Recombinant corynebacterium glutamicum and application thereof

Abstract

The invention relates to the technical field of bioengineering, in particular to recombinant corynebacterium glutamicum and application thereof. The invention discloses an acrylic acid-resistant recombinant corynebacterium glutamicum with high ammonialyase activity, the recombinant strain can catalytically convert acrylic acid into beta-alanine in a whole-cell manner, meanwhile, cells can be reused, and the cost advantage is remarkable compared with enzyme catalysis. Considering from the aspects of improving chassis cell robustness, key enzyme expression stability and cell reuse efficiency, the invention provides a high-activity aspartase mutant (ammonialyase) and a recombinant corynebacterium glutamicum with high ammonialyase activity, the conversion rate of acrylic acid is high, the selectivity of the product beta-alanine is high, and the yield of the recombinant corynebacterium glutamicum is high. And more than 20 batches of cells can be repeatedly utilized, so that the method has remarkable economical efficiency.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and relates to a recombinant Corynebacterium glutamicum capable of whole-cell catalytic synthesis of beta-alanine, a construction method thereof, and application in whole-cell catalytic synthesis of beta-alanine. Background Art

[0002] β-Alanine is the only β-amino acid that exists in nature. It is widely used in medicine, food, and chemical industries. The global demand is about 80,000 tons. It is known as one of the 12 most promising three-carbon compounds in the world in the future. At present, the production of β-alanine mainly includes chemical synthesis, microbial fermentation, and bio-enzyme conversion. Chemical synthesis is the most competitive of the three synthesis methods, but it needs to be carried out under high temperature, high pressure, strong acid, and strong alkali conditions, and the reaction conditions are relatively extreme. The β-alanine microbial fermentation method is mainly concentrated in Escherichia coli. Liu Zhiqiang's team used metabolic engineering, protein engineering, and fermentation engineering to construct a high-yield β-alanine recombinant strain with Escherichia coli W3110 as the starting strain. The product concentration is >85g / L and the yield reaches 0.24g / g. Therefore, the low product yield leads to a high cost of its raw material glucose. According to the different substrates, the bioenzyme conversion method can be divided into the nitrilase conversion method (with β-aminopropionitrile as substrate), the PanD conversion method (with L-aspartic acid as substrate) and the acrylic acid ammonia enzyme method (with acrylic acid as substrate), among which the acrylic acid ammonia enzyme method has attracted much attention due to its high atom economy and low cost. Li Ruifeng et al. disclosed an aminolyase mutant protein and its encoding gene and application in Chinese patent CN108866028A, which is based on Bacillus subtilis aspartase and realizes the preparation of (R) 1-propyl (2-amino) formic acid. Wu Bian's team applied for aspartase mutants and their preparation methods and applications (China Patent Publication No.: CN113416726A), and protected the aspartase mutant enzyme that catalyzes acrylic acid to β-alanine. It was clarified that the mutant enzyme has T187I and N326C mutations at the 187th and 326th positions of the aspartase amino acid sequence of the source Bacillus, and there are also 0-9 mutations, selected from: D20V, V75E, Q89H, L156F, T164I, Y204C, N226I, L258I, M285L, M321I, K324I, K381R, K389I, I406L, R426C and / or P456L. Therefore, it is very important to develop an acrylic acid ammonia addition method with high conversion rate, more stability and low cost. Summary of the invention

[0003] Purpose of the invention: The technical problem to be solved by the present invention is to provide a recombinant Corynebacterium glutamicum for converting acrylic acid into β-alanine in view of the deficiencies in the prior art.

[0004] The technical problem that the present invention also aims to solve is to provide a culture method and a whole-cell repeated catalytic process of the recombinant Corynebacterium glutamicum capable of efficiently converting acrylic acid to synthesize β-alanine.

[0005] In order to solve the above technical problems, the present invention discloses a recombinant Corynebacterium glutamicum and its application. The specific technical scheme is as follows:

[0006] An aspartase mutant, whose amino acid sequence is shown in SEQ ID No. 7. The aspartase mutant is obtained by performing the following five mutations on the aspartase derived from Corynebacterium glutamicum, namely V52M, R226M, T241I, N380I, and M499L.

[0007] In a second aspect, the present invention provides a nucleic acid molecule encoding the aspartase mutant described in the first aspect, and its nucleotide sequence is shown as SEQ ID No.8.

[0008] In a third aspect, the present invention provides a recombinant expression vector comprising the nucleic acid molecule described in the second aspect.

[0009] In a fourth aspect, the present invention provides a recombinant Corynebacterium glutamicum, which contains the recombinant expression vector described in the third aspect. Wherein, the starting strain of the recombinant Corynebacterium glutamicum is Corynebacterium glutamicum BACG121; the Corynebacterium glutamicum BACG121 is deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC NO: M20241240 and a deposit date of June 17, 2024. Preferably, the Corynebacterium glutamicum BACG121 is a highly acrylic acid tolerant mutant strain of Corynebacterium glutamicum. Further preferably, the Corynebacterium glutamicum BACG121 is obtained as follows: using the Corynebacterium glutamicum model strain NCIB 10025 as the starting strain, 80-120W as the radio frequency power, 10-30SLM as the gas flow rate, and 10-30s as the irradiation time, the strain is subjected to plasma mutagenesis; then an adaptive evolution strategy is adopted to gradually increase the concentration of acrylic acid in the composite culture medium to obtain a mutant strain tolerant to high concentrations of acrylic acid, and the pH is maintained at 7.5-8.0 with ammonia water during the acclimation process; the composite culture medium formula: 0.1-3g / L MgSO4, 0.5-3g / L KH2PO4, 2-7g / L NaCl, 10-30g / L corn steep liquor powder, 10-50g / L glycerol; the optimal formula is 0.5g / L MgSO4, 1g / L KH2PO4, 5g / L NaCl, 20g / L corn steep liquor powder, 30g / L glycerol; the acrylic acid concentration is gradually increased from 5g / L to 10g / L

[0010] Further preferably, the highly acrylic acid tolerant Corynebacterium glutamicum mutant strain BACG121 is used as the starting strain, and the coding gene of the aspartase mutant having ammonia addition activity is integrated into the chromosome to obtain the recombinant Corynebacterium glutamicum.

[0011] In a fifth aspect, the present invention provides the use of the aspartase mutant described in the first aspect, the nucleic acid molecule described in the second aspect, the recombinant expression vector described in the third aspect, or the recombinant Corynebacterium glutamicum described in the fourth aspect in the production of β-alanine. Preferably, ...

[0012] The substrate acrylic acid is contacted with the aspartase mutant described in the first aspect or the recombinant Corynebacterium glutamicum described in the fourth aspect to carry out a catalytic reaction to generate β-alanine.

[0013] Preferably, the recombinant Corynebacterium glutamicum described in the fourth aspect is fermented and cultured to obtain a bacterial solution, which is centrifuged to obtain wet bacterial bodies, and the wet bacterial bodies are used as whole-cell catalysts to contact with the substrate acrylic acid to perform a whole-cell catalytic reaction;

[0014] The whole-cell catalytic reaction has a reaction temperature of 40-45°C and a reaction pH of 7.5-8.0; preferably, the reaction temperature is 40°C, the reaction pH is 7.5, and the reaction is carried out until the substrate acrylic acid is completely catalyzed;

[0015] In the reaction system, the concentration of wet bacteria is 5-10 g / L, the concentration of acrylic acid is 100-150 g / L, preferably, the concentration of wet bacteria is 5 g / L, and the concentration of alanine is 2 M. Further preferably, the fermentation culture is as follows: the temperature of the recombinant Corynebacterium glutamicum cell culture process is 30-37°C, the pH is 7.5-8.0, the dissolved oxygen is controlled at 10%, and the cell density (OD 600 ) After the increase is less than 0.5 per hour, the culture is terminated and the next whole-cell catalytic process is directly carried out, or it is frozen and refrigerated for standby use. Further preferably, the pH in the fermentation process is adjusted by ammonia water, and the dissolved oxygen is achieved by adjusting the stirring, ventilation volume and / or feeding back the carbon source glycerol in the form of feed. More preferably, the dissolved oxygen is achieved by feeding back the carbon source glycerol. In the whole-cell catalytic process, wet bacteria or frozen and refrigerated cells can be directly used as whole-cell catalysts without cell disruption.

[0016] After one whole-cell catalytic reaction is completed, the wet bacteria are collected by centrifugation, nutrients are added for aerobic induction, and the whole-cell catalyst is used again as the whole-cell catalyst for the next whole-cell catalytic reaction, thereby realizing the reuse of the whole-cell catalyst, and the number of reuses of the whole-cell catalyst is more than 20 times.

[0017] The nutrients include: 0.5g / L MgSO4, 0.5g / L KH2PO4, 0.5g / L NaCl, 1.5g / L corn steep liquor powder, 2g / L glycerol and 0.5g / L acrylic acid; the aerobic induction temperature is 37°C and the induction time is 2-3h. The induction under the culture conditions between batches of cell reuse can improve the efficiency of cell reuse.

[0018] Beneficial effects:

[0019] The present invention considers improving the robustness of chassis cells, improves the tolerance of acrylic acid of chassis cells of Corynebacterium glutamicum by combining normal temperature plasma mutagenesis with adaptive evolution, and obtains mutant strain BACG121; the present invention further considers the key enzyme level, integrates the coding gene of aspartase mutant with ammonia enzyme activity into the chromosome of BACG121, and obtains recombinant Corynebacterium glutamicum that can stably catalyze acrylic acid to β-alanine; the present invention finally considers improving the efficiency of cell recycling, and introduces a substrate-induced cell activation strategy in the process of cell batch reuse. The acrylic acid-resistant Corynebacterium glutamicum BACG121 is deposited with CCTCC NO: M20241240, and the amino acid sequence of aspartase mutant with ammonia enzyme activity is shown in SEQ ID No.7. The present invention also discloses the construction method and application of the above-mentioned recombinant strain. The high ammonia enzyme activity recombinant Corynebacterium glutamicum provided by the present invention has high acrylic acid conversion rate, high product β-alanine selectivity, and can realize more than 20 batches of cell reuse, which has significant economic efficiency. The invention directly uses recombinant Corynebacterium glutamicum as a whole-cell catalyst, and the single catalytic acrylic acid conversion rate can reach more than 98%. The whole-cell catalyst can be reused more than 20 times, and the acrylic acid conversion rate can still reach more than 93%.

[0020] In general, in order to further reduce the production cost of β-alanine, the present invention screens mutant enzymes with stronger activity and better enzyme stability, and at the same time screens and constructs an expression host that can stably express the enzyme from the level of chassis microorganisms, and constructs a more economical whole-cell catalytic system, and it is imperative to reuse cells to synthesize β-alanine. Based on the above-mentioned industry pain points, the present invention constructs a recombinant Corynebacterium glutamicum strain with efficient conversion of acrylic acid to synthesize β-alanine, and establishes its culture process and whole-cell catalytic process, the production process is stable, and the technical indicators have significant economy. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0022] Figure 1 This is the gel electrophoresis diagram of colony PCR identification. DETAILED DESCRIPTION

[0023] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be purchased commercially unless otherwise specified.

[0024] Example 1 Breeding of acrylic acid-resistant Corynebacterium glutamicum

[0025] This example illustrates the breeding process of acrylic acid-resistant Corynebacterium glutamicum. First, the Corynebacterium glutamicum model strain NCIB 10025 is used as the starting strain. The strain is subjected to plasma mutagenesis using an ARTP-normal pressure room temperature plasma mutagenesis breeding instrument, with a radio frequency power of 80-120 W, a gas flow rate of 10-30 SLM, and an irradiation time of 10-30 s. The conditions for a cell lethality rate of 95% are determined to be 120 W for the radio frequency power, 10 SLM for the gas flow rate, and 30 s for the irradiation time.

[0026] The mutant strain was further bred by adaptive evolution to select high acrylic acid tolerance mutants. The acclimatization medium was 0.5g / L MgSO4, 1g / L KH2PO4, 5g / L NaCl, 20g / L corn syrup powder, 30g / L glycerol, pH 7.5, and dissolved oxygen was controlled at 10%. The initial acrylic acid concentration was controlled at 5g / L. When the cell density OD 600 After reaching 2.0, the acrylic acid concentration was increased to 6 g / L. At this time, the cell density dropped significantly. Under the selection of high concentration acrylic acid, the mutant strain evolved and grew. When the cell density OD 600 After reaching 2.0, the acrylic acid concentration was further increased until it reached 10 g / L, and the mutant strain Corynebacterium glutamicum BACG121 was obtained, which has been deposited in the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with the deposit number CCTCC NO: M20241240 and the deposit date on June 17, 2024.

[0027] Example 2 Construction of high ammonia enzyme activity and acrylic acid resistant recombinant Corynebacterium glutamicum

[0028] This example is used to illustrate the process of constructing the acrylic acid-resistant mutant strain Corynebacterium glutamicum BACG121 obtained by breeding in Example 1 using gene editing technology as the starting strain:

[0029] (1) Pick the starting strain from a fresh culture dish and inoculate it into 5 mL BHI medium, and culture it at 30°C and 200 rpm for 12 h. The next day, transfer it to 50 mL LB medium containing 3% (g / mL) glycine and 0.1% v / v Tween 80 at an inoculum volume of about 1% v / v, and culture it at 30°C and 200 rpm until the cell OD reaches 600 Reach 0.6 to make it electrocompetent.

[0030] (2) Using the genome of the acrylic acid-resistant mutant strain BACG121 as a template, a high-fidelity PCR amplification system was used to design homologous box fragments at the upstream and downstream of the insertion site, and the upstream homologous box primer and the downstream homologous box primer were used to amplify the linear DNA homologous fragment with the plasmid homology arm. The coding gene of the aspartase mutant with an amino acid sequence as shown in SEQ ID No.7 was codon-optimized, and the codon-optimized gene encoding the aspartase mutant (nucleotide sequence as shown in SEQ ID No.8) was synthesized by the company. The same method was used to use the plasmid containing the codon-optimized aspartase mutant coding gene synthesized by the company as a template, and the insertion fragment with the homology arm was amplified using the insertion fragment primer. The aspartase mutant used in the present invention is a V52M, R226M, T241I, N380I and M499L five mutations based on the original aspartase from Corynebacterium glutamicum, and its amino acid sequence is shown in SEQ ID No.7. The primer sequences used in this example are as follows:

[0031] Upstream homology box primer:

[0032] F: 5'-acgaattcgagctcggtaccCATGATGCAGGATGGAGTATCCG-3' (SEQ ID No. 1)

[0033] R: 5'-ttggcagctaATCTTTGGCGCCTAGTTGGC-3' (SEQ ID No. 2)

[0034] Insert Primers:

[0035] F: 5'-cgccaaagatTAGCTGCCAATTATTCCGGG-3' (SEQ ID No. 3)

[0036] R: 5'-ggaagtgggatcgaaaAGAGTTTGTAGAAACGCAAAAAGGC-3' (SEQ ID No. 4)

[0037] Downstream homology box primer:

[0038] F: 5'-ctctTTTCGATCCCACTTCCTGATTT-3'(SEQ ID No.5)

[0039] R: 5'-gcctgcaggtcgactctagaTGGTTCTGGGGAGGATCGA-3' (SEQ ID No. 6)

[0040] The reaction system (50 μL) is as follows:

[0041] 2 μL each of upstream and downstream primers with homology arms (100 pmol / μL);

[0042] Template DNA (100 ng / μL) 1 μL;

[0043] 2×buffer 25μL;

[0044] dNTPs mix (10 mM) 1 μL;

[0045] Phanta Max Super-Fidelity DNA Polymerase 1μL;

[0046] ddH2O 18μL.

[0047] Reaction conditions: 95°C, 5 min; 95°C, 15 s; 55°C, 15 s; 72°C, 1000 bp / min; 35 cycles; 72°C, 10 min.

[0048] (3) The recombinant plasmid was synthesized using the ClonExpress Ultra One Step Cloning Kit V3 system. The reaction system was as follows:

[0049] 2×CE Mix V3 5 μL;

[0050] The optimal amount of cloning vector (pk18mobsacB) used = [0.02 × number of base pairs of cloning vector] ng (0.03 pmol);

[0051] The optimal amount of each fragment = [0.02 × number of base pairs per fragment] ng (0.03 pmol);

[0052] Add ddH2O to make up to 10 μL.

[0053] React at 50°C for 30 min, quickly put on ice, and then transform DH5α.

[0054] (4) The recombinant plasmid prepared in step (3) was electrotransformed into the competent Corynebacterium glutamicum prepared in step (1). The electroporation conditions of the 2 mm electroporation cup were: 200Ω, 25Μf, and the electroporation voltage was 2.5kv. After electroporation, 46°C preheated BHI+sorbitol (91g / L) medium was quickly added, and the medium was bathed in a 46°C water bath for 6 minutes, and then cultured at 180r / min and 30°C for 2-4h, and then coated with BHI recovery medium containing kanamycin (kan) to screen for positive intermediates.

[0055] (5) The recombinant was verified by colony PCR using the upstream and downstream primers of the upper and lower homologous boxes (nucleotide sequences are SEQ ID No.1 and SEQ ID No.6), and the verification system is as follows: 2×Rapid Taq Master Mix 25μL; template colony; 2μL of upstream and downstream primers; ddH2O is supplemented to 50μL. Reaction system: 98℃, 8min; (95℃, 15s; 55℃, 15s; 72℃, 2000bp / min; 30 cycles); 72℃, 10min. Through the above construction method, the expression cassette of the nucleotide sequence such as SEQ ID No.9 was successfully inserted into the genome of Corynebacterium glutamicum.

[0056] (6) The positive intermediate was inoculated into a BHI test tube overnight, and then coated with a streptomycin BHI plate. The recombinant was verified by colony PCR using the upstream and downstream primers of the upper and lower homologous boxes. The reaction system and conditions were the same as in step (5). The results were as follows: Figure 1 As shown in the figure, the PCR fragment size of the positive strain with correct recombination is 3200bp, and the fragment size of the negative strain is 2040bp. It can be seen from the figure that the positive rate of random identification is 87.5%.

[0057] Example 3 Dissolved oxygen feedback feeding increases cell density and enzyme activity units

[0058] In this embodiment, the oxygen demand of Corynebacterium glutamicum is high and the dissolved oxygen is difficult to control during high-density fermentation. The fermentation process in this embodiment is as follows:

[0059] The recombinant Corynebacterium glutamicum prepared in Example 2 was streaked on an LB plate and cultured for 12 h, and a single colony was inoculated into a test tube containing 5 mL of LB liquid medium and cultured at 37° C. for 12 h. The seed solution in the test tube was then inoculated into a shake flask containing 150 mL of LB liquid medium at a 1% v / v inoculation rate and cultured at 37° C. for 6-8 h. The seed solution cultured in the shake flask was inoculated into a fermenter containing 3 L of fermentation medium at a ratio of 5% v / v.

[0060] The fermentation medium used in this embodiment is 0.5g / L MgSO4, 1g / L KH2PO4, 5g / L NaCl, 20g / L corn steep liquor powder, and a total of about 200g / L glycerol is added in batches or feedback. The pH is maintained at 7.5 with ammonia water, and the dissolved oxygen is controlled at 10%.

[0061] The specific method of adding in batches is as follows: taking a 5L bioreactor as an example, the liquid volume is 3L, and a total of 600g of glycerol is required according to the glycerol dosage of 200g / L. When the dissolved oxygen in the fermentation system is higher than 50%, glycerol is added. The one-time addition in this embodiment is to add 600g of glycerol to the fermentation liquid at one time, the two-time addition is to add 300g of glycerol to the fermentation liquid twice, and the three-time addition is to add 200g of glycerol to the fermentation liquid three times.

[0062] The feedback supplement is specifically performed as follows: taking a 5L bioreactor as an example, the liquid volume is 3L, and a total of 600g of glycerol is required at a glycerol dosage of 200g / L. The feedback supplement is performed by setting the dissolved oxygen parameter to 10%. When the dissolved oxygen is higher than 10%, the feed pump is automatically turned on to add glycerol to the fermentation liquid until no more glycerol is added.

[0063] By comparing the control of dissolved oxygen by feedback feeding, the control of dissolved oxygen by changing the stirring speed and ventilation volume under batch feeding, the results are shown in Table 1. The results show that the feedback feeding method of controlling dissolved oxygen can obtain higher cell density and higher enzyme activity, while maintaining lower synthesis of byproduct acetic acid, so higher cell density can be obtained. In addition, the feedback feeding method can reduce the stirring speed and ventilation speed, lower energy consumption, and significantly reduce production costs.

[0064] In this example, the enzyme activity 1U is defined as: catalytic conversion of 1g of acrylic acid per minute under a catalytic system of pH 7.5, temperature 40°C, and substrate acrylic acid 2 mol / L.

[0065] Table 1 Comparative study on controlling dissolved oxygen by changing stirring speed and ventilation volume under feedback feeding and batch feeding

[0066]

[0067] Example 4 Feasibility study of whole cell catalysis

[0068] This example is a feasibility study on whether Corynebacterium glutamicum can be used for product synthesis by whole-cell catalysis, and the relevant data are accumulated for repeated batches of cells, with crude enzyme solution of high-pressure cell disruption as a comparison. The bacterial solution is prepared according to the feedback feeding fermentation method described in Example 3, and the obtained bacterial solution is treated according to any of the following methods:

[0069] The preparation method of the crude cell enzyme solution is as follows: the bacterial solution is subjected to high-pressure cell disruption, the conditions of the high-pressure cell disruption are 1000 bar, 4° C. temperature control, and the disruption is repeated twice.

[0070] The whole-cell catalysis uses recombinant Corynebacterium glutamicum as a whole-cell catalyst. The preparation method of the whole-cell catalyst is as follows: after the fermentation of the recombinant bacteria is completed, the bacterial liquid is centrifuged at 8000rpm and 4°C to obtain bacterial sludge, and the bacterial sludge is resuspended with water before catalysis. After the amount of bacterial sludge is controlled, the substrate can be added to start the whole-cell catalysis.

[0071] The crude cell enzyme solution or whole cell catalyst prepared above is used to catalyze the substrate acrylic acid to synthesize β-alanine under the following catalytic conditions:

[0072] 5 g / L of recombinant Corynebacterium glutamicum wet cells were directly or broken into a reaction system (the solvent was water), the substrate acrylic acid was 2 mol / L, pH was 7.5, and the catalysis was carried out at 40°C. Samples were taken every 1 hour, and the time required for catalysis was the time when the substrate was completely catalyzed.

[0073] The differences in catalysis between the two methods are compared, and the results are shown in Table 2. The results show that both whole-cell catalysis and crude cell enzyme solution can catalyze acrylic acid to synthesize β-alanine under the conditions of only 5g / L cell dosage and 30U total enzyme activity, and the substrate conversion rate can reach more than 98%, but the whole-cell catalysis time is longer than that of enzyme catalysis. The possible reason is that it involves the transport of substrates and products. Secondly, after a whole-cell catalysis, the cells are recovered by centrifugation at 8000rpm and 4°C. The recovered cells are subjected to a second catalysis according to the above catalytic conditions. Although the cell density and enzyme activity have decreased, the substrate conversion rate remains above 95%. Therefore, whole-cell catalysis has a significant cost competitive advantage.

[0074] Table 2 Comparison of catalysis by crude enzyme solution and whole cell after cell disruption

[0075]

[0076] Example 5 Study on cell viability enhancement strategy by whole cell reuse

[0077] In this example, in the process of whole-cell catalysis of Corynebacterium glutamicum, cell apoptosis and enzyme activity decreased rapidly. Although the substrate conversion rate was high, the catalysis time was greatly prolonged and the number of repetitions was low. The strategy of using a short aerobic induction strategy between batches to improve cell viability and enzyme activity was investigated.

[0078] The strategy of aerobic induction is as follows: when a batch of whole-cell catalysis is finished, the cells are recovered by centrifugation at 8000rpm and 4°C, and nutrients are added to the recovered cells. The concentration of the nutrients added in the catalytic system is 0.5g / L MgSO4, 0.5g / L KH2PO4, 0.5g / L NaCl, 1.5g / L corn steep liquor powder, 2g / L glycerol and 0.5g / L acrylic acid (solvent is water). The dissolved oxygen is controlled to be above 10%, and the aerobic induction is carried out at 37°C for 2 to 5 hours. The effect of the induction time on the cell viability is investigated, and the results are shown in Table 3.

[0079] Table 3 Effect of aerobic induction time on cell mass and enzyme activity

[0080] Aerobic induction time (h) Cell volume (g / L) Total enzyme activity (U) 0 3.2 20 1 3.3 20 2 4.6 29 3 5.9 35 4 6.2 37 5 6.1 36

[0081] Example 6 Whole cell continuous batch catalysis study

[0082] This example investigates repeated batch studies after introducing a cell viability enhancement strategy into a process using whole-cell catalysis of Corynebacterium glutamicum.

[0083] The strategy of aerobic induction is as follows: when a batch of whole-cell catalysis is completed, the cells are centrifuged at 8000rpm and 4°C to recover the cells, and nutrients are added to the recovered cells. The concentration of nutrients added in the catalytic system is 0.5g / L MgSO4, 0.5g / L KH2PO4, 0.5g / L NaCl, 1.5g / L corn steep liquor powder, 2g / L glycerol and 0.5g / L acrylic acid (the solvent is water). The dissolved oxygen is controlled to be above 10%, and aerobic induction is performed at 37°C for 3h. The induced cell fluid is centrifuged at 8000rpm and 4°C to recover the cells, and the whole-cell catalyst is used again, and the next batch of whole-cell catalysis is carried out according to the above catalytic system. After the introduction of this strategy, 20 batches of continuous catalytic studies were carried out, and the catalytic time for each batch was 12h. The results are shown in Table 4.

[0084] Table 4 Continuous catalysis results of whole cell catalyst

[0085]

[0086] The present invention provides a kind of thinking and method of recombinant Corynebacterium glutamicum and application thereof, the method and approach of specifically realizing this technical scheme are many, the above-mentioned is only the preferred implementation mode of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and embellishments can also be made, and these improvements and embellishments should also be regarded as protection scope of the present invention. Undefined each component in the present embodiment can be realized with prior art.

Claims

1. An aspartase mutant, characterized in that: Its amino acid sequence is shown in SEQ ID No.

7.

2. A nucleic acid molecule encoding the aspartase mutant according to claim 1, wherein the nucleotide sequence is shown in SEQ ID No.

8. A recombinant expression vector comprising the nucleic acid molecule according to claim 2.

4. A recombinant Corynebacterium glutamicum, comprising the recombinant expression vector according to claim 3.

5. The recombinant Corynebacterium glutamicum according to claim 4, characterized in that The starting strain is Corynebacterium glutamicum BACG121; the Corynebacterium glutamicum BACG121 is preserved in the China Center for Type Culture Collection, with a preservation number of CCTCC NO: M20241240 and a preservation time of June 17, 2024.

6. Use of the aspartase mutant according to claim 1, the nucleic acid molecule according to claim 2, the recombinant expression vector according to claim 3 or the recombinant Corynebacterium glutamicum according to any one of claims 4 to 5 in the production of β-alanine.

7. The use according to claim 6, characterized in that: The substrate acrylic acid is contacted with the aspartase mutant according to claim 1, or with the recombinant Corynebacterium glutamicum according to claim 4 or 5, and a catalytic reaction is carried out to generate β-alanine.

8. The use according to claim 7, characterized in that: Fermenting and culturing the recombinant Corynebacterium glutamicum to obtain a bacterial liquid, centrifuging to obtain wet bacterial bodies, and using the wet bacterial bodies as whole-cell catalysts to contact with a substrate, acrylic acid, to perform a whole-cell catalytic reaction; The whole cell catalytic reaction has a reaction temperature of 40-45°C and a reaction pH of 7.5-8.0; In the reaction system, the concentration of wet bacteria is 5-10 g / L, and the concentration of acrylic acid is 100-150 g / L.

9. The use according to claim 8, characterized in that: After one whole-cell catalytic reaction is completed, the wet bacteria are collected by centrifugation, nutrients are added for aerobic induction, and the whole-cell catalyst is used again for the next whole-cell catalytic reaction to achieve the reuse of the whole-cell catalyst, and the number of reuses of the whole-cell catalyst is more than 20 times.

10. The use according to claim 9, characterized in that: The nutrients include: 0.5 g / L MgSO4, 0.5 g / L KH2PO4, 0.5 g / L NaCl, 1.5 g / L corn steep liquor powder, 2 g / L glycerol and 0.5 g / L acrylic acid; The aerobic induction has an induction temperature of 37° C. and an induction time of 2-3 h.

Images