A pyricularia oryzae glutamine transaminase mutant and use thereof

Abstract

The application provides a pyroglutamic acid transaminase mutant and application thereof, the mutant is obtained by mutation on the basis of wild-type pyroglutamic acid transaminase, and specific mutation includes at least one of the following sites: (1) the 13th isoleucine is mutated into arginine; (2) the 35th valine is mutated into arginine; (3) the 183rd asparagine is mutated into aspartic acid; and the mutant is suitable for industrial enzymatic production of L-aspartic acid and the process of amino acids taking L-aspartic acid as a precursor; especially, the I13R / V35R / N183D mutant obtained by modification has high positive catalytic activity and strict direction selectivity, and completely loses the reverse reaction activity of decomposing L-aspartic acid; the mutant is overexpressed with phosphoenolpyruvate carboxylase in L-threonine production strain THRS-6, and a high-efficiency L-threonine synthesis metabolic pathway is constructed, and the yield of L-threonine is significantly improved when glucose is used as a substrate.

Description

Technical Field

[0001] This invention relates to the fields of enzyme engineering and microbial engineering technology, and in particular to an aspartate aminotransferase mutant and its applications. Background Technology

[0002] L-Aspartic acid is an important natural amino acid with wide applications in the food, pharmaceutical, and chemical industries. With the continuous growth of market demand, developing efficient and green bio-production technologies for L-aspartic acid is of great significance.

[0003] Currently, the industrial production of L-aspartic acid mainly relies on enzymatic catalysis, specifically using aspartate aminotransferase (AAT, EC2.6.1.1), also known as glutamate-oxaloacetate transaminase (GOT), to catalyze the following reversible amino reaction: L-glutamic acid + oxaloacetic acid ⇌ α-ketoglutarate + L-aspartic acid. This method uses inexpensive L-glutamic acid and oxaloacetic acid as substrates and has advantages such as mild reaction conditions, high specificity, and low environmental pollution. Among these, the substrates derived from *Escherichia coli* (…) Escherichia coli Aspartate aminotransferase (encoding gene) AspC Due to its high expression level and high stability, it has become one of the most common enzymes in industrial applications and research.

[0004] However, wild-type Escherichia coli aspartate transaminase still has significant limitations in practical industrial applications. The catalytic efficiency (kcat / Km) of this enzyme in the forward (to L-aspartic acid) and reverse (to L-glutamate) reactions described above is similar, and its thermodynamic equilibrium constant is close to 1, resulting in an unfavorable equilibrium point for the large accumulation of L-aspartic acid. Oxaloacetic acid is unstable in the reaction system and readily undergoes spontaneous decarboxylation to generate pyruvate, which is an inhibitor of this enzyme and further affects catalytic efficiency. Therefore, the wild-type enzyme suffers from low catalytic efficiency, a long reaction cycle, and the final product concentration and yield failing to reach optimal levels, limiting further improvements in production efficiency.

[0005] To address the aforementioned issues, molecular modification of aspartate transaminase through rational design or directed evolution to obtain mutants with superior catalytic performance is a current research hotspot. Existing technologies have reported some point mutations or multi-point mutants aimed at improving enzyme stability, optimal pH adaptation, or catalytic activity against non-natural substrates. However, research on mutants specifically designed to enhance the rate of the efficient synthesis of L-aspartate using oxaloacetate as the amino acceptor remains relatively limited, and the magnitude of catalytic efficiency improvements (e.g., doubling) is still insufficient to meet the growing industrial demand for ultra-high-efficiency biocatalysts. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide an aspartate aminotransferase mutant.

[0007] Another technical problem to be solved by the present invention is to provide biomaterials related to the above-mentioned aspartate aminotransferase mutant.

[0008] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned aspartate aminotransferase mutant and biological materials.

[0009] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: A mutant aspartate aminotransferase (AST) obtained by mutation of wild-type AST (whose amino acid sequence is shown in SEQ ID NO.1), specifically including at least one of the following sites: (1) The isoleucine (I) at position 13 is mutated to arginine (R), i.e., I13R; (2) The valine (V) at position 35 is mutated to arginine (R), i.e., V35R; (3) The asparagine (N) at position 183 is mutated to aspartic acid (D), i.e., N183D; The mutant exhibits the activity of catalyzing the formation of L-aspartic acid from L-glutamic acid and oxaloacetic acid.

[0010] The amino acid sequence of the above-mentioned wild-type aspartate aminotransferase is shown in SEQ ID NO.1 of the sequence listing, and its encoding gene... AspC The nucleotide sequence is shown in the sequence listing SEQ ID NO.2.

[0011] Preferably, the above-mentioned aspartate aminotransferase mutant is as follows: Mutants with a single point mutation of I13R; V35R single-point mutation mutant; A mutant of the N183D single-point mutation; Mutants with double point mutations of I13R and V35R; Mutants with double point mutations of I13R and N183D; Mutants with double point mutations of V35R and N183D; or A mutant with a combination of I13R, V35R and N183D mutations.

[0012] Preferably, the above-mentioned aspartate aminotransferase mutant is a mutant with a three-point combination mutation of I13R, V35R, and N183D, and its amino acid sequence is shown in SEQ ID NO. 3 of the sequence listing. This specific mutant loses the reverse reaction activity catalyzing the reaction of L-aspartic acid with α-ketoglutarate to produce oxaloacetic acid and glutamate, and retains only the forward reaction activity catalyzing the reaction of oxaloacetic acid with glutamate to produce L-aspartic acid and α-ketoglutarate.

[0013] Preferably, the nucleotide sequence of the coding gene of the above-mentioned aspartate aminotransferase mutant is as shown in SEQ ID NO.4, or is a degenerate sequence encoding the same amino acid sequence.

[0014] The biological material associated with the above-mentioned aspartate aminotransferase mutant is any one of the following (a1) to (a3): (a1) The nucleic acid molecule encoding the above-mentioned aspartate aminotransferase mutant; (a2) A recombinant expression vector comprising the nucleic acid molecule described in (a1); (a3) A recombinant bacterium expressing the nucleic acid molecule described in (a1) or containing the recombinant expression vector described in (a2).

[0015] Preferably, in the above-mentioned biological material, the recombinant bacteria is the L-threonine-producing bacterium THRS-6 (THRS-6 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on August 20, 2024, with accession number CGMCC NO.31695, and classified as Escherichia coli). Escherichia coli The strain (obtainable from Qiqihar Longjiang Fufeng Biotechnology Co., Ltd.) was used as the starting strain, and it also overexpressed the enzyme encoding phosphoenolpyruvate carboxylase (PEPC). ppc Gene.

[0016] Preferably, in the above-mentioned biological material, the enzyme encoding phosphoenolpyruvate carboxylase... ppc The nucleotide sequence of the gene is shown in SEQ ID NO.5 of the sequence listing.

[0017] The application of the above-mentioned AST mutants or biomaterials in the catalytic synthesis of L-aspartic acid.

[0018] Preferably, in the above application, L-glutamic acid and oxaloacetic acid are used as substrates to produce L-aspartic acid under the catalysis of AST mutants or recombinant bacteria.

[0019] Preferably, the above application involves the following steps: using L-glutamic acid and oxaloacetic acid as substrates, and an aspartate aminotransferase mutant or recombinant bacteria as a catalyst, a catalytic reaction is carried out in a Tris-HCl buffer solution at pH 7.0-8.5 in the presence of pyridoxal coenzyme phosphate to obtain L-aspartic acid.

[0020] The above-mentioned AST mutants or biomaterials are used in the production of amino acids with L-aspartic acid as a precursor.

[0021] Preferably, in the above application, the biological material is a recombinant bacterium overexpressing an aspartate aminotransferase mutant and phosphoenolpyruvate carboxylase, which uses glucose as a substrate to synthesize amino acids with L-aspartic acid as a precursor.

[0022] Preferably, in the above applications, the amino acids with L-aspartic acid as a precursor include, but are not limited to: L-threonine, L-alanine, L-lysine, L-methionine, and L-isoleucine.

[0023] Preferably, in the above application, the amino acid with L-aspartic acid as a precursor is L-threonine.

[0024] Beneficial effects: The aforementioned AST mutant solves the technical problem of yield limitation due to reversible reaction equilibrium. It is suitable for industrial enzymatic production of L-aspartic acid and amino acids (such as L-threonine) with L-aspartic acid as a precursor. It can effectively shorten reaction time, improve substrate conversion rate and product yield, and reduce production costs. It has great application value and market potential in the large-scale biomanufacturing of L-aspartic acid and various amino acids with L-aspartic acid as a precursor. In particular, the I13R / V35R / N183D mutant, obtained by precisely modifying Escherichia coli-derived aspartate aminotransferase through site-directed mutagenesis, exhibits high forward catalytic activity and strict directional selectivity, completely losing its reverse reaction activity for decomposing L-aspartic acid. In in vitro catalytic reactions, the yield of L-aspartic acid synthesized by this mutant is more than 2 times higher than that of the wild-type enzyme. Overexpression of this mutant with phosphoenolpyruvate carboxylase (PEPC) in the L-threonine-producing strain THRS-6 constructed a highly efficient L-threonine biosynthetic pathway, increasing the yield of L-threonine to 1.3 times that of the original strain when glucose is used as a substrate. This has important application value in the biocatalytic synthesis of L-aspartic acid and its downstream products such as L-threonine. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the construction process of recombinant aspartate aminotransferase and its mutant expression vector in Example 1 of the present invention.

[0026] Figure 2 Comparative graphs of enzyme activity curves for AST and the I13R / V35R / N183D mutant. (a) Forward enzyme kinetic curve (synthesis of L-aspartic acid using oxaloacetate and glutamate as substrates); (b) Reverse enzyme kinetic curve (decomposition of L-aspartic acid using α-ketoglutarate and L-aspartic acid as substrates).

[0027] Figure 3 This is a schematic diagram of the construction process of the recombinant aspartate aminotransferase mutant-phosphoenolpyruvate carboxylase co-expression vector in Example 1 of the present invention.

[0028] The strain THRS-6 involved in this invention was deposited on August 20, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.31695, and classified as Escherichia coli. Escherichia coli . Detailed Implementation

[0029] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0030] Unless otherwise specified, all reagents involved in the embodiments of this invention are commercially available products and can be purchased through commercial channels.

[0031] In this embodiment of the invention, the sequence involved is as follows: the amino acid sequence of wild-type aspartate aminotransferase is shown in SEQ ID NO.1 of the sequence listing; the sequence encoding wild-type aspartate aminotransferase is... AspC The nucleotide sequence of the gene is shown in SEQ ID NO.2 of the sequence listing; the nucleotide sequence of the ppc gene encoding phosphoenolpyruvate carboxylase is shown in SEQ ID NO.5 of the sequence listing.

[0032] In this embodiment of the invention, the Escherichia coli hosts involved are BL21 (DE3) and THRS-6 (CGMCC NO.31695).

[0033] In this embodiment of the invention, the LB liquid culture medium consists of: 10 g / L peptone, 5 g / L yeast extract, and 10 g / L sodium chloride, with deionized water as the solvent.

[0034] In this embodiment of the invention, the LB plate medium is composed of LB liquid medium with a final concentration of 2 g / L agar added.

[0035] In this embodiment of the invention, the final concentration of the conversion liquid culture medium is: 5 g / L sodium chloride, 10 g / L tryptone, 5 g / L yeast extract, with 5 g / L oxaloacetic acid added, and deionized water as the solvent, pH 7.2-7.5 (adjusted with NaOH).

[0036] In this embodiment of the invention, the sequence information of the primers is shown in Table 1.

[0037] Table 1 Primers used

[0038] The detection methods involved in the following embodiments are as follows: The method for detecting L-aspartic acid content was as follows: a high performance liquid chromatography (HPLC) system was used, with o-phthalaldehyde (OPA) pre-column derivatization, a C18 reversed-phase column, and fluorescence detectors (λex = 360 nm and λem = 450 nm).

[0039] The method for detecting L-threonine content was as follows: a high performance liquid chromatography (HPLC) system was used, employing the 2,4-dinitrofluorobenzene (FDNB) pre-column derivatization method, a C18 reversed-phase column, and a UV detector (360 nm).

[0040] Assay for the activity of aspartate aminotransferase (AST): The activity of AST in catalyzing the reaction of oxaloacetate and glutamate to aspartate was indirectly reflected by measuring the decrease in absorbance of NADH at 340 nm using a glutamate dehydrogenase-coupled assay. The reaction system consisted of 200 μL of the following: 80 μL of 100 mM sodium phosphate buffer (pH 8.0), 20 μL of glutamate solution (50 mM), 20 μL of oxaloacetate solution (50 mM), 10 μL of NADH solution (20 mM), 0.1 μg of wild-type or mutant AST protein, and 0.3 U of glutamate dehydrogenase. The volume was adjusted to 200 μL with water. The reaction solution was added sequentially to 96-well plates and immediately placed in a multi-mode microplate reader (Infinite 2000Pro) to measure the absorbance at 340 nm. The AST activity was determined based on the decrease in absorbance. The amount of enzyme required to catalyze the production of 1 μM α-ketoglutarate from oxaloacetate per minute is defined as one unit of enzyme activity (U), and the specific enzyme activity formula is shown in Equation 1: Formula 1 Where t is the detection time; Vt is the total reaction volume; d is the detection optical path length; and ε is the molar absorptivity of NADH (6220 mol). -1 ·L·cm -1 C represents the enzyme concentration (mg / mL); Vs represents the enzyme solution volume.

[0041] Assay for the reverse activity of aspartate aminotransferase (AST): The activity of AST in catalyzing the reaction of α-ketoglutarate and L-aspartate to oxaloacetate was indirectly reflected by measuring the decrease in absorbance of NADH at 340 nm using a malate dehydrogenase-coupled assay. The total reaction volume was 200 μL, containing 80 μL of 100 mM sodium phosphate buffer (pH 8.0), 20 μL of aspartate solution (50 mM), 20 μL of α-ketoglutarate solution (50 mM), 10 μL of NADH solution (20 mM), 0.1 μg of wild-type or mutant AST protein, 1 U of malate dehydrogenase, and water to a final volume of 200 μL. After adding the reaction solution sequentially to 96-well plates, the plates were immediately placed in a multi-mode microplate reader (Infinite 2000Pro) to measure absorbance at 340 nm. The forward enzyme activity was determined based on the decrease in absorbance. The amount of enzyme required to catalyze the production of 1 μM oxaloacetate from α-ketoglutarate per minute is defined as one unit of enzyme activity (U), and the specific enzyme activity formula is shown in Equation 1.

[0042] Example 1 Construction of wild-type and mutant aspartate aminotransferase-producing genetically engineered bacteria The data from E. coli in the NCBI database ( Escherichia coli The aspartate aminotransferase gene (GenBank Accession No. X03629.1, amino acid sequence as shown in SEQ ID NO.1, nucleotide sequence as shown in SEQ ID NO.2) was synthesized in its entirety. The synthesized gene was then processed via... NdeI and Hiand III Restriction endonuclease sites were cloned into the expression vector pET-21b(+) (EMD Biosciences, Novagen) to obtain the recombinant plasmid pET-21b- AspC (Wt). The plasmid was transformed into Escherichia coli BL21(DE3) to obtain the wild-type expression strain BL21 / pET-28a- AspC (Wt), see the schematic diagram of the recombinant plasmid construction process. Figure 1 .

[0043] pET-21b- AspC Using Wt as a template, primers containing I13R, V35R, and N183D mutations were designed (see Table 1: I13R-F / I13R-R; V35R-F / V35R-R; N183D-F / N183D-R). Site-directed mutagenesis was performed using overlap extension PCR. The PCR products were digested with DpnI and transformed into DH5α competent cells. Positive clones were screened and sequenced for verification. The correctly sequenced recombinant plasmids were named pET-21b- AspCSingle mutants (including three single mutants: I13R, V35R, and N183D), pET-21b- AspC Double mutants (including three double mutants: I13R / V35R, V35R / N183D, and I13R / N183D) and pET-21b- AspC Triple mutant (I13R / V35R / N183D) (see) Figure 1 The above plasmids were transformed into Escherichia coli BL21(DE3) to obtain mutant expression strains, denoted as BL21 / pET-21b- AspC (Mut)s. The mutant pET-21b- AspC The amino acid sequence of the triple mutant (I13R / V35R / N183D) is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.4.

[0044] Example 2 Induction, expression, purification, and enzyme activity assay of recombinase The BL21 / pET-21b- constructed in Example 1 AspC (Wt) and BL21 / pET-21b- AspC (Mut)s single colonies were inoculated into LB liquid medium containing 100 μg / mL ampicillin and cultured overnight at 37°C and 220 rpm. A 2% inoculum was then transferred to fresh LB medium and cultured at 37°C until the OD600 reached approximately 0.6. IPTG was added to a final concentration of 0.1 mM, and the culture was induced at 25°C for 12 h. The bacterial cells were collected by centrifugation, resuspended in buffer, and sonicated. The supernatant was collected by centrifugation as the crude enzyme solution. The His-tagged target protein was purified using HisTrap HP column affinity chromatography, as detailed below: (1) Place the pump heads of pumps A and B of the protein purification instrument into deionized water to wash the pumps and remove ethanol from the system. After the pump washing program is completed, place the pump heads of pumps A and B into solution A (20mM sodium phosphate, 1mM DTT, 0.15M NaCl, pH=7.0) and solution B (20mM sodium phosphate, 1mM DTT, 0.15M NaCl, 500mM imidazole, pH=7.0), respectively, and turn on the pump washing program to fill the corresponding pipes with solution A and solution B. Set to 100% A and equilibrate the system with solution A for 10 minutes.

[0045] (2) Set the flow rate to 0.5 mL / min for a 1 mL column and 2.5 mL / min for a 5 mL column. Connect the HisTrap HP column and wait for the column pressure to stabilize before continuing to pump in 100% A solution to equilibrate the column. A smooth UV baseline indicates that equilibration is complete (approximately 30-40 column volumes).

[0046] (3) Sample loading: After the column is equilibrated, place pump head A into the sample to be purified, set the appropriate flow rate to 2.5 mL / min, and start the injection. Note that air should not be pumped in. Collect the flow-through sample for comparison. (4) After the injection is completed, rinse the column with 100% solution A. Once the peak returns to the baseline, the column washing is complete (about 30-40 column volumes).

[0047] (5) Pump in eluent A and B according to the gradient elution method (set 30 column volumes, 0-100% B according to the flow rate), and collect the eluted sample after the peaks are eluted; (6) After elution, continue rinsing the column with 100% B solution until the absorbance decreases to the baseline, then remove the column. Rinse the pump heads of pumps A and B in water to rinse both pumps and the tubing (10 column volumes), then place the pump heads in a 20% ethanol solution to fill the entire tubing with ethanol for storage. Prevent air bubbles from entering during the entire process.

[0048] (7) Rinse the removed HisTrap HP column with water for 30-50 column volumes, rinse with 1M NaOH for 5 column volumes, rinse with water for 30-50 column volumes to completely remove NaOH, fill with 20% ethanol solution, and store at 4°C.

[0049] (8) Samples were detected by 12% SDS-PAGE electrophoresis and Coomassie brilliant blue staining, as well as by flow-through and elution. High-purity target proteins were collected for subsequent enzyme activity assays.

[0050] According to the aforementioned enzyme activity assay method, the forward (synthesis of L-aspartic acid using oxaloacetic acid and glutamic acid as substrates) and reverse (decomposition of L-aspartic acid using α-ketoglutarate and L-aspartic acid as substrates) enzyme activities of wild-type and each mutant enzyme were measured, and the results are shown in Table 2.

[0051] Table 2. Specific enzyme activity parameters of aspartate aminotransferase and its mutants

[0052] Table 2 shows that the wild-type enzyme exhibits significant forward and reverse activities, with a reverse specific activity of 45.17 U / mg and a forward specific activity of 17.19 U / mg. The reverse activity is approximately 2.6 times (nearly 3 times) that of the forward activity. In contrast, the I13R / V35R / N183D triple mutant shows a decrease in forward activity to 10.63 U / mg and a sharp drop in reverse activity to 0.97 U / mg, with the reverse activity being less than 1 / 10 (approximately 9.1%) of the forward activity. The forward and reverse enzyme activity curves for the wild-type and I13R / V35R / N183D triple mutant are shown in Table 2. Figure 2 .

[0053] For the single mutants I13R, V35R, and N183D, the three showed similar enzyme activity characteristics: the forward enzyme activity was 14.64 U / mg, and the reverse enzyme activity was 12.56 U / mg. The reverse activity was about 0.86 times that of the forward activity, that is, the forward activity was slightly higher than the reverse activity, but the two were still in the same order of magnitude.

[0054] For the double mutants I13R / V35R, I13R / N183D, and V35R / N183D, the three showed consistent enzyme activity data: the forward enzyme activity was 12.48 U / mg, and the reverse enzyme activity was 3.49 U / mg. The ratio of reverse activity to forward activity was approximately 0.28, indicating a significant decrease in reverse activity, but still higher than that of the triple mutant. Overall, with the accumulation of mutation sites, the reverse activity showed a trend of decreasing significantly at each level, while the decrease in forward activity was relatively moderate.

[0055] Example 3 In vitro synthesis of L-aspartic acid catalyzed by AST mutant A 1 mL reaction system was prepared consisting of 100 mmol / L Tris-HCl buffer (pH 8.0), 40 mmol / L oxaloacetic acid, 80 mmol / L L-glutamate sodium, 0.2 mmol / L PLP, and the purified wild-type or mutant enzyme solution from Example 2 (final concentration 0.5 mg / mL). The reaction was carried out at 35 °C and 200 rpm for 12 hours. After the reaction, the amount of L-aspartic acid produced was detected by HPLC. The results are shown in Table 3.

[0056] Table 3. Data on the in vitro catalytic synthesis of L-aspartic acid by aspartate aminotransferase and its mutants.

[0057] Table 3 shows that in the wild-type enzyme-catalyzed reaction, the final concentration of L-aspartic acid was 18.5 mmol / L, with a conversion rate of 46.3%. However, in the reaction catalyzed by the I13R / V35R / N183D mutant, the final concentration of L-aspartic acid reached as high as 38.2 mmol / L, with a conversion rate of 95.5%, 2.06 times that of the wild-type. This indicates that the mutant completely removed the constraints of reaction equilibrium, achieving highly efficient substrate conversion.

[0058] Example 4 L-Threonine Production by Fermentation of Recombinant Bacteria Co-expressing Aspartate Transaminase Mutant and Phosphoenolpyruvate Carboxylase (1) Construction of co-expression plasmids pET-21b- AspC (Wt) / pET-21b- AspCUsing the genomes of (Mut) (I13R / V35R / N183D) and the high-threonine-producing bacterium THRS-6 as templates, PCR amplification was performed using primer pairs TH-F / RBS-TH-R (primer sequences are shown in Table 1) to obtain wild-type strains containing ribosome binding site (RBS) sequences. AspC Gene expression cassette fragment (wild-type aspartate aminotransferase expression cassette fragment, nucleotide sequence shown in SEQ ID NO.6 of the sequence listing) and I13R / V35R / N183D mutant AspC The nucleotide sequence of the gene expression cassette fragment (13R / V35R / N183D mutant aspartate aminotransferase expression cassette) is shown in SEQ ID NO.7 of the sequence listing. PCR reaction conditions: 95℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 30 s, for a total of 30 cycles; final extension at 72℃ for 5 min. The amplified product was purified by 1% agarose gel electrophoresis.

[0059] Using genomic DNA from the L-threonine-producing strain THRS-6 as a template, PCR amplification was performed using primer pair RBS-PPC-F / PPC-R (primer sequences are shown in Table 1) to obtain samples containing their own RBS sequence. ppc The gene (encoding phosphoenolpyruvate carboxylase, PEPC) expression cassette fragment (phosphoenolpyruvate carboxylase expression cassette, nucleotide sequence shown in SEQ ID NO. 8 of the sequence listing) was used. PCR reaction conditions were the same as above, with the extension time adjusted to 90 s. The amplification product was purified by agarose gel electrophoresis.

[0060] The two expression cassette fragments mentioned above were used in overlap extension PCR technology. AspC Expression box and ppc The expression cassette is then ligated. Specific procedure: The purified... AspC Expression box fragments and ppc The expression cassette fragments were mixed at a molar ratio of 1:1 as a template, and primers TH-F and PPC-R were added for a second round of PCR. Reaction conditions: 95℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 58℃ annealing for 15 s, 72℃ extension for 90 s, for a total of 25 cycles; final extension at 72℃ for 5 min. The amplified product is... AspC (Wild type or triple mutant) -RBS- ppc Serial expression boxes. Among them, AspC -RBS- ppc The nucleotide sequence is shown in SEQ ID NO.9 of the sequence listing. AspC Triple mutant-RBS- ppc The nucleotide sequence is shown in SEQ ID NO.10 of the sequence listing.

[0061] The aforementioned tandem expression cassette and plasmid vector pTrac-99A (Danaherg, Cytiva) were double-digested with restriction endonucleases BamHI and EcoRI, respectively. The digestion products were purified by agarose gel electrophoresis and then ligated overnight at 16°C using T4 DNA ligase. The ligation products were transformed into *E. coli* DH5α competent cells, and positive clones were selected for colony PCR verification. The plasmids were extracted and sequenced for confirmation. The correctly sequenced recombinant plasmids were named pTrac-99A- AspC -RBS- ppc (Co-expresses wild-type aspartate aminotransferase and PEPC); pTrac-99A- AspC Triple mutant-RBS- ppc (Co-expresses I13R / V35R / N183D mutant aspartate aminotransferase and PEPC). See the schematic diagram of the construction process. Figure 3 .

[0062] (2) Construction of recombinant strains and fermentation production of L-threonine The successfully constructed recombinant plasmids were introduced into competent cells of the L-threonine-producing strain THRS-6 via electroporation. The constructed recombinant strain THRS-6: pTrac-99A-AspC triple mutant-RBS-ppc and the control strain THRS-6: pTrac-99A- AspC RBS-ppc were inoculated into seed culture medium containing ampicillin antibiotic (see Table 4) for activation. They were then transferred to a 5L fermenter at a 10% inoculation rate. The initial glucose concentration of the fermentation medium was 50 g / L (see Table 5). Fermentation conditions: 37℃, dissolved oxygen maintained above 30%, pH controlled at 7.0 using ammonia. During fermentation, when the glucose concentration fell below 5 g / L, a 60% glucose solution was added. After 48 hours of fermentation, samples were taken to determine the L-threonine yield (three replicates were performed).

[0063] Table 4 Seed culture medium (g / L)

[0064] Table 5 Fermentation medium (g / L)

[0065] The results are shown in Table 6. Control strain THRS-6: pTrac-99A- AspC -RBS- ppc The L-threonine yield was 95.6 g / L. The recombinant strain THRS-6: pTrac-99A- expressing the I13R / V35R / N183D mutant... AspC Three Mutations - RBS - ppcThe yield of L-threonine reached 124.3 g / L, representing a 30% (1.3-fold) increase. This result demonstrates that by introducing an irreversible L-aspartic acid synthesis pathway (catalyzed by the mutant GOT I13R / V35R / N183D) and enhancing the supply of oxaloacetate catalyzed by phosphoenolpyruvate carboxylase (PEPC), the supply of L-threonine precursor L-aspartic acid was significantly enhanced, thereby effectively increasing the yield of the target product.

[0066] Table 6. Comparison of productivity of threonine-producing genetically engineered bacteria

[0067] In summary, the AST I13R / V35R / N183D mutant almost completely loses its ability to catalyze the reverse reaction of L-aspartic acid and α-ketoglutarate to produce oxaloacetate and glutamate, retaining only the forward reaction activity of oxaloacetate and glutamate. This significantly enhances the affinity and conversion rate of the substrate oxaloacetate, effectively directing the reaction towards L-aspartic acid synthesis, thereby greatly improving L-aspartic acid production efficiency and possessing significant industrial application value. Compared to the wild type, the mutant increases the yield of L-aspartic acid synthesis by more than two times. Co-expression of the mutant with phosphoenolpyruvate carboxylase in the high-yield L-threonine strain THRS-6, using glucose as a substrate for fermentation, increased the L-threonine yield to 1.3 times that of the wild-type strain.

[0068] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention. Improvements and modifications such as strain modification based on the method of the present invention or based on the method are all considered to be within the scope of protection of the present invention.

Claims

1. A glutamic-glutamyl transferase mutant, characterized in that: It was obtained by mutation of wild-type aspartate aminotransferase, the amino acid sequence of which is shown in SEQ ID NO.1 of the sequence listing. The specific mutations include at least one of the following sites: (1) The isoleucine at position 13 is mutated to arginine; (2) Valine at position 35 is mutated to arginine; (3) The asparagine at position 183 is mutated to aspartic acid.

2. The aspartate aminotransferase mutant according to claim 1, characterized in that: The mutant is: Mutants with a single point mutation of I13R; V35R single-point mutation mutant; A mutant of the N183D single-point mutation; Mutants with double point mutations of I13R and V35R; Mutants with double point mutations of I13R and N183D; Mutants with double point mutations of V35R and N183D; or A mutant with a combination of I13R, V35R and N183D mutations.

3. The aspartate aminotransferase mutant according to claim 1 or 2, characterized in that: The mutant is a combination of I13R, V35R and N183D mutations, and its amino acid sequence is shown in SEQ ID NO. 3 of the sequence listing.

4. The aspartate aminotransferase mutant according to claim 3, characterized in that: The nucleotide sequence of the gene encoding the mutant is shown in SEQ ID NO.4, or is a degenerate sequence encoding the same amino acid sequence.

5. Biomaterials related to the aspartate aminotransferase mutant according to any one of claims 1-4, characterized in that: It can be any one of the following (a1) to (a3): (a1) Nucleic acid molecule encoding an aspartate aminotransferase mutant; (a2) A recombinant expression vector comprising the nucleic acid molecule described in (a1); (a3) A recombinant bacterium expressing the nucleic acid molecule described in (a1) or containing the recombinant expression vector described in (a2).

6. The biomaterial according to claim 5, characterized in that: The recombinant bacteria, starting with the L-threonine-producing high-yield strain THRS-6, also overexpressed enzymes encoding phosphoenolpyruvate carboxylase. PPC Gene.

7. The use of the aspartate aminotransferase mutant according to any one of claims 1-4 or the biomaterial according to claim 5 or 6 in the catalytic synthesis of L-aspartic acid.

8. The application of the aspartate aminotransferase mutant according to any one of claims 1-4 or the biomaterial according to claim 5 or 6 in the production of amino acids with L-aspartic acid as a precursor.

9. The application according to claim 8, characterized in that: The biological material is a recombinant bacterium that overexpresses an aspartate aminotransferase mutant and phosphoenolpyruvate carboxylase, and uses glucose as a substrate to synthesize amino acids with L-aspartic acid as a precursor.

10. The application according to claim 9, characterized in that: The amino acid that uses L-aspartic acid as a precursor is L-threonine.

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