Method for producing L-glutamic acid

Abstract

Modified bacteria that produce L-glutamic acid are provided wherein a heterologous polynucleotide of phosphoketolase (XFP) is contained in its genome as compared to non-modified bacteria. The phosphoketolase is derived from bifidobacterium. In addition, methods of increasing the yield of L-glutamic acid using the modified bacteria are also provided.

Description

Technical Field

[0001] The present disclosure relates to a method for producing L-glutamic acid by fermentation using bacteria. Background Art

[0002] Glutamic acid is an acidic amino acid. As an important nutrient for human growth, glutamic acid not only has special physiological effects, but also has unique functions in the food industry.

[0003] Currently, the most commonly used method for producing glutamic acid is fermentation, which has the advantages of a wide range of raw material sources, low production costs, controllable product quality, and a single product. For example, L-glutamic acid is mainly produced by fermentation using L-glutamic acid-producing bacteria called coryneform bacteria belonging to the genera Brevibacterium, Corynebacterium, and Microbacterium, or mutant strains thereof.

[0004] Recombinant DNA technology can improve microbial L-amino acid production. However, current strains producing glutamate still have poor fermentation performance and unsatisfactory glutamate conversion rates. Glutamic acid is the primary raw material for monosodium glutamate production, and industrial demand for it is extremely high. Existing strains are unable to meet the needs of large-scale industrial production. Summary of the Invention

[0005] The present disclosure provides a modified bacterium producing L-glutamate, wherein the genome of the modified bacterium comprises a heterologous polynucleotide encoding phosphoketolase (XFP) compared to a non-modified bacterium.

[0006] In a specific embodiment, the bacterium is a Corynebacterium bacterium, preferably Corynebacterium glutamicum.

[0007] In a specific embodiment, the phosphoketolase is D-xylulose-5-phosphate phosphoketolase and / or D-fructose 6-phosphate phosphoketolase.

[0008] In a specific embodiment, the phosphoketolase is derived from Bifidobacterium, preferably Bifidobacterium longum, Bifidobacterium adolescentis, or Bifidobacterium animalis.

[0009] In a specific embodiment, the modified bacterium comprises more than one copy of the phosphoketolase gene, preferably three to ten copies, more preferably six copies.

[0010] In a specific embodiment, the insertion site of each copy of the phosphoketolase gene is selected from the group consisting of: within the alaT gene, within the odhA gene, between the cg3364 and cg3365 genes, between the cg2564 and cg2563 genes, between the cg3124 and cg3125 genes, between the cg3384 and cg3385 genes, between the BBD29_07270 and BBD29_07275 genes, between the cg2211 and cg2212 genes, between the cg0928 and CGTRNA_RS15430 genes, between the BBD29_07210 and BBD29_07215 genes, between the cg1512 and cg1513 genes, and between the cg2337 and cg2336 genes.

[0011] In a specific embodiment, the phosphoketolase comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 or an amino acid sequence that is at least 70% identical to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17; or the phosphoketolase comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 or a nucleic acid sequence that is at least 70% identical to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18.

[0012] In a specific embodiment, the modified bacteria further comprises a modification of the expression control sequence of the phosphoketolase gene, preferably, the modification of the expression control sequence is a promoter, more preferably, the promoter is a Ptuf promoter or a PscpB promoter.

[0013] The present disclosure provides a modified bacterium for producing L-glutamate, wherein the genome of the modified bacterium comprises a heterologous polynucleotide encoding a phosphoketolase (XFP) compared to a non-modified bacterium. The modified bacterium further comprises a modification to reduce odhA activity; an amino acid substitution of A100T in the yggB encoded protein; and / or a modification to enhance gdh activity.

[0014] In a specific embodiment, the modified bacteria further include modifications that reduce odhA activity, such as knockout of odhA or deletion of 5 base pairs at positions 544-548 (gacgt) of the odhA gene; amino acid substitution of A100T in the yggB encoded protein; and / or modifications that enhance gdh activity, such as the DNA sequence at position -35 of the gdh transcription start site is TTGTCA and the DNA sequence at position -10 of the transcription start site is TATAAT.

[0015] Those skilled in the art will appreciate that the modification methods for reducing odhA activity may be of various types, and the modification for reducing odhA activity is selected from complete knockout of odhA, truncation of odhA and simultaneous repair of the frameshift mutation to the wild type, and other truncated forms of odhA whose activity is reduced by more than 10%.

[0016] Those skilled in the art will appreciate that the gdh activity enhancement modification can be of various types, for example, using a wild-type gdh gene while using a strong promoter.

[0017] Those skilled in the art will appreciate that the amino acid substitution of A100T in the yggB encoded protein, which can effectively increase glutamate production, can also be used in the present disclosure.

[0018] In one embodiment, the modified bacteria further comprises gltA -10M-C361Y 、suc M199I 、

[0019] ach S372C 、tktA T234I 、putA A755V 、ds G171D , and / or nadA D312N .

[0020] In a specific embodiment, the modified bacterium further comprises a modification that reduces phosphofructokinase activity.

[0021] In a specific embodiment, the bacterium comprises a modification of the expression control sequence of the phosphofructokinase gene and / or a modification of the coding sequence of the phosphofructokinase gene.

[0022] In a specific embodiment, the modification of the expression control sequence of the phosphofructokinase gene is selected from: a) replacing the phosphofructokinase gene promoter with PrecA promoter; b) replacing the phosphofructokinase gene promoter with Pzwf promoter; c) replacing the phosphofructokinase gene promoter with Pgdh promoter.

[0023] The modification of the coding sequence of the phosphofructokinase gene is selected from: a) replacing the ATG start sequence of the coding nucleic acid of the phosphofructokinase gene with TTG; and / or b) replacing the protein coding sequence of the phosphofructokinase gene with E171D.

[0024] In a specific embodiment, the L-glutamic acid is ammonium L-glutamate or sodium L-glutamate.

[0025] In another aspect, provided is use of the modified bacteria of the present disclosure for increasing L-glutamic acid production.

[0026] In another aspect, provided is a method for producing L-glutamic acid, comprising culturing the modified bacterium of the present disclosure in a culture medium.

[0027] In a specific embodiment, said culture medium comprises glucose.

[0028] In a specific embodiment, the method further comprises isolating L-glutamic acid.

[0029] In another aspect, a bioreactor comprises the modified bacteria described in the present disclosure.

[0030] Beneficial effects

[0031] The genetically engineered bacteria provided by the present disclosure have a high glutamate conversion rate. DETAILED DESCRIPTION

[0032] The following description of the present disclosure is intended only to illustrate various embodiments of the present disclosure. Therefore, the specific modifications discussed should not be interpreted as limiting the scope of the present disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of the present disclosure, and it should be understood that these equivalent embodiments are intended to be included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0033] In order to enable those skilled in the art to better understand the solutions of the present disclosure, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present disclosure, rather than all of the embodiments.

[0034] Table 1. Primer list

[0035]

[0036]

[0037]

[0038]

[0039]

[0040]

[0041]

[0042] Example 1: Construction of starting strains

[0043] 1.1 Construction of ATCC13869→WJ0140 starting strain

[0044] 1.1.1 Plasmid construction

[0045] 1).pK18-Pgdh -10M Plasmid construction

[0046] PCR amplification was performed using the genome of Corynebacterium glutamicum ATCC13869 as a template using Phusion high-fidelity polymerase (New England BioLabs). PCR amplification was performed using primer pairs PE640 / PE641 and PE642 / PE643 to obtain Pgdh -10M The primer sequences for the upstream and downstream homology arms UP and DN fragments are shown in Table 1. The PCR program was as follows: denaturation at 98°C for 10 s, annealing at 55°C for 20 s, and extension at 72°C for 15 s / kb for 30 cycles, followed by a complete extension at 72°C for 10 min. The resulting fragments were purified and digested using an agarose gel recovery kit (Tiangen) and stored at -20°C until use.

[0047] Subsequently, PE640 / PE643 was used as a primer pair and the upstream and downstream homology arms were used as templates to prepare the recombinant fragment. The PCR procedure was the same as above, and the resulting recombinant fragment was purified using an agarose gel recovery kit (Tian Gen). At the same time, pK18mobsacB was digested with restriction endonucleases XbaI / EcoRI, and the recombinant fragment was ligated to the vector using T4 DNA ligase (TransGen Biotech). Trans1T1 competent cells (TransGen Biotech) were transformed, and kanamycin-resistant clones were picked. XbaI / EcoRI enzyme digestion was used to identify the positive clones in which the fragment was inserted into pK18mobsacB. The correct band size was 1061 bp. Sequencing (GENEWIZ) was further used to identify that the inserted fragment was correct, and the primers submitted for testing were PE640 / PE643. The resulting plasmid was named pK18-Pgdh -10M .

[0048] 2).pK18-yggB A100T Plasmid construction

[0049] PCR amplification was performed using the genome of Corynebacterium glutamicum ATCC13869 as a template using Phusion high-fidelity polymerase (New England BioLabs). PCR amplification using primer pairs PE646 / PE647 and PE648 / PE649 yielded the yggB A100TThe upstream and downstream homology arms UP and DN fragments, the primer sequences are shown in Table 1. Subsequently, PE646 / PE649 was used as a primer pair, and the upstream and downstream homology arms were used as templates to prepare the recombinant fragment. The PCR procedure was the same as above, and the resulting recombinant fragment was purified by an agarose gel recovery kit (Tian Gen). At the same time, pK18mobsacB was digested with XbaI / EcoRI restriction endonucleases, and the fragment was connected to the vector with T4 DNA ligase (TransGen Biotech), and the Trans1T1 competent cells (TransGenBiotech) were transformed. The kanamycin-resistant clones were picked, and the XbaI / EcoRI enzyme digestion was used to identify the positive clones in which the fragments were inserted into pK18mobsacB. The correct band size was 1036bp. The inserted fragment was further identified by sequencing (GENEWIZ), and the primers sent for testing were PE646 / PE649. The resulting plasmid was named pK18-yggB A100T .

[0050] 3). Construction of pK18-odhA ORF-5bpQS plasmid

[0051] With Corynebacterium glutamicum ATCC13869 genome as template, utilize Phusion super-fidelity polymerase (NewEngland BioLabs) to carry out PCR amplification.Be that primer pair carries out PCR amplification with PE652 / PE653, PE654 / PE655 and obtain upstream and downstream homology arm UP and DN fragment, and primer sequence is as shown in Table 1.Subsequently with PE652 / PE655 as primer pair, with upstream and downstream homology arm as template, prepare recombinant fragment, PCR program is the same, and gained recombinant fragment is purified through agarose gel recovery test kit (Tiangen).Simultaneously, pK18mobsacB is digested with XbaI / EcoRI restriction enzyme, and with T4 DNA ligase (TransGen Biotech), fragment is connected to carrier, transform Trans1T1 competent cells (TransGenBiotech), picking kana resistance clone, XbaI / EcoRI enzyme digestion identification obtains the positive clone that fragment is inserted into pK18mobsacB, and correct band size is 1039bp. The inserted fragment was further confirmed to be correct by sequencing (GENEWIZ), and the primers used were PE652 / PE655. The resulting plasmid was named pK18-odhAORF-5bpQS.

[0052] 4).pK18-gltA -10M-C361Y Plasmid construction

[0053] PCR amplification was performed using the genome of Corynebacterium glutamicum ATCC13869 as a template using Phusion high-fidelity polymerase (New England BioLabs). PCR amplification was performed using primer pairs PE656 / PE657, PE658 / PE659, and PE660 / PE661 to obtain gltA. -10M-C361Y The upstream and downstream homology arms UP and DN and the middle fragment, the primer sequences are shown in Table 1. Subsequently, PE656 / PE661 was used as a primer pair, and the upstream and downstream homology arms and the middle fragment were used as templates to prepare the recombinant fragment. The PCR procedure was the same as above, and the resulting recombinant fragment was purified by an agarose gel recovery kit (Tian Gen). At the same time, pK18mobsacB was digested with XbaI / EcoRI restriction endonucleases, and the fragment was connected to the vector with T4 DNA ligase (TransGen Biotech), and Trans1T1 competent cells (TransGen Biotech) were transformed. Kanamycin-resistant clones were picked, and XbaI / EcoRI enzyme digestion was used to identify the positive clones in which the fragments were inserted into pK18mobsacB. The correct band size was 2241bp. The inserted fragment was further identified by sequencing (GENEWIZ), and the primers sent for testing were PE656 / PE661 / PE362 / PE363. The resulting plasmid was named pK18-gltA -10M-C361Y .

[0054] 5).pK18-ach S372C Plasmid construction

[0055] With Corynebacterium glutamicum ATCC13869 genome as template, utilize Phusion super-fidelity polymerase (New England BioLabs) to carry out PCR amplification.With PE664 / PE665, PE666 / PE667 as primer pair, carry out PCR amplification and obtain upstream and downstream homology arm UP and DN fragment, and primer sequence is as shown in Table 1.With PE664 / PE667 as primer pair subsequently, with upstream and downstream homology arm fragment as template, prepare recombinant fragment, PCR program is the same as above, and gained recombinant fragment is purified through agarose gel recovery test kit (Tian Gen).Simultaneously, pK18-mobsacB is utilized XbaI / HindIII to digest, and with T4DNA ligase (TransGenBiotech), fragment and carrier are connected, transform Trans1T1 competent cells (TransGen Biotech), picking kana resistance clone, XbaI / HindIII enzyme digestion identification obtains the positive clone that fragment is inserted into pK18mobsacB, and correct band size is 1035bp. The inserted fragment was further confirmed to be correct by sequencing (GENEWIZ), and the primers used for the test were PE664 / PE667. The resulting plasmid was named pK18-achS372C .

[0056] 6).pK18-suc M199I Plasmid construction

[0057] Phusion high-fidelity polymerase (New England BioLabs) was used, 49-UP-1F / 49-UP-1R and 49-DN-2F / 49-DN-2R were used as primer pairs, and the genome of Corynebacterium glutamicum ATCC13869 was used as a template to prepare the upstream and downstream homology arm UP and DN fragments. The primer sequences are shown in Table 1. Subsequently, 49-UP-1F / 49-DN-2R was used as a primer pair and the upstream and downstream homology arms were used as templates to prepare the recombinant fragment. The PCR procedure was the same as above. The obtained recombinant fragment was purified by agarose gel recovery kit (Tiangen). At the same time, pK18-mobsacB was digested with BamHI / XbaI and the fragment was ligated to the vector using T4 DNA ligase (TransGen Biotech). Trans1T1 competent cells (TransGen Biotech) were transformed, and kanamycin-resistant clones were picked. BamHI / XbaI enzyme digestion was used to identify the positive clones with the fragment inserted into pK18mobsacB. The correct band size was 1002bp. The inserted fragment was further identified by sequencing (GENEWIZ). The primers sent for testing were 49-UP-1F / 49-DN-2R. The resulting plasmid was named pK18-suc M199I .

[0058] 7).pK18-tktA T234I Plasmid construction

[0059] Primers were designed based on the sequence of the tktA gene (GenBank NO: NCgl1512) in the NCBI database. The primer sequences are shown in Table 1. Using Phusion high-fidelity polymerase (New England BioLabs), tktA-UP-1F / tktA-UP-1R and tktA-DN-2F / tktA-DN-2R were used as primer pairs, and the genome of Corynebacterium glutamicum ATCC13869 was used as a template to prepare upstream and downstream homology arm UP and DN fragments. Subsequently, tktA-UP-1F / tktA-DN-2R was used as a primer pair, and the upstream and downstream homology arms were used as templates to prepare recombinant fragments. The PCR procedure was the same as above. The resulting recombinant fragment was purified using an agarose gel recovery kit (Tiangen) and then digested with XbaI / HindIII. pK18-mobsacB was also digested with XbaI / HindIII and ligated to the vector using T4 DNA ligase (TransGen Biotech). Trans1T1 competent cells (TransGen Biotech) were then transformed, and kanamycin-resistant clones were selected. XbaI / HindIII digestion was performed to identify positive clones with the fragment inserted into pK18mobsacB. The correct band size was 1028 bp. Sequencing (GENEWIZ) confirmed that the inserted fragment was correct. The primers used for the assay were tktA-UP-1F / tktA-DN-2R. The resulting plasmid was named pK18-tktA. T234I .

[0060] 8).pK18-putA A755V Plasmid construction

[0061] Phusion high-fidelity polymerase (New England BioLabs) was used with the putA-UP-1F / putA-UP-1R and putA-DN-2F / putA-DN-2R primer pairs, using the genome of Corynebacterium glutamicum ATCC13869 as a template to prepare upstream and downstream homology arm UP and DN fragments. The primer sequences are shown in Table 1. Subsequently, recombinant fragments were prepared using the putA-UP-1F / putA-DN-2R primer pair and the upstream and downstream homology arms as templates, following the same PCR procedure as above. The resulting recombinant fragment was purified using an agarose gel recovery kit (Tiangen) and then digested with XbaI / HindIII. pK18-mobsacB was also digested with XbaI / HindIII and ligated to the vector using T4 DNA ligase (TransGen Biotech). Trans1T1 competent cells (TransGen Biotech) were then transformed, and kanamycin-resistant clones were selected. XbaI / HindIII digestion was performed to identify positive clones with the fragment inserted into pK18mobsacB. The correct band size was 998 bp. Sequencing (GENEWIZ) confirmed that the inserted fragment was correct. The primers used for the assay were putA-UP-1F / putA-DN-2R. The resulting plasmid was named pK18-putA. A755V .

[0062] 9).pK18-nadA D312N Plasmid construction

[0063] Primers were designed based on the sequence of the nadA gene (GenBank NO: NCgl1024) in the NCBI database. Upstream and downstream homology arm UP and DN fragments were prepared using Phusion high-fidelity polymerase (New England BioLabs) and the genome of Corynebacterium glutamicum ATCC13869 as a template, using nadA-UP-1F / nadA-UP-1R and nadA-DN-2F / nadA-DN-2R as primer pairs. The primer sequences are shown in Table 1. Subsequently, nadA-UP-1F / nadA-DN-2R was used as a primer pair and the upstream and downstream homology arms were used as templates to prepare the recombinant fragment. The PCR procedure was the same as above. The resulting recombinant fragment was purified using an agarose gel recovery kit (Tiangen) and then digested with XbaI / HindIII. At the same time, pK18-mobsacB was digested with XbaI / HindIII and ligated with the vector using T4 DNA ligase (TransGen Biotech). Trans1T1 competent cells (TransGen Biotech) were transformed, and kanamycin-resistant clones were picked. XbaI / HindIII enzyme digestion was used to identify positive clones with fragments inserted into pK18mobsacB. The correct band size was 990 bp. The inserted fragment was further identified by sequencing (GENEWIZ). The primers submitted for testing were nadA-UP-1F / nadA-DN-2R. The resulting plasmid was named pK18-nadA D312N .

[0064] 10).pK18-ds G171D Plasmid construction

[0065] Primers were designed based on the sequence of the ds gene (GenBank NO: NCgl0950) in the NCBI database. The primer sequences are shown in Table 1. Using Phusion High-Fidelity Polymerase (New England BioLabs), the ds-UP-1F / ds-UP-1R and ds-DN-2F / ds-DN-2R primer pairs were used to generate upstream and downstream homology arms (UP and DN) fragments using the genome of Corynebacterium glutamicum ATCC13869 as a template. Subsequently, recombinant fragments were generated using the ds-UP-1F / ds-DN-2R primer pair and the upstream and downstream homology arms as templates, following the same PCR procedure as above. The obtained recombinant fragment was purified by agarose gel recovery kit (Tiangen), and then digested with XbaI / PstI. At the same time, pK18-mobsacB was digested with XbaI / PstI and the fragment was ligated to the vector with T4 DNA ligase (TransGenBiotech). Trans1T1 competent cells (TransGen Biotech) were transformed, and kanamycin-resistant clones were picked. XbaI / PstI enzyme digestion was used to identify the positive clones with the fragment inserted into pK18mobsacB. The correct band size was 1012bp. Sequencing (GENEWIZ) was further used to identify the correct insertion of the fragment. The primers sent for testing were ds-UP-1F / ds-DN-2R. The resulting plasmid was named pK18-ds G171D .

[0066] 1.1.2 Strain construction

[0067] 1).ATCC13869-Pgdh -10M Strain construction (WJ0138-1)

[0068] ATCC13869 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and pK18-Pgdh -10MThe recombinants were transformed into Corynebacterium glutamicum ATCC13869, and exchange recombinants were selected on selective medium containing 15 mg / L kanamycin. Kan clones were identified by colony PCR using Fast Taq DNA polymerase (TransGenBiotech) with primer pairs PE640 / P85 and P82 / PE643. The PCR program was: 94°C for 10 minutes, 94°C for 30 seconds, 50°C for 30 seconds, and extension at 72°C for 15 seconds / kb for 30 cycles, followed by a complete extension at 72°C for 10 minutes. The fragments amplified by the two primer pairs were similar in length, with clones around 1100 bp being considered positive. Selected positive clones were inoculated into antibiotic-free BHI medium and cultured for 12-14 hours. The bacterial suspension was diluted 100-1000-fold and plated onto solid BHI medium containing 10% sucrose and cultured for 36 hours. The selected strains were further verified for kanamycin resistance. KanS recombinants were selected and point mutation recombinants were verified using the identification primer pair PE642 / PE643. By exploring the annealing temperature, recombinants containing point mutations were obtained. The resulting positive recombinants were amplified and sequenced using PE640 / PE644. The correct recombinant was 2356 bp in length using the primers PE640 / PE644 / PE642 / PE643 / PE645. The strain with the correct sequence was named WJ0138-1.

[0069] 2).WJ0138-1-yggB A100T Strain construction (WJ0138-4)

[0070] WJ0138-1 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and pK18-yggB A100TChange among the Corynebacterium glutamicum WJ0138-1, on the selection medium that contains the kanamycin of 15mg / L, select to exchange recombinant.Utilize Fast Taq archaeal dna polymerase (TransGen Biotech), carry out bacterium colony PCR identification Kan clone with PE646 / P85, P82 / PE649 primer pair, PCR program is the same.Two pairs of primers are little different to the fragment length size that amplifies, and all are positive clones at the clone of about 1100bp.The positive clone that selects is inoculated into and does not have in the anti-BHI substratum and cultivated 12-14 hour, with the bacterium liquid dilution 100-1000 doubly coated on the solid BHI substratum that contains 10% sucrose and cultivated 36 hours, the bacterial strain that is screened out is further carried out that resistance phenotype verification of kanamycin, select the recombinant of KanS and utilize identification primer pair PE648 / PE649 to verify the point mutation recombinant, by groping the annealing temperature, obtain to contain the recombinant of point mutation. The positive recombinants were amplified and sequenced using PE650 / PE651. The correct recombinant was 2020 bp long. The primers used for the test were PE650 / PE651 / PE648 / PE649. The strain with the correct sequence was named WJ0138-4.

[0071] 3). Construction of WJ0138-4-odhAORF-5bpQS strain (WJ0138-7)

[0072] Prepare WJ0138-4 competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Charpter 23), pK18-odhAORF-5bpQS is changed among the Corynebacterium glutamicum WJ0138-4, on the selection medium that contains the kanamycin of 15mg / L, select to exchange recombinant.Utilize Fast Taq archaeal dna polymerase (TransGenBiotech), carry out bacterium colony PCR identification Kan clone with PE652 / P85, P82 / PE655 primer pair, PCR program is the same.Two pairs of primers are little different to the fragment length size that amplifies, and all are positive colony at the clone of about 1100bp. Selected positive clones were inoculated into antibiotic-free BHI medium and cultured for 12-14 hours. The bacterial suspension was diluted 100-1000-fold and plated onto solid BHI medium containing 10% sucrose and cultured for 36 hours. The selected strains were further verified for kanamycin resistance. KanS recombinants were selected and identified using the primer pair PE654 / PE655 to verify 5bp deletions and point mutations. The resulting positive recombinants were amplified and sequenced using PE025 / PE026. The correct recombinant was 3993bp long, using the primers PE025 / PE026 / PE021 / PE022 / PE023 / PE024 / PE655. The strain with the correct sequence was named WJ0138-7.

[0073] 4).WJ0138-7-gltA-10 zone M+gltA C361Y Strain construction (WJ0138-9)

[0074] WJ0138-7 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and pK18-gltA -10M-C361Y Change among the Corynebacterium glutamicum WJ0138-7, on the selection medium that contains the kanamycin of 15mg / L, select to exchange recombinant.Utilize Fast Taq archaeal dna polymerase (TransGenBiotech), carry out bacterium colony PCR identification Kan clone with PE656 / P85, P82 / PE661 primer pair, PCR program is the same.Two pairs of primers are significantly different to the fragment length size that amplifies, and short band is about 1100bp, and long band is a positive colony at the clone of about 2500bp.The positive colony that selects is inoculated into and does not have in the anti-BHI substratum and cultivated 12-14 hour, with the bacterium liquid dilution 100-1000 doubly coated on the solid BHI substratum that contains 10% sucrose and cultivated 36 hours, the bacterial strain that is screened out is further carried out that resistance phenotype verification of kanamycin, select the recombinant of KanS and utilize identification primer pair PE660 / PE661 to check the point mutation recombinant, by groping the annealing temperature, obtain to contain the recombinant of point mutation. The positive recombinants were amplified and sequenced using PE662 / PE663. The correct recombinant was 2439 bp long. The primers used for the test were PE662 / PE663 / PE362 / PE363. The strain with the correct sequence was named WJ0138-9.

[0075] 5).WJ0138-9-suc M199I Strain construction (WJ0139-2)

[0076] WJ0138-9 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and the plasmid pK18-suc M199IThe recombinant was transformed into Corynebacterium glutamicum WJ0138-9 competent cells using electroporation and selected for exchange on a selective culture medium containing 15 mg / L of kanamycin. Fast Taq DNA polymerase (TransGen Biotech) was used to identify the KanR clone using colony PCR with the 49-UP-1F / P85 and P82 / 49-DN-2R primer pairs. The PCR procedure was the same. The fragment lengths amplified by the two pairs of primer pairs showed little difference, with clones around 1100 bp being positive. The selected positive clones were inoculated into antibiotic-free BHI medium and cultured for 12-14 hours. The bacterial solution was diluted 100-1000 times and then spread on solid BHI medium containing 10% sucrose and cultured for 36 hours. The screened strains were further verified for kanamycin resistance phenotype. KanS recombinants were selected and point mutation recombinants were verified using the identification primer pair 49-F / 49-DN-2R. By exploring the annealing temperature, recombinants containing point mutations were obtained. The resulting positive recombinants were amplified and sequenced using ID-F / ID-R. The correct recombinant was 1124 bp in length. The primers submitted for testing were 49-ID-F / 49-ID-R. The strain with correct sequencing was named WJ0139-2.

[0077] 6).WJ0139-2-ach S372C Strain construction (WJ0139-3)

[0078] WJ0139-2 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and pK18-ach S372C The recombinants were transformed into Corynebacterium glutamicum WJ0139-2 and selected on a selective medium containing 15 mg / L kanamycin. KanR clones were identified by colony PCR using Fast Taq DNA polymerase (TransGen Biotech) with primer pairs PE664 / P85 and P82 / PE667. The PCR procedure was the same as above.

[0079] The fragment lengths amplified by the two primer pairs were similar, with clones around 1100 bp being considered positive. Selected positive clones were inoculated into antibiotic-free BHI medium and cultured for 12-14 hours. The bacterial suspension was diluted 100-1000-fold and plated onto solid BHI medium containing 10% sucrose and cultured for 36 hours. The selected strains were further verified for kanamycin resistance. KanS recombinants were selected and point mutation recombinants were verified using the identification primer pair PE666 / PE667. By exploring the annealing temperature, recombinants containing point mutations were obtained. The resulting positive recombinants were amplified and sequenced using PE667 / PE668. The correct recombinant was 1650 bp long, and the primers used for testing were PE667 / PE668 / PE664. The strain with the correct sequence was designated WJ0139-3.

[0080] 7).WJ0139-3-tktA T234I Strain construction (WJ0139-5)

[0081] WJ0139-3 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and pK18-tktA T234I The recombinants were transferred into Corynebacterium glutamicum WJ0139-3 and selected on a selective medium containing 15 mg / L kanamycin. KanR clones were identified by colony PCR using Fast Taq DNA polymerase (TransGen Biotech) with the tktA-UP-1F / P85 and P82 / tktA-DN-2R primer pairs. The PCR procedure was the same as above. The fragments amplified by the two primer pairs showed little difference in length; clones with both fragments around 1100 bp were considered positive. The selected positive clones were inoculated into antibiotic-free BHI medium and cultured for 12-14 hours. The bacterial solution was diluted 100-1000 times and then spread on solid BHI medium containing 10% sucrose and cultured for 36 hours. The screened strains were further verified for kanamycin resistance phenotype. KanS recombinants were selected and the point mutation recombinants were verified using the identification primer pair tktA-F / tktA-DN-2R. By exploring the annealing temperature, recombinants containing point mutations were obtained. The resulting positive recombinants were amplified and sequenced using tktA-ID-F / tktA-ID-R. The correct recombinant was 1223 bp in length. The primers submitted for testing were tktA-ID-F / tktA-ID-R. The strain with correct sequencing was named WJ0139-5.

[0082] 8).WJ0139-5-putA A755V Strain construction (WJ0139-8)

[0083] Prepare WJ0139-5 competent cells according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and insert pK18-putA A755V The recombinants were transformed into Corynebacterium glutamicum WJ0139-5, and exchange recombinants were selected on a selective medium containing 15 mg / L kanamycin. KanR clones were identified by colony PCR using Fast Taq DNA polymerase (TransGen Biotech) with the putA-UP-1F / P85 and P82 / putA-DN-2R primer pairs, following the same PCR procedure as above. The fragments amplified by the two primer pairs showed similar lengths, with clones around 1100 bp being considered positive. The selected positive clones were inoculated into antibiotic-free BHI medium and cultured for 12-14 hours. The bacterial solution was diluted 100-1000-fold and spread on solid BHI medium containing 10% sucrose and cultured for 36 hours. The screened strains were further verified for kanamycin resistance phenotype. KanS recombinants were selected and point mutation recombinants were verified using the identification primer pair putA-F / putA-DN-2R. By exploring the annealing temperature, recombinants containing point mutations were obtained. The resulting positive recombinants were amplified and sequenced using ID-F / ID-R. The correct recombinant was 1214 bp in length. The primers used for testing were putA-ID-F / putA-ID-R. The strain with the correct sequence was named WJ0139-8.

[0084] 9).WJ0139-8-ds G171D Strain construction (WJ0139-9)

[0085] WJ0139-8 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and pK18-ds G171D The strains were transferred into Corynebacterium glutamicum WJ0139-8 and exchange recombinants were selected on a selective medium containing 15 mg / L of kanamycin. The KanR clones were identified by colony PCR using Fast Taq DNA polymerase (TransGen Biotech) with ds-UP-1F / P85 and P82 / ds-DN-2R primers. The PCR procedure was the same as above. The fragment lengths amplified by the two primer pairs were not much different, and clones around 1100 bp were considered positive clones. The selected positive clones were inoculated into non-resistant BHI medium and cultured for 12-14 hours. The bacterial solution was diluted 100-1000 times and then spread on a solid BHI medium containing 10% sucrose and cultured for 36 hours. The strains screened were further verified for kanamycin resistance phenotype, and KanR clones were selected. SThe recombinant was verified by using the identification primer pair ds-F / ds-DN-2R to verify the point mutation recombinant. By exploring the annealing temperature, the recombinant containing the point mutation was obtained. The obtained positive recombinant was amplified and sequenced using ds-ID-F / ds-ID-R. The correct recombinant was 1172bp long. The primers submitted for testing were ds-ID-F / ds-ID-R. The strain with correct sequencing was named WJ0139-9.

[0086] 10).WJ0139-9-nadA D312N Strain construction (WJ0140)

[0087] WJ0139-9 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and pK18-nadA D312N The recombinants were transformed into Corynebacterium glutamicum WJ0139-9 and selected on a selective medium containing 15 mg / L kanamycin. KanR clones were identified by colony PCR using Fast Taq DNA polymerase (TransGen Biotech) with the nadA-UP-1F / P85 and P82 / nadA-DN-2R primer pairs, using the same PCR procedure as above. The fragments amplified by the two primer pairs showed little difference in length; clones with both fragments around 1100 bp were considered positive. The selected positive clones were inoculated into antibiotic-free BHI medium and cultured for 12-14 hours. The bacterial solution was diluted 100-1000 times and then spread on solid BHI medium containing 10% sucrose and cultured for 36 hours. The screened strains were further verified for kanamycin resistance phenotype. KanS recombinants were selected and the point mutation recombinants were verified using the identification primer pair nadA-F / nadA-DN-2R. By exploring the annealing temperature, recombinants containing point mutations were obtained. The resulting positive recombinants were amplified and sequenced using nadA-ID-F / nadA-ID-R. The correct recombinant was 1116 bp in length. The primers submitted for testing were nadA-ID-F / nadA-ID-R. The strain with correct sequencing was named WJ0140.

[0088] 1.1.3 Shake flask verification

[0089] The recombinant Corynebacterium glutamicum constructed was fermented to verify its glutamic acid production performance, specifically as follows: the bacterial strain frozen in a -80 ℃ glycerol tube was inoculated in the above-mentioned slant medium and activated, grown a bacterial lawn after culturing at 31.5 ℃ for 24 hours, picked a bacterial lawn from the freshly activated slant, inoculated in the above-mentioned seed culture medium, and cultured with shaking at 31.5 ℃, 220 rpm to the middle and late stages of logarithmic growth for 12 hours to obtain a seed solution, which was inoculated into a 500 mL shake flask equipped with 20 mL of fermentation medium with an inoculum size of 10%, and cultured with shaking at 31.5 ℃, 220 rpm for 16 hours. After glucose was completely consumed, the concentration of L-glutamic acid accumulated in the culture medium was determined by HPLC.

[0090] The culture medium formula is as follows:

[0091] 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°C, 0.1 MPa for 30 min;

[0092] 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 cake hydrolyzate 22 mL / L, pH 7.0-7.2, sterilized at 121°C, 0.1 MPa for 15 min;

[0093] Fermentation medium: 60 g / L glucose, 15 g / L ammonium sulfate, 1.0 g / L KH2PO4, 0.4 g / L MgSO4·7H2O, 1.0 mg / L FeSO4·7H2O, 1.0 mg / L MnSO4·5H2O, 200 μg / L VB1, 300 μg / L biotin, 0.48 g / L soybean hydrolyzate. Adjust pH to 7.2-7.5 with NaOH, sterilize at 121°C, 0.1 MPa, for 15 minutes, and then add 0.8 g heat-sterilized calcium carbonate. The fermentation results are shown in the table below.

[0094] Table 2. Glutamate content detection of recombinant strains

[0095] Strain number genotype Fermentation OD 562nm Glutamic acid g / L Conversion Rate % 13869 wild type 60.2 1.62 2.7 WJ0138-1 <![CDATA[13869-Pgdh -10M ]]> 57.8 4.14 6.9 WJ0138-4 <![CDATA[WJ0138-1-yggB A100T ]]> 55.3 10.98 18.3 WJ0138-7 WJ0138-4-odhA-5bpQS 49.6 15.78 26.3 WJ0138-9 <![CDATA[WJ0138-7-gltA -10M-C361Y ]]> 52.1 19.8 33 WJ0139-2 <![CDATA[WJ0138-9-suc M199I ]]> 53.0 20.46 34.1 WJ0139-3 <![CDATA[WJ0139-2-ach S372C ]]> 52.8 22.02 36.7 WJ0139-5 <![CDATA[WJ0139-3-tktA T234I ]]> 54.5 22.8 38 WJ0139-8 <![CDATA[WJ0139-5-putA A755V ]]> 51.2 25.26 42.1 WJ0139-9 <![CDATA[WJ0139-8-ds G171D ]]> 51.6 27.84 46.4 WJ0140 <![CDATA[WJ0139-9-nadA D312N ]]> 49.3 29.1 48.5

[0096] These shake flask results demonstrate that superimposing 10 genes on ATCC 13869 significantly improves glutamate production and conversion efficiency. Specifically, the conversion rate of WJ0140 increased from 2.7% to 48.5%, without affecting growth or sugar consumption.

[0097] 1.2 Construction of ATCC13032→MH13032-10 starting strain

[0098] 1.2.1 Plasmid construction

[0099] 1).pK18-Pgdh -10M (13032)Plasmid construction

[0100] PCR amplification was performed using the genome of Corynebacterium glutamicum ATCC13032 as a template using Phusion high-fidelity polymerase (New England BioLabs). PCR amplification was performed using PE640 / PE641 and PE642 / PE643 as primer pairs to obtain the upstream and downstream homology arms UP and DN fragments of Pgdh-10M. The primer sequences are shown in Table 1. Subsequently, PE640 / PE643 was used as a primer pair and the upstream and downstream homology arms were used as templates to prepare recombinant fragments. The PCR program was: 94°C for 10 minutes, 94°C for 30 seconds, 50°C for 30 seconds, 72°C for 15 seconds / kb extension, for a total of 30 cycles, and 72°C for a complete extension of 10 minutes. The resulting recombinant fragment was purified using an agarose gel recovery kit (Tian Gen). At the same time, pK18mobsacB was digested with XbaI / EcoRI restriction endonucleases, and the recombinant fragment was ligated to the vector using T4 DNA ligase (TransGen Biotech). Trans1T1 competent cells (TransGen Biotech) were then transformed, and kanamycin-resistant clones were selected. XbaI / EcoRI digestion was performed to identify positive clones with the fragment inserted into pK18mobsacB. The correct band size was 1061 bp. Sequencing (GENEWIZ) was further performed to confirm the correct insertion of the fragment. The primers used for the test were PE640 / PE643. The resulting plasmid was named pK18-Pgdh -10M (13032).

[0101] 2).pK18-yggB A100T (13032)Plasmid construction

[0102] PCR amplification was performed using the genome of Corynebacterium glutamicum ATCC13032 as a template using Phusion high-fidelity polymerase (New England BioLabs). PCR amplification using primer pairs PE646 / PE647 and PE648 / PE649 was performed to obtain yggB. A100T The primer sequences for the upstream and downstream homology arms UP and DN fragments are shown in Table 1. The PCR procedure was the same as above. The resulting fragments were purified and digested using an agarose gel recovery kit (Tiangen) and stored at -20°C until use.

[0103] Subsequently, PE646 / PE649 was used as a primer pair and the upstream and downstream homology arms were used as templates to prepare a recombinant fragment. The PCR procedure was the same as above, and the resulting recombinant fragment was purified using an agarose gel recovery kit (Tian Gen). At the same time, pK18mobsacB was digested with XbaI / EcoRI restriction endonucleases, and the fragment was ligated to the vector using T4 DNA ligase (TransGen Biotech). Trans1T1 competent cells (TransGen Biotech) were transformed, and kanamycin-resistant clones were picked. XbaI / EcoRI enzyme digestion was used to identify positive clones in which the fragment was inserted into pK18mobsacB. The correct band size was 1036 bp. Sequencing (GENEWIZ) was further used to identify that the inserted fragment was correct, and the primers submitted for testing were PE646 / PE649. The resulting plasmid was named pK18-yggB A100T (13032).

[0104] 3). Construction of pK18-odhA ORF-5bpQS(13032) plasmid

[0105] PCR amplification was performed using the genome of Corynebacterium glutamicum ATCC13032 as a template using Phusion High-Fidelity Polymerase (New England BioLabs). PCR amplification of the upstream and downstream homology arms (UP and DN) of the odhAORF-5bpQS fragment was performed using the primer pairs PE652 / PE653 and PE654 / PE655. The primer sequences are shown in Table 1. Subsequently, recombinant fragments were prepared using the PE652 / PE655 primer pair and the upstream and downstream homology arms as templates. The PCR procedure was the same as above, and the resulting recombinant fragment was purified using an agarose gel recovery kit (Tiangen). pK18mobsacB was digested with XbaI / EcoRI restriction enzymes, and the fragment was ligated to the vector using T4 DNA ligase (TransGen Biotech). Trans1T1 competent cells (TransGen Biotech) were then transformed, and kanamycin-resistant clones were selected. Positive clones showing insertion of the fragment into pK18mobsacB were identified by XbaI / EcoRI digestion. The correct band size was 1039 bp. Sequencing (GENEWIZ) confirmed the correct insertion of the fragment using primers PE652 / PE655. The resulting plasmid was named pK18-odhA ORF-5bpQS(13032).

[0106] 4).pK18-gltA -10M、C361Y (13032)Plasmid construction

[0107] The genome of Corynebacterium glutamicum ATCC13032 was used as a template and PCR amplification was performed using Phusion high-fidelity polymerase (New England BioLabs). The gltA-10 region M+gltA was obtained by PCR amplification using primer pairs PE656 / PE657 and PE658 / PE661. C361Y The upstream and downstream homology arms UP and fragments, the primer sequences are shown in Table 1. Subsequently, PE656 / PE661 was used as a primer pair, and the upstream homology arms and fragments were used as templates to prepare the recombinant fragment. The PCR procedure was the same as above, and the resulting recombinant fragment was purified by an agarose gel recovery kit (Tian Gen). At the same time, pK18mobsacB was digested with XbaI / EcoRI restriction endonucleases, and the fragment was connected to the vector with T4 DNA ligase (TransGen Biotech), and Trans1T1 competent cells (TransGen Biotech) were transformed. Kanamycin-resistant clones were picked, and XbaI / EcoRI enzyme digestion was used to identify the positive clones in which the fragments were inserted into pK18mobsacB. The correct band size was 2241bp. The inserted fragment was further identified by sequencing (GENEWIZ Company), and the primers sent for testing were PE656 / PE661 / PE362 / PE363. The resulting plasmid was named pK18-gltA -10M-C361Y (13032).

[0108] 5).pK18-ach S372C (13032)Plasmid construction

[0109] The genome of Corynebacterium glutamicum ATCC13032 was used as a template and PCR amplification was performed using Phusion high-fidelity polymerase (New England BioLabs). PCR amplification was performed using primer pairs PE664 / P14-3 and P14-4 / PE667 to obtain the ach S372CThe upstream and downstream homology arm UP and DN fragments, the primer sequences are shown in Table 1. Subsequently, PE664 / PE667 was used as a primer pair, and the upstream and downstream homology arm fragments were used as templates to prepare the recombinant fragment. The PCR procedure was the same as above, and the resulting recombinant fragment was purified by an agarose gel recovery kit (Tian Gen). At the same time, pK18-mobsacB was digested with XbaI / HindIII, and the fragment was connected to the vector with T4 DNA ligase (TransGen Biotech), and the Trans1T1 competent cells (TransGen Biotech) were transformed. The kanamycin-resistant clones were picked, and the XbaI / HindIII enzyme digestion was used to identify the positive clones in which the fragments were inserted into pK18mobsacB. The correct band size was 1035bp. The inserted fragment was further identified by sequencing (GENEWIZ Company), and the primers sent for testing were PE664 / PE667. The resulting plasmid was named pK18-ach S372C (13032).

[0110] 6).pK18-suc M199I (13032)Plasmid construction

[0111] Phusion high-fidelity polymerase (New England BioLabs) was used, 49-UP-1F / P13-2 and P13-3 / 49-DN-2R were used as primer pairs, and the genome of Corynebacterium glutamicum ATCC13032 was used as a template to prepare the upstream and downstream homology arm UP and DN fragments. The primer sequences are shown in Table 1. Subsequently, 49-UP-1F / 49-DN-2R was used as a primer pair and the upstream and downstream homology arms were used as templates to prepare the recombinant fragment. The PCR procedure was the same as above. The obtained recombinant fragment was purified by agarose gel recovery kit (Tiangen). At the same time, pK18-mobsacB was digested with BamHI / XbaI and the fragment was ligated to the vector using T4 DNA ligase (TransGen Biotech). Trans1T1 competent cells (TransGen Biotech) were transformed, and kanamycin-resistant clones were picked. BamHI / XbaI enzyme digestion was used to identify the positive clones with the fragment inserted into pK18mobsacB. The correct band size was 1002bp. The inserted fragment was further identified by sequencing (GENEWIZ Company). The primers sent for testing were 49-UP-1F / 49-DN-2R. The resulting plasmid was named pK18-suc M199I (13032).

[0112] 7).pK18-tktA T234I (13032)Plasmid construction

[0113] Primers were designed using the genome of Corynebacterium glutamicum ATCC13032 as a template, and the primer sequences are shown in Table 1. Utilizing Phusion ultra-fidelity polymerase (New England BioLabs), tktA-UP-1F / tktA-UP-1R, tktA-DN-2F / tktA-DN-2R were used as primer pairs, and the genome of Corynebacterium glutamicum ATCC13032 was used as a template to prepare upstream and downstream homology arms UP and DN fragments. Subsequently, tktA-UP-1F / tktA-DN-2R was used as a primer pair, and upstream and downstream homology arms were used as templates to prepare recombinant fragments, and the PCR procedure was the same as above. The resulting recombinant fragment was purified using an agarose gel recovery kit (Tiangen) and then digested with XbaI / HindIII. pK18-mobsacB was also digested with XbaI / HindIII and ligated to the vector using T4 DNA ligase (TransGen Biotech). Trans1T1 competent cells (TransGen Biotech) were then transformed, and kanamycin-resistant clones were selected. XbaI / HindIII digestion was performed to identify positive clones with the fragment inserted into pK18mobsacB. The correct band size was 1028 bp. Sequencing (GENEWIZ) confirmed the correct insertion of the fragment. The primers used for the assay were tktA-UP-1F / tktA-DN-2R. The resulting plasmid was named pK18-tktA. T234I (13032).

[0114] 8).pK18-putA A755V (13032)Plasmid construction

[0115] Phusion high-fidelity polymerase (New England BioLabs) was used with the putA-UP-1F / putA-UP-1R and putA-DN-2F / putA-DN-2R primer pairs, using the genome of Corynebacterium glutamicum ATCC13032 as a template to prepare upstream and downstream homology arm UP and DN fragments. The primer sequences are shown in Table 1. Subsequently, recombinant fragments were prepared using the putA-UP-1F / putA-DN-2R primer pair and the upstream and downstream homology arms as templates, following the same PCR procedure as above. The resulting recombinant fragment was purified using an agarose gel recovery kit (Tiangen) and then digested with XbaI / HindIII. pK18-mobsacB was also digested with XbaI / HindIII and ligated to the vector using T4 DNA ligase (TransGen Biotech). Trans1T1 competent cells (TransGen Biotech) were then transformed, and kanamycin-resistant clones were selected. XbaI / HindIII digestion was performed to identify positive clones with the fragment inserted into pK18mobsacB. The correct band size was 998 bp. Sequencing (GENEWIZ) confirmed that the inserted fragment was correct. The primers used for the assay were putA-UP-1F / putA-DN-2R. The resulting plasmid was named pK18-putA. A755V (13032).

[0116] 9).pK18-nadA D312N (13032)Plasmid construction

[0117] Primers were designed using the genome of Corynebacterium glutamicum ATCC13032 as a template. Using Phusion ultra-fidelity polymerase (New England BioLabs), P13-7 / nadA-UP-1R and P13-8 / nadA-DN-2R were used as primer pairs, and the genome of Corynebacterium glutamicum ATCC13032 was used as a template to prepare upstream and downstream homology arm UP and DN fragments. The primer sequences are shown in Table 1. Subsequently, P13-7 / nadA-DN-2R was used as a primer pair and the upstream and downstream homology arms were used as templates to prepare the recombinant fragment. The PCR procedure was the same as above. The resulting recombinant fragment was purified using an agarose gel recovery kit (Tiangen) and then digested with XbaI / HindIII. At the same time, pK18-mobsacB was digested with XbaI / HindIII and the fragment was ligated to the vector using T4 DNA ligase (TransGen Biotech). Trans1T1 competent cells (TransGen Biotech) were transformed, and kanamycin-resistant clones were picked. XbaI / HindIII enzyme digestion was used to identify positive clones with the fragment inserted into pK18mobsacB. The correct band size was 990 bp. Sequencing (GENEWIZ) was further used to identify the correct insertion of the fragment. The primers submitted for testing were P13-7 / nadA-DN-2R. The resulting plasmid was named pK18-nadA D312N (13032).

[0118] 10).pK18-ds G171D (13032)Plasmid construction

[0119] Primers were designed using the genome of Corynebacterium glutamicum ATCC13032 as a template, and the primer sequences are shown in Table 1. Using Phusion ultra-fidelity polymerase (New England BioLabs), ds-UP-1F / ds-UP-1R and ds-DN-2F / ds-DN-2R were used as primer pairs, and the genome of Corynebacterium glutamicum ATCC13032 was used as a template to prepare upstream and downstream homology arms UP and DN fragments. Subsequently, ds-UP-1F / ds-DN-2R was used as a primer pair, and the upstream and downstream homology arms were used as templates to prepare recombinant fragments. The PCR procedure was the same as above. The resulting recombinant fragment was purified using an agarose gel recovery kit (Tiangen), then digested with XbaI / PstI. pK18-mobsacB was also digested with XbaI / PstI, and the fragment was ligated to the vector using T4 DNA ligase (TransGen Biotech). Trans1T1 competent cells (TransGen Biotech) were then transformed, and kanamycin-resistant clones were selected. XbaI / PstI digestion was performed to identify positive clones with fragments inserted into pK18mobsacB. The correct band size was 1012 bp. Sequencing (GENEWIZ) was further used to identify the correct insertion of the fragment. The primers used for the assay were ds-UP-1F / ds-DN-2R. The resulting plasmid was named pK18-ds G171D (13032).

[0120] 1.2.2 Construction of ATCC 13032 starting strain

[0121] 1).ATCC13032-Pgdh -10M Strain construction (MH13032-1)

[0122] ATCC13032 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and pK18-Pgdh -10M(13032) was transformed into Corynebacterium glutamicum ATCC13032, and exchange recombinants were selected on selective medium containing 15 mg / L kanamycin. Kan clones were identified by colony PCR using Fast Taq DNA polymerase (TransGenBiotech) with primer pairs PE640 / P85 and P82 / PE643. The PCR program was: 94°C for 10 minutes, 94°C for 30 seconds, 50°C for 30 seconds, and extension at 72°C for 15 seconds / kb for 30 cycles, followed by a complete extension at 72°C for 10 minutes. The lengths of the fragments amplified by the two primer pairs were similar, with clones around 1100 bp being considered positive. Selected positive clones were inoculated into antibiotic-free BHI medium and cultured for 12-14 hours. The bacterial suspension was diluted 100-1000-fold and plated onto solid BHI medium containing 10% sucrose and cultured for 36 hours. The selected strains were further verified for kanamycin resistance. KanS recombinants were selected and point mutation recombinants were verified using the identification primer pair PE642 / PE643. By exploring the annealing temperature, recombinants containing point mutations were obtained. The resulting positive recombinants were amplified and sequenced using PE640 / PE644. The correct recombinant was 2356 bp in length using the primers PE640 / PE644 / P14-2 / PE643 / PE645. The strain with the correct sequence was designated MH13032-1.

[0123] 2).MH13032-1-yggB A100T Strain construction (MH13032-2)

[0124] Prepare MH13032-1 competent cells according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and insert pK18-yggB A100T(13032) was transferred into Corynebacterium glutamicum MH13032-1 and exchange recombinants were selected on a selective medium containing 15 mg / L of kanamycin. Using Fast Taq DNA polymerase (TransGenBiotech), colony PCR was performed with primer pairs PE646 / P85 and P82 / PE649 to identify Kan clones. The PCR procedure was the same as above. The fragment lengths amplified by the two primer pairs were not significantly different, with clones around 1100 bp being considered positive clones. The selected positive clones were inoculated into non-resistance BHI medium and cultured for 12-14 hours. The bacterial solution was diluted 100-1000 times and then plated onto solid BHI medium containing 10% sucrose and cultured for 36 hours. The selected strains were further subjected to kanamycin resistance phenotype verification. KanS recombinants were selected and point mutation recombinants were verified using the identification primer pair PE648 / PE649. By exploring the annealing temperature, recombinants containing point mutations were obtained. The positive recombinants were amplified and sequenced using PE650 / PE651. The correct recombinant was 2020 bp long. The primers used for the test were PE650 / PE651 / PE648 / PE649. The strain with the correct sequence was named MH13032-2.

[0125] 3). Construction of MH13032-2-odhAORF-5bpQS strain (MH13032-3)

[0126] Prepare MH13032-2 competent cells according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Charpter 23), pK18-odhAORF-5bpQS (13032) is changed among the Corynebacterium glutamicum MH13032-2, on the selection medium that contains the kanamycin of 15mg / L, select to exchange recombinant.Utilize Fast Taq archaeal dna polymerase (TransGen Biotech), carry out bacterium colony PCR identification Kan clone with PE652 / P85, P82 / PE655 primer pair, PCR program is the same.Two pairs of primers are little different to the fragment length size that amplifies, and all are positive colonies at the clone of about 1100bp. The selected positive clones were inoculated into antibiotic-free BHI medium and cultured for 12-14 hours. The bacterial solution was diluted 100-1000 times and then spread on solid BHI medium containing 10% sucrose and cultured for 36 hours. The selected strains were further verified for kanamycin resistance phenotype, and KanS recombinants were selected and identified using the PE654 / PE655 primer pair to verify 5bp deletion and point mutation recombinants.

[0127] The positive recombinants were amplified and sequenced using P13-1 / PE026. The correct recombinant was 3993 bp long. The primers used for the test were P13-1 / PE026 / PE020 / PE021 / PE022 / PE023 / PE024 / PE655. The strain with the correct sequence was named MH13032-3.

[0128] 4).MH13032-3-gltA -10M +gltA C361Y Strain construction (MH13032-4)

[0129] MH13032-3 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and pK18-gltA -10M-C361Y (13032) was transformed into Corynebacterium glutamicum MH13032-3, and exchange recombinants were selected on selective medium containing 15 mg / L kanamycin. Kan clones were identified by colony PCR using Fast Taq DNA polymerase (TransGen Biotech) with primer pairs PE656 / P85 and P82 / PE661, following the same PCR procedure as above. The fragments amplified by the two primer pairs showed significant differences in length: clones with a short band of approximately 1100 bp and a long band of approximately 2500 bp were considered positive clones. Selected positive clones were inoculated into antibiotic-free BHI medium and cultured for 12-14 hours. The bacterial suspension was diluted 100-1000-fold and plated onto solid BHI medium containing 10% sucrose and cultured for 36 hours. The selected strains were further verified for kanamycin resistance. KanS recombinants were selected and point mutation recombinants were verified using the identification primer pair PE660 / PE661. By exploring the annealing temperature, recombinants containing point mutations were obtained. The resulting positive recombinants were amplified and sequenced using PE662 / PE663. The correct recombinant was 2439 bp in length, using the primers PE662 / PE663 / PE362 / PE363. The strain with the correct sequence was designated MH13032-4.

[0130] 5).MH13032-4-suc M199I Strain construction (MH13032-5)

[0131] MH13032-4 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and the plasmid pK18-suc M199I(13032) was transformed into competent cells of Corynebacterium glutamicum MH13032-4 by electroporation, and exchange recombinants were selected on selective medium containing 15 mg / L kanamycin. KanR clones were identified by colony PCR using Fast Taq DNA polymerase (TransGen Biotech) with the primer pairs 49-UP-1F / P85 and P82 / 49-DN-2R, following the same PCR procedure as above. The fragments amplified by the two primer pairs showed similar lengths, with clones around 1100 bp being considered positive clones. The selected positive clones were inoculated into antibiotic-free BHI medium and cultured for 12-14 hours. The bacterial solution was diluted 100-1000 times and then spread on solid BHI medium containing 10% sucrose and cultured for 36 hours. The screened strains were further verified for kanamycin resistance phenotype. KanS recombinants were selected and point mutation recombinants were verified using the identification primer pair P13-4 / 49-DN-2R. By exploring the annealing temperature, recombinants containing point mutations were obtained. The resulting positive recombinants were amplified and sequenced using 49-ID-F / IP13-5. The correct recombinant was 1124 bp in length. The primers submitted for testing were 49-ID-F / P13-5. The strain with correct sequencing was named MH13032-5.

[0132] 6).MH13032-5-ach S372C Strain construction (MH13032-6)

[0133] MH13032-5 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and pK18-ach S372CThe recombinant strain (13032) was transformed into Corynebacterium glutamicum MH13032-5, and exchange recombinants were selected on a selective medium containing 15 mg / L kanamycin. KanR clones were identified by colony PCR using Fast Taq DNA polymerase (TransGenBiotech) with primer pairs PE664 / P85 and P82 / PE667, following the same PCR procedure as above. The lengths of the fragments amplified by the two primer pairs were similar, with clones around 1100 bp being considered positive clones. The selected positive clones were inoculated into antibiotic-free BHI medium and cultured for 12-14 hours. The bacterial solution was diluted 100-1000 times and then spread on solid BHI medium containing 10% sucrose and cultured for 36 hours. The screened strains were further verified for kanamycin resistance phenotype. KanS recombinants were selected and the point mutation recombinants were verified using the identification primer pair PE666 / PE667. By exploring the annealing temperature, recombinants containing point mutations were obtained. The obtained positive recombinants were amplified and sequenced using PE667 / PE668. The correct recombinant was 1650bp long. The primers submitted for testing were PE667 / PE668 / PE664. The strain with correct sequencing was named MH13032-6.

[0134] 7).MH13032-6-tktA T234I Strain construction (MH13032-7)

[0135] MH13032-6 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and pK18-tktA T234IThe recombinant strain (13032) was transformed into Corynebacterium glutamicum MH13032-6, and exchange recombinants were selected on selective medium containing 15 mg / L kanamycin. KanR clones were identified by colony PCR using Fast Taq DNA polymerase (TransGenBiotech) with the primer pairs tktA-UP-1F / P85 and P82 / tktA-DN-2R. The PCR procedure was the same as above. The lengths of the fragments amplified by the two primer pairs were similar, with clones around 1100 bp being considered positive clones. The selected positive clones were inoculated into antibiotic-free BHI medium and cultured for 12-14 hours. The bacterial solution was diluted 100-1000 times and then spread on solid BHI medium containing 10% sucrose and cultured for 36 hours. The screened strains were further verified for kanamycin resistance phenotype. KanS recombinants were selected and the point mutation recombinants were verified using the identification primer pair tktA-F / tktA-DN-2R. By exploring the annealing temperature, recombinants containing point mutations were obtained. The resulting positive recombinants were amplified and sequenced using tktA-ID-F / tktA-ID-R. The correct recombinant was 1223 bp in length. The primers submitted for testing were tktA-ID-F / tktA-ID-R. The strain with correct sequencing was named MH13032-7.

[0136] 8).MH13032-7-putA-cg0129 A755V Strain construction (MH13032-8)

[0137] Prepare MH13032-7 competent cells according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and insert pK18-putA A755V(13032) was transformed into Corynebacterium glutamicum MH13032-7, and exchange recombinants were selected on selective medium containing 15 mg / L kanamycin. KanR clones were identified by colony PCR using Fast Taq DNA polymerase (TransGenBiotech) with the putA-UP-1F / P85 and P82 / putA-DN-2R primer pairs, following the same PCR procedure as above. The lengths of the fragments amplified by the two primer pairs were similar, with clones around 1100 bp being considered positive clones. The selected positive clones were inoculated into antibiotic-free BHI medium and cultured for 12-14 hours. The bacterial solution was diluted 100-1000 times and spread on solid BHI medium containing 10% sucrose and cultured for 36 hours. The screened strains were further verified for kanamycin resistance phenotype. KanS recombinants were selected and point mutation recombinants were verified using the identification primer pair putA-F / putA-DN-2R. By exploring the annealing temperature, recombinants containing point mutations were obtained. The resulting positive recombinants were amplified and sequenced using P13-6 / putA-ID-R. The correct recombinant was 1214 bp in length. The primers submitted for testing were P13-6 / putA-ID-R. The strain with correct sequencing was named MH13032-8.

[0138] 9).MH13032-8-ds G171D Strain construction (MH13032-9)

[0139] MH13032-8 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and pK18-ds G171D (13032) was transferred into Corynebacterium glutamicum MH13032-8, and the exchange recombinants were selected on a selection medium containing 15 mg / L of kanamycin. Using Fast Taq DNA polymerase (TransGenBiotech), colony PCR was performed with ds-UP-1F / P85 and P82 / ds-DN-2R primer pairs to identify KanR clones. The PCR procedure was the same as above. The lengths of the fragments amplified by the two primer pairs were not much different, and clones around 1100 bp were considered positive clones. The selected positive clones were inoculated into non-antibiotic BHI medium and cultured for 12-14 hours. The bacterial solution was diluted 100-1000 times and then spread on solid BHI medium containing 10% sucrose and cultured for 36 hours. The selected strains were further verified for kanamycin resistance phenotype, and Kan was selected. SThe recombinant was verified by identifying the point mutation recombinant using the primer pair ds-F / ds-DN-2R. By exploring the annealing temperature, the recombinant containing the point mutation was obtained. The positive recombinant was amplified and sequenced using ds-ID-F / ds-ID-R. The correct recombinant was 1172bp long. The primers submitted for testing were ds-ID-F / ds-ID-R. The strain with correct sequencing was named MH13032-9.

[0140] 10).MH13032-9-nadA D312N Strain construction (MH13032-10)

[0141] MH13032-9 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and pK18-nadA D312N (13032) was transformed into Corynebacterium glutamicum MH13032-9, and exchange recombinants were selected on selective medium containing 15 mg / L kanamycin. KanR clones were identified by colony PCR using Fast Taq DNA polymerase (TransGenBiotech) with the primer pairs P13-7 / P85 and P82 / nadA-DN-2R, following the same PCR procedure as above. The lengths of the fragments amplified by the two primer pairs were similar, with clones around 1100 bp being considered positive clones. The selected positive clones were inoculated into antibiotic-free BHI medium and cultured for 12-14 hours. The bacterial solution was diluted 100-1000 times and then spread on solid BHI medium containing 10% sucrose and cultured for 36 hours. The screened strains were further verified for kanamycin resistance phenotype. KanS recombinants were selected and the point mutation recombinants were verified using the identification primer pair nadA-F / nadA-DN-2R. By exploring the annealing temperature, recombinants containing point mutations were obtained. The resulting positive recombinants were amplified and sequenced using nadA-ID-F / P13-9. The correct recombinant was 1116 bp in length. The primers submitted for testing were nadA-ID-F / P13-9. The strain with correct sequencing was named MH13032-10.

[0142] 1.2.3 ATCC13032 starting strain shake flask verification

[0143] The recombinant Corynebacterium glutamicum constructed above was fermented to verify its glutamic acid production performance. The shake flask method was referred to 1.1.3. The specific fermentation results are shown in the following table.

[0144] Table 3. Glutamate content detection of recombinant strains starting from ATCC13032

[0145] Strain number genotype Fermentation OD 562nm Glutamic acid g / L Conversion Rate % ATCC13032 wild type 66.6 0.24 0.4 MH13032-1 <![CDATA[MH13032-Pgdh -10M ]]> 65.1 6.06 10.1 MH13032-2 <![CDATA[MH13032-1-yggB A100T ]]> 62.7 9.72 16.2 MH13032-3 MH13032-2-odhAORF-5bpQS 58.4 12.06 20.1 MH13032-4 MH13032-3-gltA-10M-C361Y 54.6 15.12 25.2 MH13032-5 MH13032-4-sucM199I 55.5 16.68 27.8 MH13032-6 MH13032-5-achS372C 54.2 17.28 28.8 MH13032-7 <![CDATA[MH13032-6-tktA T234I ]]> 53.6 17.58 29.3 MH13032-8 <![CDATA[MH13032-7-putA A755V ]]> 53.2 18.12 30.2 MH13032-9 <![CDATA[MH13032-8-ds G171D ]]> 52.1 19.56 32.6 MH13032-10 <![CDATA[MH13032-9-nadA D312N ]]> 51.4 20.1 33.5

[0146] These shake flask results demonstrate that superimposing the 10 genes onto the wild-type strain 13032 significantly impacted glutamate production, continuously improving glutamate conversion efficiency. Specifically, the glutamate conversion efficiency of MH13032-10 increased from 0.4% of the wild-type strain to 33.5%, while maintaining growth and sugar consumption.

[0147] 1.3 Construction of ATCC14067→MH14067-10 starting strain

[0148] 1.3.1 Plasmid construction

[0149] 1).pK18-Pgdh -10M (14067)Plasmid construction

[0150] PCR amplification was performed using the genome of Corynebacterium glutamicum ATCC14067 as a template using Phusion high-fidelity polymerase (New England BioLabs). PCR amplification was performed using primer pairs PE640 / P14-1 and P14-2 / PE643 to obtain Pgdh- 10M The primer sequences for the upstream and downstream homology arms UP and DN fragments are shown in Table 1. Subsequently, recombinant fragments were prepared using the PE640 / PE643 primer pair and the upstream and downstream homology arms as templates. The PCR program was as follows: 94°C for 10 minutes, 94°C for 30 seconds, 50°C for 30 seconds, and 72°C for 15 seconds / kb extension for 30 cycles, followed by a complete extension at 72°C for 10 minutes. The resulting recombinant fragment was purified using an agarose gel extraction kit (Tiangen). Simultaneously, pK18mobsacB was digested with XbaI / EcoRI restriction enzymes and ligated to the vector using T4 DNA ligase (TransGen Biotech). Trans1T1 competent cells (TransGen Biotech) were then transformed, and kanamycin-resistant clones were selected and identified by XbaI / EcoRI digestion to identify positive clones with the fragment inserted into pK18mobsacB. The correct band size was 1061 bp. The inserted fragment was further identified by sequencing (GENEWIZ Company) and the primers used for the test were PE640 / PE643. The resulting plasmid was named pK18-Pgdh -10M (14067).

[0151] 2).pK18-yggB A100T (14067)Plasmid construction

[0152] PCR amplification was performed using the genome of Corynebacterium glutamicum ATCC14067 as a template using Phusion high-fidelity polymerase (New England BioLabs). PCR amplification using primer pairs PE646 / PE647 and PE648 / PE649 yielded the yggB A100T The upstream and downstream homology arms UP and DN fragments, the primer sequences are shown in Table 1. Subsequently, PE646 / PE649 was used as a primer pair, and the upstream and downstream homology arms were used as templates to prepare the recombinant fragment. The PCR procedure was the same as above, and the resulting recombinant fragment was purified by an agarose gel recovery kit (Tian Gen). At the same time, pK18mobsacB was digested with XbaI / EcoRI restriction endonucleases, and the fragment was connected to the vector with T4 DNA ligase (TransGen Biotech), and the Trans1T1 competent cells (TransGenBiotech) were transformed. Kanamycin-resistant clones were picked, and XbaI / EcoRI enzyme digestion was used to identify the positive clones in which the fragments were inserted into pK18mobsacB. The correct band size was 1036bp. The inserted fragment was further identified by sequencing (GENEWIZ Company) to be correct, and the primers sent for testing were PE646 / PE649. The resulting plasmid was named pK18-yggB A100T (14067).

[0153] 3). Construction of plasmid pK18-odhA ORF-5bpQS(14067)

[0154] PCR amplification was performed using the genome of Corynebacterium glutamicum ATCC14067 as a template using Phusion High-Fidelity Polymerase (New England BioLabs). PCR amplification of the upstream and downstream homology arms (UP and DN) of the odhAORF-5bpQS fragment was performed using primer pairs PE652 / PE653 and PE654 / PE655. The primer sequences are shown in Table 1. Subsequently, recombinant fragments were prepared using the PE652 / PE655 primer pair and the upstream and downstream homology arms as templates. The PCR procedure was the same as above, and the resulting recombinant fragment was purified using an agarose gel recovery kit (Tiangen). pK18mobsacB was digested with XbaI / EcoRI restriction enzymes, and the fragment was ligated to the vector using T4 DNA ligase (TransGen Biotech). Trans1T1 competent cells (TransGen Biotech) were then transformed, and kanamycin-resistant clones were selected. Positive clones showing the fragment inserted into pK18mobsacB were identified by XbaI / EcoRI digestion. The correct band size was 1039 bp. Sequencing (GENEWIZ) confirmed the correct insertion of the fragment using primers PE652 / PE655. The resulting plasmid was named pK18-odhA ORF-5bpQS(14067).

[0155] 4).pK18-gltA -10M、C361Y (14067)Plasmid construction

[0156] PCR amplification was performed using the genome of Corynebacterium glutamicum ATCC14067 as a template using Phusion high-fidelity polymerase (New England BioLabs). PCR amplification was performed using primer pairs PE656 / PE657 and PE658 / PE661 to obtain gltA. -10M , gltA C361YThe upstream and downstream homology arms UP and fragments, the primer sequences are shown in Table 1. Subsequently, PE656 / PE661 was used as a primer pair, and the upstream homology arms and fragments were used as templates to prepare the recombinant fragment. The PCR procedure was the same as above, and the resulting recombinant fragment was purified by an agarose gel recovery kit (Tian Gen). At the same time, pK18mobsacB was digested with XbaI / EcoRI restriction endonucleases, and the fragment was connected to the vector with T4 DNA ligase (TransGen Biotech), and Trans1T1 competent cells (TransGen Biotech) were transformed. Kanamycin-resistant clones were picked, and XbaI / EcoRI enzyme digestion was used to identify the positive clones in which the fragments were inserted into pK18mobsacB. The correct band size was 2241bp. The inserted fragment was further identified by sequencing (GENEWIZ Company), and the primers sent for testing were PE656 / PE661 / PE362 / PE363. The resulting plasmid was named pK18-gltA -10M、C361Y (14067).

[0157] 5).pK18-ach S372C (14067)Plasmid construction

[0158] The genome of Corynebacterium glutamicum ATCC14067 was used as a template and PCR amplification was performed using Phusion high-fidelity polymerase (New England BioLabs). PCR amplification was performed using primer pairs PE664 / P14-3 and P14-4 / PE667 to obtain the ach S372C The upstream and downstream homology arm UP and DN fragments, the primer sequences are shown in Table 1. Subsequently, PE664 / PE667 was used as a primer pair, and the upstream and downstream homology arm fragments were used as templates to prepare the recombinant fragment. The PCR procedure was the same as above, and the resulting recombinant fragment was purified by an agarose gel recovery kit (Tian Gen). At the same time, pK18-mobsacB was digested with XbaI / HindIII, and the fragment was connected to the vector with T4 DNA ligase (TransGen Biotech), and the Trans1T1 competent cells (TransGen Biotech) were transformed. The kanamycin-resistant clones were picked, and the XbaI / HindIII enzyme digestion was used to identify the positive clones in which the fragments were inserted into pK18mobsacB. The correct band size was 1035bp. The inserted fragment was further identified by sequencing (GENEWIZ Company), and the primers sent for testing were PE664 / PE667. The resulting plasmid was named pK18-ach S372C (14067).

[0159] 6).pK18-suc M199I (14067)Plasmid construction

[0160] Phusion high-fidelity polymerase (New England BioLabs) was used, 49-UP-1F / 49-UP-1R and 49-DN-2F / 49-DN-2R were used as primer pairs, and the genome of Corynebacterium glutamicum ATCC14067 was used as a template to prepare the upstream and downstream homology arm UP and DN fragments. The primer sequences are shown in Table 1. Subsequently, 49-UP-1F / 49-DN-2R was used as a primer pair and the upstream and downstream homology arms were used as templates to prepare the recombinant fragment. The PCR procedure was the same as above. The resulting recombinant fragment was purified by an agarose gel recovery kit (Tiangen). At the same time, pK18-mobsacB was digested with BamHI / XbaI and the fragment was ligated to the vector using T4 DNA ligase (TransGen Biotech). Trans1T1 competent cells (TransGen Biotech) were transformed, and kanamycin-resistant clones were picked. BamHI / XbaI enzyme digestion was used to identify positive clones with fragments inserted into pK18mobsacB. The correct band size was 1002 bp. Sequencing (GENEWIZ) was further used to identify the correct insertion of the fragment. The primers sent for testing were 49-UP-1F / 49-DN-2R. The resulting plasmid was named pK18-suc-NCgl0049. M199I (14067).

[0161] 7).pK18-tktA T234I (14067)Plasmid construction

[0162] Primers were designed using the genome of Corynebacterium glutamicum ATCC14067 as a template, and the primer sequences are shown in Table 1. Utilizing Phusion ultra-fidelity polymerase (New England BioLabs), tktA-UP-1F / tktA-UP-1R, tktA-DN-2F / tktA-DN-2R were used as primer pairs, and the genome of Corynebacterium glutamicum ATCC14067 was used as a template to prepare upstream and downstream homology arms UP and DN fragments. Subsequently, tktA-UP-1F / tktA-DN-2R was used as a primer pair, and upstream and downstream homology arms were used as templates to prepare recombinant fragments, and the PCR procedure was the same as above. The resulting recombinant fragment was purified using an agarose gel recovery kit (Tiangen) and then digested with XbaI / HindIII. pK18-mobsacB was also digested with XbaI / HindIII and ligated to the vector using T4 DNA ligase (TransGen Biotech). Trans1T1 competent cells (TransGen Biotech) were then transformed, and kanamycin-resistant clones were selected. XbaI / HindIII digestion was performed to identify positive clones with the fragment inserted into pK18mobsacB. The correct band size was 1028 bp. Sequencing (GENEWIZ) confirmed the correct insertion of the fragment. The primers used for the assay were tktA-UP-1F / tktA-DN-2R. The resulting plasmid was named pK18-tktA. T234I (14067).

[0163] 8).pK18-putA A755V (14067)Plasmid construction

[0164] Phusion high-fidelity polymerase (New England BioLabs) was used with the primer pairs putA-UP-1F / putA-UP-1R and putA-DN-2F / putA-DN-2R, using the genome of Corynebacterium glutamicum ATCC14067 as a template to prepare the upstream and downstream homology arms UP and DN fragments. The primer sequences are shown in Table 1. Subsequently, recombinant fragments were prepared using the primer pair putA-UP-1F / putA-DN-2R and the upstream and downstream homology arms as templates, following the same PCR procedure as above. The resulting recombinant fragment was purified using an agarose gel recovery kit (Tiangen) and then digested with XbaI / HindIII. pK18-mobsacB was also digested with XbaI / HindIII and ligated to the vector using T4 DNA ligase (TransGen Biotech). Trans1T1 competent cells (TransGen Biotech) were then transformed, and kanamycin-resistant clones were selected. XbaI / HindIII digestion was performed to identify positive clones with the fragment inserted into pK18mobsacB. The correct band size was 998 bp. Sequencing (GENEWIZ) confirmed that the inserted fragment was correct. The primers used for the assay were putA-UP-1F / putA-DN-2R. The resulting plasmid was named pK18-putA. A755V (14067).

[0165] 9).pK18-nadA D312N (14067)Plasmid construction

[0166] Primers were designed using the genome of Corynebacterium glutamicum ATCC14067 as a template. Using Phusion high-fidelity polymerase (New England BioLabs), nadA-UP-1F / nadA-UP-1R and nadA-DN-2F / nadA-DN-2R were used as primer pairs, and the genome of Corynebacterium glutamicum ATCC14067 was used as a template to prepare upstream and downstream homology arm UP and DN fragments. The primer sequences are shown in Table 1. Subsequently, nadA-UP-1F / nadA-DN-2R was used as a primer pair and the upstream and downstream homology arms were used as templates to prepare a recombinant fragment. The PCR procedure was the same as above. The resulting recombinant fragment was purified using an agarose gel recovery kit (Tiangen) and then digested with XbaI / HindIII. At the same time, pK18-mobsacB was digested with XbaI / HindIII and ligated with the vector using T4 DNA ligase (TransGenBiotech). Trans1T1 competent cells (TransGen Biotech) were transformed, and kanamycin-resistant clones were picked. XbaI / HindIII enzyme digestion was used to identify positive clones with fragments inserted into pK18mobsacB. The correct band size was 990 bp. Sequencing (GENEWIZ) confirmed that the inserted fragment was correct. The primers sent for testing were nadA-UP-1F / nadA-DN-2R. The resulting plasmid was named pK18-nadA D312N (14067).

[0167] 10).pK18-ds G171D (14067)Plasmid construction

[0168] Primers were designed using the genome of Corynebacterium glutamicum ATCC14067 as a template, and the primer sequences are shown in Table 1. Using Phusion ultra-fidelity polymerase (New England BioLabs), ds-UP-1F / ds-UP-1R and ds-DN-2F / ds-DN-2R were used as primer pairs, and the genome of Corynebacterium glutamicum ATCC14067 was used as a template to prepare upstream and downstream homology arms UP and DN fragments. Subsequently, ds-UP-1F / ds-DN-2R was used as a primer pair, and the upstream and downstream homology arms were used as templates to prepare recombinant fragments, and the PCR procedure was the same as above. The resulting recombinant fragment was purified using an agarose gel recovery kit (Tiangen), then digested with XbaI / PstI. pK18-mobsacB was also digested with XbaI / PstI, and the fragment was ligated to the vector using T4 DNA ligase (TransGen Biotech). Trans1T1 competent cells (TransGen Biotech) were then transformed, and kanamycin-resistant clones were selected. XbaI / PstI digestion was performed to identify positive clones with fragments inserted into pK18mobsacB. The correct band size was 1012 bp. Sequencing (GENEWIZ) was further used to identify the correct insertion of the fragment. The primers used for the assay were ds-UP-1F / ds-DN-2R. The resulting plasmid was named pK18-ds G171D (14067).

[0169] 1.3.2 Construction of ATCC14067 starting strain

[0170] 1).ATCC14067-Pgdh -10M Strain construction (MH14067-1)

[0171] ATCC14067 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and pK18-Pgdh -10M(14067) was transformed into Corynebacterium glutamicum ATCC14067, and exchange recombinants were selected on selective medium containing 15 mg / L kanamycin. Kan clones were identified by colony PCR using Fast Taq DNA polymerase (TransGenBiotech) with primer pairs PE640 / P85 and P82 / PE643. The PCR program was: 94°C for 10 minutes, 94°C for 30 seconds, 50°C for 30 seconds, and extension at 72°C for 15 seconds / kb for 30 cycles, followed by a complete extension at 72°C for 10 minutes. The lengths of the fragments amplified by the two primer pairs were similar, with clones around 1100 bp being considered positive. Selected positive clones were inoculated into antibiotic-free BHI medium and cultured for 12-14 hours. The bacterial suspension was diluted 100-1000-fold and plated onto solid BHI medium containing 10% sucrose and cultured for 36 hours. The selected strains were further verified for kanamycin resistance. KanS recombinants were selected and point mutation recombinants were verified using the identification primer pair PE642 / PE643. By exploring the annealing temperature, recombinants containing point mutations were obtained. The resulting positive recombinants were amplified and sequenced using PE640 / PE644. The correct recombinant was 2356 bp in length using the primers PE640 / PE644 / P14-2 / PE643 / PE645. The strain with the correct sequence was designated MH14067-1.

[0172] 2).MH14067-1-yggB A100T Strain construction (MH14067-2)

[0173] Prepare MH14067-1 competent cells according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and transfect pK18-yggB A100T(14067) was transferred into Corynebacterium glutamicum MH14067-1 and exchange recombinants were selected on a selective medium containing 15 mg / L of kanamycin. Utilizing Fast Taq DNA polymerase (TransGenBiotech), colony PCR was performed using primer pairs PE646 / P85 and P82 / PE649 to identify Kan clones. The PCR procedure was the same as above. The fragment lengths amplified by the two pairs of primer pairs were not much different, and clones with lengths of about 1100 bp were considered positive clones. The selected positive clones were inoculated into non-resistant BHI medium and cultured for 12-14 hours. The bacterial solution was diluted 100-1000 times and then applied onto solid BHI medium containing 10% sucrose and cultured for 36 hours. The selected strains were further subjected to kanamycin resistance phenotype verification. Recombinants with KanS were selected and point mutation recombinants were verified using primer pair PE648 / PE649. Recombinants containing point mutations were obtained by groping for the annealing temperature. The positive recombinants were amplified and sequenced using PE650 / PE651. The correct recombinant was 2020 bp long. The primers used for the test were PE650 / PE651 / PE648 / PE649. The strain with the correct sequence was named MH14067-2.

[0174] 3). Construction of MH14067-2-odhAORF-5bpQS strain (MH14067-3)

[0175] Prepare MH14067-2 competent cells according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Charpter 23), pK18-odhAORF-5bpQS (14067) is changed among the Corynebacterium glutamicum MH14067-2, on the selection medium that contains the kanamycin of 15mg / L, select to exchange recombinant.Utilize Fast Taq archaeal dna polymerase (TransGen Biotech), carry out bacterium colony PCR identification Kan clone with PE652 / P85, P82 / PE655 primer pair, PCR program is the same.Two pairs of primers are little different to the fragment length size that amplifies, and all are positive colonies at the clone of about 1100bp. The selected positive clones were inoculated into antibiotic-free BHI medium and cultured for 12-14 hours. The bacterial solution was diluted 100-1000 times and then spread on solid BHI medium containing 10% sucrose and cultured for 36 hours. The selected strains were further verified for kanamycin resistance phenotype, and KanS recombinants were selected and identified using the PE654 / PE655 primer pair to verify 5bp deletion and point mutation recombinants.

[0176] The positive recombinant obtained was amplified and sequenced using PE025 / PE026. The correct recombinant was 3993 bp long. The primers submitted for testing were PE025 / PE026 / PE020 / PE021 / PE022 / PE023 / PE024 / PE655. The strain with correct sequencing was named MH14067-3.

[0177] 4).MH14067-3-gltA -10M、C361Y Strain construction (MH14067-4)

[0178] MH14067-3 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and pK18-gltA -10M、C361Y (14067) was transformed into Corynebacterium glutamicum MH14067-3, and exchange recombinants were selected on a selective medium containing 15 mg / L kanamycin. Kan clones were identified by colony PCR using Fast Taq DNA polymerase (TransGen Biotech) with primer pairs PE656 / P85 and P82 / PE661, following the same PCR procedure as above. The fragments amplified by the two primer pairs showed significant differences in length: clones with a short band of approximately 1100 bp and a long band of approximately 2500 bp were considered positive clones. Selected positive clones were inoculated into antibiotic-free BHI medium and cultured for 12-14 hours. The bacterial suspension was diluted 100-1000-fold and plated onto solid BHI medium containing 10% sucrose and cultured for 36 hours. The selected strains were further verified for kanamycin resistance. KanS recombinants were selected and point mutation recombinants were verified using the identification primer pair PE660 / PE661. By exploring the annealing temperature, recombinants containing point mutations were obtained. The resulting positive recombinants were amplified and sequenced using PE662 / PE663. The correct recombinant was 2439 bp in length, using the primers PE662 / PE663 / PE362 / PE363. The strain with the correct sequence was designated MH14067-4.

[0179] 5).MH14067-4-suc M199I Strain construction (MH14067-5)

[0180] MH14067-4 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and the plasmid pK18-suc M199I(14067) was transformed into competent cells of Corynebacterium glutamicum MH14067-4 by electroporation, and exchange recombinants were selected on selective medium containing 15 mg / L kanamycin. KanR clones were identified by colony PCR using Fast Taq DNA polymerase (TransGen Biotech) with the primer pairs 49-UP-1F / P85 and P82 / 49-DN-2R, following the same PCR procedure as above. The fragments amplified by the two primer pairs showed similar lengths, with clones around 1100 bp being considered positive clones. The selected positive clones were inoculated into antibiotic-free BHI medium and cultured for 12-14 hours. The bacterial solution was diluted 100-1000 times and then spread on solid BHI medium containing 10% sucrose and cultured for 36 hours. The screened strains were further verified for kanamycin resistance phenotype. KanS recombinants were selected and point mutation recombinants were verified using the identification primer pair 49-F / 49-DN-2R. By exploring the annealing temperature, recombinants containing point mutations were obtained. The resulting positive recombinants were amplified and sequenced using 49-ID-F / 49-ID-R. The correct recombinant was 1124 bp in length. The primers submitted for testing were 49-ID-F / 49-ID-R. The strain with correct sequencing was named MH14067-5.

[0181] 6).MH14067-5-ach S372C Strain construction (MH14067-6)

[0182] Prepare MH14067-5 competent cells according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and transfect pK18-ach S372CThe recombinant strain (14067) was transformed into Corynebacterium glutamicum MH14067-5, and exchange recombinants were selected on a selective medium containing 15 mg / L kanamycin. KanR clones were identified by colony PCR using Fast Taq DNA polymerase (TransGenBiotech) with primer pairs PE664 / P85 and P82 / PE667, following the same PCR procedure as above. The lengths of the fragments amplified by the two primer pairs were similar, with clones around 1100 bp being considered positive clones. The selected positive clones were inoculated into antibiotic-free BHI medium and cultured for 12-14 hours. The bacterial solution was diluted 100-1000 times and then spread on solid BHI medium containing 10% sucrose and cultured for 36 hours. The screened strains were further verified for kanamycin resistance phenotype. KanS recombinants were selected and the point mutation recombinants were verified using the identification primer pair PE666 / PE667. By exploring the annealing temperature, recombinants containing point mutations were obtained. The obtained positive recombinants were amplified and sequenced using PE667 / PE668. The correct recombinant was 1650bp long. The primers submitted for testing were PE667 / PE668 / PE664. The strain with correct sequencing was named MH14067-6.

[0183] 7).MH14067-6-tktA T234I Strain construction (MH14067-7)

[0184] MH14067-6 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and pK18-tktA T234I(14067) was transformed into Corynebacterium glutamicum MH14067-6, and exchange recombinants were selected on selective medium containing 15 mg / L kanamycin. KanR clones were identified by colony PCR using Fast Taq DNA polymerase (TransGenBiotech) with the primer pairs tktA-UP-1F / P85 and P82 / tktA-DN-2R. The PCR procedure was the same as above. The lengths of the fragments amplified by the two primer pairs were similar, with clones around 1100 bp being considered positive clones. The selected positive clones were inoculated into antibiotic-free BHI medium and cultured for 12-14 hours. The bacterial solution was diluted 100-1000 times and then spread on solid BHI medium containing 10% sucrose and cultured for 36 hours. The screened strains were further verified for kanamycin resistance phenotype. KanS recombinants were selected and the point mutation recombinants were verified using the identification primer pair tktA-F / tktA-DN-2R. By exploring the annealing temperature, recombinants containing point mutations were obtained. The resulting positive recombinants were amplified and sequenced using tktA-ID-F / tktA-ID-R. The correct recombinant was 1223 bp in length. The primers submitted for testing were tktA-ID-F / tktA-ID-R. The strain with correct sequencing was named MH14067-7.

[0185] 8).MH14067-7-putA-cg0129 A755V Strain construction (MH14067-8)

[0186] Prepare MH14067-7 competent cells according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and transfect pK18-putA-cg0129 A755V(14067) was transformed into Corynebacterium glutamicum MH14067-7, and exchange recombinants were selected on selective medium containing 15 mg / L kanamycin. KanR clones were identified by colony PCR using Fast Taq DNA polymerase (TransGen Biotech) with the putA-UP-1F / P85 and P82 / putA-DN-2R primer pairs, following the same PCR procedure as above. The lengths of the fragments amplified by the two primer pairs were similar, with clones around 1100 bp being considered positive clones. The selected positive clones were inoculated into antibiotic-free BHI medium and cultured for 12-14 hours. The bacterial solution was diluted 100-1000 times and spread on solid BHI medium containing 10% sucrose and cultured for 36 hours. The screened strains were further verified for kanamycin resistance phenotype. KanS recombinants were selected and point mutation recombinants were verified using the identification primer pair putA-F / putA-DN-2R. By exploring the annealing temperature, recombinants containing point mutations were obtained. The resulting positive recombinants were amplified and sequenced using P14-5 / putA-ID-R. The correct recombinant was 1214 bp in length. The primers submitted for testing were P14-5 / putA-ID-R. The strain with correct sequencing was named MH14067-8.

[0187] 9).MH14067-8-ds G171D Strain construction (MH14067-9)

[0188] MH14067-8 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and pK18-ds G171D (14067) was transferred into Corynebacterium glutamicum MH14067-8, and the exchange recombinants were selected on a selection medium containing 15 mg / L of kanamycin. Using Fast Taq DNA polymerase (TransGenBiotech), colony PCR was performed with ds-UP-1F / P85 and P82 / ds-DN-2R primer pairs to identify KanR clones. The PCR procedure was the same as above. The lengths of the fragments amplified by the two primer pairs were not much different, and clones around 1100 bp were considered positive clones. The selected positive clones were inoculated into non-antibiotic BHI medium and cultured for 12-14 hours. The bacterial solution was diluted 100-1000 times and then spread on solid BHI medium containing 10% sucrose and cultured for 36 hours. The selected strains were further verified for kanamycin resistance phenotype, and Kan was selected. SThe recombinant was verified as a point mutation recombinant using the identification primer pair ds-F / ds-DN-2R. By exploring the annealing temperature, a recombinant containing a point mutation was obtained. The positive recombinant was amplified and sequenced using ds-ID-F / ds-ID-R. The correct recombinant was 1172bp long. The primers submitted for testing were ds-ID-F / ds-ID-R. The strain with correct sequencing was named MH14067-9.

[0189] 10).MH14067-9-nadA D312N Strain construction (MH14067-10)

[0190] MH14067-9 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and pK18-nadA D312N (14067) was transformed into Corynebacterium glutamicum MH14067-9, and exchange recombinants were selected on selective medium containing 15 mg / L kanamycin. KanR clones were identified by colony PCR using Fast Taq DNA polymerase (TransGenBiotech) with the primer pairs nadA-UP-1F / P85 and P82 / nadA-DN-2R, following the same PCR procedure as above. The lengths of the fragments amplified by the two primer pairs were similar, with clones around 1100 bp being considered positive clones. The selected positive clones were inoculated into antibiotic-free BHI medium and cultured for 12-14 hours. The bacterial solution was diluted 100-1000 times and then spread on solid BHI medium containing 10% sucrose and cultured for 36 hours. The screened strains were further verified for kanamycin resistance phenotype. KanS recombinants were selected and the point mutation recombinants were verified using the identification primer pair nadA-F / nadA-DN-2R. By exploring the annealing temperature, recombinants containing point mutations were obtained. The resulting positive recombinants were amplified and sequenced using nadA-ID-F / nadA-ID-R. The correct recombinant was 1116 bp in length. The primers submitted for testing were nadA-ID-F / nadA-ID-R. The strain with correct sequencing was named MH14067-10.

[0191] 1.3.3 Shake flask verification of ATCC14067 starting strain

[0192] The recombinant Corynebacterium glutamicum constructed above was fermented to verify its glutamic acid production performance. The shake flask method was referred to 1.1.3. The specific fermentation results are shown in the following table.

[0193] Table 4. Glutamate content detection of recombinant strains

[0194] Strain number genotype Fermentation OD 562nm Glutamic acid g / L Conversion Rate % ATCC 14067 wild type 58.2 0.12 0.2 MH14067-1 <![CDATA[MH14067-Pgdh -10M ]]> 56.4 3.42 5.7 MH14067-2 <![CDATA[MH14067-1-yggB A100T ]]> 55.5 7.14 11.9 MH14067-3 MH14067-2-odhAORF-5bpQS 56.2 12.24 20.4 MH14067-4 <![CDATA[MH14067-3-gltA -10M-C361Y ]]> 54.6 13.5 22.5 MH14067-5 <![CDATA[MH14067-4-suc M199I ]]> 55.7 14.22 23.7 MH14067-6 <![CDATA[MH14067-5-ach S372C ]]> 53.3 14.52 24.2 MH14067-7 <![CDATA[MH14067-6-tktA T234I ]]> 52.9 15.24 25.4 MH14067-8 <![CDATA[MH14067-7-putA A755V ]]> 53.7 16.14 26.9 MH14067-9 <![CDATA[MH14067-8-ds G171D ]]> 52.5 16.86 28.1 MH14067-10 <![CDATA[MH14067-9-nadA D312N ]]> 51.8 17.82 29.7

[0195] These shake flask results demonstrate that superimposing these 10 gene modifications on wild-type strain 14067 significantly improves glutamate production, with a continuous increase in glutamate conversion efficiency. Specifically, the glutamate conversion efficiency of MH14067-10 increased from 0.2% of the wild-type strain to 29.7%, without affecting growth or sugar consumption. Combined with the shake flask results in 1.1.3 and 1.2.3, this demonstrates that superimposing these 10 gene modifications significantly improves glutamate conversion efficiency, regardless of whether the starting strain is C. glutamicum ATCC13869, ATCC13032, or ATCC14067.

[0196] Example 2: Construction of xfp and pfk modified plasmids

[0197] 2.1 Construction of ATCC13869 route XFP and PFK transformation plasmids

[0198] 2.1.1 Construction of pK18-odhA-PcspB-Bl_xfp plasmid

[0199] Using the WJ0140 genome as a template, primers PE617-UP-1F / PE618-UP-1R were used to amplify the upstream homology arm (500 bp), and primers PE623-DN-4F / PE624-DN-4R were used to amplify the downstream homology arm DN (500 bp). Using the pXMJ19-PcspB-Bl-xfp plasmid (the xfp expression plasmid was synthesized by GENEWIZ and assembled with the provided pXMJ19-PcspB vector) as a template, and primers PE934-Bm_xfp-F and PE937-Bl_xfp-R as a pair, PcspB-Bl_xfp was obtained by reverse PCR amplification. The resulting fragment was purified using an agarose gel extraction kit (Tiangen) and stored frozen at -20°C until further use. pK18mobsacB was digested with XbaI / HindIII restriction enzymes and gel-cleaved to obtain the plasmid backbone.

[0200] The amplified plasmid backbone and fragments were mixed in the appropriate proportions using the ClonExpress II Exnase One-Step Cloning Kit (Novozymes Biotech Co., Ltd.), pipetted to mix thoroughly, and then placed in a metal bath (Golden Ginkgo Biotechnology Co., Ltd.) for ligation at 37°C for 30 minutes. After completion of the reaction, Trans1T1 competent cells (TransGenBiotech) were transformed, and kanamycin-resistant clones were selected and identified using the primer pair P81 and P86. The correct band size was 4417 bp. The correct insertion was further verified by sequencing (GENEWIZ) using the primers P81, P86, PE934-Bm_xfp-F, PE937-Bl_xfp-R, PE962-Bl-check-F1, and PE963-Bl-check-F2. The resulting plasmid was named pK18-odhA-PcspB-Bl_xfp.

[0201] 2.1.2 Construction of pK18-alaT-Ptuf-Bl_xfp plasmid

[0202] Using the WJ0140 genome as a template, the upstream homology arm (539 bp) was amplified using the PE552-UP-1F / PE569-UP-1R primer pair, and the downstream homology arm (506 bp) was amplified using the PE574-DN-4F / PE559-DN-4R primer pair. The Ptuf promoter was amplified using the Corynebacterium glutamicum ATCC13032 (GenBank NO: NC_003450.3) genome as a template, using the PE999-alaT-tuf-F and PE1000-alaT-tuf-R primer pair. The Bl_xfp fragment was amplified by PCR using the pXMJ19-PcspB-Bl-xfp plasmid (the xfp expression plasmid was synthesized by GENEWIZ and assembled with the provided pXMJ19-PcspB vector) as a template and primers PE1001-alaT-BL-F and PE1002-alaT-BL-R. The resulting fragment was purified using an agarose gel extraction kit (Tiangen) and stored frozen at -20°C until further use. pK18mobsacB was digested with XbaI / HindIII restriction enzymes and the plasmid backbone was recovered from the gel.

[0203] The amplified plasmid backbone, promoter, and gene fragment were mixed in the appropriate proportions using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech Co., Ltd.). After pipetting to mix thoroughly, the fragment was placed in a metal bath (Golden Ginkgo Biotechnology Co., Ltd.) for ligation at 37°C for 30 minutes. After completion of the reaction, the cells were transformed into Trans1T1 competent cells (TransGen Biotech). Kanamycin-resistant clones were selected and identified using the primer pair P81 and P86. The correct band size was 4115 bp. The correct insertion was further verified by sequencing (GENEWIZ) using the primers P81, P86, PE962-Bl-check-F1, PE963-Bl-check-F2, PE999-alaT-tuf-F, and PE1002-alaT-BL-R. The resulting plasmid was named pK18-alaT-Ptuf-Bl-xfp.

[0204] 2.1.3 Construction of pK18-odhA-Ptuf-Bl_xfp plasmid

[0205] The genome of Corynebacterium glutamicum WJ0140 was used as a template, and primers PE1006-odhA-BL-UP-F / PE1007-odhA-BL-UP-R, PE1008-odhA-BL-DN-F / PE1009-odhA-BL-DN-R, and PE1010-odhA-tuf-F / PE1000-alaT-tuf-R were used to amplify the upstream and downstream homology arms and the Ptuf promoter. The Bl_xfp fragment was obtained by amplification using pK18-odhA-PcspB-Bl_xfp as a template and primers PE1001-alaT-BL-F and PE1011-odhA-BL-R. The resulting fragment was purified using an agarose gel extraction kit (Tiangen) and stored frozen at -20°C until further use.

[0206] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified xfp, UP, DN, and Ptuf fragments were mixed with the double-digested vector in the appropriate proportions using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech). After pipetting to mix thoroughly, the cells were placed in a metal bath (Golden Ginkgo Biotechnology Co., Ltd.) at 37°C for 30 minutes for ligation. After the reaction, Trans1T1 competent cells (TransGenBiotech) were transformed, and kanamycin-resistant clones were selected. Using the primer pair P81 and P86, the correct band size was 4475 bp. The correct insertion was further verified by sequencing (GENEWIZ) using the following primers: P81, P86, PE962-Bl-check-F1, PE963-Bl-check-F2, PE1009-odhA-BL-DN-R, and PE1010-odhA-tuf-F. The resulting plasmid was named pK18-odhA-Ptuf-Bl_xfp.

[0207] 2.1.4 Construction of pK18-cg3384-Ptuf-Bl_xfp plasmid

[0208] The genome of Corynebacterium glutamicum ATCC13869 was used as a template, and primers PE1101-3384-UP-1F / PE1102-3384-UP-1R and PE1103-3384-DN-2F / PE1104-3384-DN-2R were used to amplify the upstream and downstream homology arms. The Ptuf-Bl_xfp fragment was obtained by amplification using pK18-odhA-Ptuf-Bl_xfp as a template and primers PE1105-xfp-F and PE1106-xfp-R. The resulting fragment was purified using an agarose gel extraction kit (Tiangen) and stored frozen at -20°C until further use.

[0209] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP, DN, and Ptuf-Bl_xfp fragments were mixed with the double-digested vector in the appropriate proportions using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech). After pipetting to mix thoroughly, the cells were placed in a metal bath (Golden Ginkgo Biotechnology Co., Ltd.) at 37°C for 30 minutes for ligation. After completion of the reaction, Trans1T1 competent cells (TransGenBiotech) were transformed. Kanamycin-resistant clones were selected and identified using the primer pair P81 and P86. The correct band size was 4143 bp. The correct insertion was further verified by sequencing (GENEWIZ) using the following primers: P81, P86, PE962-Bl-check-F1, PE963-Bl-check-F2, PE1105-xfp-F, and PE1106-xfp-R. The resulting plasmid was named pK18-cg3384-Ptuf-Bl_xfp.

[0210] 2.1.5 Construction of pK18-cg3364-Ptuf-Bl_xfp plasmid

[0211] The genome of Corynebacterium glutamicum ATCC13869 was used as a template, and primers PE1117-3364-UP-1F, PE1118-3364-UP-1R / PE1119-3364-DN-2F, and PE1120-3364-DN-2R were used to amplify the upstream and downstream homology arms. The Ptuf-Bl_xfp fragment was obtained by amplification using pK18-odhA-Ptuf-Bl_xfp as a template and primers PE1121-xfp-F and PE1122-xfp-R. The resulting fragment was purified using an agarose gel recovery kit (Tiangen) and stored frozen at -20°C until further use.

[0212] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP, DN, and Ptuf-Bl_xfp fragments were mixed with the double-digested vector in the appropriate proportions using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech). After pipetting and mixing, the cells were placed in a metal bath (Golden Ginkgo Biotechnology Co., Ltd.) at 37°C for 30 minutes for ligation. After completion of the reaction, Trans1T1 competent cells (TransGenBiotech) were transformed, and kanamycin-resistant clones were selected and identified using the primer pair P81 and P86. The correct band size was 4128 bp. The correct insertion was further verified by sequencing (GENEWIZ) using the following primers: P81, P86, PE962-Bl-check-F1, PE963-Bl-check-F2, PE1121-xfp-F, and PE1122-xfp-R. The resulting plasmid was named pK18-cg3364-Ptuf-Bl_xfp.

[0213] 2.1.6 Construction of pK18-cg0928-Ptuf-Bl_xfp plasmid

[0214] The genome of Corynebacterium glutamicum ATCC13869 was used as a template, and primers PE1125-0928-UP-1F / PE1126-0928-UP-1R and PE1127-0928-DN-2F / PE1128-0928-DN-2R were used to amplify the upstream and downstream homology arms. The Ptuf-Bl_xfp fragment was obtained by amplification using pK18-odhA-Ptuf-Bl_xfp as a template and primers PE1129-xfp-F and PE1130-xfp-R. The resulting fragment was purified using an agarose gel recovery kit (Tiangen) and stored frozen at -20°C until further use.

[0215] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP, DN, and Ptuf-Bl_xfp fragments were mixed with the double-digested vector in the appropriate proportions using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech). After pipetting and mixing, the cells were placed in a metal bath (Golden Ginkgo Biotechnology Co., Ltd.) at 37°C for 30 minutes for ligation. After completion of the reaction, Trans1T1 competent cells (TransGenBiotech) were transformed, and kanamycin-resistant clones were selected and identified using the primer pair P81 and P86. The correct band size was 4154 bp. The correct insertion was further verified by sequencing (GENEWIZ) using the following primers: P81, P86, PE962-Bl-check-F1, PE963-Bl-check-F2, PE1129-xfp-F, and PE1130-xfp-R. The resulting plasmid was named pK18-cg0928-Ptuf-Bl_xfp.

[0216] 2.1.7 Construction of pK18-cg2211-Ptuf-Bl_xfp plasmid

[0217] The genome of Corynebacterium glutamicum ATCC13869 was used as a template, and primers PE1339-2211-UP-1F / PE1377-2211-UP-1R2 and PE1342-2211-DN-2R / PE1378-2211-DN-2F2 were used to amplify the upstream and downstream homology arms. The Ptuf-Bl_xfp fragment was obtained by amplification using pK18-odhA-Ptuf-Bl_xfp as a template and primers PE1379-XFP-F and PE1380-XFP-R. The resulting fragment was purified using an agarose gel extraction kit (Tiangen) and stored frozen at -20°C until further use.

[0218] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP, DN, and Ptuf-Bl_xfp fragments were mixed with the double-digested vector in the appropriate proportions using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech). After pipetting and mixing, the cells were placed in a metal bath (Golden Ginkgo Biotechnology Co., Ltd.) at 37°C for 30 minutes for ligation. After completion of the reaction, Trans1T1 competent cells (TransGenBiotech) were transformed. Kanamycin-resistant clones were selected and identified using the primer pair P81 and P86. The correct band size was 4143 bp. The correct insertion was further verified by sequencing (GENEWIZ) using the following primers: P81, P86, PE962-Bl-check-F1, PE963-Bl-check-F2, PE1379-XFP-F, and PE1380-XFP-R. The resulting plasmid was named pK18-cg2211-Ptuf-Bl_xfp.

[0219] 2.1.8 Construction of pK18-cg3125-Ptuf-Bl_xfp plasmid

[0220] The genome of Corynebacterium glutamicum ATCC13869 was used as a template, and primers PE1357-3125-UP-1F / PE1385-3125-UP-1R2 and PE1386-3125-DN-2F2 / PE1360-3125-DN-2R were used to amplify the upstream and downstream homology arms. The Ptuf-Bl_xfp fragment was obtained by amplification using the primers PE1387-XFP-F and PE1388-XFP-R, using pK18-odhA-Ptuf-Bl_xfp as a template. The resulting fragment was purified using an agarose gel extraction kit (Tiangen) and stored frozen at -20°C until further use.

[0221] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP, DN, and Ptuf-Bl_xfp fragments were mixed with the double-digested vector in the appropriate proportions using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech). After pipetting to mix thoroughly, the cells were placed in a metal bath (Golden Ginkgo Biotechnology Co., Ltd.) at 37°C for 30 minutes for ligation. After completion of the reaction, Trans1T1 competent cells (TransGenBiotech) were transformed. Kanamycin-resistant clones were selected and identified using the primer pair P81 and P86. The correct band size was 4137 bp. The correct insertion was further verified by sequencing (GENEWIZ) using the following primers: P81, P86, PE962-Bl-check-F1, PE963-Bl-check-F2, PE1387-XFP-F, and PE1388-XFP-R. The resulting plasmid was named pK18-cg3125-Ptuf-Bl_xfp.

[0222] 2.1.9 Construction of pK18-cg2564-Ptuf-Bl_xfp plasmid

[0223] The genome of Corynebacterium glutamicum ATCC13869 was used as a template, and primers PE1351-2564-UP-1F / PE1352-2564-UP-1R and PE1353-2564-DN-2F / PE1354-2564-DN-2R were used to amplify the upstream and downstream homology arms. The Ptuf-Bl_xfp fragment was obtained by amplification using pK18-odhA-Ptuf-Bl_xfp as a template and primers PE1355-xfp-F and PE1356-xfp-R. The resulting fragment was purified using an agarose gel recovery kit (Tiangen) and stored frozen at -20°C until further use.

[0224] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP, DN, and Ptuf-Bl_xfp fragments were mixed with the double-digested vector in the appropriate proportions using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech). After pipetting and mixing, the cells were placed in a metal bath (Golden Ginkgo Biotechnology Co., Ltd.) at 37°C for 30 minutes for ligation. After completion of the reaction, Trans1T1 competent cells (TransGenBiotech) were transformed. Kanamycin-resistant clones were selected and identified using the primer pair P81 and P86. The correct band size was 4138 bp. The correct insertion was further verified by sequencing (GENEWIZ) using the following primers: P81, P86, PE962-Bl-check-F1, PE963-Bl-check-F2, PE1355-xfp-F, and PE1356-xfp-R. The resulting plasmid was named pK18-cg2564-Ptuf-Bl_xfp.

[0225] 2.1.10 Construction of pK18-BBD29-07270-Ptuf-Bl_xfp Plasmid

[0226] The genome of Corynebacterium glutamicum ATCC13869 was used as a template, and primers PE1407-7270-UP-1F, PE1408-7270-UP-1R / PE1409-7270-DN-2F, and PE1410-7270-DN-2R were used to amplify the upstream and downstream homology arms. The Ptuf-Bl_xfp fragment was obtained by amplification using pK18-odhA-Ptuf-Bl_xfp as a template and primers PE1411-xfp-F and PE1412-xfp-R. The resulting fragment was purified using an agarose gel extraction kit (Tiangen) and stored frozen at -20°C until further use.

[0227] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP, DN, and Ptuf-Bl_xfp fragments were mixed with the double-digested vector in the appropriate proportions using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech). After pipetting to mix thoroughly, the cells were placed in a metal bath (Golden Ginkgo Biotechnology Co., Ltd.) at 37°C for 30 minutes for ligation. After completion of the reaction, Trans1T1 competent cells (TransGenBiotech) were transformed. Kanamycin-resistant clones were selected and identified using the primer pair P81 and P86. The correct band size was 4130 bp. The correct insertion was further verified by sequencing (GENEWIZ) using the following primers: P81, P86, PE962-Bl-check-F1, PE963-Bl-check-F2, PE1411-xfp-F, and PE1412-xfp-R. The resulting plasmid was named pK18-BBD29-07270-Ptuf-Bl_xfp.

[0228] 2.1.11 Construction of pK18-pfk-A1T Plasmid

[0229] Primers were designed based on the pfk gene sequence in the NCBI database (GenBank NO: BBD29_06675). The primer sequences are shown in Table 1. PCR amplification was performed using Phusion Super-Fidelity Polymerase (New England BioLabs). Upstream and downstream homology arms were obtained by reverse PCR amplification using primer pairs PE878-pfk-TTG-UP-1F / PE879-pfk-TTG-UP-1R and PE880-pfk-TTG-DN-2F / PE881-pfk-UP-2R. The resulting fragment was purified using an agarose gel extraction kit (Tiangen) and stored at -20°C until further use.

[0230] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP and DN fragments were mixed with the double-digested vector in the appropriate ratio using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech). After pipetting and mixing, the cells were placed in a metal bath (Golden Ginkgo Biotechnology Co., Ltd.) at 37°C for 30 minutes for ligation. After the reaction, Trans1T1 competent cells (TransGen Biotech) were transformed, and kanamycin-resistant clones were selected and identified using the primer pair P81 and P86. The correct band size was 1316 bp. The correct insertion was further confirmed by sequencing (GENEWIZ) using the primers P81 and P86. The resulting plasmid was named pK18-pfk-A1T.

[0231] 2.1.12 Construction of pK18-Pzwf-pfk Plasmid

[0232] Primers were designed based on the gene sequence of pfk (GenBank NO: BBD29_06675) in the NCBI database. The primer sequences are shown in Table 1. PCR amplification was performed using Phusion high-fidelity polymerase (New England BioLabs). PE882-pfk-UP-1F / PE883-pfk-UP-1R and PE884-pfk-DN-2F / PE885-pfk-UP-2R were used as primer pairs, and upstream and downstream homology arms were obtained by PCR reverse amplification. The Pzwf promoter was amplified using the genome of Corynebacterium glutamicum ATCC13032 (GenBank NO: NC_003450.3) as a template and PE886-Pzwf-F / PE887-Pzwf-R as a primer pair. The obtained fragment was purified and recovered using an agarose gel recovery kit (Tiangen) and stored at -20°C for later use.

[0233] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP, DN, and Pzwf promoters were then mixed with the double-digested vector in the appropriate proportions using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech). After pipetting and mixing, the cells were placed in a metal bath (Ginko Biotechnology Co., Ltd.) at 37°C for 30 minutes for ligation. After the reaction, Trans1T1 competent cells (TransGenBiotech) were transformed, and kanamycin-resistant clones were selected and identified using the primer pair P81 and P86. The correct band size was 1749 bp. The correct insertion was further verified by sequencing (GENEWIZ) using the primers P81, P86, and PE886-Pzwf-F. The resulting plasmid was named pK18-Pzwf-pfk.

[0234] 2.1.13 Construction of pK18-PrecA-pfk Plasmid

[0235] Primers were designed based on the gene sequence of pfk (GenBank NO: BBD29_06675) in the NCBI database. The primer sequences are shown in Table 1. PCR amplification was performed using Phusion high-fidelity polymerase (New England BioLabs). PE882-pfk-UP-1F / PE883-pfk-UP-1R and PE884-pfk-DN-2F / PE885-pfk-UP-2R were used as primer pairs, and upstream and downstream homology arms were obtained by PCR reverse amplification. The PrecA promoter was amplified using the genome of Corynebacterium glutamicum ATCC13032 (GenBank NO: NC_003450.3) as a template and PE889-PrecA-F / PE890-PrecA-R as a primer pair. The obtained fragment was purified and recovered using an agarose gel recovery kit (Tiangen) and stored at -20°C for later use.

[0236] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP, DN, and PrecA promoters were then mixed with the double-digested vector in the appropriate proportions using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech). After pipetting and mixing, the cells were placed in a metal bath (Golden Ginkgo Biotechnology Co., Ltd.) at 37°C for 30 minutes for ligation. After the reaction, Trans1T1 competent cells (TransGenBiotech) were transformed, and kanamycin-resistant clones were selected and identified using the primer pair P81 and P86. The correct band size was 1765 bp. The correct insertion of the fragment was further confirmed by sequencing (GENEWIZ) using the primers P81, P86, and PE889-PrecA-F. The resulting plasmid was named pK18-PrecA-pfk.

[0237] 2.1.14 Construction of pK18-pfk-E171K plasmid

[0238] Primers were designed based on the pfk gene sequence in the NCBI database (GenBank NO: BBD29_06675). The primer sequences are shown in Table 1. PCR amplification was performed using Phusion Super-Fidelity Polymerase (New England BioLabs). Upstream and downstream homology arms were obtained by reverse PCR amplification using primer pairs PE892-pfk-E171K-UP-1F / PE893-pfk-E171K-UP-1R and PE894-pfk-E171K-DN-2F / PE895-pfk-E171K-DN-2R. The resulting fragment was purified using an agarose gel extraction kit (Tiangen) and stored at -20°C until further use.

[0239] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP and DN fragments were mixed with the double-digested vector in a specific ratio using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech). After pipetting and mixing, the cells were placed in a metal bath (Golden Ginkgo Biotechnology Co., Ltd.) at 37°C for 30 minutes for ligation. After completion of the reaction, Trans1T1 competent cells (TransGen Biotech) were transformed, and kanamycin-resistant clones were selected and identified using the primer pair P81 and P86. The correct band size was 1327 bp. Sequencing (GENEWIZ) was further performed to confirm the correct insertion of the fragment, using primers P81 and P86. The resulting plasmid was named pK18-pfk-E171K.

[0240] 2.1.15 Construction of pK18-pfk-E171D plasmid

[0241] Primers were designed based on the pfk gene sequence in the NCBI database (GenBank NO: BBD29_06675). The primer sequences are shown in Table 1. PCR amplification was performed using Phusion Super-Fidelity Polymerase (New England BioLabs). Upstream and downstream homology arms were obtained by reverse PCR amplification using primer pairs PE892-pfk-E171K-UP-1F / PE893-pfk-E171K-UP-1R and PE897-pfk-E171D-DN-2F / PE895-pfk-E171K-DN-2R. The resulting fragment was purified using an agarose gel extraction kit (Tiangen) and stored at -20°C until further use.

[0242] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP and DN fragments were mixed with the double-digested vector in the appropriate ratio using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech). After pipetting and mixing, the fragments were placed in a metal bath (Golden Ginkgo Biotechnology Co., Ltd.) at 37°C for 3 minutes for ligation. After the reaction, Trans1T1 competent cells (TransGen Biotech) were transformed, and kanamycin-resistant clones were selected and identified using the primer pair P81 and P86. The correct band size was 1327 bp. Sequencing (GENEWIZ) was further performed to confirm the correct insertion of the fragment, using primers P81 and P86. The resulting plasmid was named pK18-pfk-E171D.

[0243] 2.1.16 Construction of pXMJ19-PcspB-xfp expression plasmid

[0244] The xfp expression plasmids pXMJ19-PcspB-Bm_XFP, pXMJ19-PcspB-Bl_XFP, pXMJ19-PcspB-Ll_XFP, pXMJ19-PcspB-Lm_XFP, pXMJ19-PcspB-Pa_XFP, pXMJ19-PcspB-Bab_XFP, pXMJ19-PcspB-An_XFP, pXMJ19-PcspB-Ban_XFP, and pXMJ19-PcspB-Bb_XFP were all obtained by whole gene synthesis in GENEWIZ and assembled with the provided pXMJ19-PcspB vector.

[0245] Table 5. XFP from different sources

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254] 2.2 Construction of XFP multi-copy and PFK modified plasmids based on ATCC13032

[0255] 2.2.1. Construction of pK18-alaT-Ptuf-Bl_xfp(13032) plasmid

[0256] The upper homology arm UP was obtained by PCR amplification using the C. glutamicum ATCC13032 genome as a template and the primer pair P13-10 / PE997-rev-alaT-R. The lower homology arm DN was obtained by PCR amplification using the primer pair PE998-rev-alaT-R / PE1022-alaT-DN-2R. The Ptuf promoter was amplified using the C. glutamicum ATCC13032 genome as a template and the primer pair PE999-alaT-tuf-F / PE1000-alaT-tuf-R. The Bl_xfp fragment was amplified by PCR using the pXMJ19-PcspB-Bl-xfp plasmid (the xfp expression plasmid was synthesized by GENEWIZ and assembled with the provided pXMJ19-PcspB vector) as a template and the primer pair PE1001-alaT-BL-F / PE1002-alaT-BL-R. The resulting fragment was purified using an agarose gel extraction kit (Tiangen) and stored at -20°C until further use.

[0257] The amplified UP, DN, promoter, and gene fragments were mixed in the specified proportions using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech Co., Ltd.), pipetted to mix thoroughly, and then placed in a metal bath (Golden Ginkgo Biotechnology Co., Ltd.) for ligation at 37°C for 30 minutes. After completion of the reaction, the cells were transformed into Trans1T1 competent cells (TransGen Biotech). Kanamycin-resistant clones were selected and identified using the primer pair P81 / P86. The correct band size was 4115 bp. The correct insertion was further verified by sequencing (GENEWIZ) using the primers P81 / P86 / PE962-Bl-check-F1 / PE963-Bl-check-F2 / PE999-alaT-tuf-F / PE1002-alaT-BL-R. The resulting plasmid was named pK18-alaT-Ptuf-Bl-xfp(13032).

[0258] 2.2.2 Construction of pK18-cg3384-Ptuf-Bl_xfp(13032) plasmid

[0259] The upper homology arm UP was amplified using the genome of Corynebacterium glutamicum ATCC13032 as a template using the primer pair PE1101-3384-UP-1F / PE1102-3384-UP-1R. The lower homology arm DN was amplified using the primer pair P14-14 / PE1104-3384-DN-2R. The Ptuf-Bl_xfp fragment was obtained by amplification using the primer pair PE1105-xfp-F / P14-13 using pK18-odhA-Ptuf-Bl_xfp as a template. The resulting fragment was purified and recovered using an agarose gel recovery kit (Tiangen) and stored at -20°C until further use.

[0260] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP, DN, and Ptuf-Bl_xfp fragments were mixed with the double-digested vector in the appropriate proportions using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech). After pipetting to mix thoroughly, the cells were placed in a metal bath (Golden Ginkgo Biotechnology Co., Ltd.) at 37°C for 30 minutes for ligation. After completion of the reaction, Trans1T1 competent cells (TransGenBiotech) were transformed. Kanamycin-resistant clones were selected and identified using the primer pair P81 / P86. The correct band size was 4143 bp. The correct insertion was further verified by sequencing (GENEWIZ) using the primers P81 / P86 / PE962-Bl-check-F1 / PE963-Bl-check-F2 / PE1105-xfp-F / P14-13. The resulting plasmid was named pK18-cg3384-Ptuf-Bl_xfp(13032).

[0261] 2.2.3 Construction of pK18-cg3364-Ptuf-Bl_xfp(13032) plasmid

[0262] The genome of Corynebacterium glutamicum ATCC13032 was used as a template, and primer pairs PE1117-3364-UP-1F / PE1118-3364-UP-1R and PE1119-3364-DN-2F / PE1120-3364-DN-2R were used to amplify the upstream and downstream homology arms. The Ptuf-Bl_xfp fragment was obtained by amplification using the primer pair PE1121-xfp-F / PE1122-xfp-R, using pK18-odhA-Ptuf-Bl_xfp as a template. The resulting fragment was purified using an agarose gel recovery kit (Tiangen) and stored frozen at -20°C until further use.

[0263] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP, DN, and Ptuf-Bl_xfp fragments were mixed with the double-digested vector in the appropriate proportions using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech). After pipetting to mix thoroughly, the cells were placed in a metal bath (Golden Ginkgo Biotechnology Co., Ltd.) at 37°C for 30 minutes for ligation. After completion of the reaction, Trans1T1 competent cells (TransGenBiotech) were transformed. Kanamycin-resistant clones were selected and identified using the primer pair P81 / P86. The correct band size was 4128 bp. The correct insertion was further verified by sequencing (GENEWIZ) using the primers P81 / P86 / PE962-Bl-check-F1 / PE963-Bl-check-F2 / PE1121-xfp-F / PE1122-xfp-R. The resulting plasmid was named pK18-cg3364-Ptuf-Bl_xfp(13032).

[0264] 2.2.4 Construction of pK18-cg0928-Ptuf-Bl_xfp(13032) plasmid

[0265] The genome of Corynebacterium glutamicum ATCC13032 was used as a template, and primers PE1125-0928-UP-1F / P13-12 and P13-15 / P14-17 were used to amplify the upstream and downstream homology arms. The Ptuf-Bl_xfp fragment was obtained by amplification using the primer pair P13-13 / P13-14, using pK18-odhA-Ptuf-Bl_xfp as a template. The resulting fragment was purified using an agarose gel recovery kit (Tiangen) and stored frozen at -20°C until further use.

[0266] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP, DN, and Ptuf-Bl_xfp fragments were mixed with the double-digested vector in the appropriate proportions using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech). After pipetting to mix thoroughly, the cells were placed in a metal bath (Golden Ginkgo Biotechnology Co., Ltd.) at 37°C for 30 minutes for ligation. After completion of the reaction, Trans1T1 competent cells (TransGenBiotech) were transformed. Kanamycin-resistant clones were selected and identified using the primer pair P81 / P86. The correct band size was 4154 bp. The correct insertion was further verified by sequencing (GENEWIZ) using the primers P81 / P86 / PE962-Bl-check-F1 / PE963-Bl-check-F2 / P13-13 / P13-14. The resulting plasmid was named pK18-cg0928-Ptuf-Bl_xfp(13032).

[0267] 2.2.5 Construction of pK18-cg3125-Ptuf-Bl_xfp(13032) plasmid

[0268] The genome of Corynebacterium glutamicum ATCC13032 was used as a template, and primers PE1357-3125-UP-1F / PE1358-3125-UP-1R and PE1359-3125-DN-2F / PE1360-3125-DN-2R were used to amplify the upstream and downstream homology arms. The Ptuf-Bl_xfp fragment was obtained by amplification using pK18-odhA-Ptuf-Bl_xfp as a template and primers PE1361-XFP-F / PE1362-XFP-R. The resulting fragment was purified using an agarose gel extraction kit (Tiangen) and stored frozen at -20°C until further use.

[0269] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP, DN, and Ptuf-Bl_xfp fragments were mixed with the double-digested vector in the appropriate proportions using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech). After pipetting to mix thoroughly, the cells were placed in a metal bath (Golden Ginkgo Biotechnology Co., Ltd.) at 37°C for 30 minutes for ligation. After completion of the reaction, Trans1T1 competent cells (TransGenBiotech) were transformed. Kanamycin-resistant clones were selected and identified using the primer pair P81 / P86. The correct band size was 4137 bp. The correct insertion was further verified by sequencing (GENEWIZ) using the primers P81 / P86 / PE962-Bl-check-F1 / PE963-Bl-check-F2 / PE1361-XFP-F / PE1362-XFP-R. The resulting plasmid was named pK18-cg3125-Ptuf-Bl_xfp(13032).

[0270] 2.2.6 Construction of pK18-cg2564-Ptuf-Bl_xfp(13032) plasmid

[0271] The genome of Corynebacterium glutamicum ATCC13032 was used as a template, and primer pairs PE1351-2564-UP-1F / PE1352-2564-UP-1R and PE1353-2564-DN-2F / PE1354-2564-DN-2R were used to amplify the upstream and downstream homology arms. The Ptuf-Bl_xfp fragment was obtained by amplification using the primer pair PE1355-xfp-F and PE1356-xfp-R using pK18-odhA-Ptuf-Bl_xfp as a template. The resulting fragment was purified using an agarose gel recovery kit (Tiangen) and stored frozen at -20°C until further use.

[0272] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP, DN, and Ptuf-Bl_xfp fragments were mixed with the double-digested vector in the appropriate proportions using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech). After pipetting to mix thoroughly, the cells were placed in a metal bath (Golden Ginkgo Biotechnology Co., Ltd.) at 37°C for 30 minutes for ligation. After completion of the reaction, Trans1T1 competent cells (TransGenBiotech) were transformed. Kanamycin-resistant clones were selected and identified using the primer pair P81 / P86. The correct band size was 4138 bp. The correct insertion was further verified by sequencing (GENEWIZ) using the primers P81 / P86 / PE962-Bl-check-F1 / PE963-Bl-check-F2 / PE1355-xfp-F / PE1356-xfp-R. The resulting plasmid was named pK18-cg2564-Ptuf-Bl_xfp(13032).

[0273] 2.2.7 Construction of pK18-BBD29-07270-Ptuf-Bl_xfp(13032) plasmid

[0274] The genome of Corynebacterium glutamicum ATCC13032 was used as a template, and primers PE1407-7270-UP-1F / PE1408-7270-UP-1R and PE1409-7270-DN-2F / PE1410-7270-DN-2R were used to amplify the upstream and downstream homology arms. The Ptuf-Bl_xfp fragment was obtained by amplification using pK18-odhA-Ptuf-Bl_xfp as a template and primers PE1411-xfp-F / PE1412-xfp-R. The resulting fragment was purified using an agarose gel extraction kit (Tiangen) and stored frozen at -20°C until further use.

[0275] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP, DN, and Ptuf-Bl_xfp fragments were mixed with the double-digested vector in the appropriate proportions using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech). After pipetting and mixing, the cells were placed in a metal bath (Golden Ginkgo Biotechnology Co., Ltd.) at 37°C for 30 minutes for ligation. After the reaction, Trans1T1 competent cells (TransGenBiotech) were transformed, and kanamycin-resistant clones were selected and identified using the primer pair P81 / P86. The correct band size was 4130 bp. The correct insertion was further verified by sequencing (GENEWIZ) using the primers P81 / P86 / PE962-Bl-check-F1 / PE963-Bl-check-F2 / PE1411-xfp-F / PE1412-xfp-R. The resulting plasmid was named pK18-BBD29-07270-Ptuf-Bl_xfp(13032).

[0276] 2.2.8 Construction of pK18-pfk-A1T(13032) plasmid

[0277] Primers were designed using the genome of Corynebacterium glutamicum ATCC13032 as a template. The primer sequences are shown in Table 1. PCR amplification was performed using Phusion Super-Fidelity Polymerase (New England BioLabs). Upstream and downstream homology arms were obtained by reverse PCR amplification using primer pairs PE878-pfk-TTG-UP-1F / PE879-pfk-TTG-UP-1R and PE880-pfk-TTG-DN-2F / PE881-pfk-UP-2R, respectively. The resulting fragments were purified and recovered using an agarose gel recovery kit (Tiangen) and stored at -20°C until further use.

[0278] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP and DN fragments were mixed with the double-digested vector in the appropriate ratio using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech). After pipetting and mixing, the cells were placed in a metal bath (Golden Ginkgo Biotechnology Co., Ltd.) at 37°C for 30 minutes for ligation. After the reaction, Trans1T1 competent cells (TransGen Biotech) were transformed, and kanamycin-resistant clones were selected and identified using the primer pair P81 / P86. The correct band size was 1316 bp. The correct insertion was further confirmed by sequencing (GENEWIZ) using the primers P81 / P86. The resulting plasmid was named pK18-pfk-A1T(13032).

[0279] 2.2.9 Construction of pK18-Pzwf-pfk(13032) plasmid

[0280] Primers were designed using the genome of Corynebacterium glutamicum ATCC13032 as a template, and the primer sequences are shown in Table 1. PCR amplification was performed using Phusion ultra-fidelity polymerase (New England BioLabs). PE882-pfk-UP-1F / PE883-pfk-UP-1R and PE884-pfk-DN-2F / PE885-pfk-UP-2R were used as primer pairs, and upstream and downstream homology arms were obtained by PCR reverse amplification. Using the genome of Corynebacterium glutamicum ATCC13032 as a template, PE886-Pzwf-F / PE887-Pzwf-R were used as primer pairs to amplify the Pzwf promoter. The resulting fragment was purified and recovered using an agarose gel recovery kit (Tian Gen) and stored at -20°C for future use.

[0281] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP, DN, and Pzwf promoters were then mixed with the double-digested vector in the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech Co., Ltd.) in the appropriate proportions. After pipetting and mixing, the cells were placed in a metal bath (Golden Ginkgo Biotechnology Co., Ltd.) at 37°C for 30 minutes for ligation. After the reaction, Trans1T1 competent cells (TransGenBiotech) were transformed, and kanamycin-resistant clones were selected and identified using the primer pair P81 / P86. The correct band size was 1749 bp. The correct insertion was further confirmed by sequencing (GENEWIZ) using the primers P81 / P86 / PE886-Pzwf-F. The resulting plasmid was named pK18-Pzwf-pfk(13032).

[0282] 2.2.10 Construction of pK18-PrecA-pfk(13032) Plasmid

[0283] Primers were designed using the genome of Corynebacterium glutamicum ATCC13032 as a template, and the primer sequences are shown in Table 1. PCR amplification was performed using Phusion ultra-fidelity polymerase (New England BioLabs). PE882-pfk-UP-1F / PE883-pfk-UP-1R and PE884-pfk-DN-2F / PE885-pfk-UP-2R were used as primer pairs, and upstream and downstream homology arms were obtained by PCR reverse amplification. The PrecA promoter was amplified using the genome of Corynebacterium glutamicum ATCC13032 as a template and PE889-PrecA-F / PE890-PrecA-R as primer pairs. The resulting fragment was purified and recovered using an agarose gel recovery kit (Tiangen) and stored at -20°C for future use.

[0284] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP, DN, and PrecA promoters were then mixed with the double-digested vector in the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech Co., Ltd.) in the appropriate proportions. After pipetting and mixing, the cells were placed in a metal bath (Golden Ginkgo Biotechnology Co., Ltd.) at 37°C for 30 minutes for ligation. After the reaction, Trans1T1 competent cells (TransGenBiotech) were transformed, and kanamycin-resistant clones were selected and identified using the primer pair P81 / P86. The correct band size was 1765 bp. Sequencing (GENEWIZ) was further performed to confirm the correct insertion of the fragment, using the primers P81 / P86 / PE889-PrecA-F. The resulting plasmid was named pK18-PrecA-pfk(13032).

[0285] 2.2.11 Construction of pK18-pfk-E171D(13032) plasmid

[0286] Primers were designed using the genome of Corynebacterium glutamicum ATCC13032 as a template. The primer sequences are shown in Table 1. PCR amplification was performed using Phusion high-fidelity polymerase (New England BioLabs). Using PE892-pfk-E171K-UP-1F / PE893-pfk-E171K-UP-1R and PE897-pfk-E171D-DN-2F / PE895-pfk-E171K-DN-2R as primer pairs, upstream and downstream homology arms were obtained by reverse PCR amplification. The resulting fragments were purified and recovered using an agarose gel recovery kit (Tiangen) and stored at -20°C for later use.

[0287] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP and DN fragments were mixed with the double-digested vector in a specific ratio using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech). After pipetting and mixing, the cells were placed in a metal bath (Golden Ginkgo Biotechnology Co., Ltd.) at 37°C for 3 minutes for ligation. After the reaction, Trans1T1 competent cells (TransGen Biotech) were transformed. Kanamycin-resistant clones were selected and identified using the primer pair P81 / P86. The correct band size was 1327 bp. Sequencing (GENEWIZ) was further performed to confirm the correct insertion of the fragment, using the primers P81 / P86. The resulting plasmid was named pK18-pfk-E171D (13032).

[0288] 2.3 Starting from ATCC14067, construction of XFP multi-copy and PFK transformation plasmids

[0289] 2.3.1 Construction of pK18-alaT-Ptuf-Bl_xfp(14067) plasmid

[0290] UP was obtained by PCR amplification using the genome of Corynebacterium glutamicum ATCC14067 as a template and the primer pair PE1021-alaT-UP-1F / PE997-rev-alaT-R. DN was obtained by PCR amplification using the primer pair PE998-rev-alaT-R / PE1022-alaT-DN-2R. The Ptuf promoter was obtained by PCR amplification using the genome of Corynebacterium glutamicum ATCC13032 as a template and the primer pair PE999-alaT-tuf-F / PE1000-alaT-tuf-R. The Bl_xfp fragment was amplified by PCR using the pXMJ19-PcspB-Bl-xfp plasmid (the xfp expression plasmid was synthesized by GENEWIZ and assembled with the provided pXMJ19-PcspB vector) as a template and the primer pair PE1001-alaT-BL-F / PE1002-alaT-BL-R. The resulting fragment was purified using an agarose gel extraction kit (Tiangen) and stored at -20°C until further use.

[0291] The amplified UP, DN, promoter, and gene fragments were mixed in the specified proportions using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech Co., Ltd.), pipetted to mix thoroughly, and then placed in a metal bath (Golden Ginkgo Biotechnology Co., Ltd.) for ligation at 37°C for 30 minutes. After completion of the reaction, the cells were transformed into Trans1T1 competent cells (TransGen Biotech). Kanamycin-resistant clones were selected and identified using the primer pair P81 / P86. The correct band size was 4115 bp. The correct insertion was further verified by sequencing (GENEWIZ) using the primers P81 / P86 / PE962-Bl-check-F1 / PE963-Bl-check-F2 / PE999-alaT-tuf-F / PE1002-alaT-BL-R. The resulting plasmid was named pK18-alaT-Ptuf-Bl-xfp(14067).

[0292] 2.3.2 Construction of pK18-cg3384-Ptuf-Bl_xfp(14067) plasmid

[0293] The upper homology arm UP was amplified using the genome of Corynebacterium glutamicum ATCC14067 as a template using the primer pair PE1101-3384-UP-1F / PE1102-3384-UP-1R. The lower homology arm DN was amplified using the primer pair P14-14 / PE1104-3384-DN-2R. The Ptuf-Bl_xfp fragment was amplified using the primer pair PE1105-xfp-F / P14-13 using pK18-odhA-Ptuf-Bl_xfp as a template. The resulting fragment was purified using an agarose gel recovery kit (Tiangen) and stored frozen at -20°C until further use.

[0294] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP, DN, and Ptuf-Bl_xfp fragments were mixed with the double-digested vector in the appropriate proportions using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech). After pipetting to mix thoroughly, the cells were placed in a metal bath (Golden Ginkgo Biotechnology Co., Ltd.) at 37°C for 30 minutes for ligation. After completion of the reaction, Trans1T1 competent cells (TransGenBiotech) were transformed. Kanamycin-resistant clones were selected and identified using the primer pair P81 / P86. The correct band size was 4143 bp. The correct insertion was further verified by sequencing (GENEWIZ) using the primers P81 / P86 / PE962-Bl-check-F1 / PE963-Bl-check-F2 / PE1105-xfp-F / P14-13. The resulting plasmid was named pK18-cg3384-Ptuf-Bl_xfp(14067).

[0295] 2.3.3 Construction of pK18-cg3364-Ptuf-Bl_xfp(14067) plasmid

[0296] The genome of Corynebacterium glutamicum ATCC14067 was used as a template, and primer pairs PE1117-3364-UP-1F / PE1118-3364-UP-1R and PE1119-3364-DN-2F / PE1120-3364-DN-2R were used to amplify the upstream and downstream homology arms. The Ptuf-Bl_xfp fragment was obtained by amplification using the primer pair PE1121-xfp-F / PE1122-xfp-R, using pK18-odhA-Ptuf-Bl_xfp as a template. The resulting fragment was purified using an agarose gel recovery kit (Tiangen) and stored frozen at -20°C until further use.

[0297] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP, DN, and Ptuf-Bl_xfp fragments were mixed with the double-digested vector in the appropriate proportions using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech). After pipetting to mix thoroughly, the cells were placed in a metal bath (Golden Ginkgo Biotechnology Co., Ltd.) at 37°C for 30 minutes for ligation. After completion of the reaction, Trans1T1 competent cells (TransGenBiotech) were transformed. Kanamycin-resistant clones were selected and identified using the primer pair P81 / P86. The correct band size was 4128 bp. The correct insertion was further verified by sequencing (GENEWIZ) using the primers P81 / P86 / PE962-Bl-check-F1 / PE963-Bl-check-F2 / PE1121-xfp-F / PE1122-xfp-R. The resulting plasmid was named pK18-cg3364-Ptuf-Bl_xfp(14067).

[0298] 2.3.4 Construction of pK18-cg0928-Ptuf-Bl_xfp(14067) plasmid

[0299] The genome of Corynebacterium glutamicum ATCC14067 was used as a template, and primers PE1125-0928-UP-1F / P14-15 and PE1127-0928-DN-2F / P14-17 were used to amplify the upstream and downstream homology arms. The Ptuf-Bl_xfp fragment was obtained by amplification using the primers P14-16 / PE1130-xfp-R and pK18-odhA-Ptuf-Bl_xfp template. The resulting fragment was purified using an agarose gel recovery kit (Tiangen) and stored frozen at -20°C until further use.

[0300] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP, DN, and Ptuf-Bl_xfp fragments were mixed with the double-digested vector in the appropriate proportions using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech). After pipetting to mix thoroughly, the cells were placed in a metal bath (Golden Ginkgo Biotechnology Co., Ltd.) at 37°C for 30 minutes for ligation. After completion of the reaction, Trans1T1 competent cells (TransGenBiotech) were transformed. Kanamycin-resistant clones were selected and identified using the primer pair P81 / P86. The correct band size was 4154 bp. The correct insertion was further verified by sequencing (GENEWIZ) using the primers P81 / P86 / PE962-Bl-check-F1 / PE963-Bl-check-F2 / P14-16 / PE1130-xfp-R. The resulting plasmid was named pK18-cg0928-Ptuf-Bl_xfp(14067).

[0301] 2.3.5 Construction of pK18-cg3125-Ptuf-Bl_xfp(14067) plasmid

[0302] The genome of Corynebacterium glutamicum ATCC14067 was used as a template, and the primer pairs P14-7 / PE1358-3125-UP-1R and P14-9 / P14-10 were used to amplify the upstream and downstream homology arms. The Ptuf-Bl_xfp fragment was obtained by amplification using the primer pair PE1361-XFP-F / P14-8, using pK18-odhA-Ptuf-Bl_xfp as a template. The resulting fragment was purified using an agarose gel recovery kit (Tiangen) and stored frozen at -20°C until further use.

[0303] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP, DN, and Ptuf-Bl_xfp fragments were mixed with the double-digested vector in the appropriate proportions using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech). After pipetting and mixing, the cells were placed in a metal bath (Golden Ginkgo Biotechnology Co., Ltd.) at 37°C for 30 minutes for ligation. After the reaction, Trans1T1 competent cells (TransGenBiotech) were transformed, and kanamycin-resistant clones were selected and identified using the primer pair P81 / P86. The correct band size was 4137 bp. The correct insertion was further verified by sequencing (GENEWIZ) using the primers P81 / P86 / PE962-Bl-check-F1 / PE963-Bl-check-F2 / PE1361-XFP-F / P14-8. The resulting plasmid was named pK18-cg3125-Ptuf-Bl_xfp(14067).

[0304] 2.3.6 Construction of pK18-cg2564-Ptuf-Bl_xfp(14067) plasmid

[0305] The genome of Corynebacterium glutamicum ATCC14067 was used as a template, and primer pairs PE1351-2564-UP-1F / PE1352-2564-UP-1R and PE1353-2564-DN-2F / PE1354-2564-DN-2R were used to amplify the upstream and downstream homology arms. The Ptuf-Bl_xfp fragment was obtained by amplification using the primer pair PE1355-xfp-F and PE1356-xfp-R using pK18-odhA-Ptuf-Bl_xfp as a template. The resulting fragment was purified using an agarose gel recovery kit (Tiangen) and stored frozen at -20°C until further use.

[0306] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP, DN, and Ptuf-Bl_xfp fragments were mixed with the double-digested vector in the appropriate proportions using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech). After pipetting to mix thoroughly, the cells were placed in a metal bath (Golden Ginkgo Biotechnology Co., Ltd.) at 37°C for 30 minutes for ligation. After completion of the reaction, Trans1T1 competent cells (TransGenBiotech) were transformed. Kanamycin-resistant clones were selected and identified using the primer pair P81 / P86. The correct band size was 4138 bp. The correct insertion was further verified by sequencing (GENEWIZ) using the primers P81 / P86 / PE962-Bl-check-F1 / PE963-Bl-check-F2 / PE1355-xfp-F / PE1356-xfp-R. The resulting plasmid was named pK18-cg2564-Ptuf-Bl_xfp(14067).

[0307] 2.3.7 Construction of pK18-BBD29-07270-Ptuf-Bl_xfp(14067) plasmid

[0308] The genome of Corynebacterium glutamicum ATCC14067 was used as a template, and primers PE1407-7270-UP-1F / PE1408-7270-UP-1R and PE1409-7270-DN-2F / PE1410-7270-DN-2R were used to amplify the upstream and downstream homology arms. The Ptuf-Bl_xfp fragment was obtained by amplification using pK18-odhA-Ptuf-Bl_xfp as a template and primers PE1411-xfp-F / PE1412-xfp-R. The resulting fragment was purified using an agarose gel extraction kit (Tiangen) and stored frozen at -20°C until further use.

[0309] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP, DN, and Ptuf-Bl_xfp fragments were mixed with the double-digested vector in the appropriate proportions using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech). After pipetting and mixing, the cells were placed in a metal bath (Golden Ginkgo Biotechnology Co., Ltd.) at 37°C for 30 minutes for ligation. After the reaction, Trans1T1 competent cells (TransGenBiotech) were transformed, and kanamycin-resistant clones were selected and identified using the primer pair P81 / P86. The correct band size was 4130 bp. The correct insertion was further verified by sequencing (GENEWIZ) using the primers P81 / P86 / PE962-Bl-check-F1 / PE963-Bl-check-F2 / PE1411-xfp-F / PE1412-xfp-R. The resulting plasmid was named pK18-BBD29-07270-Ptuf-Bl_xfp(14067).

[0310] 2.3.8 Construction of pK18-pfk-A1T(14067) plasmid

[0311] Primers were designed using the genome of Corynebacterium glutamicum ATCC14067 as a template. The primer sequences are shown in Table 1. PCR amplification was performed using Phusion high-fidelity polymerase (New England BioLabs). Upstream and downstream homology arms were obtained by reverse PCR amplification using primer pairs PE878-pfk-TTG-UP-1F / PE879-pfk-TTG-UP-1R and PE880-pfk-TTG-DN-2F / PE881-pfk-UP-2R, respectively. The resulting fragments were purified and recovered using an agarose gel recovery kit (Tiangen) and stored at -20°C for later use.

[0312] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP and DN fragments were mixed with the double-digested vector in the appropriate ratio using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech). After pipetting and mixing, the mixture was placed in a metal bath (Ginko Biotech) at 37°C for 30 minutes for ligation. After the reaction, Trans1T1 competent cells (TransGen Biotech) were transformed, and kanamycin-resistant clones were selected and identified using the primer pair P81 / P86. The correct band size was 1316 bp. The correct insertion was further confirmed by sequencing (GENEWIZ) using the primers P81 / P86. The resulting plasmid was named pK18-pfk-A1T(14067).

[0313] 2.3.9 Construction of pK18-Pzwf-pfk(14067) plasmid

[0314] Primers were designed using the genome of Corynebacterium glutamicum ATCC14067 as a template, and the primer sequences are shown in Table 1. PCR amplification was performed using Phusion ultra-fidelity polymerase (New England BioLabs). PE882-pfk-UP-1F / PE883-pfk-UP-1R and PE884-pfk-DN-2F / PE885-pfk-UP-2R were used as primer pairs, and upstream and downstream homology arms were obtained by PCR reverse amplification. The Pzwf promoter was amplified using the genome of Corynebacterium glutamicum ATCC13032 (GenBank NO: NC_003450.3) as a template and PE886-Pzwf-F / PE887-Pzwf-R as a primer pair. The resulting fragment was purified and recovered using an agarose gel recovery kit (Tian Gen) and stored at -20°C for later use.

[0315] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP, DN, and Pzwf promoters were then mixed with the double-digested vector in the appropriate proportions using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech). After pipetting and mixing, the cells were placed in a metal bath (Ginko Biotechnology Co., Ltd.) at 37°C for 30 minutes for ligation. After the reaction, Trans1T1 competent cells (TransGenBiotech) were transformed, and kanamycin-resistant clones were selected and identified using the primer pair P81 / P86. The correct band size was 1749 bp. The correct insertion was further confirmed by sequencing (GENEWIZ) using the primers P81 / P86 / PE886-Pzwf-F. The resulting plasmid was named pK18-Pzwf-pfk(14067).

[0316] 2.3.10 Construction of pK18-PrecA-pfk(14067) Plasmid

[0317] Primers were designed using the genome of Corynebacterium glutamicum ATCC14067 as a template, and the primer sequences are shown in Table 1. PCR amplification was performed using Phusion ultra-fidelity polymerase (New England BioLabs). PE882-pfk-UP-1F / PE883-pfk-UP-1R and PE884-pfk-DN-2F / PE885-pfk-UP-2R were used as primer pairs, and upstream and downstream homology arms were obtained by PCR reverse amplification. The PrecA promoter was amplified using the genome of Corynebacterium glutamicum ATCC13032 (GenBank NO: NC_003450.3) as a template and PE889-PrecA-F / PE890-PrecA-R as primer pairs. The resulting fragment was purified and recovered using an agarose gel recovery kit (Tian Gen) and stored at -20°C for later use.

[0318] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP, DN, and PrecA promoters were mixed with the double-digested vector in the appropriate proportions using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech). After pipetting and mixing, the cells were placed in a metal bath (Golden Ginkgo Biotechnology Co., Ltd.) at 37°C for 30 minutes for ligation. After the reaction, Trans1T1 competent cells (TransGenBiotech) were transformed, and kanamycin-resistant clones were selected and identified using the primer pair P81 / P86. The correct band size was 1765 bp. Sequencing (GENEWIZ) was further performed to confirm the correct insertion of the fragment using the primers P81 / P86 / PE889-PrecA-F. The resulting plasmid was named pK18-PrecA-pfk(14067).

[0319] 2.3.11 Construction of pK18-pfk-E171D(14067) plasmid

[0320] Primers were designed using the genome of Corynebacterium glutamicum ATCC14067 as a template. The primer sequences are shown in Table 1. PCR amplification was performed using Phusion high-fidelity polymerase (New England BioLabs). Upstream and downstream homology arms were obtained by reverse PCR amplification using primer pairs PE892-pfk-E171K-UP-1F / PE893-pfk-E171K-UP-1R and PE897-pfk-E171D-DN-2F / P14-18. The resulting fragments were purified and recovered using an agarose gel recovery kit (Tiangen) and stored at -20°C until further use.

[0321] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP and DN fragments were mixed with the double-digested vector in the appropriate ratio using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech). After pipetting and mixing, the cells were placed in a metal bath (Golden Ginkgo Biotechnology Co., Ltd.) at 37°C for 3 minutes for ligation. After the reaction, Trans1T1 competent cells (TransGen Biotech) were transformed. Kanamycin-resistant clones were selected and identified using the primer pair P81 / P86. The correct band size was 1327 bp. Sequencing (GENEWIZ) was further performed to confirm the correct insertion of the fragment, using the primers P81 / P86. The resulting plasmid was named pK18-pfk-E171D (14067).

[0322] Example 3: Construction of strains overexpressing xfp from different sources and their glutamate conversion rates

[0323] 3.1WJ0140-pXMJ19-PcspB-Ll-xfp

[0324] Prepare WJ0140 competent cells according to the Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), the recombinant plasmid pXMJ19-PcspB-L1-xfp is changed into Corynebacterium glutamicum WJ0140 competent cells with the electrotransformation method, and the recombinant is screened on the selective medium containing 5mg / L chloramphenicol. By PCR amplification of the target sequence (PE813-L1-XFP-1F, PE814-L1-XFP-2R), nucleotide sequencing analysis, obtain importing the plasmid recombinant microorganism strain containing the target gene.

[0325] 3.2WJ0140-pXMJ19-PcspB-Pa-xfp

[0326] WJ0140 competent cells were prepared according to the classical method for Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23). The recombinant plasmid pXMJ19-PcspB-Pa-xfp was transformed into Corynebacterium glutamicum WJ0140 competent cells by electroporation, and recombinants were screened on a selective medium containing 5 mg / L chloramphenicol. The target sequences (PE817-Pa-XFP-1F and PE818-Pa-XFP-2R) were amplified by PCR and analyzed by nucleotide sequencing to obtain a recombinant microbial strain containing the plasmid containing the target gene.

[0327] 3.3WJ0140-pXMJ19-PcspB-Bb-xfp

[0328] WJ0140 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23). The recombinant plasmid pXMJ19-PcspB-Bb-xfp was transformed into Corynebacterium glutamicum WJ0140 competent cells by electroporation, and recombinants were screened on a selective medium containing 5 mg / L chloramphenicol. The target sequences (PE823-Bb-XFP-1F and PE824-Bb-XFP-2R) were amplified by PCR and analyzed by nucleotide sequencing to obtain a recombinant microbial strain containing the plasmid containing the target gene.

[0329] 3.4WJ0140-pXMJ19-PcspB-Bm-xfp

[0330] Prepare WJ0140 competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Charpter 23), recombinant plasmid pXMJ19-PcspB-Bm-xfp is changed into Corynebacterium glutamicum WJ0140 competent cell with electrotransformation method, on the selection medium that contains the chloramphenicol of 5mg / L, screen recombinant.By pcr amplification target sequence (PE807-Bm-XFP-1F, PE810-Bm-XFP-2R), sending and testing primer is PE807-Bm-XFP-1F / PE810-Bm-XFP-2R / PE808-Bm-XFP-1R / PE809-Bm-XFP-2F.By nucleotide sequencing analysis, obtain and import the plasmid recombinant microorganism strain that contains target gene.

[0331] 3.5WJ0140-pXMJ19-PcspB-Lm-xfp

[0332] WJ0140 competent cells were prepared according to the classical method for Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23). The recombinant plasmid pXMJ19-PcspB-Lm-xfp was transformed into Corynebacterium glutamicum WJ0140 competent cells by electroporation, and recombinants were screened on a selective medium containing 5 mg / L chloramphenicol. The target sequences (PE815-Lm-XFP-1F and PE816-Lm-XFP-2R) were amplified by PCR and analyzed by nucleotide sequencing to obtain a recombinant microbial strain containing the plasmid containing the target gene.

[0333] 3.6WJ0140-pXMJ19-PcspB-Bab-xfp

[0334] WJ0140 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23). The recombinant plasmid pXMJ19-PcspB-Bab-xfp was transformed into Corynebacterium glutamicum WJ0140 competent cells by electroporation, and recombinants were screened on a selective medium containing 5 mg / L chloramphenicol. The target sequences (PE807-Bm-XFP-1F and PE810-Bm-XFP-2R) were amplified by PCR and analyzed by nucleotide sequencing to obtain a recombinant microbial strain containing the plasmid containing the target gene.

[0335] 3.7WJ0140-pXMJ19-PcspB-An-xfp

[0336] WJ0140 competent cells were prepared according to the classical method for Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23). The recombinant plasmid pXMJ19-PcspB-An-xfp was transformed into Corynebacterium glutamicum WJ0140 competent cells by electroporation, and recombinants were screened on a selective medium containing 5 mg / L chloramphenicol. The target sequences (PE819-An-XFP-1F and PE820-An-XFP-2R) were amplified by PCR and analyzed by nucleotide sequencing to obtain a recombinant microbial strain containing the plasmid containing the target gene.

[0337] 3.8WJ0140-pXMJ19-PcspB-Ban-xfp

[0338] WJ0140 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23). The recombinant plasmid pXMJ19-PcspB-Ban-xfp was transformed into Corynebacterium glutamicum WJ0140 competent cells by electroporation, and recombinants were screened on a selective medium containing 5 mg / L chloramphenicol. The target sequences (PE821-Ban-XFP-1F and PE822-Ban-XFP-2R) were amplified by PCR and analyzed by nucleotide sequencing to obtain a recombinant microbial strain containing the plasmid containing the target gene.

[0339] 3.9WJ0140-pXMJ19-PcspB-Bl-xfp

[0340] WJ0140 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23). The recombinant plasmid pXMJ19-PcspB-Bl-xfp was transferred into Corynebacterium glutamicum WJ0140 competent cells by electroporation, and recombinants were screened on a selective medium containing 5 mg / L chloramphenicol. The target sequences (PE811-Bl-XFP-1F and PE812-Bl-XFP-2R) were amplified by PCR and analyzed by nucleotide sequencing to obtain a recombinant microbial strain containing the plasmid containing the target gene.

[0341] 3.10WJ0140-pXMJ19 no load

[0342] WJ0140 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and the empty plasmid pXMJ19 was transformed into Corynebacterium glutamicum WJ0140 competent cells by electroporation, and screened on a selective medium containing 5 mg / L chloramphenicol.

[0343] 3.11 Shake flask validation

[0344] We performed shake flask fermentation on the above 10 expression bacteria to verify their glutamic acid production performance. For specific shake flask method, please refer to the shake flask fermentation method in Section 1.1.3.

[0345] The specific fermentation results are shown in the table below. Compared to the empty vector, expression of phosphoketolase from different sources increased glutamate production and conversion efficiency to varying degrees. Compared to a reported sequence (Bmxfp), expression of Bl-xfp resulted in better strain growth and a glutamate conversion rate of 59.3%, so this sequence was selected for subsequent transformation.

[0346] Table 6. Conversion rate of glutamate in strains with different sources of xfp

[0347]

[0348] Example 4: Construction of xfp multi-copy strains and their glutamate conversion efficiency starting from WJ0140

[0349] 4.1WJ0140-alaT-Ptuf-Bl_xfp(SME762a)xfp one copy

[0350] Prepare WJ0140 competent cells according to the classical method of Corynebacterium glutamicum (C.glutamicum Handbook, Chapter 23). Recombinant plasmid pK18-alaT-Ptuf-Bl_xfp is transferred into Corynebacterium glutamicum WJ0140 competent cells with electroporation method. Cultures are carried out on BHI medium flat board containing 15mg / L kanamycin for 48 hours. The primary identification primers are P81 / PE1022-alaT-DN-2R and PE1021-alaT-UP-1F / P86. The recombinant obtained by screening is inoculated into non-anti-BHI medium and cultured for 12-14 hours. The culture temperature is 30 ℃, and the culture is shaken on a rotary shaker at 220rpm. After the bacterial liquid is diluted 100-1000 times, it is coated on the solid BHIS medium containing 10% sucrose and cultured for 36 hours. The secondary identification primers are PE1023-alaT-ID-F and PE1024-alaT-ID-R. The correct size was 4054 bp, and the recombinant strain with the correct identification band size was sent to Jinweizhi for sequencing. Subsequently, nucleotide sequencing analysis was performed, and the recombinant microbial strain with the correct sequencing was named SME762a.

[0351] 4.2SME762a-cg3364-Ptuf-Bl_xfp(SME768a)xfp second copy

[0352] Prepare SME762a competent cells according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), recombinant plasmid pK18-cg3364-Ptuf-Bl_xfp is changed into Corynebacterium glutamicum SME762a competent cells with electrotransformation method, on the selection medium that contains the kanamycin of 15mg / L, screen a recombinant.Primary identification primer is P86 / PE1117-3364-UP-1F, PE1120-3364-DN-2R / P81.The recombinant that sieves is inoculated into and cultivated 12-14 hour in no anti-BHI substratum, cultivation temperature is 30 ℃, rotary shaker 220rpm shaking culture.Bacterial liquid is diluted 100-1000 times after being coated on the solid BHIS substratum that contains 10% sucrose and cultivated 36 hours.Secondary identification primer is PE1123-ID-F, PE1124-ID-R. The recombinant bacteria with the correct identification band size were sent to Jinweizhi Company for sequencing, followed by nucleotide sequencing analysis, and the recombinant microbial strain with the correct sequencing was named SME768a.

[0353] 4.3SME768a-cg2564-Ptuf-Bl_xfp (SME777a) xfp three copies

[0354] Prepare SME768a competent cells according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), recombinant plasmid pK18-cg2564-Ptuf-Bl_xfp is changed into Corynebacterium glutamicum SME768a competent cells with electrotransformation method, on the selection medium that contains the kanamycin of 15mg / L, screen a recombinant.Primary identification primer is P86 / PE1351-2564-UP-1F, PE1354-2564-DN-2R / P81.The recombinant that sieves is inoculated into and cultivated 12-14 hour in no anti-BHI substratum, cultivation temperature is 30 ℃, rotary shaker 220rpm shaking culture.Bacterial liquid is diluted 100-1000 times after being coated on the solid BHIS substratum that contains 10% sucrose and cultivated 36 hours.Secondary identification primer is PE1369-ID-F, PE1370-ID-R. The recombinant bacteria with the correct identification band size were sent to Jinweizhi Company for sequencing, followed by nucleotide sequencing analysis, and the recombinant microbial strain with the correct sequencing was named SME777a.

[0355] 4.4SME777a-cg3125-Ptuf-Bl_xfp(SME778a)xfp four copies

[0356] Prepare SME777a competent cells according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), recombinant plasmid pK18-cg3125-Ptuf-Bl_xfp is changed into Corynebacterium glutamicum SME777a competent cells with electrotransformation method, on the selection medium that contains the kanamycin of 15mg / L, screen a recombinant.Primary identification primer is P86 / PE1357-3125-UP-1F, PE1360-3125-DN-2R / P81.The recombinant that sieves is inoculated into and cultivated 12-14 hour in no anti-BHI substratum, cultivation temperature is 30 ℃, rotary shaker 220rpm shaking culture.Bacterial liquid is diluted 100-1000 times after being coated on the solid BHIS substratum that contains 10% sucrose and cultivated 36 hours.Secondary identification primer is PE1371-ID-F, PE1372-ID-R. The recombinant bacteria with the correct identification band size were sent to Jinweizhi Company for sequencing, followed by nucleotide sequencing analysis, and the recombinant microbial strain with the correct sequencing was named SME778a.

[0357] 4.5SME778a-cg3384-Ptuf-Bl_xfp(SME783a)xfp five copies

[0358] Prepare SME778a competent cells according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), recombinant plasmid pK18-cg3384-Ptuf-Bl_xfp is changed into Corynebacterium glutamicum SME778a competent cells with electrotransformation method, on the selection medium that contains the kanamycin of 15mg / L, screen a recombinant.Primary identification primer is P86 / PE1101-3384-UP-1F, PE1104-3384-DN-2R / P81.The recombinant that sieves is inoculated into and cultivated 12-14 hour in no anti-BHI substratum, cultivation temperature is 30 ℃, rotary shaker 220rpm shaking culture.Bacterial liquid is diluted 100-1000 times after being coated on the solid BHIS substratum that contains 10% sucrose and cultivated 36 hours.Secondary identification primer is PE1107-ID-F, PE1108-ID-R. The recombinant bacteria with the correct identification band size were sent to Jinweizhi Company for sequencing, followed by nucleotide sequencing analysis, and the recombinant microbial strain with the correct sequencing was named SME783a.

[0359] 4.6SME783a-BBD29_7270-Ptuf-Bl_xfp(SME787a)xfp six copies

[0360] Prepare SME783a competent cells according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), recombinant plasmid pK18-BBD29_7270-Ptuf-Bl_xfp is changed into Corynebacterium glutamicum SME783a competent cells with electrotransformation method, on the selection medium that contains the kanamycin of 15mg / L, screen a recombinant. Primary identification primer is P86 / PE1407-7270-UP-1F, PE1410-7270-DN-2R / P81. The recombinant that sieves is inoculated into and cultivated 12-14 hour in no anti-BHI substratum, cultivation temperature is 30 ℃, rotary shaker 220rpm shaking culture. After the bacterial liquid is diluted 100-1000 times, be coated on the solid BHIS substratum that contains 10% sucrose and cultivated 36 hours. Secondary identification primer is PE1425-ID-F, PE1426-ID-R. The recombinant bacteria with the correct identification band size were sent to Jinweizhi Company for sequencing, followed by nucleotide sequencing analysis, and the recombinant microbial strain with the correct sequencing was named SME787a.

[0361] 4.7SME787a-cg0928-Ptuf-Bl_xfp(SME789a)xfp seven copies

[0362] Prepare SME787a competent cells according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), recombinant plasmid pK18-cg0928-Ptuf-Bl_xfp is changed into Corynebacterium glutamicum SME787a competent cells with electrotransformation method, on the selection medium that contains the kanamycin of 15mg / L, screen a recombinant.Primary identification primer is P86 / PE1125-0928-UP-1F, PE1128-0928-DN-2R / P81.The recombinant that sieves is inoculated into and cultivated 12-14 hour in no anti-BHI substratum, cultivation temperature is 30 ℃, rotary shaker 220rpm shaking culture.Bacterial liquid is diluted 100-1000 times after being coated on the solid BHIS substratum that contains 10% sucrose and cultivated 36 hours.Secondary identification primer is PE1131-ID-F, PE1132-ID-R. The recombinant bacteria with the correct identification band size were sent to Jinweizhi Company for sequencing, followed by nucleotide sequencing analysis, and the recombinant microbial strain with the correct sequencing was named SME789a.

[0363] 4.8SME789a-cg2337-Ptuf-Bl_xfp(SME870)xfp eight copies

[0364] Prepare SME789a competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Charpter 23), recombinant plasmid pK18-cg2337-Ptuf-Bl_xfp is changed in the Corynebacterium glutamicum SME789a competent cell with the electrotransformation method, on the selection medium that contains the kanamycin of 15mg / L, screen a recombinant, wherein target gene is inserted in the karyomit(e) because homologous recombination.Once identification primer is PE1345-2337-UP-1F / P86 and P81 / PE1348-2337-DN-2R.The recombinant that sieves is inoculated into and cultivates 12-14h in no anti-YB substratum, and cultivation temperature is 30 ℃, rotary shaking table 220rpm shaking culture.In this culturing process, reorganization takes place second time in transformant, and by homologous recombination exchange, carrier sequence is removed from genome. The bacterial suspension was diluted 100-1000-fold and plated on solid YBS medium containing 10% sucrose and cultured for 36 hours. Strains grown on sucrose medium do not carry the inserted vector sequence in their genomes. The secondary identification primers were PE1367-ID-F / PE1368-ID-R, with a band size of 4101 bp. Recombinant strains with the correct identification band size were sent to Jinweizhi for sequencing using the following primers: PE1367-ID-F / PE1368-ID-R / PE962-Bl-check-F1 / PE963-Bl-check-F2 / PE1349-xfp-F / PE1350-xfp-R. Nucleotide sequencing analysis was then performed, and the recombinant strain identified with the correct sequence was named SME870.

[0365] 4.9SME870a-BBD29-7210-Ptuf-Bl_xfp(SME879)xfp nine copies

[0366] Prepare SME870a competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Charpter 23), recombinant plasmid pK18-cg7210-Ptuf-Bl_xfp is changed in Corynebacterium glutamicum SME870a competent cell with electrotransformation method, on the selection medium that contains the kanamycin of 15mg / L, screen a recombinant, wherein target gene is inserted into karyomit(e) due to homologous recombination.Once identification primer is PPE1389-7210-UP-1F / P86 and P81 / PE1392-7210-DN-2R.The recombinant that sieves is inoculated into and does not have to cultivate 12-14h in the anti-YB substratum, and cultivation temperature is 30 ℃, rotary shaking table 220rpm shaking culture.In this culturing process, transformant second time reorganization takes place, exchanges the carrier sequence from genome, and is removed. The bacterial suspension was diluted 100-1000 times and plated on solid YBS medium containing 10% sucrose and cultured for 36 hours. Strains grown on sucrose medium do not carry the inserted vector sequence in their genomes. The secondary identification primers were PE1419-ID-F / PE1420-ID-R, with a band size of 4101 bp. Recombinant strains with the correct identification band size were sent to Jinweizhi for sequencing using the following primers: PE1419-ID-F / PE1420-ID-R / PE962-Bl-check-F1 / PE963-Bl-check-F2 / PE1393-xfp-F / PE1394-xfp-R. Nucleotide sequencing analysis was then performed, and the recombinant strain identified with the correct sequence was named SME879.

[0367] 4.10SME879a-odhA-Ptuf-Bl_xfp(SME886)xfp ten copies

[0368] Prepare SME879a competent cells according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), recombinant plasmid pK18-odhA-Ptuf-Bl_xfp is changed into Corynebacterium glutamicum SME879a competent cells with electrotransformation method, on the selection medium that contains the kanamycin of 15mg / L, screen a recombinant.One time identification primer is P81 / PE1009-odhA-BL-DN-R, PE1006-odhA-BL-UP-F / P86.The recombinant that sieves is inoculated into and cultivated 12-14 hour in no anti-BHI substratum, and cultivation temperature is 30 ℃, rotary shaking table 220rpm shaking culture.Bacterium liquid is diluted 100-1000 times after being coated on the solid BHIS substratum that contains 10% sucrose and cultivated 36 hours. The secondary identification primers were PE1009-odhA-BL-DN-R / PE1006-odhA-BL-UP-F. Recombinant bacteria with the correct identification band size were sent to Jinweizhi for sequencing, followed by nucleotide sequencing analysis. The recombinant microbial strain with the correct sequence was named SME886.

[0369] 4.11 Shake flask validation

[0370] We conducted shake flask fermentations with the 10 phosphoketolase-expressing strains described above to verify their glutamate production performance. For detailed shake flask methods, refer to the shake flask fermentation method in Section 1.1.3. The fermentation results are shown in the table below. Compared to the starting strain WJ0140, glutamate yield and conversion efficiency varied with expression of different phosphoketolase copies. This generally correlated positively with the number of copies expressed. The sixth copy had the highest conversion rate, reaching 50.64%.

[0371] Table 7. Conversion rate of glutamate in strains overexpressing xfp

[0372]

[0373] Example 5: Construction of xfp multi-copy strains based on WJ 0140 and their glutamate conversion efficiency

[0374] 5.1odhA-Ptuf-Bl_xfp, xfp one copy

[0375] Prepare WJ0140 competent cells according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), recombinant plasmid pK18-odhA-Ptuf-Bl_xfp is changed into Corynebacterium glutamicum WJ0140 competent cells with electrotransformation method, on the selection medium that contains the kanamycin of 15mg / L, screen a recombinant.One time identification primer is P81 / PE1009-odhA-BL-DN-R, PE1006-odhA-BL-UP-F / P86.The recombinant that sieves is inoculated into and cultivated 12-14 hour in no anti-BHI substratum, and cultivation temperature is 30 ℃, rotary shaking table 220rpm shaking culture.Bacterial liquid is diluted 100-1000 times after being coated on the solid BHIS substratum that contains 10% sucrose and cultivated 36 hours. The secondary identification primers were PE1009-odhA-BL-DN-R / PE1006-odhA-BL-UP-F. Recombinant bacteria with the correct identification band size were sent to Jinweizhi for sequencing, followed by nucleotide sequencing analysis.

[0376] 5.2alaT-Ptuf-Bl_xfp, xfp second copy

[0377] The recombinant plasmid pK18-alaT-Ptuf-Bl_xfp was further transformed into the aforementioned Corynebacterium glutamicum by electroporation, and recombinants were screened for primary recombinant activity on a selective medium containing 15 mg / L kanamycin. The primary identification primers were P81 / PE1022-alaT-DN-2R and PE1021-alaT-UP-1F / P86. The screened recombinants were inoculated into antibiotic-free BHI medium and cultured for 12-14 hours at 30°C on a rotary shaker at 220 rpm. The bacterial solution was diluted 100-1000-fold and plated onto solid BHIS medium containing 10% sucrose and cultured for 36 hours. The secondary identification primers were PE1023-alaT-ID-F and PE1024-alaT-ID-R. Recombinant bacteria with the correct identification band size were sent to Jinweizhi for sequencing.

[0378] 5.3odhA-PcspB-Bl_xfp, three copies of xfp

[0379] The recombinant plasmid pK18-odhA-PcspB-Bl_xfp was further transformed into Corynebacterium glutamicum by electroporation, and recombinants were screened for primary expression on a selective medium containing 15 mg / L kanamycin. The primary identification primers were P86 / PE617-UP-1F and PE624-DN-4R / P81. The selected recombinants were inoculated into antibiotic-free BHI medium and cultured for 12-14 hours at 30°C on a rotary shaker at 220 rpm. The bacterial solution was diluted 100-1000-fold and plated on solid BHIS medium containing 10% sucrose and cultured for 36 hours. The secondary identification primers were PE625-ID-F / PE626-ID-R. Recombinant bacteria with the correct identification band size were sent to Jinweizhi for sequencing, followed by nucleotide sequencing analysis.

[0380] 5.4cg3384-Ptuf-Bl_xfp, four copies of xfp

[0381] The recombinant plasmid pK18-cg3384-Ptuf-Bl_xfp was further transformed into Corynebacterium glutamicum SME751b competent cells by electroporation, and recombinants were screened for primary recombinant activity on selective medium containing 15 mg / L kanamycin. The primary identification primers were P86 / PE1101-3384-UP-1F and PE1104-3384-DN-2R / P81. The selected recombinants were inoculated into antibiotic-free BHI medium and cultured for 12-14 hours at 30°C on a rotary shaker at 220 rpm. The bacterial solution was diluted 100-1000-fold and plated on solid BHIS medium containing 10% sucrose and cultured for 36 hours. The secondary identification primers were PE1107-ID-F / PE1108-ID-R. Recombinant bacteria with the correct identification band size were sent to Jinweizhi for sequencing, followed by nucleotide sequencing analysis.

[0382] 5.5cg2564-Ptuf-Bl_xfp, five copies of xfp

[0383] The recombinant plasmid pK18-cg2564-Ptuf-Bl_xfp was further transformed into competent cells of Corynebacterium glutamicum SME753a by electroporation, and recombinants were screened for primary recombinant activity on a selective medium containing 15 mg / L kanamycin. The primary identification primers were P86 / PE1351-2564-UP-1F and PE1354-2564-DN-2R / P81. The selected recombinants were inoculated into antibiotic-free BHI medium and cultured for 12-14 hours at 30°C on a rotary shaker at 220 rpm. The bacterial solution was diluted 100-1000-fold and plated on solid BHIS medium containing 10% sucrose and cultured for 36 hours. The secondary identification primers were PE1369-ID-F / PE1370-ID-R. Recombinant bacteria with the correct identification band size were sent to Jinweizhi for sequencing, followed by nucleotide sequencing analysis.

[0384] 5.6cg2211-Ptuf-Bl_xfp, six copies of xfp

[0385] The recombinant plasmid pK18-cg2211-Ptuf-Bl_xfp was transformed into Corynebacterium glutamicum by electroporation, and recombinants were screened for primary recombinant activity on selective medium containing 15 mg / L kanamycin. The primary identification primers were P86 / PE1339-2211-UP-1F and PE1342-2211-DN-2R / P81. The selected recombinants were inoculated into antibiotic-free BHI medium and cultured for 12-14 hours at 30°C on a rotary shaker at 220 rpm. The bacterial solution was diluted 100-1000-fold and plated onto solid BHIS medium containing 10% sucrose and cultured for 36 hours. The secondary identification primers were PE1365-ID-F / PE1366-ID-R. Recombinant bacteria with the correct identification band size were sent to Jinweizhi for sequencing, followed by nucleotide sequencing analysis. 5.7cg3364-Ptuf-Bl_xfp, seven copies of xfp

[0386] The recombinant plasmid pK18-cg3364-Ptuf-Bl_xfp was transformed into Corynebacterium glutamicum by electroporation, and recombinants were screened for primary recombinant activity on selective medium containing 15 mg / L kanamycin. The primary identification primers were P86 / PE1117-3364-UP-1F and PE1120-3364-DN-2R / P81. The selected recombinants were inoculated into antibiotic-free BHI medium and cultured for 12-14 hours at 30°C on a rotary shaker at 220 rpm. The bacterial suspension was diluted 100-1000-fold and plated onto solid BHIS medium containing 10% sucrose and cultured for 36 hours. The secondary identification primers were PE1123-ID-F and PE1124-ID-R. Recombinant strains with the correct identification band size were sent to GeneVision for sequencing, followed by nucleotide sequencing analysis. 5.8cg1512-Ptuf-Bl_xfp, eight copies of xfp

[0387] The recombinant plasmid pK18-cg1512-Ptuf-Bl_xfp was transformed into Corynebacterium glutamicum by electroporation. Recombinants were screened on a selective culture medium containing 15mg / L of kanamycin. The target gene was inserted into the chromosome due to homologous recombination. The primers for primary identification were PPE1133-1512-UP-1F / P86 and P81 / PE1136-1512-DN-2R. The recombinants screened were inoculated into non-anti-YB culture medium and cultured for 12-14h at 30°C on a rotary shaker at 220rpm. During this incubation, a second recombination took place in the transformant, and the vector sequence was removed from the genome by homologous recombination exchange. The bacterial solution was diluted 100-1000 times and then coated on a solid YBS culture medium containing 10% sucrose for 36h. The bacterial strain grown on the sucrose culture medium did not carry the vector sequence of insertion in its genome. The secondary identification primers were PE1371-ID-F / PE1372-ID-R, and the band size was 4101 bp. Recombinant strains with the correct identification band size were sent to Jinweizhi for sequencing using the following primers: PE1371-ID-F / PE1372-ID-R / PE962-Bl-check-F1 / PE963-Bl-check-F2 / PE1137-xfp-F / PE1138-xfp-R. Nucleotide sequencing analysis was then performed.

[0388] 5.9cg3125-Ptuf-Bl_xfp, xfp nine copies

[0389] The recombinant plasmid pK18-cg3125-Ptuf-Bl_xfp was transformed into competent cells of Corynebacterium glutamicum SME824a by electroporation, and recombinants were screened for primary recombinant activity on selective medium containing 15 mg / L kanamycin. The primary identification primers were P86 / PE1357-3125-UP-1F and PE1360-3125-DN-2R / P81. The selected recombinants were inoculated into antibiotic-free BHI medium and cultured for 12-14 hours at 30°C on a rotary shaker at 220 rpm. The bacterial suspension was diluted 100-1000-fold and plated onto solid BHIS medium containing 10% sucrose and cultured for 36 hours. The secondary identification primers were PE1371-ID-F and PE1372-ID-R. Recombinant cells with the correct identification band size were sent to Jinweizhi for sequencing, followed by nucleotide sequencing analysis.

[0390] 5.10cg0928-Ptuf-Bl_xfp, xfp ten copies

[0391] The recombinant plasmid pK18-cg0928-Ptuf-Bl_xfp is transferred into Corynebacterium glutamicum SME871a competent cells with the electroporation method. On the selective culture medium containing 15mg / L of kanamycin, a recombinant is screened. The target gene is inserted into the chromosome due to homologous recombination. The primers for primary identification are P86, PE1125-0928-UP-1F / PE1128-0928-DN-2R, and P81. The recombinant obtained by screening is inoculated into the non-anti-YB culture medium and cultivated for 12-14h. The cultivation temperature is 30 ℃ and the culture is shaken on a rotary shaker at 220rpm. In this culture process, a second recombination takes place in the transformant, and the vector sequence is removed from the genome by homologous recombination exchange. After the bacterial liquid is diluted 100-1000 times, it is coated on the solid YBS culture medium containing 10% sucrose and cultivated for 36h. The bacterial strain grown on the sucrose culture medium does not carry the vector sequence of insertion in its genome. The secondary identification primers were PE1131-ID-F and PE1132-ID-R, and the band size was 4101 bp. Recombinant strains with the correct identification band size were sent to Jinweizhi for sequencing using the following primers: PE1131-ID-F, PE1132-ID-R, PE962-Bl-check-F1, PE963-Bl-check-F2, PE1129-xfp-F, and PE1130-xfp-R. Nucleotide sequencing analysis was then performed.

[0392] 5.11 Shake flask validation

[0393] We conducted shake flask fermentations with the seven phosphoketolase-expressing strains described above to verify their glutamate production performance. For detailed shake flask methods, refer to the shake flask fermentation method in Section 1.1.3. The fermentation results are shown in the table below. Compared to the starting strain SME666b, expressing different copies of phosphoketolase increased glutamate yield and conversion efficiency to varying degrees. The sixth copy achieved the highest conversion rate of 61.65%, while adding xfp to 10 copies had no significant effect.

[0394] Table 8. Conversion rate of glutamate in strains overexpressing xfp

[0395]

[0396] Example 6: Construction of xfp multi-copy strains and their glutamate conversion efficiency starting from WJ0140

[0397] 6.1 Plasmid construction

[0398] 6.1.1 Construction of pK18-2337-Ptuf-Bl_xfp Plasmid

[0399] The genome of Corynebacterium glutamicum ATCC13869 (PRJNA305665) was used as a template, and primers PE1345-2337-UP-1F / PE1346-2337-UP-1R were used to amplify the upstream homology arm. Primers PE1347-2337-DN-2F / PE1348-2337-DN-2R were used to amplify the downstream homology arm. Ptuf_xfp was amplified using the pk18-odhA-BL_xfp plasmid as a template and primers PE1349-xfp-F / PE1350-xfp-R. The PCR program was as follows: denaturation at 98°C for 10 s, annealing at 55°C for 20 s, and extension at 72°C for 15 s / kb for 30 cycles, followed by complete extension at 72°C for 10 min. The resulting fragment was purified using an agarose gel extraction kit (Tiangen) and stored frozen at -20°C until further use. pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP, DN, and Ptuf-Bl_xfp fragments were mixed with the double-digested vector in the appropriate proportions using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech). After pipetting to mix thoroughly, the cells were placed in a metal bath (Golden Ginkgo Biotechnology Co., Ltd.) at 37°C for 30 minutes for ligation. After completion of the reaction, Trans1T1 competent cells (TransGen Biotech) were transformed. Kanamycin-resistant clones were selected and identified using the primer pair P81 and P86. The correct band size was 4128 bp. The correct insertion was further verified by sequencing (GENEWIZ) using the following primers: P81 / P86 / PE962-Bl-check-F1 / PE963-Bl-check-F2 / PE1349-xfp-F / PE1350-xfp-R. The resulting plasmid was named pK18-2337-Ptuf-Bl_xfp.

[0400] 6.1.2 Construction of pK18-1512-Ptuf-Bl_xfp Plasmid

[0401] The genome of Corynebacterium glutamicum ATCC13869 (PRJNA305665) was used as a template, and the upstream homology arm was amplified using primers PE1133-1512-UP-1F / PE1134-1512-UP-1R. The downstream homology arm was amplified using primers PE1135-1512-DN-2F / PE1136-1512-DN-2R. tuf_xfp was amplified using the pk18-odhA-BL_xfp plasmid as a template and primers PE1137-xfp-F / PE1138-xfp-R. The PCR procedure was the same as above. pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP, DN, and Ptuf-Bl_xfp fragments were mixed with the double-digested vector in the appropriate proportions using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech). After pipetting and mixing, the cells were placed in a metal bath (Golden Ginkgo Biotechnology Co., Ltd.) at 37°C for 30 minutes for ligation. After completion of the reaction, Trans1T1 competent cells (TransGen Biotech) were transformed. Kanamycin-resistant clones were selected and identified using the primer pair P81 and P86. The correct band size was 4154 bp. The correct insertion was further verified by sequencing (GENEWIZ) using the following primers: P81 / P86 / PE962-Bl-check-F1 / PE963-Bl-check-F2 / PE1137-xfp-F / PE1138-xfp-R. The resulting plasmid was named pK18-1512-Ptuf-Bl_xfp.

[0402] 6.1.3 Construction of pK18-7210-Ptuf-Bl_xfp Plasmid

[0403] The genome of Corynebacterium glutamicum ATCC13869 (PRJNA305665) was used as a template, and primers PE1389-7210-UP-1F / PE1390-7210-UP-1R were used to amplify the upstream homology arm. The downstream homology arm was amplified using primers PE1391-7210-DN-2F / PE1392-7210-DN-2R. tuf_xfp was amplified using the pk18-0928-xfp plasmid as a template and primers PE1393-xfp-F / PE1394-xfp-R. The PCR procedure was the same as above. pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP, DN, and Ptuf-Bl_xfp fragments were mixed with the double-digested vector in the appropriate proportions using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech). After pipetting and mixing, the cells were placed in a metal bath (Golden Ginkgo Biotechnology Co., Ltd.) at 37°C for 30 minutes for ligation. After completion of the reaction, Trans1T1 competent cells (TransGen Biotech) were transformed. Kanamycin-resistant clones were selected and identified using the primer pair P81 and P86. The correct band size was 4154 bp. The correct insertion was further verified by sequencing (GENEWIZ) using the following primers: P81 / P86 / PE962-Bl-check-F1 / PE963-Bl-check-F2 / PE1393-xfp-F / PE1394-xfp-R. The resulting plasmid was named pK18-7210-Ptuf-Bl_xfp.

[0404] 6.2 Strain Construction

[0405] 6.2.1pK18-odhA-Ptuf-Bl_xfp, four copies of xfp

[0406] The competent cell that prepares embodiment 4.3 part makes according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Charpter 23), recombinant plasmid pK18-odhA-Ptuf-Bl_xfp is changed in Corynebacterium glutamicum with electrotransformation method, on the selection culture medium that contains the kanamycin of 15mg / L, screen a recombinant, wherein target gene is inserted in the karyomit(e) because homologous recombination.Once identification primer is P81, PE1009-odhA-BL-DN-R / PE1006-odhA-BL-UP-F, P86.The recombinant that sieve is inoculated into does not have to cultivate 12-14h in the anti-YB substratum, and cultivation temperature is 30 ℃, rotary shaking table 220rpm shaking culture.In this culturing process, second time reorganization takes place in transformant, and by homologous recombination exchange, carrier sequence is removed from genome. The bacterial suspension was diluted 100-1000-fold and plated on solid YBS medium containing 10% sucrose and cultured for 36 hours. Strains grown on sucrose medium do not carry the inserted vector sequence in their genomes. Secondary identification primers were PE1009-odhA-BL-DN-R / PE1006-odhA-BL-UP-F, yielding a band size of 4017 bp. Recombinant strains with the correct band size were sent to GeneVision for sequencing using primers PE962-Bl-check-F1, PE963-Bl-check-F2, PE1009-odhA-BL-DN-R, PE1006-odhA-BL-UP-F, and PE1010-odhA-tuf-F. Nucleotide sequencing analysis was then performed.

[0407] 6.2.2pK18-cg0928-Ptuf-Bl_xfp, five copies of xfp

[0408] The recombinant plasmid pK18-cg0928-Ptuf-Bl_xfp was further transformed into Corynebacterium glutamicum using an electroporation method. Recombinants were screened on a selective medium containing 15 mg / L of kanamycin. The target gene was inserted into the chromosome due to homologous recombination. The primers for primary identification were P86, PE1125-0928-UP-1F / PE1128-0928-DN-2R, and P81. The recombinants screened were inoculated into an anti-antibody YB medium and cultured for 12-14 hours at 30°C on a rotary shaker at 220 rpm. During this culture, a second recombination occurred in the transformants, and the vector sequence was removed from the genome through homologous recombination exchange. The bacterial solution was diluted 100-1000 times and then coated on a solid YBS medium containing 10% sucrose and cultured for 36 hours. The bacterial strain grown on the sucrose medium did not carry the inserted vector sequence in its genome. The secondary identification primers were PE1131-ID-F and PE1132-ID-R, and the band size was 4101 bp. Recombinant strains with the correct identification band size were sent to Jinweizhi for sequencing using the following primers: PE1131-ID-F, PE1132-ID-R, PE962-Bl-check-F1, PE963-Bl-check-F2, PE1129-xfp-F, and PE1130-xfp-R. Nucleotide sequencing analysis was then performed.

[0409] 6.2.3pK18-cg2337-Ptuf-Bl_xfp, six copies of xfp

[0410] Further, the recombinant plasmid pK18-cg2337-Ptuf-Bl_xfp was transformed into Corynebacterium glutamicum by electroporation. On a selective culture medium containing 15mg / L of kanamycin, a recombinant was screened. The target gene was inserted into the chromosome due to homologous recombination. The primers for primary identification were PE1345-2337-UP-1F / P86 and P81 / PE1348-2337-DN-2R. The recombinant obtained by screening was inoculated into non-anti-YB culture medium and cultured for 12-14 hours at 30°C on a rotary shaker at 220rpm. During this culture, a second recombination took place in the transformant, and the vector sequence was removed from the genome by homologous recombination exchange. After the bacterial solution was diluted 100-1000 times, it was coated on a solid YBS culture medium containing 10% sucrose and cultured for 36 hours. The bacterial strain grown on the sucrose culture medium did not carry the inserted vector sequence in its genome. The secondary identification primers were PE1367-ID-F / PE1368-ID-R, and the band size was 4101 bp. Recombinant strains with the correct identification band size were sent to Jinweizhi for sequencing using the following primers: PE1367-ID-F / PE1368-ID-R / PE962-Bl-check-F1 / PE963-Bl-check-F2 / PE1349-xfp-F / PE1350-xfp-R. Nucleotide sequencing analysis was then performed.

[0411] 6.2.4pK18-cg1512-Ptuf-Bl_xfp, seven copies of xfp

[0412] The recombinant plasmid pK18-cg1512-Ptuf-Bl_xfp was further transformed into Corynebacterium glutamicum using an electroporation method. Recombinants were screened on a selective medium containing 15 mg / L of kanamycin. The target gene was inserted into the chromosome due to homologous recombination. The primers for primary identification were PPE1133-1512-UP-1F / P86 and P81 / PE1136-1512-DN-2R. The recombinants screened were inoculated into an anti-antibody-free YB medium and cultured for 12-14 hours at 30°C on a rotary shaker at 220 rpm. During this culture process, a second recombination occurred in the transformants, and the vector sequence was removed from the genome through homologous recombination exchange. The bacterial solution was diluted 100-1000 times and then coated on a solid YBS medium containing 10% sucrose and cultured for 36 hours. The strain grown on the sucrose medium did not carry the inserted vector sequence in its genome. The secondary identification primers were PE1371-ID-F / PE1372-ID-R, and the band size was 4101 bp. Recombinant strains with the correct identification band size were sent to Jinweizhi for sequencing using the following primers: PE1371-ID-F / PE1372-ID-R / PE962-Bl-check-F1 / PE963-Bl-check-F2 / PE1137-xfp-F / PE1138-xfp-R. Nucleotide sequencing analysis was then performed.

[0413] 6.2.5pK18-cg0928-Ptuf-Bl_xfp, five copies of xfp

[0414] According to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Charpter 23), prepare 5.4 part xfp four copies competent cells, recombinant plasmid pK18-cg0928-Ptuf-Bl_xfp is changed in Corynebacterium glutamicum with electrotransformation method, on the selection medium containing the kanamycin of 15mg / L, screen a recombinant, wherein target gene is inserted in karyomit(e) due to homologous recombination.Once identification primer is P86, PE1125-0928-UP-1F / PE1128-0928-DN-2R, P81.The recombinant that sieve is inoculated into does not cultivate 12-14h in anti-YB substratum, and cultivation temperature is 30 ℃, rotary shaking table 220rpm shaking culture.In this culturing process, transformant second time reorganization takes place, and by homologous recombination exchange, carrier sequence is removed from genome. The bacterial suspension was diluted 100-1000-fold and plated on solid YBS medium containing 10% sucrose and cultured for 36 hours. Strains grown on sucrose medium do not carry the inserted vector sequence in their genome. Secondary identification primers were PE1131-ID-F and PE1132-ID-R, and the band size was 4101 bp. Recombinant strains with the correct identification band size were sent to Jinweizhi for sequencing using the following primers: PE1131-ID-F, PE1132-ID-R, PE962-Bl-check-F1, PE963-Bl-check-F2, PE1129-xfp-F, and PE1130-xfp-R. Nucleotide sequencing analysis was then performed.

[0415] 6.2.6pK18-BBD29-7210-Ptuf-Bl_xfp, six copies of xfp

[0416] The recombinant plasmid pK18-cg7210-Ptuf-Bl_xfp was further transformed into Corynebacterium glutamicum by electroporation. Recombinants were screened on a selective medium containing 15 mg / L of kanamycin. The target gene was inserted into the chromosome due to homologous recombination. The primers for primary identification were PPE1389-7210-UP-1F / P86 and P81 / PE1392-7210-DN-2R. The recombinants screened were inoculated into non-antibiotic YB culture medium and cultured for 12-14 hours at 30°C on a rotary shaker at 220 rpm. During this culture, a second recombination occurred in the transformants, and the vector sequence was removed from the genome through homologous recombination exchange. The bacterial solution was diluted 100-1000 times and then coated on a solid YBS medium containing 10% sucrose and cultured for 36 hours. The bacterial strain grown on the sucrose medium did not carry the inserted vector sequence in its genome. The secondary identification primers were PE1419-ID-F / PE1420-ID-R, and the band size was 4101 bp. Recombinant strains with the correct identification band size were sent to Jinweizhi for sequencing using the following primers: PE1419-ID-F / PE1420-ID-R / PE962-Bl-check-F1 / PE963-Bl-check-F2 / PE1393-xfp-F / PE1394-xfp-R. Nucleotide sequencing analysis was then performed.

[0417] 6.2.7pK18-odhA-Ptuf-Bl_xfp(SME882)xfp seven copies

[0418] The recombinant plasmid pK18-odhA-Ptuf-Bl_xfp was further transformed into Corynebacterium glutamicum using an electroporation method. Recombinants were screened on a selective medium containing 15 mg / L of kanamycin. The target gene was inserted into the chromosome due to homologous recombination. The primary identification primers were P81, PE1009-odhA-BL-DN-R / PE1006-odhA-BL-UP-F, and P86. The screened recombinants were inoculated into an antibiotic-free YB medium and cultured for 12-14 hours at 30°C on a rotary shaker at 220 rpm. During this culture process, the transformants underwent a second recombination, and the vector sequence was removed from the genome through homologous recombination exchange. The bacterial solution was diluted 100-1000 times and then coated on a solid YBS medium containing 10% sucrose and cultured for 36 hours. The strain grown on the sucrose medium did not carry the inserted vector sequence in its genome. The secondary identification primers were PE1009-odhA-BL-DN-R / PE1006-odhA-BL-UP-F, and the band size was 4017 bp. Recombinant strains with the correct identification band size were sent to Jinweizhi for sequencing using the following primers: PE962-Bl-check-F1, PE963-Bl-check-F2, PE1009-odhA-BL-DN-R, PE1006-odhA-BL-UP-F, and PE1010-odhA-tuf-F. Nucleotide sequencing analysis was then performed.

[0419] 6.3 Shake flask validation

[0420] We performed shake flask fermentation on the phosphoketolase-expressing strain to verify its glutamate production performance. For details on the shake flask fermentation method, refer to the shake flask fermentation method in Section 1.1.3. The fermentation results are shown in the table below.

[0421] Table 9. Glutamate content detection of recombinant strains

[0422]

[0423]

[0424] The above shake flask results show that whether randomly stacking from three copies of 0140-xfp to seven copies or randomly stacking from four copies to seven copies, the glutamate conversion rate reaches the highest at six copies, regardless of the order of insertion sites.

[0425] Example 7: Construction of xfp multi-copy strains and their glutamate conversion efficiency

[0426] 7.1 Starting from ATCC13032, xfp multiple copies

[0427] 7.1.1 MH13032-10-alaT-Ptuf-Bl_xfp strain construction (MH13032-11)

[0428] Prepare MH13032-10 competent cells according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), recombinant plasmid pK18-alaT-Ptuf-Bl_xfp (13032) is changed into Corynebacterium glutamicum MH13032-10 competent cells with electrotransformation method, on the BHI substratum flat board that contains 15mg / L kanamycin, cultivate 48h.One time identification primer is P81 / PE1022-alaT-DN-2R, P13-10 / P86. The recombinant that will sieve is inoculated into and cultivated 12-14 hour in the non-anti-BHI substratum, and cultivation temperature is 30 ℃, rotary shaker 220rpm shaking culture.Bacterium liquid is diluted 100-1000 doubly after being coated on the solid BHIS substratum that contains 10% sucrose and cultivated 36 hours. The secondary identification primers were PE1023-alaT-ID-F and PE1024-alaT-ID-R. The correct band size was 4054 bp. Recombinant strains with the correct identification band size were sent to GENEWIZ for sequencing. Subsequently, nucleotide sequencing analysis was performed, and the recombinant strain with the correct sequence was named MH13032-11.

[0429] 7.1.2 MH13032-11-cg3364-Ptuf-Bl_xfp strain construction (MH13032-12)

[0430] Prepare MH13032-11 competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Charter 23), recombinant plasmid pK18-cg3364-Ptuf-Bl_xfp (13032) is changed into Corynebacterium glutamicum MH13032-11 competent cell with the electrotransformation method, on the selection medium that contains the kanamycin of 15mg / L, screen a recombinant.Once identification primer is P86 / PE1117-3364-UP-1F, PE1120-3364-DN-2R / P81.The recombinant that sieves is inoculated into and cultivated 12-14 hour in the anti-BHI substratum, and cultivation temperature is 30 ℃, rotary shaking table 220rpm shaking culture.Bacterium liquid is diluted 100-1000 doubly after being coated on the solid BHIS substratum that contains 10% sucrose and cultivated 36 hours. The secondary identification primers were P13-11 and PE1124-ID-R. The recombinant bacteria with the correct identification band size were sent to GENEWIZ for sequencing, followed by nucleotide sequencing analysis. The recombinant microbial strain with the correct sequence was named MH13032-12.

[0431] 7.1.3 MH13032-12-cg2564-Ptuf-Bl_xfp strain construction (MH13032-13)

[0432] Prepare MH13032-12 competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Charter 23), recombinant plasmid pK18-cg2564-Ptuf-Bl_xfp (13032) is changed into Corynebacterium glutamicum MH13032-12 competent cell with the electrotransformation method, on the selection medium that contains the kanamycin of 15mg / L, screen a recombinant.Once identification primer is P86 / PE1351-2564-UP-1F, PE1354-2564-DN-2R / P81.The recombinant that sieves is inoculated into and cultivated 12-14 hour in the anti-BHI substratum without, and cultivation temperature is 30 ℃, rotary shaking table 220rpm shaking culture.Bacterium liquid is diluted 100-1000 doubly after being coated on the solid BHIS substratum that contains 10% sucrose and cultivated 36 hours. The secondary identification primers were PE1369-ID-F and PE1370-ID-R. Recombinant bacteria with the correct identification band size were sent to GENEWIZ for sequencing, followed by nucleotide sequencing analysis. The recombinant microbial strain with the correct sequence was named MH13032-13.

[0433] 7.1.4 MH13032-13-cg3125-Ptuf-Bl_xfp strain construction (MH13032-14)

[0434] Prepare MH13032-13 competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Charter 23), recombinant plasmid pK18-cg3125-Ptuf-Bl_xfp (13032) is changed into Corynebacterium glutamicum MH13032-13 competent cell with the electrotransformation method, on the selection medium that contains the kanamycin of 15mg / L, screen a recombinant.Once identification primer is P86 / PE1357-3125-UP-1F, PE1360-3125-DN-2R / P81.The recombinant that sieves is inoculated into and cultivated 12-14 hour in the anti-BHI substratum, and cultivation temperature is 30 ℃, rotary shaking table 220rpm shaking culture.Bacterium liquid is diluted 100-1000 doubly after being coated on the solid BHIS substratum that contains 10% sucrose and cultivated 36 hours. The secondary identification primers were PE1371-ID-F / P14-12. The recombinant bacteria with the correct identification band size were sent to GENEWIZ for sequencing, followed by nucleotide sequencing analysis. The recombinant microbial strain with the correct sequence was named MH13032-14.

[0435] 7.1.5 MH13032-14-cg3384-Ptuf-Bl_xfp strain construction (MH13032-15)

[0436] Prepare MH13032-14 competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Charter 23), recombinant plasmid pK18-cg3384-Ptuf-Bl_xfp (13032) is changed into Corynebacterium glutamicum MH13032-14 competent cell with the electrotransformation method, on the selection medium that contains the kanamycin of 15mg / L, screen a recombinant.Once identification primer is P86 / PE1101-3384-UP-1F, PE1104-3384-DN-2R / P81.The recombinant that sieves is inoculated into and cultivated 12-14 hour in the anti-BHI substratum without, and cultivation temperature is 30 ℃, rotary shaking table 220rpm shaking culture.Bacterium liquid is diluted 100-1000 doubly after being coated on the solid BHIS substratum that contains 10% sucrose and cultivated 36 hours. The secondary identification primers were PE1107-ID-F / PE1108-ID-R. Recombinant bacteria with the correct identification band size were sent to GENEWIZ for sequencing, followed by nucleotide sequencing analysis. The recombinant microbial strain with the correct sequence was named MH13032-15.

[0437] 7.1.6 MH13032-15-BBD29_7270-Ptuf-Bl_xfp strain construction (MH13032-16)

[0438] Prepare MH13032-15 competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Charter 23), recombinant plasmid pK18-BBD29_7270-Ptuf-Bl_xfp (13032) is changed into Corynebacterium glutamicum MH13032-15 competent cell with the electrotransformation method, on the selection medium that contains the kanamycin of 15mg / L, screen a recombinant.Once identification primer is P86 / PE1407-7270-UP-1F, PE1410-7270-DN-2R / P81.The recombinant that sieves is inoculated into and cultivated 12-14 hour in the anti-BHI substratum, and cultivation temperature is 30 ℃, rotary shaking table 220rpm shaking culture.Bacterium liquid is diluted 100-1000 doubly after being coated on the solid BHIS substratum that contains 10% sucrose and cultivated 36 hours. The secondary identification primers were PE1425-ID-F / PE1426-ID-R. Recombinant bacteria with the correct identification band size were sent to GENEWIZ for sequencing, followed by nucleotide sequencing analysis. The recombinant microbial strain with the correct sequence was named MH13032-16.

[0439] 7.1.7 MH13032-16-cg0928-Ptuf-Bl_xfp strain construction (MH13032-17)

[0440] Prepare MH13032-16 competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Charter 23), recombinant plasmid pK18-cg0928-Ptuf-Bl_xfp (13032) is changed into Corynebacterium glutamicum MH13032-16 competent cell with the electrotransformation method, on the selection culture medium that contains the kanamycin of 15mg / L, screen a recombinant.Once identification primer is P86 / PE1125-0928-UP-1F, P14-17 / P81.The recombinant that sieves is inoculated into and cultivated 12-14 hour in the anti-BHI substratum, and cultivation temperature is 30 ℃, rotary shaking table 220rpm shaking culture.Bacterium liquid is diluted 100-1000 doubly after being coated on the solid BHIS substratum that contains 10% sucrose and cultivated 36 hours. The secondary identification primers were PE1131-ID-F and PE1132-ID-R. Recombinant bacteria with the correct identification band size were sent to GENEWIZ for sequencing, followed by nucleotide sequencing analysis. The recombinant microbial strain with the correct sequence was named MH13032-17.

[0441] 7.1.8 13032 XFP multi-copy strain shake flask verification

[0442] The recombinant Corynebacterium glutamicum constructed above was fermented to verify its glutamic acid production performance. For the specific shake flask method, refer to the shake flask fermentation method in Section 1.1.3.

[0443] The table above shows the results of shake flask experiments using multiple copies of the Bifidobacterium longum phosphoketolase (XFP) superimposed on MH13032-10. The results show that glutamate conversion efficiency increased overall with the addition of 1 to 7 copies of XFP, reaching a peak of 39.7% at 6 copies. While there was an improvement with 7 copies, glutamate conversion decreased compared to 6 copies.

[0444] Table 10. Glutamate content detection of recombinant strains

[0445]

[0446] 7.2 Starting from ATCC14067, xfp multiple copies

[0447] 7.2.1 MH14067-10-alaT-Ptuf-Bl_xfp strain construction (MH14067-11)

[0448] Prepare MH14067-10 competent cells according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), recombinant plasmid pK18-alaT-Ptuf-Bl_xfp (14067) is changed into Corynebacterium glutamicum MH14067-10 competent cells with electrotransformation method, on the BHI culture medium flat board that contains 15mg / L kanamycin, cultivate 48h. One-time identification primer is P81 / PE1022-alaT-DN-2R, PE1021-alaT-UP-1F / P86. The recombinant that sieves is inoculated into and cultivated 12-14 hour in no anti-BHI culture medium, and cultivation temperature is 30 ℃, rotary shaker 220rpm shaking culture. Bacterial liquid is diluted 100-1000 times after being coated on the solid BHIS culture medium that contains 10% sucrose and cultivated 36 hours. The secondary identification primers were PE1023-alaT-ID-F and PE1024-alaT-ID-R. The correct band size was 4054 bp. Recombinant strains with the correct identification band size were sent to GENEWIZ for sequencing. Subsequently, nucleotide sequencing analysis was performed, and the recombinant strain with the correct sequence was named MH14067-11.

[0449] 7.2.2 MH14067-11-cg3364-Ptuf-Bl_xfp strain construction (MH14067-12)

[0450] Prepare MH14067-11 competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Charter 23), recombinant plasmid pK18-cg3364-Ptuf-Bl_xfp (14067) is changed into Corynebacterium glutamicum MH14067-11 competent cell with the electrotransformation method, on the selection medium that contains the kanamycin of 15mg / L, screen a recombinant.Once identification primer is P86 / PE1117-3364-UP-1F, PE1120-3364-DN-2R / P81.The recombinant that sieves is inoculated into and cultivated 12-14 hour in the anti-BHI substratum without, and cultivation temperature is 30 ℃, rotary shaking table 220rpm shaking culture.Bacterium liquid is diluted 100-1000 doubly after being coated on the solid BHIS substratum that contains 10% sucrose and cultivated 36 hours. The secondary identification primers were PE1123-ID-F and PE1124-ID-R. Recombinant bacteria with the correct identification band size were sent to GENEWIZ for sequencing, followed by nucleotide sequencing analysis. The recombinant microbial strain with the correct sequence was named MH14067-12.

[0451] 7.2.3 MH14067-12-cg2564-Ptuf-Bl_xfp strain construction (MH14067-13)

[0452] Prepare MH14067-12 competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Charter 23), recombinant plasmid pK18-cg2564-Ptuf-Bl_xfp (14067) is changed into Corynebacterium glutamicum MH14067-12 competent cell with the electrotransformation method, on the selection medium that contains the kanamycin of 15mg / L, screen a recombinant.Once identification primer is P86 / PE1351-2564-UP-1F, PE1354-2564-DN-2R / P81.The recombinant that sieves is inoculated into and cultivated 12-14 hour in the anti-BHI substratum without, and cultivation temperature is 30 ℃, rotary shaking table 220rpm shaking culture.Bacterium liquid is diluted 100-1000 doubly after being coated on the solid BHIS substratum that contains 10% sucrose and cultivated 36 hours. The secondary identification primers were PE1369-ID-F and PE1370-ID-R. Recombinant bacteria with the correct identification band size were sent to GENEWIZ for sequencing, followed by nucleotide sequencing analysis. The recombinant microbial strain with the correct sequence was named MH14067-13.

[0453] 7.2.4 MH14067-13-cg3125-Ptuf-Bl_xfp strain construction (MH14067-14)

[0454] Prepare MH14067-13 competent cells according to the Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), recombinant plasmid pK18-cg3125-Ptuf-Bl_xfp (14067) is changed into Corynebacterium glutamicum MH14067-13 competent cells with the electrotransformation method, on the selection medium that contains the kanamycin of 15mg / L, screen a recombinant. The primary identification primer is P86 / P14-7, P14-10 / P81. The recombinant that sieves is inoculated into and cultivated 12-14 hour in no anti-BHI substratum, and the cultivation temperature is 30 ℃, rotary shaker 220rpm shaking culture. After the bacterial liquid is diluted 100-1000 times, be coated on the solid BHIS substratum that contains 10% sucrose and cultivated 36 hours. The secondary identification primer is P14-11, P14-12. The recombinant bacteria with the correct identification band size were sent to GENEWIZ for sequencing, followed by nucleotide sequencing analysis. The recombinant microbial strain with the correct sequencing was named MH14067-14.

[0455] 7.2.5 MH14067-14-cg3384-Ptuf-Bl_xfp strain construction (MH14067-15)

[0456] Prepare MH14067-14 competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Charter 23), recombinant plasmid pK18-cg3384-Ptuf-Bl_xfp (14067) is changed into Corynebacterium glutamicum MH14067-14 competent cell with the electrotransformation method, on the selection medium that contains the kanamycin of 15mg / L, screen a recombinant.Once identification primer is P86 / PE1101-3384-UP-1F, PE1104-3384-DN-2R / P81.The recombinant that sieves is inoculated into and cultivated 12-14 hour in the anti-BHI substratum, and cultivation temperature is 30 ℃, rotary shaking table 220rpm shaking culture.Bacterium liquid is diluted 100-1000 doubly after being coated on the solid BHIS substratum that contains 10% sucrose and cultivated 36 hours. The secondary identification primers were PE1107-ID-F and PE1108-ID-R. Recombinant bacteria with the correct identification band size were sent to GENEWIZ for sequencing, followed by nucleotide sequencing analysis. The recombinant microbial strain with the correct sequence was named MH14067-15.

[0457] 7.2.6 MH14067-15-BBD29_7270-Ptuf-Bl_xfp strain construction (MH14067-16)

[0458] Prepare MH14067-15 competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Charter 23), recombinant plasmid pK18-BBD29_7270-Ptuf-Bl_xfp (14067) is changed into Corynebacterium glutamicum MH14067-15 competent cell with the electrotransformation method, on the selection medium that contains the kanamycin of 15mg / L, screen a recombinant.Once identification primer is P86 / PE1407-7270-UP-1F, PE1410-7270-DN-2R / P81.The recombinant that sieves is inoculated into and cultivated 12-14 hour in the anti-BHI substratum, and cultivation temperature is 30 ℃, rotary shaking table 220rpm shaking culture.Bacterium liquid is diluted 100-1000 doubly after being coated on the solid BHIS substratum that contains 10% sucrose and cultivated 36 hours. The secondary identification primers were PE1425-ID-F and PE1426-ID-R. Recombinant bacteria with the correct identification band size were sent to GENEWIZ for sequencing, followed by nucleotide sequencing analysis. The recombinant microbial strain with the correct sequence was named MH14067-16.

[0459] 7.2.7 MH14067-16-cg0928-Ptuf-Bl_xfp strain construction (MH14067-17)

[0460] Prepare MH14067-16 competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Charter 23), recombinant plasmid pK18-cg0928-Ptuf-Bl_xfp (14067) is changed into Corynebacterium glutamicum MH14067-16 competent cell with the electrotransformation method, on the selection medium that contains the kanamycin of 15mg / L, screen a recombinant.Once identification primer is P86 / PE1125-0928-UP-1F, P14-17 / P81.The recombinant that will sieve is inoculated into and cultivated 12-14 hour in the anti-BHI substratum without, and cultivation temperature is 30 ℃, rotary shaking table 220rpm shaking culture.Bacterium liquid is diluted 100-1000 doubly after being coated on the solid BHIS substratum that contains 10% sucrose and cultivated 36 hours. The secondary identification primers were PE1131-ID-F and PE1132-ID-R. Recombinant bacteria with the correct identification band size were sent to GENEWIZ for sequencing, followed by nucleotide sequencing analysis. The recombinant microbial strain with the correct sequence was named MH14067-17.

[0461] 7.2.8 XFP Multi-Copy Shake Flask Verification of ATCC14067

[0462] The recombinant Corynebacterium glutamicum constructed above was fermented to verify its glutamic acid production performance. For the specific shake flask method, refer to the shake flask fermentation method in Section 1.1.3.

[0463] The table above shows the results of shake flask experiments using multiple copies of the Bifidobacterium longum phosphoketolase (XFP) superimposed on MH14067-10. The results show that glutamate conversion efficiency increased overall with the addition of 1 to 7 copies of XFP, reaching a peak of 35.4% at 6 copies. While there was an improvement with 7 copies, glutamate conversion decreased compared to 6 copies.

[0464] Table 11. Glutamate content detection of recombinant strains

[0465]

[0466] Example 8: Construction of a strain with weakened pfk and its glutamate conversion rate

[0467] 8.1 Starting from the six-copy xfp bacteria, weakening pfk

[0468] 8.1.1pfk-A1T

[0469] According to the classical method of Corynebacterium glutamicum (C.glutamicum Handbook, Chapter 23), six copies of xfp competent cells described in Example 4 were prepared. The recombinant plasmid pK18-pfk-A1T was transferred into Corynebacterium glutamicum by electroporation and recombinants were screened on a selective culture medium containing 15 mg / L of kanamycin. The primary identification primers were P86 / PE878-pfk-TTG-UP-1F and PE881-pfk-UP-2R / P81. The recombinants screened were inoculated into non-antibiotic BHI culture medium and cultured for 12-14 hours at 30°C on a rotary shaker at 220 rpm. The bacterial solution was diluted 100-1000 times and then coated on a solid BHIS culture medium containing 10% sucrose and cultured for 36 hours. The secondary identification primers were PE898-A1T-ID-F and PE899-A1T-ID-R. The recombinant bacteria with the correct identification band size were sent to Jinweizhi Company for sequencing, followed by nucleotide sequencing analysis.

[0470] 8.1.2PrecA-pfk

[0471] According to the Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), six copies of xfp competent cells described in Example 4 were prepared. Recombinant plasmid pK18-PrecA-pfk was transferred to Corynebacterium glutamicum with electroporation and screened for a recombinant on a selective culture medium containing 15mg / L of kanamycin. The primary identification primers were P86 / PE882-pfk-UP-1F and PE885-pfk-DN-2R / P81. The recombinant obtained by screening was inoculated into the non-anti-BHI culture medium and cultivated for 12-14 hours. The cultivation temperature was 30 ℃ with a rotary shaker at 220rpm. The bacterial liquid was diluted 100-1000 times and then coated on a solid BHIS culture medium containing 10% sucrose and cultivated for 36 hours. The secondary identification primers were PE891-PrecA-check-F and PE899-A1T-ID-R. The recombinant bacteria with the correct identification band size were sent to Jinweizhi Company for sequencing, followed by nucleotide sequencing analysis.

[0472] 8.1.3pfk-E171D

[0473] According to the Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23) prepare xfp six copy competent cells described in the embodiment 4, recombinant plasmid pK18-pfk-E171D is changed in the Corynebacterium glutamicum with the electrotransformation method, on the selection medium that contains the kanamycin of 15mg / L, screen a recombinant. An identification primer is P86 / PE892-pfk-E171K-UP-1F, PE895-pfk-E171K-DN-2R / P81. The recombinant that sieves is inoculated into and cultivated 12-14 hour in no anti-BHI substratum, and cultivation temperature is 30 ℃, rotary shaker 220rpm shaking culture. After the bacterium liquid is diluted 100-1000 times, be coated on the solid BHIS substratum that contains 10% sucrose and cultivated 36 hours. The secondary identification primers were PE946-E171-ID-F and PE947-E171-ID-R. Recombinant bacteria with the correct identification band size were sent to Jinweizhi for sequencing, followed by nucleotide sequencing analysis.

[0474] 8.1.4pK18-Pzwf-pfk

[0475] According to the classical method of Corynebacterium glutamicum (C.glutamicum Handbook, Chapter 23), six copies of xfp competent cells were prepared in Example 4. Recombinant plasmid pK18-Pzwf-pfk was transferred into Corynebacterium glutamicum with electroporation and screened for recombinants on a selective culture medium containing 15mg / L of kanamycin. The primary identification primers were P86 / PE882-pfk-UP-1F and PE885-pfk-DN-2R / P81. The recombinants were inoculated into the non-anti-BHI culture medium and cultured for 12-14 hours at 30°C on a rotary shaker at 220rpm. The bacterial solution was diluted 100-1000 times and then coated on a solid BHIS culture medium containing 10% sucrose and cultured for 36 hours. The secondary identification primers were PE888-zwf-check-F and PE899-A1T-ID-R. The recombinant bacteria with the correct identification band size were sent to Jinweizhi Company for sequencing, followed by nucleotide sequencing analysis.

[0476] 8.1.5 Shake flask validation

[0477] The recombinant Corynebacterium glutamicum constructed above was fermented to verify its glutamic acid production performance. For the specific shake flask method, refer to the shake flask fermentation method in Section 1.1.3.

[0478] Based on the six-copy xfp competent cells described in Example 4, the pfk gene was attenuated in various ways. With the exception of E171D, all attenuation methods improved glutamate conversion to varying degrees. Attenuation of the PrecA promoter resulted in a 61.76% conversion rate, but the fermentation cycle was extended by 24 hours. Attenuation of the Pzwf promoter resulted in a 56.46% conversion rate, with no impact on the fermentation cycle or sugar consumption.

[0479] Table 12. Conversion rate of glutamate in pfk-modified strains

[0480]

[0481] 8.2 Starting from the six-copy xfp bacteria, weakening pfk

[0482] 8.2.1Pzwf-pfk

[0483] According to the Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), six copies of xfp competent cells described in Example 5 were prepared. Recombinant plasmid pK18-Pzwf-pfk was transferred to Corynebacterium glutamicum with electroporation and screened for a recombinant on a selective culture medium containing 15mg / L of kanamycin. The primary identification primers were P86, PE882-pfk-UP-1F / PE885-pfk-DN-2R, and P81. The recombinant obtained by screening was inoculated into the non-anti-BHI culture medium and cultivated for 12-14 hours. The cultivation temperature was 30°C with a rotary shaker at 220rpm. The bacterial liquid was diluted 100-1000 times and then coated on a solid BHIS culture medium containing 10% sucrose and cultivated for 36 hours. The secondary identification primers were PE888-zwf-check-F and PE899-A1T-ID-R. The recombinant bacteria with the correct identification band size were sent to Jinweizhi Company for sequencing, followed by nucleotide sequencing analysis.

[0484] 8.2.2PrecA-pfk

[0485] According to the Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), six copies of xfp competent cells described in Example 5 were prepared. Recombinant plasmid pK18-PrecA-pfk was transferred to Corynebacterium glutamicum with the electroporation method. A recombinant was screened on the selective culture medium containing 15mg / L of kanamycin. The primary identification primers were P86 / PE882-pfk-UP-1F and PE885-pfk-DN-2R / P81. The recombinant obtained by screening was inoculated into the non-anti-BHI culture medium and cultivated for 12-14 hours. The cultivation temperature was 30 ℃, and the culture was shaken on a rotary shaker at 220rpm. The bacterial liquid was diluted 100-1000 times and then coated on the solid BHIS culture medium containing 10% sucrose and cultivated for 36 hours. The secondary identification primers were PE891-PrecA-check-F and PE899-A1T-ID-R. The recombinant bacteria with the correct identification band size were sent to Jinweizhi Company for sequencing, followed by nucleotide sequencing analysis.

[0486] 8.2.3pfk-E171K

[0487] According to the Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23) prepare xfp six copy competent cells described in the embodiment 5, recombinant plasmid pK18-pfk-E171K is changed in the Corynebacterium glutamicum with the electrotransformation method, on the selection medium that contains the kanamycin of 15mg / L, screen a recombinant. An identification primer is P86 / PE892-pfk-E171K-UP-1F, PE895-pfk-E171K-DN-2R / P81. The recombinant that sieves is inoculated into and cultivated 12-14 hour in no anti-BHI substratum, and cultivation temperature is 30 ℃, rotary shaker 220rpm shaking culture. After the bacterium liquid is diluted 100-1000 times, be coated on the solid BHIS substratum that contains 10% sucrose and cultivated 36 hours. The secondary identification primers were PE946-E171-ID-F and PE947-E171-ID-R. Recombinant bacteria with the correct identification band size were sent to Jinweizhi for sequencing, followed by nucleotide sequencing analysis.

[0488] 8.2.4 Shake flask validation

[0489] The recombinant Corynebacterium glutamicum constructed above was fermented to verify its glutamic acid production performance. For the specific shake flask method, refer to the shake flask fermentation method in Section 1.1.3.

[0490] Based on the six copies of xfp described in Example 5, different attenuation methods for the pfk gene were used, and it was found that all of these methods improved glutamate conversion efficiency to varying degrees. In particular, the attenuated Pzwf promoter strain 764a achieved a conversion rate of 59.0%, with no impact on fermentation cycle or sugar consumption. However, the attenuated pfk-E171K variant disclosed in patent (CN200580027259) exhibited reduced sugar consumption, resulting in a 24-hour extension of the fermentation cycle.

[0491] Table 13. Conversion rate of glutamate in pfk-modified strains

[0492]

[0493] 8.3 Starting from ATCC13032, weakening pfk

[0494] 8.3.1 Construction of MH13032-17-pfk-A1T Strain (MH13032-18)

[0495] Prepare MH13032-17 competent cells according to the Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), recombinant plasmid pK18-pfk-A1T (13032) is changed into Corynebacterium glutamicum MH13032-17 competent cells with the electrotransformation method, screen a recombinant on the selection medium that contains the kanamycin of 15mg / L. An identification primer is P86 / PE878-pfk-TTG-UP-1F, PE881-pfk-UP-2R / P81. The recombinant that sieves is inoculated into and cultivated 12-14 hour in the non-anti-BHI substratum, and the cultivation temperature is 30 ℃, rotary shaker 220rpm shaking culture. After the bacterial liquid is diluted 100-1000 times, be coated on the solid BHIS substratum that contains 10% sucrose and cultivated 36 hours. The secondary identification primers were PE898-A1T-ID-F and PE899-A1T-ID-R. Recombinant strains with the correct identification band size were sent to GENEWIZ for sequencing, followed by nucleotide sequencing analysis. The recombinant strain with the correct sequence was named MH13032-18.

[0496] 8.3.2 Construction of MH13032-18-PrecA-pfk strain (MH13032-19)

[0497] Prepare MH13032-18 competent cells according to the Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), recombinant plasmid pK18-PrecA-pfk (13032) is changed into Corynebacterium glutamicum MH13032-18 competent cells with the electrotransformation method, on the selection medium that contains the kanamycin of 15mg / L, screen a recombinant. An identification primer is P86 / PE882-pfk-UP-1F, PE885-pfk-DN-2R / P81. The recombinant that sieves is inoculated into and cultivated 12-14 hour in no anti-BHI substratum, and cultivation temperature is 30 ℃, rotary shaking table 220rpm shaking culture. Bacterial liquid is diluted 100-1000 times after being coated on the solid BHIS substratum that contains 10% sucrose and cultivated 36 hours. The secondary identification primers were PE891-PrecA-check-F and PE899-A1T-ID-R. Recombinant strains with the correct identification band size were sent to GENEWIZ for sequencing, followed by nucleotide sequencing analysis. The recombinant strain with the correct sequence was named MH13032-19.

[0498] 8.3.3 Construction of MH13032-19-pfk-E171D strain (MH13032-20)

[0499] Prepare MH13032-19 competent cells according to the Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), recombinant plasmid pK18-pfk-E171D (13032) is changed into Corynebacterium glutamicum MH13032-19 competent cells with the electrotransformation method, on the selection medium that contains the kanamycin of 15mg / L, screen a recombinant. An identification primer is P86 / PE892-pfk-E171K-UP-1F, PE895-pfk-E171K-DN-2R / P81. The recombinant that sieves is inoculated into and cultivated 12-14 hour in no anti-BHI substratum, and the cultivation temperature is 30 ℃, rotary shaking table 220rpm shaking culture. After the bacterium liquid is diluted 100-1000 times, be coated on the solid BHIS substratum that contains 10% sucrose and cultivated 36 hours. The secondary identification primers were P14-19 / P14-20. The recombinant bacteria with the correct identification band size were sent to GENEWIZ for sequencing, followed by nucleotide sequencing analysis. The recombinant microbial strain with the correct sequencing was named MH13032-20.

[0500] 8.3.4 Construction of MH13032-20-Pzwf-pfk strain (MH13032-21)

[0501] Prepare MH13032-20 competent cells according to the Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), recombinant plasmid pK18-Pzwf-pfk (13032) is changed into Corynebacterium glutamicum MH13032-20 competent cells with the electrotransformation method, on the selection medium that contains the kanamycin of 15mg / L, screen a recombinant. An identification primer is P86 / PE882-pfk-UP-1F, PE885-pfk-DN-2R / P81. The recombinant that sieves is inoculated into and cultivated 12-14 hour in no anti-BHI substratum, and the cultivation temperature is 30 ℃, rotary shaking table 220rpm shaking culture. Bacterial liquid is diluted 100-1000 times after being coated on the solid BHIS substratum that contains 10% sucrose and cultivated 36 hours. The secondary identification primers were PE888-zwf-check-F and PE899-A1T-ID-R. Recombinant strains with the correct identification band size were sent to GENEWIZ for sequencing, followed by nucleotide sequencing analysis. The recombinant strain with the correct sequence was named MH13032-21.

[0502] The recombinant Corynebacterium glutamicum constructed above was fermented to verify its glutamic acid production performance. The specific shake flask method refers to the shake flask fermentation method in Section 1.1.3. PFK was weakened based on MH13032-16 (six copies of xfp), involving a total of four schemes, and the shake flask results are shown in the table. The results showed that, with the exception of the pfk-E171D scheme, the remaining three schemes were all effective. And when Ppfk was replaced with the Pzwf scheme, the glutamic acid conversion rate reached a maximum of 41.7%.

[0503] Table 14. Glutamate content detection of recombinant strains

[0504]

[0505] 8.4 Starting from ATCC14067, weakening pfk

[0506] 8.4.1 Construction of MH14067-17-pfk-A1T Strain (MH14067-18)

[0507] Prepare MH14067-17 competent cells according to the Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), recombinant plasmid pK18-pfk-A1T (14067) is changed into Corynebacterium glutamicum MH14067-17 competent cells with the electrotransformation method, on the selection medium that contains the kanamycin of 15mg / L, screen a recombinant. An identification primer is P86 / PE878-pfk-TTG-UP-1F, PE881-pfk-UP-2R / P81. The recombinant that sieves is inoculated into and cultivated 12-14 hour in no anti-BHI substratum, and the cultivation temperature is 30 ℃, rotary shaker 220rpm shaking culture. After the bacterial liquid is diluted 100-1000 times, be coated on the solid BHIS substratum that contains 10% sucrose and cultivated 36 hours. The secondary identification primers were PE898-A1T-ID-F and PE899-A1T-ID-R. Recombinant strains with the correct identification band size were sent to GENEWIZ for sequencing, followed by nucleotide sequencing analysis. The recombinant strain with the correct sequence was named MH14067-18.

[0508] 8.4.2 Construction of MH14067-18-PrecA-pfk strain (MH14067-19)

[0509] Prepare MH14067-18 competent cells according to the Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), recombinant plasmid pK18-PrecA-pfk (14067) is changed into Corynebacterium glutamicum MH14067-18 competent cells with the electrotransformation method, on the selection medium that contains the kanamycin of 15mg / L, screen a recombinant. An identification primer is P86 / PE882-pfk-UP-1F, PE885-pfk-DN-2R / P81. The recombinant that sieves is inoculated into and cultivated 12-14 hour in no anti-BHI substratum, and cultivation temperature is 30 ℃, rotary shaking table 220rpm shaking culture. Bacterial liquid is diluted 100-1000 times after being coated on the solid BHIS substratum that contains 10% sucrose and cultivated 36 hours. The secondary identification primers were PE891-PrecA-check-F and PE899-A1T-ID-R. Recombinant strains with the correct identification band size were sent to GENEWIZ for sequencing, followed by nucleotide sequencing analysis. The recombinant strain with the correct sequence was named MH14067-19.

[0510] 8.4.3 Construction of MH14067-19-pfk-E171D strain (MH14067-20)

[0511] Prepare MH14067-19 competent cells according to the classical method of Corynebacterium glutamicum (C.glutamicum Handbook, Chapter 23). Recombinant plasmid pK18-pfk-E171D (14067) is transferred into Corynebacterium glutamicum MH14067-19 competent cells with the electroporation method. On the selection medium containing 15mg / L kanamycin, screen the recombinant. The primary identification primers are P86 / PE892-pfk-E171K-UP-1F and P14-18 / P81. The recombinant that is sieved is inoculated into the non-anti-BHI substratum and cultivated for 12-14 hours. The cultivation temperature is 30 ℃, and the shaking culture of rotary shaker 220rpm is carried out. After the bacterial liquid is diluted 100-1000 times, it is coated on the solid BHIS substratum that contains 10% sucrose and cultivated for 36 hours. The secondary identification primer is P14-19 / P14-20. The recombinant bacteria with the correct identification band size were sent to GENEWIZ for sequencing, followed by nucleotide sequencing analysis. The recombinant microbial strain with the correct sequencing was named MH14067-20.

[0512] 8.4.4 Construction of MH14067-20-Pzwf-pfk Strain (MH14067-21)

[0513] Prepare MH14067-20 competent cells according to the Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), recombinant plasmid pK18-Pzwf-pfk (14067) is changed into Corynebacterium glutamicum MH14067-20 competent cells with the electrotransformation method, on the selection medium that contains the kanamycin of 15mg / L, screen a recombinant. An identification primer is P86 / PE882-pfk-UP-1F, PE885-pfk-DN-2R / P81. The recombinant that sieves is inoculated into and cultivated 12-14 hour in no anti-BHI substratum, and the cultivation temperature is 30 ℃, rotary shaking table 220rpm shaking culture. Bacterial liquid is diluted 100-1000 times after being coated on the solid BHIS substratum that contains 10% sucrose and cultivated 36 hours. The secondary identification primers were PE888-zwf-check-F and PE899-A1T-ID-R. Recombinant strains with the correct identification band size were sent to GENEWIZ for sequencing, followed by nucleotide sequencing analysis. The recombinant strain with the correct sequence was named MH14067-21.

[0514] The recombinant C. glutamicum constructed above was fermented to verify its glutamate production performance. For the specific shake flask method, refer to the shake flask fermentation method in Section 1.1.3. Four schemes were used to weaken pfk based on MH14067-16. The shake flask results are shown in the table above. The results showed that, with the exception of the pfk-E171D scheme, the remaining three schemes were effective. Furthermore, the scheme where Ppfk was replaced with Pzwf achieved a maximum glutamate conversion rate of 36.2%.

[0515] Table 15. Glutamate content detection of recombinant strains

[0516]

[0517]

[0518] Example 9: Construction of xfp multi-copy strains and their glutamate conversion efficiency based on WJ0138-7

[0519] We performed multi-copy xfp genome expression based on WJ0138-7 (gdh / odhA / yggB combination). We completed the transformation of 7 copies of Bl-xfp and found that the integration of the 6th copy had the best bacterial performance. The insertion sites of the 7-copy xfp transformation are prioritized in the following order, but are not limited to these 7 insertion sites:

[0520] First copy: Ptuf-Bl_xfp is inserted at odhA;

[0521] Second copy: insert Ptuf-Bl_xfp at alaT;

[0522] The third copy: PcspB-Bl_xfp is inserted into odhA;

[0523] The fourth copy: Ptuf-Bl_xfp was inserted between cg3384 and cg3385 genes;

[0524] The fifth copy: Ptuf-Bl_xfp was inserted between cg2564 and cg2563 genes;

[0525] The sixth copy: Ptuf-Bl_xfp was inserted between cg2211 and cg2212 genes;

[0526] The 7th copy: Ptuf-Bl_xfp was inserted between cg3364 and cg3365 genes;

[0527] The optional insertion sites are as follows:

[0528] Within the alaT gene, within the odhA gene, between cg3364 and cg3365 genes, between cg2564 and cg2563 genes, between cg3124 and cg3125 genes, between cg3384 and cg3385 genes, between BBD29_07270 and BBD29_07275 genes, between cg2211 and cg2212 genes, between cg0928 and CGTRNA_RS15430 genes, between BBD29_07210 and BBD29_07215 genes, between cg1512 and cg1513 genes, and between cg2337 and cg2336 genes.

[0529] Other modifications to reduce odhA activity included complete odhA knockout, odhA truncation with simultaneous frameshift repair to wild-type, and other truncated forms of odhA with activity reduced by more than 10%. Modifications to enhance gdh activity also involved enhancing gdh activity using a strong promoter. All of these substitutions yielded similar results.

[0530] Incorporated by Reference

[0531] Each patent and scientific document mentioned herein is incorporated by reference in its entirety for all purposes.

[0532] Equivalence

[0533] The present disclosure may be embodied in other specific forms without departing from its spirit or essential characteristics. Therefore, the above-described embodiments should be considered in all cases as illustrative rather than limiting of the invention described herein. The scope of the present disclosure is therefore indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalents of the claims are intended to be embraced therein.

Claims

1. A modified bacterium producing L-glutamic acid, wherein the genome of the modified bacterium comprises a heterologous polynucleotide encoding phosphoketolase (XFP) compared to a non-modified bacterium.

2. The modified bacterium according to claim 1, wherein the bacterium is a Corynebacterium, preferably Corynebacterium glutamicum.

3. The modified bacterium according to claim 1 or 2, wherein the phosphoketolase is D-xylulose-5-phosphate phosphoketolase and / or D-fructose 6-phosphate phosphoketolase.

4. The modified bacterium according to any one of claims 1 to 3, wherein the phosphoketolase is derived from Bifidobacterium, preferably Bifidobacterium longum, Bifidobacterium adolescentis, or Bifidobacterium animalis.

5. The modified bacterium according to any one of claims 1 to 4, comprising more than one copy of the phosphoketolase gene, preferably three to ten copies, more preferably six copies.

6. The modified bacterium of any one of claims 1 to 5, wherein the insertion site of each copy of the phosphoketolase gene is selected from the group consisting of: within the alaT gene, within the odhA gene, between the cg3364 and cg3365 genes, between the cg2564 and cg2563 genes, between the cg3124 and cg3125 genes, between the cg3384 and cg3385 genes, between the BBD29_07270 and BBD29_07275 genes, between the cg2211 and cg2212 genes, between the cg0928 and CGTRNA_RS15430 genes, between the BBD29_07210 and BBD29_07215 genes, between the cg1512 and cg1513 genes, and between the cg2337 and cg2336 genes.

7. The modified bacterium according to any one of claims 1 to 6, wherein the phosphoketolase comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17, or an amino acid sequence that is at least 70% identical to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17; or the phosphoketolase comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18, or a nucleic acid sequence that is at least 70% identical to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18.

8. The modified bacterium according to any one of claims 1 to 7, further comprising a modification of the expression control sequence of the phosphoketolase gene, preferably, the modification of the expression control sequence is a promoter, more preferably, the promoter is a Ptuf promoter or a PscpB promoter.

9. The modified bacterium according to any one of claims 1 to 8, further comprising a modification that reduces odhA activity, a mutation in the yggB encoded protein that increases glutamate production, such as an amino acid substitution of A100T in the yggB encoded protein, and / or a modification that enhances gdh activity; preferably, the modification that reduces odhA activity is selected from the group consisting of complete odhA knockout, odhA truncation and simultaneous repair of the frameshift mutation to the wild type, or other truncated forms of odhA with activity reduced by more than 10%, and the modification that enhances gdh activity is the use of a strong promoter.

10. The modified bacterium of any one of claims 1 to 9, further comprising gltA -10M-C361Y 、suc M199I ach S372C 、tktA T234I 、putA A755V 、ds G171D , and / or nadA D312N .

11. The modified bacterium of any one of claims 1 to 10, wherein the bacterium further comprises a modification that reduces phosphofructokinase activity.

12. The modified bacterium according to claim 11, wherein the bacterium comprises a modification of an expression control sequence of the phosphofructokinase gene and / or a modification of a coding sequence of the phosphofructokinase gene.

13. The modified bacterium of claim 12, wherein the modification of the expression control sequence of the phosphofructokinase gene is selected from the group consisting of: a) replacing the phosphofructokinase gene promoter with a PrecA promoter; b) replacing the phosphofructokinase gene promoter with a Pzwf promoter; c) replacing the phosphofructokinase gene promoter with a Pgdh promoter.

14. The modified bacterium according to claim 12, wherein the modification of the coding sequence of the phosphofructokinase gene is selected from: a) replacing the ATG start sequence of the coding nucleic acid of the phosphofructokinase gene with TTG; and / or b) replacing the protein coding sequence of the phosphofructokinase gene with E171D.

15. The modified bacterium according to any one of claims 1 to 14, wherein the L-glutamic acid is ammonium L-glutamate or sodium L-glutamate.

16. Use of the modified bacterium according to any one of claims 1 to 15 for increasing L-glutamic acid production.

17. A method for producing L-glutamic acid, comprising culturing the modified bacterium according to any one of claims 1 to 16 in a culture medium.

18. The method of claim 17, wherein the culture medium comprises glucose.

19. The method of claim 17 or claim 18, further comprising isolating L-glutamic acid.

20. A bioreactor comprising the modified bacteria of claims 1-16.

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