Method for increasing glutamic acid fermentation yield

Abstract

The application provides a method for improving the yield of glutamic acid fermentation, and the yield and conversion rate of L-glutamic acid in an L-glutamic acid production strain are significantly improved by mutating threonine at the 37th position of a protein encoded by a wild-type BBD29_07680 gene of corynebacterium glutamicum into alanine or leucine, thereby providing an effective means for glutamic acid fermentation production.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and more specifically, relates to a method for increasing the yield of glutamic acid fermentation. Background Technology

[0002] Glutamic acid, one of the 20 natural amino acids, is widely used in food, pharmaceuticals, biochemistry, and animal husbandry, in addition to its role in protein synthesis, neurotransmitter signaling, and unique culinary flavor. Originally isolated by German chemists through acid hydrolysis of wheat gluten, glutamate is now primarily produced through microbial fermentation. Corynebacterium glutamicum is the mainstay of industrial microbial fermentation for glutamate production, and due to its high-efficiency amino acid production capacity, it is also widely used in the production of lysine, valine, leucine, threonine, and other amino acids.

[0003] Glutamate is synthesized in a one-step enzymatic reaction catalyzed by glutamate dehydrogenase (GDH) from α-ketoglutarate, an intermediate in the tricarboxylic acid (TCA) cycle. Naturally occurring Corynebacterium glutamicum does not produce high levels of glutamate; necessary genetic modifications are crucial for achieving high glutamate production. Recent decades of research have led to considerably high glutamate production levels, and genes related to glutamate synthesis have been extensively explored and studied. However, the complexity of cellular metabolic networks means that these related genes cannot further increase glutamate production. Therefore, further research is needed to identify genes unrelated to glutamate synthesis to support increased glutamate production. Summary of the Invention

[0004] The purpose of this invention is to provide a method for increasing the yield of glutamic acid fermentation.

[0005] To achieve the objectives of this invention, in a first aspect, this invention provides a protein mutant encoded by the BBD29_07680 gene, with the amino acid sequence encoded by the wild-type BBD29_07680 gene of Corynebacterium glutamicum as a reference sequence. The protein mutant encoded by the BBD29_07680 gene contains a mutation at position 37 from T to A or L, preferably T37A.

[0006] In a second aspect, the present invention provides a nucleic acid molecule encoding the protein mutant or biological material containing the nucleic acid molecule.

[0007] The biomaterials include, but are not limited to, recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, or engineered bacteria.

[0008] Thirdly, the present invention provides the application of the nucleic acid molecule or the biological material in improving the yield, production intensity or conversion rate of glutamic acid in microorganisms.

[0009] Fourthly, the present invention provides a recombinant microorganism that expresses the protein mutant.

[0010] Preferably, the recombinant microorganism is constructed by mutating the BBD29_07680 gene in Corynebacterium glutamicum into a gene encoding the protein mutant.

[0011] Furthermore, the starting strain of the recombinant microorganism is Corynebacterium glutamicum, which is capable of accumulating glutamate.

[0012] Preferably, the starting strain is MHZ-0112-8 (Corynebacterium glutamicum MHZ-0112-8 preservation number CGMCCNo.11941, see CN105695383A).

[0013] Fifthly, the present invention provides a method for constructing the recombinant microorganism, which involves introducing the gene of the protein mutant into the microorganism via a plasmid or integrating it into the microbial chromosome via genetic engineering, or mutating the BBD29_07680 gene in Corynebacterium glutamicum to encode the protein mutant of claim 1.

[0014] Sixthly, the present invention provides the application of the recombinant microorganism in the fermentation production of glutamic acid.

[0015] In a seventh aspect, the present invention provides a method for increasing glutamic acid fermentation yield, comprising the step of fermenting and culturing the recombinant microorganism.

[0016] Preferably, the culture medium used for fermentation is: glucose 50-60 g / L, ammonium sulfate 10-20 g / L, KH2PO4 0.5-1.5 g / L, MgSO4·7H2O 0.2-0.5 g / L, FeSO4·7H2O 0.05-0.15 mg / L, MnSO4·5H2O 0.05-0.15 mg / L, VB1 150-250 μg / L, biotin 250-350 μg / L, soybean meal hydrolysate 0.3-0.6 g / L, pH 7.2-7.5.

[0017] This invention obtains a specific mutant by mutating threonine at position 37 of the protein encoded by the wild-type BBD29_07680 gene of Corynebacterium glutamicum to alanine or leucine, which significantly improves the yield and conversion rate of L-glutamic acid in L-glutamic acid producing strains. Detailed Implementation

[0018] The present invention aims to provide a protein mutant, a recombinant microorganism expressing the protein mutant, a method for preparing the same, and its applications.

[0019] The present invention adopts the following technical solution:

[0020] This invention provides a protein mutant encoded by the BBD29_07680 gene and a method for L-glutamic acid production, wherein the L-glutamic acid producing strain expresses the protein mutant encoded by the BBD29_07680 gene. The protein mutant, with the amino acid sequence encoded by the wild-type BBD29_07680 gene of the L-glutamic acid producing strain as a reference sequence, contains a mutation in which the threonine at position 37 is replaced by another amino acid.

[0021] Preferably, the protein mutant encoded by the BBD29_07680 gene contains a mutation in which threonine at position 37 is replaced by alanine or leucine, with alanine being preferred.

[0022] The term "mutation" refers to an alteration in the amino acid sequence of a protein-encoding gene caused by the addition, deletion, or substitution of one or more bases, thereby changing the protein's functional activity. The mutation method can be selected from mutagenesis, site-directed mutagenesis by PCR, and / or homologous recombination. In this invention, homologous recombination is preferred, specifically mutating threonine at position 37 of the protein encoded by the BBD29_07680 gene to alanine.

[0023] In this invention, the mutant protein encoded by the BBD29_07680 gene has the amino acid sequence shown in SEQ ID NO:1.

[0024] The nucleic acid encoding the protein mutant has the sequence shown in SEQ ID NO:2.

[0025] Those skilled in the art should understand that adding a tagged protein to the N-terminus or C-terminus of the above-mentioned protein mutant sequence or fusing it with other proteins to form a fusion protein, without changing the activity of the above-mentioned mutant protein itself, the tagged protein or fusion protein is also within the scope of protection of this invention.

[0026] Based on the amino acid sequence encoded by the BBD29_07680 gene provided above, those skilled in the art can obtain the sequence of the nucleic acid it encodes. Due to the degeneracy of codons, the nucleic acid sequence encoding the above amino acid sequence is not unique, and all nucleic acids capable of encoding the above protein mutants are within the protection scope of this invention.

[0027] In this invention, the amino acid producing strain is a Corynebacterium microorganism, preferably Corynebacterium glutamicum.

[0028] Based on the function of the protein mutant encoded by the BBD29_07680 gene, this invention provides the application of the protein mutant encoded by the BBD29_07680 gene or its encoded nucleic acid in increasing the L-glutamic acid yield in L-glutamic acid producing strains.

[0029] The present invention also provides a recombinant microorganism that expresses a protein mutant encoded by the BBD29_07680 gene, the protein mutant being described above; the starting strain of the recombinant microorganism is Corynebacterium glutamicum MHZ-0112-8, and its accession number is CGMCC No.11941.

[0030] Preferably, the original BBD29_07680 gene on the chromosome of the starting strain is mutated into the coding gene of the BBD29_07680 protein mutant.

[0031] The present invention provides a recombinant microorganism expressing the mutant protein encoded by the BBD29_07680 gene, which significantly improves the yield and conversion rate of L-glutamic acid in industrial L-glutamic acid production strains.

[0032] The present invention also provides a method for constructing the above-mentioned recombinant microorganism, which includes: mutating the gene encoding BBD29_07680 in the starting strain into a gene encoding a BBD29_07680 protein mutant, wherein the BBD29_07680 protein mutant is as described above.

[0033] In one specific embodiment of the present invention, it includes the step of introducing a recombinant plasmid carrying a gene encoding the BBD29_07680 protein mutant into a starting strain.

[0034] Preferably, the recombinant plasmid is a plasmid that can undergo homologous recombination in bacterial cells, and the coding gene of BBD29_07680 on the plasmid is exchanged with a homologous gene on the chromosome.

[0035] Furthermore, the construction method includes:

[0036] (1) Construct a recombinant plasmid containing the gene encoding the mutant BBD29_07680 protein;

[0037] (2) Transform the recombinant plasmid into the starting strain and screen the transformants using a selective medium containing antibiotics;

[0038] (3) Screening for recombinant microorganisms that have undergone the target mutation in positive transformants.

[0039] The present invention also provides a method for producing glutamic acid, which includes the step of fermentation culture with the above-mentioned recombinant microorganism, wherein the starting strain of the recombinant microorganism is Corynebacterium glutamicum MHZ-0112-8.

[0040] In this invention, the fermentation medium used for fermentation culture comprises the following components: glucose 50-60 g / L, ammonium sulfate 10-20 g / L, KH2PO4 0.5-1.5 g / L, MgSO4·7H2O 0.2-0.5 g / L, FeSO4·7H2O 0.5-1.5 mg / L, MnSO4·5H2O 0.8-1.2 mg / L, VB1 150-250 μg / L, biotin 250-350 μg / L, soybean meal hydrolysate 0.3-0.6 g / L, calcium carbonate 40-80 g / L, pH 7.2-7.5.

[0041] Specifically, the method for producing a protein mutant encoded by the BBD29_07680 gene and its use in L-glutamic acid production includes: inoculating the recombinant microorganism into a slant culture medium for slant culture, picking the bacterial growth on the slant culture medium and inoculating it into a seed culture medium for seed culture, and then transferring the seed culture into a fermentation medium for fermentation.

[0042] Preferably, the slant culture medium used is: 5 g / L yeast extract, 10 g / L beef extract, 10 g / L peptone, 10 g / L sodium chloride, 2.5 g / L agar powder, pH 7.0-7.2.

[0043] The seed culture medium used was: glucose 25 g / L, urea 3.0 g / L, K2HPO4·3H2O 2.2 g / L, MgSO4·7H2O 0.9 g / L, corn steep liquor 33 mL / L, soybean meal hydrolysate 22 mL / L, pH 7.0-7.2.

[0044] The fermentation medium used was: glucose 60 g / L, ammonium sulfate 15 g / L, KH2PO4 1.0 g / L, MgSO4·7H2O 0.4 g / L, FeSO4·7H2O 1.0 mg / L, MnSO4·5H2O 1.0 mg / L, V B1 200 μg / L, biotin 300 μg / L, soybean meal hydrolysate 0.48 g / L, calcium carbonate 50 g / L, pH 7.2-7.5.

[0045] Seed culture was carried out at 31.5℃ and 220 rpm with shaking until the mid-to-late logarithmic growth stage, with a culture time of 10-14 h.

[0046] Fermentation was carried out at 31.5℃ with shaking at 220 rpm for 12-20 hours.

[0047] The preparation method of the soybean meal hydrolysate used in this invention can be found in the literature "Research on the process optimization of soybean meal hydrolysis by response surface methodology, China Food Additives, 2018, No. 2".

[0048] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the examples are conducted under conventional experimental conditions, such as those described in Sambrook et al., Molecular Cloning: a Laboratory Manual (Sambrook J & Russell DW, 2001), or as recommended by the manufacturer's instructions.

[0049] The primer names and sequence information involved in the following examples are shown in Table 1.

[0050] Table 1 Primer sequences

[0051]

[0052]

[0053] Unless otherwise specified in the following examples, the techniques or conditions described in the literature in this field, or the product instructions, shall be followed. Reagents or instruments whose manufacturers are not specified can be obtained through legitimate commercial channels.

[0054] The strains involved in the following examples are as follows:

[0055] The originating strain MHZ-0112-8 is *Corynebacterium glutamicum*. A pure culture of *Corynebacterium glutamicum* MHZ-0112-8 was deposited on December 25, 2015, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), with accession number CGMCC No. 11941. *Corynebacterium glutamicum* MHZ-0112-8 has been published in CN105695383A.

[0056] Example 1: Construction of recombinant plasmid pK18-△odhA

[0057] Primers were designed based on the gene sequence of odhA (GenBank NO: BBD29_06050) in the NCBI database, and the primer sequences are shown in Table 1. Using Phusion superfidelity polymerase (New England BioLabs), odhA-UP-1F / odhA-UP-1R and odhA-DN-2F / odhA-DN-2R were used as primer pairs. Recombinant fragments UP and DN were prepared using the genome of Corynebacterium glutamicum ATCC13869 as a template. The PCR program was as follows: denaturation at 98℃ for 10 s, annealing at 55℃ for 20 s, extension at 72℃ for 30 s, repeated 30 times, followed by a final extension at 72℃ for 10 min. The resulting fragments were purified and recovered using an agarose gel extraction kit (Tiangen) and stored at -20℃ for later use. pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP / DN fragment was mixed with the double-digested vector in a certain ratio using the ClonExpress MultiS One Step Cloning Kit (Novizan Biotechnology Co., Ltd.). After mixing, the mixture was placed in a metal bath (Ginkgo Biloba Biotechnology Co., Ltd.) at 37°C for 30 min. After the reaction, the cells were transformed into Trans1T1 competent cells (TransGen Biotech). Kanamycin-resistant clones were picked, and positive clones with pK18mobsacB inserted were identified by XbaI / NheI restriction enzyme digestion. The insertion of the fragment was further confirmed by sequencing (GENEWIZ). The obtained plasmid was named pK18-△odhA.

[0058] Example 2: Construction of recombinant plasmids pK18-BBD29_07680(T37A) and pK18-BBD29_07680(T37L)

[0059] Primers were designed based on the sequence of the BBD29_07680 gene (GenBank NO: BBD29_07680) in the NCBI database, and the primer sequences are shown in Table 1. Using Phusion superfidelity polymerase (New England BioLabs), primer pairs P39 / P40, P41 / P42, P39 / P44, and P43 / P42 were used as primers. Recombinant fragments UP1, DN2, UP3, and DN4 were prepared using the genome of *Corynebacterium glutamicum* MHZ-0112-08 as a template. The PCR program was as follows: denaturation at 98℃ for 10 s, annealing at 55℃ for 20 s, extension at 72℃ for 30 s, 30 cycles, and a final extension at 72℃ for 10 min. The resulting fragments were purified and recovered using an agarose gel extraction kit (Tiangen) and stored at -20℃ for later use. pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP1 / DN2 fragments were mixed with the double-digested vector in a certain ratio using the ClonExpressMultiS One Step Cloning Kit (Novizan Biotechnology Co., Ltd.). At the same time, the UP3 / DN4 fragments were mixed with the double-digested vector in a certain ratio. After mixing, the mixture was placed in a metal bath (Ginkgo Biloba Biotechnology Co., Ltd.) at 37°C for 30 min. After the reaction, the cells were transformed into Trans1T1 competent cells (TransGen Biotech). Kanamycin-resistant clones were picked, and positive clones with pK18mobsacB inserted were identified by XbaI / NheI restriction enzyme digestion. The insertion of the fragments was further confirmed by sequencing (GENEWIZ). The obtained plasmids were named pK18-BBD29_07680(T37A) (containing recombinant fragments UP1 and DN2) and pK18-BBD29_07680(T37L) (containing recombinant fragments UP3 and DN4).

[0060] Example 3: Deletion of the odhA gene in wild-type Corynebacterium glutamicum ATCC13869

[0061] ATCC13869 competent cells were prepared according to the classic method for *Corynebacterium glutamicum* (*C. glutamicum* Handbook, Charter 23). The recombinant plasmid pK18-ΔodhA was electroporated into *Corynebacterium glutamicum* ATCC13869 competent cells. Exchange recombinants were selected on selective medium containing 15 mg / L kanamycin. Colony PCR was performed using Fast Taq DNA polymerase (TransGen Biotech) with primer pairs odhA-UP-1F / P86 and P81 / odhA-DN-2R to identify the Kansas cell line. RCloning was performed using the following PCR parameters: 94℃ for 30 seconds, 55℃ for 30 seconds, 72℃ for 40 seconds, for a total of 30 cycles, followed by a complete extension at 72℃ for 10 minutes. Clones amplifying one long and one short fragment (1258 bp and 640 bp) using two primer pairs were considered positive clones. The selected positive clones were inoculated into antibiotic-free BHI medium and cultured for 12-14 hours. The bacterial culture was then diluted 100-1000 times and plated onto solid BHI medium containing 10% sucrose and cultured for 36 hours. The selected strains were further subjected to kanamycin resistance phenotype verification to select kanamycin-resistant strains. S The recombinants were verified using the identification primer pair odhA-ID-F / odhA-ID-R. Recombinants with a 455bp deletion were identified by PCR amplification; these were considered correct recombinants, with a length of 1198bp. The positive recombinants were then amplified and sequenced using odhA-ID-F / odhA-ID-R. The correctly sequenced strain was named 13869-△odhA.

[0062] Example 4: Recombinant strain 13869-△odhA introduces a point mutation in the BBD29_07680 gene.

[0063] 13869-△odhA competent cells were prepared according to the classic method for *Corynebacterium glutamicum* (*C. glutamicum* Handbook, Charter 23). Recombinant plasmids pK18-BBD29_07680(T37A) and pK18-BBD29_07680(T37L) were electroporated into *Corynebacterium glutamicum* 13869-△odhA competent cells. Exchange recombinants were selected on selective medium containing 15 mg / L kanamycin. Colony PCR was performed using Fast Taq DNA polymerase (TransGen Biotech) with primer pairs P39 / P86 and P81 / P42 to identify the Kansas cells. R Cloning and PCR parameters were as follows: 94℃ for 30s, 55℃ for 50s, 72℃ for 45s, for a total of 30 cycles, followed by a complete extension at 72℃ for 10min. Clones amplifying fragments of 1245bp and 1127bp with two primer pairs were considered positive clones. The selected positive clones were inoculated into antibiotic-free BHI medium and cultured for 12-14 hours. The bacterial culture was then diluted 100-1000 times and plated onto solid BHI medium containing 10% sucrose and cultured for 36 hours. The selected strains were further subjected to kanamycin resistance phenotype verification to select kanamycin-resistant strains. SThe recombinants were verified using primer pairs P47 / P46 to confirm the T37A point mutation recombinant and P48 / P46 to confirm the T37L point mutation recombinant. By adjusting the annealing temperature, recombinants containing point mutations were obtained. The positive recombinants were amplified and sequenced using P45 / P46. Correct recombinants were 1210 bp in length, and the correctly sequenced strains were named 13869-△odhA-T37A and 13869-△odhA-T37L.

[0064] Example 5: Introducing a point mutation in the BBD29_07680 gene into strain MHZ-0112-8

[0065] MHZ-0112-8 competent cells were prepared according to the classic method for *Corynebacterium glutamicum* (*C. glutamicum* Handbook, Charter 23). Recombinant plasmids pK18-BBD29_07680(T37A) and pK18-BBD29_07680(T37L) were electroporated into *Corynebacterium glutamicum* MHZ-0112-8 competent cells. Exchange recombinants were selected on selective medium containing 15 mg / L kanamycin. Colony PCR was performed using Fast Taq DNA polymerase (TransGen Biotech) with primer pairs P39 / P86 and P81 / P42 to identify the kanamycin. R Cloning and PCR parameters were as follows: 94℃ for 30s, 55℃ for 50s, 72℃ for 45s, for a total of 30 cycles, followed by a complete extension at 72℃ for 10min. Clones amplifying fragments of 1245bp and 1127bp with two primer pairs were considered positive clones. The selected positive clones were inoculated into antibiotic-free BHI medium and cultured for 12-14 hours. The bacterial culture was then diluted 100-1000 times and plated onto solid BHI medium containing 10% sucrose and cultured for 36 hours. The selected strains were further subjected to kanamycin resistance phenotype verification to select kanamycin-resistant strains. S The recombinants were verified using primer pairs P47 / P46 to confirm the T37A point mutation recombinant and P48 / P46 to confirm the T37L point mutation recombinant. By adjusting the annealing temperature, recombinants containing point mutations were obtained. The positive recombinants were amplified and sequenced using P45 / P46. Correct recombinants were 1210 bp in length, and the correctly sequenced strains were named MHZ-0112-46 and MHZ-0112-47.

[0066] Example 6: Performance verification of glutamic acid production by recombinant strains 13869-△odhA-T37A, 13869-△odhA-T37L, MHZ-0112-46, and MHZ-0112-47

[0067] The recombinant Corynebacterium glutamicum constructed in Examples 4 and 5 were fermented to verify its glutamic acid production performance, as detailed below:

[0068] The strain, frozen in glycerol tubes at -80℃, was activated by inoculating it onto agar slant medium. After culturing at 31.5℃ for 24 h, mycelial growth occurred. Mycelial growth was collected from the freshly activated slant and inoculated into seed culture medium. The culture was then incubated at 31.5℃ with shaking at 220 rpm until the mid-to-late logarithmic growth stage, for 12 h, yielding a seed culture. A 10% inoculum of this seed culture was added to a 500 ml shake flask containing 20 ml of fermentation medium and incubated at 31.5℃ with shaking at 220 rpm for 16 h. After complete glucose consumption, the concentration of L-glutamic acid accumulated in the culture medium was determined by HPLC. The results are shown in Table 2.

[0069] The culture medium formula used is as follows:

[0070] Slant culture medium: yeast powder 5.0 g / L, beef extract 10 g / L, peptone 10 g / L, sodium chloride 10 g / L, agar powder 2.5 g / L, pH 7.0-7.2, sterilized at 121℃ and 0.1 MPa for 30 min.

[0071] Seed culture medium: glucose 25 g / L, urea 3.0 g / L, K2HPO4·3H2O 2.2 g / L, MgSO4·7H2O 0.9 g / L, corn steep liquor 33 mL / L, soybean meal hydrolysate 22 mL / L, pH 7.0-7.2, sterilized at 121℃ and 0.1 MPa for 15 min.

[0072] Fermentation medium: glucose 60 g / L, ammonium sulfate 15 g / L, KH₂PO₄ 1.0 g / L, MgSO₄·7H₂O 0.4 g / L, FeSO₄·7H₂O 1.0 mg / L, MnSO₄·5H₂O 1.0 mg / L, V B1 200 μg / L, biotin 300 μg / L, soybean meal hydrolysate 0.48 g / L, calcium carbonate 50 g / L, adjust pH to 7.2-7.5 with NaOH, sterilize at 121℃ and 0.1 MPa for 15 min.

[0073] Table 2. Detection of glutamic acid content in mutant strains

[0074] 13869-△odhA 0.618±0.23 2.2±0.04 3.7±0.11 13869-△odhA-T37A 0.609±0.11 5.4±0.02 9.0±0.08 13869-△odhA-T37L 0.616±0.09 4.1±0.07 6.8±0.16 MHZ-0112-8 0.423±0.12 34.5±0.05 57.5±0.09 MHZ-0112-46 0.436±0.03 38.1±0.21 63.5±0.35 MHZ-0112-47 0.418±0.02 36.8±0.06 61.3±0.10

[0075] Note: OD 600Table 2 shows the turbidity of the culture medium diluted 100 times at 600 nm, representing cell mass. Glu (g / L) represents the amount of accumulated L-glutamate. As shown in Table 2, in the 13869-△odhA-T37A gene, after the 37th amino acid of the protein encoded by the BBD29_07680 gene was mutated from threonine (T) to alanine (A), i.e., ACG to GCG, the glutamate concentration increased from 2.2 g / L to 9.0 g / L, and the glutamate conversion rate increased by 5.3%. Conversely, in the 13869-△odhA-T37L gene, after the 37th amino acid of the protein encoded by the BBD29_07680 gene was mutated from threonine (T) to leucine (L), i.e., ACG to CTT, the glutamate concentration increased from 3.7 g / L to 6.8 g / L, and the glutamate conversion rate increased by 3.1%.

[0076] In MHZ-0112-46, the 37th amino acid of the protein encoded by the BBD29_07680 gene was mutated from threonine (T) to alanine (A), i.e., ACG mutated to GCG. This resulted in an increase in glutamate concentration from 34.5 g / L to 38.1 g / L, and a 6.0% increase in glutamate conversion rate. In MHZ-0112-47, the 37th amino acid of the protein encoded by the BBD29_07680 gene was mutated from threonine (T) to leucine (L), i.e., ACG mutated to CTT. This resulted in an increase in glutamate concentration from 34.5 g / L to 36.8 g / L, and a 3.8% increase in glutamate conversion rate. These findings indicate that mutating the 37th amino acid of the protein encoded by the BBD29_07680 gene to alanine or leucine can improve the synthesis and conversion rate of glutamate.

[0077] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A recombinant microorganism, characterized in that, The microorganism expresses a protein mutant encoded by the mutant gene of the BBD29_07680 gene; The amino acid sequence of the protein mutant is shown in SEQ ID NO:1, or the 37th position of the amino acid sequence shown in SEQ ID NO:1 is replaced by L instead of A; The starting strain of the recombinant microorganism is Corynebacterium glutamicum, which is capable of accumulating glutamate.

2. The recombinant microorganism according to claim 1, characterized in that, The starting strain was MHZ-0112-8.

3. The method for constructing the recombinant microorganism according to claim 1 or 2, characterized in that, The mutant gene of BBD29_07680 was introduced into Corynebacterium glutamicum via plasmid or integrated into the chromosome of Corynebacterium glutamicum through genetic engineering. The amino acid sequence of the protein mutant encoded by the mutant gene is shown in SEQ ID NO:1, or the 37th position of the amino acid sequence shown in SEQ ID NO:1 is replaced by L instead of A; The starting strain of Corynebacterium glutamicum is a Corynebacterium glutamicum capable of accumulating glutamate.

4. The application of the recombinant microorganisms described in claim 1 or 2 in the fermentation production of glutamic acid.

5. A method for increasing glutamic acid fermentation yield, characterized in that, It includes the step of fermenting and culturing the recombinant microorganisms described in claim 1 or 2.

6. The method according to claim 5, characterized in that, The culture medium used for fermentation is as follows: glucose 50-60 g / L, ammonium sulfate 10-20 g / L, KH2PO4 0.5-1.5 g / L, MgSO4•7H2O 0.2-0.5 g / L, FeSO4•7H2O 0.05-0.15 mg / L, MnSO4·5H2O 0.05-0.15 mg / L, VB1 150-250 μg / L, biotin 250-350 μg / L, soybean meal hydrolysate 0.3-0.6 g / L, pH 7.2-7.5.

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