10 mM, 15 mM, 20 mM, or any value or range between these values, preferably 10 mM.

[0044] In some embodiments, the above-described catalytic reaction includes catalyzing the reaction of 5-aminovaleric acid with any of the recombinant cells described above to obtain glutaric acid;

[0045] Specifically, the recombinant cells described above can be used as catalysts for whole-cell catalytic production of glutaric acid. The amount of catalyst used is 0.5-2 mg cells / mM 5-aminovaleric acid, preferably 1 mg cells / mM 5-aminovaleric acid.

[0046] It should be understood that the 4-aminobutyric acid transaminase or its mutants described in this invention can be used in whole-cell engineered bacteria, in unpurified crude enzyme form, or in partially or completely purified enzyme form. Furthermore, the 4-aminobutyric acid transaminase or its mutants of this invention can be prepared into immobilized enzymes or catalysts in immobilized cell form using immobilization techniques known in the art.

[0047] The 4-aminobutyric acid transaminase and its mutant of the present invention can efficiently catalyze the synthesis of glutaric acid. In particular, the mutant, under suitable conditions, with 5-aminovaleric acid as a substrate, has a conversion rate of 98.8% and an enzyme activity of 986.8 U / L, which is 1.87 times that of the original enzyme, and its stability is also better than that of the original enzyme. Detailed Implementation

[0048] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the techniques used in the embodiments are conventional practices in the art, or experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, the reagents and biological materials used in the embodiments are commercially available.

[0049] The Japanese fission yeast (Schizosaccharomyces japonicus yFS275) was disclosed in the literature “Ren L, Mclean J R, Hazbun TR, et al. Systematic Two-Hybrid and Comparative Proteomic Analyses Reveal Novel Yeast Pre-mRNA Splicing Factors Connected to Prp19[J]. Plos One, 2011, 6(2): e16719-e16719” and is available to the public from Wanhua Chemical Group Co., Ltd.

[0050] pET-28a(+) is a product of Sangon Biotech (Shanghai) Co., Ltd., with product catalog number B540183.

[0051] Example 1: Obtaining the gene sequence of 4-aminobutyric acid transaminase

[0052] 1. Genomic DNA was extracted from *Schizosaccharomyces japonicus* yFS275.

[0053] 2. Using the genomic DNA obtained in step 1 as a template, PCR was performed using primer 1 (5'-tctagaatgactgctgcacctttcttc-3', SEQ ID NO:5) and primer 2 (5'-ggatccctaagcagagaagaccttgtc-3', SEQ ID NO:6) to obtain a PCR amplification fragment containing the 4-aminobutyric acid transaminase gene. The nucleotide sequence of the 4-aminobutyric acid transaminase gene is shown in SEQ ID NO:2, and the amino acid sequence of the 4-aminobutyric acid transaminase it encodes is shown in SEQ ID NO:1.

[0054] Example 2: Obtaining the gene sequence of the 4-aminobutyric acid transaminase mutant using error-prone PCR technology

[0055] Error-prone PCR methods modify and increase the natural error rate of polymerases based on standard PCR. Taq polymerase is the most commonly used error-prone PCR polymerase. Compared to the basic PCR reaction (1.5 mM), error-prone PCR reactions typically contain a higher concentration of magnesium chloride (7 mM) to stabilize non-complementary pairs. Additionally, manganese chloride can be added to further increase the error rate. This example utilizes error-prone PCR technology to obtain a mutant of the 4-aminobutyric acid transaminase gene.

[0056] Using the PCR amplification fragment from Example 1 as a template, and primers 1 and 2 as primer pairs, the following error-prone PCR was performed, resulting in 96 mutants of the 4-aminobutyric acid transaminase gene.

[0057] Error-prone PCR reaction system: 5 μl of 10× amplification buffer, 4 μl each of the four dNTP mixtures (2.5 mmol / L), 50 pmol of each primer, 1.5 μg of template DNA, 0.5 μL of Taq DNA polymerase, Mg 2+ 7 mmol / L, add double-distilled water to 50 μl.

[0058] Example 3: Cloning and Construction of Strains Expressing the 4-Aminobutyrate Transaminase Gene (SJAG)

[0059] 1. The PCR amplification fragment containing the 4-aminobutyric acid transaminase gene obtained in Example 1 was digested with XbaI and BamHI to obtain the gene fragment; pET-28a(+) was digested with XbaI and BamHI to obtain the vector fragment; the gene fragment and the vector fragment were ligated to obtain the recombinant expression plasmid, which was named pET-SJAG. The plasmid was sent for sequencing, and the results were consistent with expectations.

[0060] 2. The recombinant expression plasmid pET-SJAG obtained in step 1 was heat-shocked into E. coli DH5α competent cells and plated on LB solid medium containing 25 μg / mL kanamycin. The corresponding single clone strain was obtained and named H. After amplification and plasmid extraction, the pET-SJAG plasmid was obtained. The obtained plasmid was chemically transformed into E. coli BL21(DE3) and plated on LB solid medium containing 25 μg / mL kanamycin for screening to obtain recombinant strain W expressing 4-aminobutyric acid transaminase.

[0061] Example 4: Cloning and Construction of Expression Strains of 4-Aminobutyrate Transaminase Gene Mutants

[0062] Following the method of Example 3, recombinant plasmids pET-ΔSJAG1 to pET-ΔSJAG96 were constructed using 96 mutants of the 4-aminobutyric acid transaminase gene obtained in Example 2, and recombinant bacteria 1 to recombinant bacteria 96 expressing 4-aminobutyric acid transaminase mutants were obtained.

[0063] Example 5: Obtaining a 4-aminobutyric acid transaminase mutant with high catalytic efficiency using high-throughput screening.

[0064] The recombinant bacteria from Examples 3 and 4, which were verified by PCR, were cultured in 5 mL of LB medium, and IPTG was added to induce protein expression. After 12 h, the OD of the bacterial culture was measured. 600 The value was adjusted, and the bacterial solution was diluted with water to achieve the desired OD value.600 The value was approximately 2. The diluted bacterial culture was added to 96-well plates for reaction. The reaction system consisted of 200 μL of 200 mM 5-aminovaleric acid, 150 mM α-ketoglutarate, 0.06% (v / v) polysorbate 80, and 10 mM NAD+. The initial pH was adjusted to 8.5 using 500 mM Tris base. Cells were added to the reaction system at a ratio of 1 mg cells / mM 5-aminovaleric acid. The prepared reaction system was incubated at 30 °C and 120 rpm for 24 hours using a shaker. After the reaction was completed, the reaction was terminated by heating at 95 °C for 5 min.

[0065] After the reaction was complete, 30 μl of the reaction solution was added to 40 μl of 2M 2,4-dinitrophenyl ether aqueous solution, and the mixture was placed in a 37℃ incubator for 20 min. Then, 100 μl of 0.3M sodium hydroxide aqueous solution was added, and the reaction system was diluted with water to 200 μl. After the reaction was complete, the mixture was cooled to room temperature in a water bath, and the absorbance was measured at a selected wavelength of 380 nm.

[0066] The strain corresponding to the reaction solution with the lowest absorbance (recombinant strain 42) underwent plasmid extraction. Sequencing of the extracted plasmid yielded the gene sequence encoding the 4-aminobutyric acid (GABA) transaminase mutant, as shown in SEQ ID NO:4. The amino acid sequence of the GABA transaminase mutant encoded by this mutant is shown in SEQ ID NO:3. Compared to the amino acid sequence of GABA transaminase shown in SEQ ID NO:1, this GABA transaminase mutant exhibits the following mutations: Vla at position 114 is mutated to Ala; Tyr at position 345 is mutated to His; and Glu at position 389, Arg at position 403, Glu at position 416, and Cys at position 438 are all mutated to Gly.

[0067] Example 6: Enzyme preparation and enzyme activity assay

[0068] The recombinant strain W obtained in Example 3 and the recombinant strain 42 screened in Example 4 were respectively subjected to scale-up culture. After scale-up culture, the fermentation broth was subjected to conventional treatments such as centrifugation (8000 rpm, 10 min), cell disruption, and freeze drying to prepare lyophilized powders of 4-aminobutyric acid transaminase (original enzyme) and its mutant (mutant enzyme) and stored at -80℃.

[0069] The enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the production of 1 μmol of glutaric acid per minute or the amount of enzyme required to consume 1 μmol of substrate 5-aminovaleric acid per minute under the reaction conditions of Example 5.

[0070] The enzyme activities of the original enzyme and the mutant enzyme were 526.4 U / L and 986.8 U / L, respectively. Compared with the original enzyme, the enzyme activity of the mutant enzyme was 1.87 times. The conversion rates of the two enzymes were 78.8% and 98.8%, respectively, by HPLC analysis of the residual amount of 5-aminovaleric acid.

[0071] The specific methods of HPLC are as follows:

[0072] (1) Take 1 mL of fermentation broth and centrifuge it at 120,000 rpm for 1 min;

[0073] (2) After centrifugation, carefully aspirate 500 μL of the supernatant into a 2 mL centrifuge tube and add 500 μL of ultrapure water;

[0074] (3) Use a 1mL syringe to draw 600-800μL of the diluted fermentation broth supernatant, filter it through a 0.22μm filter membrane and add it to the corresponding liquid phase bottle;

[0075] (4) Place the HPLC bottle on the sample rack, set up the method, and begin analysis. The specific procedure for HPLC analysis is as follows: Shimadzu LC-20A high-performance liquid chromatography system, equipped with Bio-Rad Aminex HPX-87N column, initial column temperature at room temperature, gradually increasing the flow rate as the column temperature increases, column temperature during analysis at 65℃, flow rate at 0.6 mL / min, mobile phase at 0.5 mM H2SO4 solution, differential refractive index detector, injection volume at 10 μL, glutaric acid elution time at 16.6 min, and 5-aminovaleric acid elution time at 19.9 min.

[0076] Example 7: Stability Test

[0077] Take a small amount of the lyophilized powder of the original enzyme and mutant enzyme obtained in Example 6, and react it according to the reaction conditions in Example 5. After the reaction is completed, centrifuge to recover the enzyme and react it again. The conversion rates of the two enzymes in each batch are shown in Table 1.

[0078] Table 1

[0079]

[0080] The results showed that the mutant enzyme had good reusability, and the conversion rate could still reach over 95% after 6 batches of reaction. Compared with the original enzyme, it had better stability. After 6 batches of reaction, the activity of the original enzyme decreased by 17.2%, while the activity of the recombinant enzyme only decreased by 2.7%.

[0081] sequence

[0082] SEQ ID NO:1

[0083] MTAAPFFPNEPKGPKIVTEEIPGPQSKAAVAEMTKYIDTSATKLVVDYEKSIGNYLVDADGNVYLDVYAQIASIAVGYNNPTLIKAAKSDEVATLMMNRPALGNFPPKEWARIVKEGLIDNAPKGQKYAYVQMSGSDANESAFKIAFIHLAAQKRKGAGFSEEDLVSVMNNQAPGSPEVAILSFRRAFHGRLFGSLSTTRSKPIHKLDIPLFPWPQADFPALKYPLEDHVEENAAEEQRCIDQVDQILSTHHCPVAACIVEPIQSEGGDNHASPEFFHKLQATLKKHGVLFIVDEVQTGVCATGNMWAHEAWNLPYPPDMVTFSKKFQVAGFFYSDLLLRPALAYRHFNTWMGDPIRVVQAKYICQEIRDHNLLQNTIEVGNYVYQGLEKLAAKYPGKINNLRGKNKGTFIAFDCESPEARDKFCADMKHEGVNIGGCGPIGIRLRPMLVFQKHHADIMLAAIDKVFSA

[0084] SEQ ID NO:2

[0085]

[0086] SEQ ID NO:3

[0087] MTAAPFFPNEPKGPKIVTEEIPGPQSKAAVAEMTKYIDTSATKLVVDYEKSIGNYLVDADGNVYLDVYAQIASIAVGYNNPTLIKAAKSDEVATLMMNRPALGNFPPKEWARIAKEGLIDNAPKGQKYAYVQMSGSDANESAFKIAFIHLAAQKRKGAGFSEEDLVSVMNNQAPGSPEVAILSFRRAFHGRLFGSLSTTRSKPIHKLDIPLFPWPQADFPALKYPLEDHVEENAAEEQRCIDQVDQILSTHHCPVAACIVEPIQSEGGDNHASPEFFHKLQATLKKHGVLFIVDEVQTGVCATGNMWAHEAWNLPYPPDMVTFSKKFQVAGFFYSDLLLRPALAHRHFNTWMGDPIRVVQAKYICQEIRDHNLLQNTIEVGNYVYQGLGKLAAKYPGKINNLGGKNKGTFIAFDCGSPEARDKFCADMKHEGVNIGGGGPIGIRLRPMLVFQKHHADIMLAAIDKVFSA

[0088] SEQ ID NO:4

[0089]

Claims

1. A 4-aminobutyric acid transaminase mutant, the amino acid sequence of which is shown in SEQ ID NO:

3.

2. A nucleic acid molecule encoding the 4-aminobutyric acid transaminase mutant of claim 1, the nucleotide sequence of which is shown in SEQ ID NO:

4.

3. A recombinant vector comprising the nucleic acid molecule of claim 2.

4. A recombinant cell comprising the recombinant vector of claim 3.

5. A method for preparing a 4-aminobutyric acid transaminase mutant, comprising the following steps: The recombinant cells described in claim 4 were induced and cultured to obtain a culture. The 4-aminobutyric acid transaminase mutant of claim 1 was isolated from the culture.

6. The application of the 4-aminobutyric acid transaminase mutant of claim 1, the nucleic acid molecule of claim 2, the recombinant vector of claim 3, the recombinant cell of claim 4, and / or the 4-aminobutyric acid transaminase mutant prepared by the preparation method of claim 5 in the preparation of glutaric acid.

7. A method for preparing glutaric acid, comprising the following steps: using the 4-aminobutyric acid transaminase mutant of claim 1, the recombinant cells of claim 4, and / or the 4-aminobutyric acid transaminase mutant prepared by the method of claim 5 as catalysts, catalyzing the reaction of 5-aminovaleric acid to obtain glutaric acid.

8. The preparation method according to claim 7, characterized in that: In the catalytic reaction, the temperature is 20-40°C; the initial pH is 7-9; and / or The catalytic reaction comprises 5-aminovaleric acid, α-ketoglutaric acid, polysorbate 80, and NAD+; The concentration of 5-aminovaleric acid is 200-500 mM; The concentration of α-ketoglutaric acid is 100-300 mM; The volume percentage of polysorbate 80 is 0.01-1%; The concentration of NAD+ is 5-20 mM.

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