Method for producing l-glutamic acid

Abstract

Provided is a modified bacterium for producing L-glutamic acid, which contains a heterologous polynucleotide encoding a phosphoketolases (XFP) in the genome thereof compared to a non-modified bacterium. The phosphoketolases is derived from Bifidobacterium. In addition, further provided is a method for increasing the yield of the L-glutamic acid by means of using the modified bacterium.

Description

A method for producing L-glutamic acid

[0001] priority

[0002] This application claims the benefit of and priority to Chinese Application No. 2024101859904, filed on February 19, 2024, the entire contents of which are incorporated herein by reference in their entirety for all purposes. Technical Field

[0003] The present disclosure relates to a method for producing L-glutamic acid by fermentation using bacteria. Background Art

[0004] 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.

[0005] 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.

[0006] 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

[0007] 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.

[0008] In a specific embodiment, the bacterium is a Corynebacterium bacterium, preferably Corynebacterium glutamicum.

[0009] In a specific embodiment, the phosphoketolase is D-xylulose-5-phosphate phosphoketolase and / or D-fructose 6-phosphate phosphoketolase.

[0010] In a specific embodiment, the phosphoketolase is derived from Bifidobacterium, preferably Bifidobacterium longum, Bifidobacterium adolescentis, or Bifidobacterium animalis.

[0011] 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; a mutation in the yggB encoding protein to increase glutamate production; and / or a modification to enhance gdh activity.

[0012] In a specific embodiment, the modified bacteria further include modifications that reduce odhA activity, such as knockout or partial knockout of odhA, such as deletion of 5 base pairs at positions 544-548 (gacgt) of the odhA gene; mutations in the yggB encoding protein, such as amino acid substitution of A100T, amino acid substitution of A111V, substitution of leucine at amino acid position 93 to alanine, substitution of leucine at amino acid position 109 to alanine, mutations in the region encoding amino acid residues 419-533 of the wild-type YggB protein, mutations in the region encoding the transmembrane domain of the wild-type YggB 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.

[0013] Those skilled in the art will appreciate that the modification method for reducing the activity of odhA can be of various types, and the modification for reducing the activity of odhA is selected from complete or partial knockout of odhA, wherein the activity is reduced by more than 10%, for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0014] Those skilled in the art will appreciate that the gdh activity-enhancing modification methods may be various, for example, using a wild-type gdh and a strong promoter at the same time, for example, a DNA sequence at position -35 of the gdh transcription start site is TTGTCA and a DNA sequence at position -10 of the transcription start site is TATAAT, or other promoter mutations that enhance gdh activity.

[0015] Those skilled in the art will appreciate that the amino acid substitution of A100T or A111V in the yggB encoded protein, as well as substitutions that can effectively increase glutamate production, can also be used in the present disclosure, such as the amino acid substitution of A111V in the yggB encoded protein, substitution of leucine at amino acid position 93 with alanine, substitution of leucine at amino acid position 109 with alanine, mutations in the region encoding amino acid residues 419-533 of the wild-type YggB protein, and mutations in the region encoding the transmembrane domain of the wild-type YggB protein.

[0016] 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 PcspB promoter.

[0017] In a specific embodiment, the modified bacterium further comprises a modification that reduces phosphofructokinase activity.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] In a specific embodiment, the phosphoketolase comprises an amino acid sequence selected from the group consisting of an amino acid sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 98%, or 99% identity to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17; or the phosphoketolase comprises an amino acid sequence selected from the group consisting of an amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 98%, or 99% identity to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 are nucleic acid sequences in the group consisting of nucleic acid sequences with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99% or more identity.

[0024] In one embodiment, the modified bacteria further comprises gltA -10M -C361Y、suc M199I ach S372C 、tktA T234I 、putA A755V 、ds G171D , and / or nadA D312N .

[0025] In a specific embodiment, the L-glutamic acid is ammonium L-glutamate or sodium L-glutamate.

[0026] In another aspect, provided is use of the modified bacteria of the present disclosure for increasing L-glutamic acid production.

[0027] 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.

[0028] In a specific embodiment, said culture medium comprises glucose.

[0029] In a specific embodiment, the method further comprises isolating L-glutamic acid.

[0030] In another aspect, a bioreactor comprises the modified bacteria described in the present disclosure. 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] Example 1: Construction of starting strains

[0036] 1.1 Construction of ATCC13869→WJ0140 starting strain

[0037] 1.1.1 Plasmid construction

[0038] 1).pK18-Pgdh -10M Plasmid construction

[0039] The genome of Corynebacterium glutamicum ATCC13869 (GenBank: GCA_001687645.1, the same below) 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 PE640 / PE641 and PE642 / PE643 to obtain Pgdh -10MThe primer sequences for the upstream and downstream homology arms (UP and DN) are shown in Table 1. The PCR procedure 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 minutes. The resulting fragments were purified and digested using an agarose gel recovery kit (Tian Gen) and stored frozen at -20°C until further use. Subsequently, recombinant fragments were prepared using the PE640 / PE643 primer pair and the upstream and downstream homology arms as templates. The PCR procedure was the same as above, and the resulting recombinant fragments were 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 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 assay were PE640 / PE643. The resulting plasmid was named pK18-Pgdh -10M .

[0040] 2).pK18-yggB A100T Plasmid construction

[0041] 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 was performed to obtain 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 (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 1036bp. The inserted fragment was further identified by sequencing (GENEWIZ Company), and the primers sent for testing were PE646 / PE649. The resulting plasmid was named pK18-yggB A100T .

[0042] 3). Construction of pK18-odhA ORF-5bpQS plasmid

[0043] With Corynebacterium glutamicum ATCC13869 genome as template, utilize Phusion super-fidelity polymerase (New England BioLabs) to carry out PCR amplification.Be that primer pair carries out PCR amplification and obtains upstream and downstream homology arm UP and DN fragment with PE652 / PE653, PE654 / PE655, 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 T4DNA ligase (TransGen Biotech), fragment is connected to carrier, transform Trans1T1 competent cells (TransGen Biotech), picking kana resistance clone, XbaI / EcoRI enzyme digestion identification obtains the positive clone that fragment inserts pK18mobsacB, and correct band size is 1039bp. The inserted fragment was further confirmed to be correct by sequencing (GENEWIZ), using primers PE652 / PE655. The resulting plasmid was named pK18-odhAORF-5bpQS.

[0044] 4).pK18-gltA -10M-C361Y Plasmid construction

[0045] 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-C361YThe 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 Company), and the primers sent for testing were PE656 / PE661 / PE362 / PE363. The resulting plasmid was named pK18-gltA -10M-C361Y .

[0046] 5).pK18-ach S372C Plasmid construction

[0047] 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 (Tiangen).Simultaneously, pK18-mobsacB is utilized XbaI / HindIII to digest, and with T4DNA ligase (TransGen Biotech), 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-ach S372C .

[0048] 6).pK18-suc M199I Plasmid construction

[0049] 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 Company). The primers sent for testing were 49-UP-1F / 49-DN-2R. The resulting plasmid was named pK18-suc M199I .

[0050] 7).pK18-tktA T234I Plasmid construction

[0051] 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 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 .

[0052] 8).pK18-putA-cg0129 A755V (13869)Plasmid construction

[0053] 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 identified positive clones with the fragment inserted into pK18mobsacB. The correct band size was 998 bp. Sequencing (GENEWIZ) confirmed the correct insertion of the fragment using the putA-UP-1F / putA-DN-2R primers. The resulting plasmid was named pK18-putA-cg0129. A755V (13869).

[0054] 9).pK18-nadA D312N Plasmid construction

[0055] 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 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 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 fragments 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 sent for testing were nadA-UP-1F / nadA-DN-2R. The resulting plasmid was named pK18-nadA D312N .

[0056] 10).pK18-ds G171D Plasmid construction

[0057] 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 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 .

[0058] 1.1.2 Strain construction

[0059] 1).ATCC13869-Pgdh -10M Strain construction (WJ0138-1)

[0060] ATCC13869 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and pK18-Pgdh -10MThe plasmid was transformed into Corynebacterium glutamicum ATCC13869, and exchange recombinants were selected on BHI medium containing 15 mg / L kanamycin. Kan clones were identified by colony PCR using Fast Taq DNA polymerase (TransGen Biotech) 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 72°C for 15 seconds / kb extension for 30 cycles, followed by a complete extension at 72°C for 10 minutes. The fragment lengths amplified by the two primer pairs were similar, with clones around 1100 bp being considered positive. The selected positive clones were inoculated into antibiotic-free BHI medium (Oxoid, product number CM1135B, the same below) 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, and the KanS recombinants were selected and the point mutation recombinants were verified using the identification primer pair PE642 / PE643. The obtained positive recombinants were amplified and sequenced using PE640 / PE644. The correct recombinant was 2356 bp long, and the primers submitted for testing were PE640 / PE644 / PE642 / PE643 / PE645. The strain with correct sequencing was named WJ0138-1.

[0061] 2).WJ0138-1-yggB A100T Strain construction (WJ0138-4)

[0062] Prepare WJ0138-1 competent cells according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and insert pK18-yggB A100TThe plasmid was transformed into Corynebacterium glutamicum WJ0138-1, and recombinants were selected for exchange on BHI medium containing 15 mg / L kanamycin. Kan clones were identified by colony PCR using Fast Taq DNA polymerase (TransGen Biotech) with primer pairs PE646 / P85 and P82 / PE649, using the same PCR procedure. The fragment lengths amplified by the two primer pairs showed little difference, 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 PE648 / PE649. The obtained positive recombinants were amplified and sequenced using PE650 / PE651. The correct recombinant was 2020 bp in length. The primers submitted for testing were PE650 / PE651 / PE648 / PE649. The strain with correct sequencing was named WJ0138-4.

[0063] 3). Construction of WJ0138-4-odhAORF-5bpQS strain (WJ0138-7)

[0064] Prepare WJ0138-4 competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), pK18-odhAORF-5bpQS plasmid is changed among the Corynebacterium glutamicum WJ0138-4, on the BHI substratum 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 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.

[0065] 4).WJ0138-7-gltA -10M +gltA C361Y Strain construction (WJ0138-9)

[0066] Prepare WJ0138-7 competent cells according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and insert pK18-gltA -10M-C361Y Plasmid was transferred into Corynebacterium glutamicum WJ0138-7 and recombinants were selected for exchange on a BHI culture medium containing 15 mg / L of kanamycin. Utilizing Fast Taq DNA polymerase (TransGen Biotech), colony PCR was performed to identify Kan clones using the PE656 / P85 and P82 / PE661 primer pairs. The PCR procedure was the same. The two pairs of primer pairs had a significant difference in the length of the fragments amplified. The short band was about 1100 bp, and the long band was a positive clone at about 2500 bp. 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 PE660 / PE661. The obtained positive recombinants were amplified and sequenced using PE662 / PE663. The correct recombinant was 2439 bp in length. The primers submitted for testing were PE662 / PE663 / PE362 / PE363. The strain with correct sequencing was named WJ0138-9.

[0067] 5).WJ0138-9-suc M199I Strain construction (WJ0139-2)

[0068] Prepare WJ0138-9 competent cells according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and transfect pK18-suc M199IThe plasmid was transformed into Corynebacterium glutamicum WJ0138-9 competent cells by electroporation, and recombinants were selected for exchange on BHI medium containing 15 mg / L of kanamycin. Using Fast Taq DNA polymerase (TransGen Biotech), colony PCR was performed to identify KanR clones using the 49-UP-1F / P85 and P82 / 49-DN-2R primer pairs. 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 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 49-F / 49-DN-2R. 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.

[0069] 6).WJ0139-2-ach S372C Strain construction (WJ0139-3)

[0070] Prepare WJ0139-2 competent cells according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and insert pK18-ach S372C The plasmid was transformed into Corynebacterium glutamicum WJ0139-2, and recombinants were selected on BHI 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.

[0071] 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. The resulting positive recombinants were amplified and sequenced using PE667 / PE668. The correct recombinant was 1650 bp long, using the primers PE667 / PE668 / PE664. The strain with the correct sequence was designated WJ0139-3.

[0072] 7).WJ0139-3-tktA T234I Strain construction (WJ0139-5)

[0073] Prepare WJ0139-3 competent cells according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and insert pK18-tktA T234I The plasmid was transformed into Corynebacterium glutamicum WJ0139-3, and recombinants were selected for exchange on BHI medium containing 15 mg / L kanamycin. KanR clones were identified by colony PCR using Fast Taq DNA polymerase (TransGen Biotech) with the primer pairs tktA-UP-1F / P85 and P82 / tktA-DN-2R. 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. 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.

[0074] 8).WJ0139-5-putA-cg0129 A755V Strain construction (WJ0139-8)

[0075] Prepare WJ0139-5 competent cells according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and insert pK18-putA-cg0129 A755VThe plasmid (13869) was transformed into Corynebacterium glutamicum WJ0139-5, and exchange recombinants were selected on BHI medium supplemented with 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. 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 putA-F / putA-DN-2R. The resulting positive recombinants were amplified and sequenced using ID-F / ID-R. The correct recombinant was 1214 bp in length. The primers submitted for testing were putA-ID-F / putA-ID-R. The strain with correct sequencing was named WJ0139-8.

[0076] 9).WJ0139-8-ds G171D Strain construction (WJ0139-9)

[0077] WJ0139-8 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and pK18-ds G171D The plasmid was transferred into Corynebacterium glutamicum WJ0139-8, and the exchange recombinants were selected on BHI medium containing 15 mg / L of kanamycin. Using Fast Taq DNA polymerase (TransGen Biotech), colony PCR was performed with ds-UP-1F / P85 and P82 / ds-DN-2R primers to identify KanR clones. The PCR procedure was the same as above. The fragment lengths amplified by the two pairs of primers 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 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. S The recombinant was verified as a point mutation recombinant using the identification primer pair ds-F / ds-DN-2R. The positive recombinant was amplified and sequenced using ds-ID-F / ds-ID-R. The correct recombinant was 1172 bp long. The primers submitted for testing were ds-ID-F / ds-ID-R. The strain with correct sequencing was named WJ0139-9.

[0078] 10).WJ0139-9-nadA D312N Strain construction (WJ0140)

[0079] Prepare WJ0139-9 competent cells according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and insert pK18-nadA D312N The plasmid was transformed into Corynebacterium glutamicum WJ0139-9, and recombinants were selected on BHI 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, 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 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. 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.

[0080] 1.1.3 Shake flask verification

[0081] The recombinant Corynebacterium glutamicum constructed was fermented to verify its glutamic acid production performance, specifically as follows: the strain frozen in a -80°C glycerol tube was inoculated in a slant medium for activation, grown a lawn after culturing at 31.5°C for 24 hours, picked a lawn from the freshly activated slant, inoculated into the above-mentioned seed culture medium, and cultured with shaking at 31.5°C, 220rpm to the middle and late stages of logarithmic growth for 12 hours to obtain a seed solution, which was inoculated into a 500mL shake flask equipped with 20mL of fermentation medium with an inoculum size of 10%, and cultured with shaking at 31.5°C, 220rpm for 16 hours. After glucose was completely consumed, the concentration of L-glutamic acid accumulated in the culture medium was determined by HPLC.

[0082] The culture medium formula is as follows:

[0083] 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;

[0084] 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;

[0085] 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.

[0086] Table 2. Glutamate content detection of recombinant strains

[0087] 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.

[0088] 1.2 Construction of ATCC13032→MH13032-10 starting strain

[0089] 1.2.1 Plasmid construction

[0090] 1).pK18-Pgdh -10M (13032)Plasmid construction

[0091] Glutamibacterium ATCC13032 genome (GenBank NO: NC_003450.3, the same below) 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 PE640 / PE641 and PE642 / PE643 to obtain Pgdh -10MThe 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. Positive clones with the fragment inserted into pK18mobsacB were identified by XbaI / EcoRI digestion. 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 (13032).

[0092] 2).pK18-yggB A100T (13032)Plasmid construction

[0093] 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 yielded the 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.

[0094] 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 connected 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 sent for testing were PE646 / PE649. The resulting plasmid was named pK18-yggB A100T (13032).

[0095] 3). Construction of pK18-odhA ORF-5bpQS(13032) plasmid

[0096] 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 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(13032).

[0097] 4).pK18-gltA -10M-C361Y (13032)Plasmid construction

[0098] 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 primer pairs PE656 / PE657 and PE658 / PE661 to obtain gltA. -10M-C361Y The upstream homology arm UP and fragment, the primer sequence is shown in Table 1. Subsequently, PE656 / PE661 was used as a primer pair, and the upstream homology arm and 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 Company), and the primers sent for testing were PE656 / PE661 / PE362 / PE363. The resulting plasmid was named pK18-gltA -10M-C361Y (13032).

[0099] 5).pK18-ach S372C (13032)Plasmid construction

[0100] 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 primer sequences of the upstream and downstream homology arm UP and DN fragments 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). Trans1T1 competent cells (TransGen Biotech) were transformed, and kanamycin-resistant clones were picked. 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).

[0101] 6).pK18-suc M199I (13032)Plasmid construction

[0102] 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).

[0103] 7).pK18-tktA T234I (13032)Plasmid construction

[0104] 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).

[0105] 8).pK18-putA-cg0129 A755V (13032)Plasmid construction

[0106] 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 identified positive clones with the fragment inserted into pK18mobsacB. The correct band size was 998 bp. Sequencing (GENEWIZ) confirmed the correct insertion of the fragment using the putA-UP-1F / putA-DN-2R primers. The resulting plasmid was named pK18-putA-cg0129. A755V (13032).

[0107] 9).pK18-nadA D312N (13032)Plasmid construction

[0108] 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 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 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 fragments 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).

[0109] 10).pK18-ds G171D (13032)Plasmid construction

[0110] 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).

[0111] 1.2.2 Construction of glutamate-producing strains based on ATCC 13032

[0112] 1).ATCC13032-Pgdh -10M Strain construction (MH13032-1)

[0113] ATCC13032 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and pK18-Pgdh -10MThe plasmid (13032) was transformed into Corynebacterium glutamicum ATCC13032, and exchange recombinants were selected on BHI medium containing 15 mg / L kanamycin. Kan clones were identified by colony PCR using Fast Taq DNA polymerase (TransGen Biotech) 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. 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 PE642 / PE643. The obtained positive recombinants were amplified and sequenced using PE640 / PE644. The correct recombinant was 2356 bp in length. The primers submitted for testing were PE640 / PE644 / P14-2 / PE643 / PE645. The strain with correct sequencing was named MH13032-1.

[0114] 2).MH13032-1-yggB A100T Strain construction (MH13032-2)

[0115] Prepare MH13032-1 competent cells according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and insert pK18-yggB A100TThe plasmid (13032) was transformed into Corynebacterium glutamicum MH13032-1, and exchange recombinants were selected on BHI medium containing 15 mg / L kanamycin. Kan clones were identified by colony PCR using Fast Taq DNA polymerase (TransGen Biotech) with primer pairs PE646 / P85 and P82 / PE649, 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 PE648 / PE649. The obtained positive recombinants were amplified and sequenced using PE650 / PE651. The correct recombinant was 2020 bp long. The primers submitted for testing were PE650 / PE651 / PE648 / PE649. The strain with correct sequencing was named MH13032-2.

[0116] 3). Construction of MH13032-2-odhAORF-5bpQS strain (MH13032-3)

[0117] Prepare MH13032-2 competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), pK18-odhAORF-5bpQS (13032) plasmid is changed among the Corynebacterium glutamicum MH13032-2, on the BHI substratum 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 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 P13-1 / PE026. The correct recombinant was 3993bp long, and the primers used for the test were P13-1 / PE026 / PE020 / PE021 / PE022 / PE023 / PE024 / PE655. The strain with the correct sequence was designated MH13032-3.

[0118] 4).MH13032-3-gltA -10M-C361Y Strain construction (MH13032-4)

[0119] MH13032-3 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and pK18-gltA -10M-C361YThe plasmid (13032) was transformed into Corynebacterium glutamicum MH13032-3, and exchange recombinants were selected on BHI 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. 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 PE660 / PE661. The obtained positive recombinants were amplified and sequenced using PE662 / PE663. The correct recombinant was 2439 bp in length. The primers submitted for testing were PE662 / PE663 / PE362 / PE363. The strain with correct sequencing was named MH13032-4.

[0120] 5).MH13032-4-suc M199I Strain construction (MH13032-5)

[0121] Prepare MH13032-4 competent cells according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and transfect pK18-suc M199IThe (13032) plasmid was transformed into competent cells of Corynebacterium glutamicum MH13032-4 by electroporation, and recombinants were selected on BHI medium supplemented with 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. 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 P13-4 / 49-DN-2R. The obtained 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.

[0122] 6).MH13032-5-ach S372C Strain construction (MH13032-6)

[0123] Prepare MH13032-5 competent cells according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and insert pK18-ach S372C The plasmid (13032) was transformed into Corynebacterium glutamicum MH13032-5, and recombinants were selected on BHI medium supplemented with 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, 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 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. The obtained positive recombinants were amplified and sequenced using PE667 / PE668. The correct recombinant was 1650 bp in length. The primers submitted for testing were PE667 / PE668 / PE664. The strain with correct sequencing was named MH13032-6.

[0124] 7).MH13032-6-tktA T234I Strain construction (MH13032-7)

[0125] MH13032-6 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and pK18-tktA T234I The plasmid (13032) was transformed into Corynebacterium glutamicum MH13032-6, and recombinants were selected on BHI medium supplemented with 15 mg / L kanamycin. KanR clones were identified by colony PCR using Fast Taq DNA polymerase (TransGen Biotech) with the primer pairs tktA-UP-1F / P85 and P82 / tktA-DN-2R. The PCR procedure was the same as above. The fragments amplified by the two primer pairs were similar in length, 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 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. 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.

[0126] 8).MH13032-7-putA-cg0129 A755V Strain construction (MH13032-8)

[0127] Prepare MH13032-7 competent cells according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and insert pK18-putA-cg0129 A755VThe plasmid (13032) was transformed into Corynebacterium glutamicum MH13032-7, and recombinants were selected on BHI medium supplemented with 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. 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 putA-F / putA-DN-2R. 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.

[0128] 9).MH13032-8-ds G171D Strain construction (MH13032-9)

[0129] Prepare MH13032-8 competent cells according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and insert pK18-ds G171D The plasmid (13032) was transformed into Corynebacterium glutamicum MH13032-8, and exchange recombinants were selected on BHI medium supplemented with 15 mg / L kanamycin. KanR clones were identified by colony PCR using Fast Taq DNA polymerase (TransGen Biotech) with the primer pairs ds-UP-1F / P85 and P82 / ds-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. 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 the point mutation recombinants were verified using the identification primer pair ds-F / ds-DN-2R. The resulting positive recombinants were amplified and sequenced using ds-ID-F / ds-ID-R. The correct recombinant was 1172 bp in length. The primers submitted for testing were ds-ID-F / ds-ID-R. The strain with correct sequencing was named MH13032-9.

[0130] 10).MH13032-9-nadA D312N Strain construction (MH13032-10)

[0131] MH13032-9 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and pK18-nadA D312N The plasmid (13032) was transformed into Corynebacterium glutamicum MH13032-9, and exchange recombinants were selected on BHI medium supplemented with 15 mg / L kanamycin. KanR clones were identified by colony PCR using Fast Taq DNA polymerase (TransGen Biotech) with the primer pairs P13-7 / P85 and P82 / nadA-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 nadA-F / nadA-DN-2R. 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.

[0132] 1.2.3 Shake flask validation of glutamate production strain ATCC13032

[0133] 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.

[0134] Table 3. Glutamate content detection of recombinant strains starting from ATCC13032

[0135] 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.

[0136] 1.3 Construction of ATCC14067→MH14067-10 starting strain

[0137] 1.3.1 Plasmid construction

[0138] 1).pK18-Pgdh -10M (14067)Plasmid construction

[0139] Glutamibacterium ATCC14067 genome (Genbank: GCA_002243555.1, the same below) 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 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. Positive clones with the fragment inserted into pK18mobsacB were identified by XbaI / EcoRI digestion. 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).

[0140] 2).pK18-yggB A100T (14067)Plasmid construction

[0141] 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 PE646 / PE647 and PE648 / PE649 to obtain 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 (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 1036bp. The inserted fragment was further identified by sequencing (GENEWIZ Company), and the primers sent for testing were PE646 / PE649. The resulting plasmid was named pK18-yggB A100T (14067).

[0142] 3). Construction of plasmid pK18-odhA ORF-5bpQS(14067)

[0143] 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 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(14067).

[0144] 4).pK18-gltA -10M-C361Y (14067)Plasmid construction

[0145] 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 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 (14067).

[0146] 5).pK18-ach S372C (14067)Plasmid construction

[0147] 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 S372CThe primer sequences of the upstream and downstream homology arm UP and DN fragments 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). Trans1T1 competent cells (TransGen Biotech) were transformed, and kanamycin-resistant clones were picked. 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).

[0148] 6).pK18-suc-NCgl0049 M199I (14067)Plasmid construction

[0149] 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 using 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).

[0150] 7).pK18-tktA T234I (14067)Plasmid construction

[0151] 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).

[0152] 8).pK18-putA-cg0129 A755V (14067)Plasmid construction

[0153] 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 ATCC14067 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 identified positive clones with the fragment inserted into pK18mobsacB. The correct band size was 998 bp. Sequencing (GENEWIZ) confirmed the correct insertion of the fragment using the putA-UP-1F / putA-DN-2R primers. The resulting plasmid was named pK18-putA-cg0129. A755V (14067).

[0154] 9).pK18-nadA D312N (14067)Plasmid construction

[0155] 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 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 fragments 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 sent for testing were nadA-UP-1F / nadA-DN-2R. The resulting plasmid was named pK18-nadA D312N (14067).

[0156] 10).pK18-ds G171D (14067)Plasmid construction

[0157] 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).

[0158] 1.3.2 Construction of glutamate-producing strains starting with ATCC14067

[0159] 1).ATCC14067-Pgdh -10M Strain construction (MH14067-1)

[0160] ATCC14067 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and pK18-Pgdh -10MThe plasmid (14067) was transformed into Corynebacterium glutamicum ATCC14067, and exchange recombinants were selected on BHI medium containing 15 mg / L kanamycin. Kan clones were identified by colony PCR using Fast Taq DNA polymerase (TransGen Biotech) 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. 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 PE642 / PE643. The obtained positive recombinants were amplified and sequenced using PE640 / PE644. The correct recombinant was 2356 bp in length. The primers submitted for testing were PE640 / PE644 / P14-2 / PE643 / PE645. The strain with correct sequencing was named MH14067-1.

[0161] 2).MH14067-1-yggB A100T Strain construction (MH14067-2)

[0162] Prepare MH14067-1 competent cells according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and insert pK18-yggB A100TThe plasmid (14067) was transformed into Corynebacterium glutamicum MH14067-1, and exchange recombinants were selected on BHI medium containing 15 mg / L kanamycin. Kan clones were identified by colony PCR using Fast Taq DNA polymerase (TransGen Biotech) with primer pairs PE646 / P85 and P82 / PE649, 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 the point mutation recombinants were verified using the identification primer pair PE648 / PE649. The obtained positive recombinants were amplified and sequenced using PE650 / PE651. The correct recombinant was 2020 bp long. The primers submitted for testing were PE650 / PE651 / PE648 / PE649. The strain with correct sequencing was named MH14067-2.

[0163] 3). Construction of MH14067-2-odhAORF-5bpQS strain (MH14067-3)

[0164] Prepare MH14067-2 competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), pK18-odhAORF-5bpQS (14067) plasmid is changed among the Corynebacterium glutamicum MH14067-2, on the BHI substratum 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 colony 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.

[0165] 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.

[0166] 4).MH14067-3-gltA -10M-C361Y Strain construction (MH14067-4)

[0167] Prepare MH14067-3 competent cells according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and insert pK18-gltA -10M-C361Y The plasmid (14067) was transformed into Corynebacterium glutamicum MH14067-3, and exchange recombinants were selected on BHI 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. 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 PE660 / PE661. The obtained positive recombinants were amplified and sequenced using PE662 / PE663. The correct recombinant was 2439 bp in length. The primers submitted for testing were PE662 / PE663 / PE362 / PE363. The strain with correct sequencing was named MH14067-4.

[0168] 5).MH14067-4-suc M199I Strain construction (MH14067-5)

[0169] Prepare MH14067-4 competent cells according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and transfect pK18-suc M199IThe plasmid (14067) was transformed into competent cells of Corynebacterium glutamicum MH14067-4 by electroporation, and recombinants were selected on BHI medium supplemented with 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. 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 49-F / 49-DN-2R. The obtained 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.

[0170] 6).MH14067-5-ach S372C Strain construction (MH14067-6)

[0171] Prepare MH14067-5 competent cells according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and insert pK18-ach S372C The plasmid (14067) was transformed into Corynebacterium glutamicum MH14067-5, and exchange recombinants were selected on BHI 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, following the same PCR procedure as above. The fragment lengths amplified by the two primer pairs were similar, 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 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, and the KanS recombinants were selected and the point mutation recombinants were verified using the identification primer pair PE666 / PE667. The obtained positive recombinants were amplified and sequenced using PE667 / PE668. The correct recombinant was 1650 bp in length, and the primers submitted for testing were PE667 / PE668 / PE664. The strain with correct sequencing was named MH14067-6.

[0172] 7).MH14067-6-tktA T234I Strain construction (MH14067-7)

[0173] Prepare MH14067-6 competent cells according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and insert pK18-tktA T234I The plasmid (14067) was transformed into Corynebacterium glutamicum MH14067-6, and exchange recombinants were selected on BHI medium containing 15 mg / L kanamycin. KanR clones were identified by colony PCR using Fast Taq DNA polymerase (TransGen Biotech) with the primer pairs tktA-UP-1F / P85 and P82 / tktA-DN-2R. The PCR procedure was the same as above. The fragments amplified by the two primer pairs were similar in length, 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 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. 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.

[0174] 8).MH14067-7-putA-cg0129 A755V Strain construction (MH14067-8)

[0175] Prepare MH14067-7 competent cells according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and insert pK18-putA-cg0129 A755VThe plasmid (14067) was transformed into Corynebacterium glutamicum MH14067-7, and recombinants were selected on BHI medium supplemented with 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. 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 putA-F / putA-DN-2R. 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.

[0176] 9).MH14067-8-ds G171D Strain construction (MH14067-9)

[0177] MH14067-8 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and pK18-ds G171D The plasmid (14067) was transferred into Corynebacterium glutamicum MH14067-8, and the exchange recombinants were selected on BHI medium containing 15 mg / L kanamycin. Using Fast Taq DNA polymerase (TransGen Biotech), colony PCR was performed with primer pairs ds-UP-1F / P85 and P82 / ds-DN-2R 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. The positive recombinant was amplified and sequenced using ds-ID-F / ds-ID-R. The correct recombinant was 1172 bp long. The primers submitted for testing were ds-ID-F / ds-ID-R. The strain with correct sequencing was named MH14067-9.

[0178] 10).MH14067-9-nadA D312N Strain construction (MH14067-10)

[0179] Prepare MH14067-9 competent cells according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23), and insert pK18-nadA D312N The plasmid (14067) was transformed into Corynebacterium glutamicum MH14067-9, and exchange recombinants were selected on BHI medium supplemented with 15 mg / L kanamycin. KanR clones were identified by colony PCR using Fast Taq DNA polymerase (TransGen Biotech) with the primer pairs nadA-UP-1F / P85 and P82 / nadA-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. 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. The obtained 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.

[0180] 1.3.3 Shake flask validation of glutamate production strain ATCC14067

[0181] 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.

[0182] Table 4. Glutamate content detection of recombinant strains

[0183] 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.

[0184] Example 2: Construction of xfp and pfk modified plasmids

[0185] 2.1 Construction of ATCC13869 route XFP and PFK transformation plasmids

[0186] 2.1.1 Construction of pK18-odhA-PcspB-Bl_xfp plasmid

[0187] Using the WJ0140 genome (constructed in Example 1.1.2 of this patent) as a template, PE617-UP-1F / PE618-UP-1R were used as primers to amplify the upstream homology arm UP (500bp), and PE623-DN-4F / PE624-DN-4R were used as primers to amplify the downstream homology arm DN (500bp). Using the pXMJ19-PcspB-Bl-xfp plasmid (the xfp expression plasmid was obtained by GENEWIZ full gene synthesis and then assembled with the provided pXMJ19-PcspB vector, as described in 2.1.16 of this patent) as a template, PE934-Bm_xfp-F / PE937-Bl_xfp-R were used as primer pairs to obtain PcspB-Bl_xfp by PCR reverse amplification. The resulting fragment was purified and recovered using an agarose gel recovery kit (Tian Gen) and frozen at -20°C for later use. pK18mobsacB was digested with XbaI / HindIII restriction endonucleases and then recovered from gel to obtain the plasmid backbone.

[0188] The amplified plasmid backbone and fragments were mixed in the specified ratio 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, the clones 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 4417 bp. Sequencing (GENEWIZ) confirmed the correct insertion of the fragment using the primers P81 / P86 / PE934-Bm_xfp-F / PE937-Bl_xfp-R / PE962-Bl-check-F1 / PE963-Bl-check-F2. The resulting plasmid was named pK18-odhA-PcspB-Bl_xfp.

[0189] 2.1.2 Construction of pK18-alaT-Ptuf-Bl_xfp plasmid

[0190] Using the WJ0140 genome as a template, the upstream homology arm UP (539 bp) was amplified using the PE552-UP-1F / PE569-UP-1R primer pair, and the downstream homology arm DN (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 / PE1000-alaT-tuf-R primer pair. Using the pXMJ19-PcspB-Bl-xfp plasmid (the xfp expression plasmid was synthesized by GENEWIZ and assembled with the provided pXMJ19-PcspB vector, as described in 2.1.16 of this patent) as a template and the primer pair PE1001-alaT-BL-F / PE1002-alaT-BL-R, the Bl_xfp fragment was amplified by PCR. The resulting fragment was purified and recovered using an agarose gel recovery kit (Tiangen) and stored frozen at -20°C until further use. pK18mobsacB was digested with XbaI / HindIII restriction endonucleases and the plasmid backbone was recovered from the gel.

[0191] The amplified plasmid backbone, promoter, and gene fragment were mixed in the appropriate proportions using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech). 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 / P86. The correct band size was 4115 bp. Sequencing (GENEWIZ) confirmed the correct insertion of the fragment 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.

[0192] 2.1.3 Construction of pK18-odhA-Ptuf-Bl_xfp plasmid

[0193] 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 UP, DN, and Ptuf promoters. The Bl_xfp fragment was obtained by amplification using pK18-odhA-PcspB-Bl_xfp as a template and primers PE1001-alaT-BL-F / 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.

[0194] 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 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. Using the primer pair P81 / P86, the correct band size was 4475 bp. The correct insertion was further verified by sequencing (GENEWIZ) using the primers P81 / P86 / PE962-Bl-check-F1 / PE963-Bl-check-F2 / PE1009-odhA-BL-DN-R / PE1010-odhA-tuf-F. The resulting plasmid was named pK18-odhA-Ptuf-Bl_xfp.

[0195] 2.1.4 Construction of pK18-cg3384-Ptuf-Bl_xfp plasmid

[0196] 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 UP and DN. The Ptuf-Bl_xfp fragment was obtained by amplification using pK18-odhA-Ptuf-Bl_xfp as a template and primers PE1105-xfp-F / PE1106-xfp-R. The resulting fragment was purified using an agarose gel extraction kit (Tiangen) and stored frozen at -20°C until further use.

[0197] 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 / 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 / PE1106-xfp-R. The resulting plasmid was named pK18-cg3384-Ptuf-Bl_xfp.

[0198] 2.1.5 Construction of pK18-cg3364-Ptuf-Bl_xfp plasmid

[0199] The genome of Corynebacterium glutamicum ATCC13869 was used as a template, and primers 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 UP and DN. The Ptuf-Bl_xfp fragment was obtained by amplification using pK18-odhA-Ptuf-Bl_xfp as a template and primers PE1121-xfp-F / PE1122-xfp-R. The resulting fragment was purified using an agarose gel extraction kit (Tiangen) and stored frozen at -20°C until further use.

[0200] 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 / 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.

[0201] 2.1.6 Construction of the pK18-cg0928-Ptuf-Bl_xfp(13869) plasmid

[0202] The genome of Corynebacterium glutamicum ATCC13869 was used as a template, and PE1125-0928-UP-1F / PE1126-0928-UP-1R and PE1127-0928-DN-2F / PE1128-0928-DN-2R were used as primer pairs for amplification to obtain the upstream and downstream homology arms UP and DN. The pK18-odhA-Ptuf-Bl_xfp plasmid (constructed in Example 2.1.3 of this patent, the same below) was used as a template, and PE1129-xfp-F / PE1130-xfp-R was used as a primer pair for amplification to obtain the Ptuf-Bl_xfp fragment. The obtained fragment was purified and recovered by an agarose gel recovery kit (Tian Gen) and frozen at -20°C for later use.

[0203] 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 / 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 / PE1129-xfp-F / PE1130-xfp-R. The resulting plasmid was named pK18-cg0928-Ptuf-Bl_xfp(13869).

[0204] 2.1.7 Construction of pK18-cg2211-Ptuf-Bl_xfp plasmid

[0205] 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 UP and DN. The Ptuf-Bl_xfp fragment was obtained by amplification using pK18-odhA-Ptuf-Bl_xfp as a template and primers PE1379-XFP-F / PE1380-XFP-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 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 / 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 / PE1379-XFP-F / PE1380-XFP-R. The resulting plasmid was named pK18-cg2211-Ptuf-Bl_xfp.

[0207] 2.1.8 Construction of pK18-cg3125-Ptuf-Bl_xfp plasmid

[0208] 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 UP and DN. The Ptuf-Bl_xfp fragment was obtained by amplification using pK18-odhA-Ptuf-Bl_xfp as a template and primers PE1387-XFP-F / PE1388-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 ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech Co., Ltd.) at the specified ratios. 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 / 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 / PE1387-XFP-F / PE1388-XFP-R. The resulting plasmid was named pK18-cg3125-Ptuf-Bl_xfp.

[0210] 2.1.9 Construction of pK18-cg2564-Ptuf-Bl_xfp plasmid

[0211] 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 UP and DN. The Ptuf-Bl_xfp fragment was obtained by amplification using pK18-odhA-Ptuf-Bl_xfp as a template and primers PE1355-xfp-F / PE1356-xfp-R. The resulting fragment was purified using an agarose gel extraction 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 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 / 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.

[0213] 2.1.10 Construction of pK18-BBD29-07270-Ptuf-Bl_xfp plasmid

[0214] The genome of Corynebacterium glutamicum ATCC13869 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 UP and DN. 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.

[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 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 / 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.

[0216] 2.1.11 Construction of pK18-pfk-A1T plasmid

[0217] 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). The upstream and downstream homology arms (UP) and DN 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 fragments were purified using an agarose gel extraction kit (Tiangen) and stored at -20°C until further use.

[0218] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP and DN fragments were mixed with the double-digested vector in the ClonExpress MultiS One-Step Cloning Kit (Novagen Biotech Co., Ltd.) in the appropriate ratios. 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. Kanamycin-resistant clones were selected and identified using the primer pair P81 / P86. The correct band size was 1316 bp. Sequencing (GENEWIZ) was further performed to confirm the correct insertion, using the primers P81 / P86. The resulting plasmid was named pK18-pfk-A1T.

[0219] 2.1.12 Construction of pK18-Pzwf-pfk plasmid

[0220] Primers were designed based on the gene sequence of pfk (GenBank NO: BBD29_06675) in the NCBI database, and 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 the upstream and downstream homology arms UP and DN were obtained by PCR reverse amplification. The Pzwf promoter was amplified using the genome of Corynebacterium glutamicum ATCC13032 as a template and PE886-Pzwf-F / PE887-Pzwf-R as a primer pair. The obtained fragment was purified and recovered by an agarose gel recovery kit (Tian Gen) and frozen at -20°C for later use.

[0221] 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 completion of 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 1749 bp. Sequencing (GENEWIZ) was performed to confirm the correct insertion, using the primers P81 / P86 / PE886-Pzwf-F. The resulting plasmid was named pK18-Pzwf-pfk.

[0222] 2.1.13 Construction of pK18-PrecA-pfk plasmid

[0223] Primers were designed based on the gene sequence of pfk (GenBank NO: BBD29_06675) in the NCBI database, and 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 the upstream and downstream homology arms UP and DN 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 a primer pair. The obtained fragment was purified and recovered by an agarose gel recovery kit (Tiangen) and frozen at -20°C for later use.

[0224] 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 (TransGen Biotech) 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) confirmed the correct insertion of the fragment using the primers P81 / P86 / PE889-PrecA-F. The resulting plasmid was named pK18-PrecA-pfk.

[0225] 2.1.14 Construction of pK18-pfk-E171K plasmid

[0226] 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 High-Fidelity Polymerase (New England BioLabs). The upstream and downstream homology arms (UP) and DN 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 fragments were purified using an agarose gel extraction kit (Tiangen) and stored at -20°C until further use.

[0227] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP and DN fragments were mixed with the double-digested vector in the ClonExpress MultiS One-Step Cloning Kit (Novagen 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 completion of 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) confirmed the correct insertion of the fragment using the primers P81 / P86. The resulting plasmid was named pK18-pfk-E171K.

[0228] 2.1.15 Construction of pK18-pfk-E171D 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 High-Fidelity Polymerase (New England BioLabs). The upstream and downstream homology arms (UP) and DN 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 fragments were 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 ClonExpress MultiS One-Step Cloning Kit (Novagen Biotech Co., Ltd.) in the appropriate proportions. After pipetting and mixing, the mixture was 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) confirmed the correct insertion of the fragment using the primer pair P81 / P86. The resulting plasmid was named pK18-pfk-E171D.

[0231] 2.1.16 Construction of pXMJ19-PcspB-xfp expression plasmid

[0232] 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.

[0233] Table 5. XFP from different sources

[0234] 2.2 Construction of XFP multi-copy and PFK modified plasmids based on ATCC13032

[0235] 2.2.1. Construction of pK18-alaT-Ptuf-Bl_xfp(13032) plasmid

[0236] Using Corynebacterium glutamicum ATCC13032 genome as template, using P13-10 / PE997-rev-alaT-R as primer pair, upper homology arm UP is obtained by PCR amplification. Using PE998-rev-alaT-R / PE1022-alaT-DN-2R as primer pair, lower homology arm DN is obtained by PCR amplification. Using Corynebacterium glutamicum ATCC13032 genome as template, using PE999-alaT-tuf-F / PE1000-alaT-tuf-R as primer pair amplification obtains Ptuf promoter. Using Bifidobacterium longum NCC2705-xfp gene (GenBank:AE014295.3) as template, after codon optimized, obtain Bl.xfp sequence, synthesized by KingSher, using PE1001-alaT-BL-F / PE1002-alaT-BL-R as primer pair, obtain Bl_xfp fragment by PCR amplification. The obtained fragment was purified and recovered using an agarose gel recovery kit (Tiangen) and stored at -20°C for future use.

[0237] The amplified UP, DN, promoter, and gene fragments were mixed in the specified proportions using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech). After pipetting to mix thoroughly, the fragments were 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. Sequencing (GENEWIZ) confirmed the correct insertion of the fragment 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).

[0238] 2.2.2 Construction of pK18-cg3384-Ptuf-Bl_xfp(13032) plasmid

[0239] 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.

[0240] 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 / 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).

[0241] 2.2.3 Construction of pK18-cg3364-Ptuf-Bl_xfp(13032) plasmid

[0242] 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 UP and DN, respectively. 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 extraction kit (Tiangen) and stored at -20°C until further use.

[0243] 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 / 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).

[0244] 2.2.4 Construction of pK18-cg0928-Ptuf-Bl_xfp(13032) plasmid

[0245] 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 UP and DN. The Ptuf-Bl_xfp fragment was obtained by amplification using pK18-odhA-Ptuf-Bl_xfp as a template and primers P13-13 / P13-14. The resulting fragment was purified using an agarose gel recovery kit (Tiangen) and stored frozen at -20°C until further use.

[0246] 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 / 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).

[0247] 2.2.5 Construction of pK18-cg3125-Ptuf-Bl_xfp(13032) plasmid

[0248] 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 UP and DN. 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.

[0249] 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 / 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).

[0250] 2.2.6 Construction of pK18-cg2564-Ptuf-Bl_xfp(13032) plasmid

[0251] 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 UP and DN, respectively. The Ptuf-Bl_xfp fragment was obtained by amplification using the primer pair PE1355-xfp-F / PE1356-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.

[0252] 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 / 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).

[0253] 2.2.7 Construction of pK18-BBD29-07270-Ptuf-Bl_xfp(13032) plasmid

[0254] 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 UP and DN. 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.

[0255] 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 / 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).

[0256] 2.2.8 Construction of pK18-pfk-A1T(13032) plasmid

[0257] 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). PE878-pfk-TTG-UP-1F / PE879-pfk-TTG-UP-1R and PE880-pfk-TTG-DN-2F / PE881-pfk-UP-2R were used as primer pairs, respectively, and upstream and downstream homology arms UP and DN 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.

[0258] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP and DN fragments were mixed with the double-digested vector in the ClonExpress MultiS One-Step Cloning Kit (Novagen Biotech Co., Ltd.) in the appropriate ratios. After pipetting and mixing, the mixture was 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 / P86. The correct band size was 1316 bp. Sequencing (GENEWIZ) was further performed to confirm the correct insertion, using the primers P81 / P86. The resulting plasmid was named pK18-pfk-A1T(13032).

[0259] 2.2.9 Construction of pK18-Pzwf-pfk(13032) plasmid

[0260] 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 the upstream and downstream homology arms UP and DN were obtained by PCR reverse amplification. Using the genome of Corynebacterium glutamicum ATCC13032 as a template, the Pzwf promoter was amplified using 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 future use.

[0261] 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 (TransGen Biotech) were transformed. Kanamycin-resistant clones were selected and identified using the primer pair P81 / P86. The correct band size was 1749 bp. Sequencing (GENEWIZ) was further performed to confirm the correct insertion, using the primers P81 / P86 / PE886-Pzwf-F. The resulting plasmid was named pK18-Pzwf-pfk(13032).

[0262] 2.2.10 Construction of pK18-PrecA-pfk(13032) plasmid

[0263] 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 the upstream and downstream homology arms UP and DN were obtained by PCR reverse amplification. Using the genome of Corynebacterium glutamicum ATCC13032 as a template, the PrecA promoter was amplified using PE889-PrecA-F / PE890-PrecA-R as a primer pair. The resulting fragment was purified and recovered using an agarose gel recovery kit (Tiangen) and stored at -20°C for later use.

[0264] 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 (TransGen Biotech) were transformed. Kanamycin-resistant clones were selected and identified using the primer pair P81 / P86. The correct band size was 1765 bp. Sequencing (GENEWIZ) confirmed the correct insertion of the fragment using the primers P81 / P86 / PE889-PrecA-F. The resulting plasmid was named pK18-PrecA-pfk(13032).

[0265] 2.2.11 Construction of pK18-pfk-E171D(13032) plasmid

[0266] 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). PE892-pfk-E171K-UP-1F / PE893-pfk-E171K-UP-1R and PE897-pfk-E171D-DN-2F / PE895-pfk-E171K-DN-2R were used as primer pairs to obtain upstream and downstream homology arms UP and DN 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.

[0267] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP and DN fragments were mixed with the double-digested vector in the ClonExpress MultiS One-Step Cloning Kit (Novagen Biotech Co., Ltd.) in the appropriate ratios. After pipetting and mixing, the mixture was 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) confirmed the correct insertion of the fragment using the primers P81 / P86. The resulting plasmid was named pK18-pfk-E171D (13032).

[0268] 2.3 Construction of XFP multi-copy and PFK modified plasmids based on ATCC14067

[0269] 2.3.1 Construction of pK18-alaT-Ptuf-Bl_xfp(14067) plasmid

[0270] 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 described in 2.1.16 of this patent) as a template and the primer pair PE1001-alaT-BL-F / PE1002-alaT-BL-R. The resulting fragment was purified using an agarose gel recovery kit (Tiangen) and stored frozen at -20°C until further use.

[0271] The amplified UP, DN, promoter, and gene fragments were mixed in the specified proportions using the ClonExpress MultiS One-Step Cloning Kit (Novozymes Biotech). After pipetting to mix thoroughly, the fragments were 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. Sequencing (GENEWIZ) confirmed the correct insertion of the fragment 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).

[0272] 2.3.2 Construction of pK18-cg3384-Ptuf-Bl_xfp(14067) plasmid

[0273] 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.

[0274] 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 / 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).

[0275] 2.3.3 Construction of pK18-cg3364-Ptuf-Bl_xfp(14067) plasmid

[0276] 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 UP and DN, respectively. 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 extraction kit (Tiangen) and stored frozen at -20°C until further use.

[0277] 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 / 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).

[0278] 2.3.4 Construction of pK18-cg0928-Ptuf-Bl_xfp(14067) plasmid

[0279] 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 UP and DN. The Ptuf-Bl_xfp fragment was obtained by amplification using pK18-odhA-Ptuf-Bl_xfp as a template and primers P14-16 / PE1130-xfp-R. The resulting fragment was purified using an agarose gel extraction kit (Tiangen) and stored frozen at -20°C until further use.

[0280] 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 / 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).

[0281] 2.3.5 Construction of pK18-cg3125-Ptuf-Bl_xfp(14067) plasmid

[0282] 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 UP and DN. 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 extraction kit (Tiangen) and stored frozen at -20°C until further use.

[0283] 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 (TransGen Biotech) 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 / P14-8. The resulting plasmid was named pK18-cg3125-Ptuf-Bl_xfp(14067).

[0284] 2.3.6 Construction of the pK18-cg2564-Ptuf-Bl_xfp(14067) plasmid

[0285] 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 UP and DN, respectively. The Ptuf-Bl_xfp fragment was obtained by amplification using the primer pair PE1355-xfp-F / PE1356-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.

[0286] 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 / 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).

[0287] 2.3.7 Construction of pK18-BBD29-07270-Ptuf-Bl_xfp(14067) plasmid

[0288] 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 UP and DN. 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.

[0289] 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 / 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).

[0290] 2.3.8 Construction of pK18-pfk-A1T(14067) plasmid

[0291] 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). PE878-pfk-TTG-UP-1F / PE879-pfk-TTG-UP-1R and PE880-pfk-TTG-DN-2F / PE881-pfk-UP-2R were used as primer pairs, respectively, and upstream and downstream homology arms UP and DN 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.

[0292] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP and DN fragments were mixed with the double-digested vector in the ClonExpress MultiS One-Step Cloning Kit (Novagen Biotech Co., Ltd.) in the appropriate ratios. 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. Kanamycin-resistant clones were selected and identified using the primer pair P81 / P86. The correct band size was 1316 bp. Sequencing (GENEWIZ) was further performed to confirm the correct insertion, using the primers P81 / P86. The resulting plasmid was named pK18-pfk-A1T (14067).

[0293] 2.3.9 Construction of pK18-Pzwf-pfk(14067) plasmid

[0294] 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 the upstream and downstream homology arms UP and DN 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.

[0295] 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, Inc.) at the specified ratios. 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. Kanamycin-resistant clones were selected and identified using the primer pair P81 / P86. The correct band size was 1749 bp. Sequencing (GENEWIZ) was performed to confirm the correct insertion, using the primers P81 / P86 / PE886-Pzwf-F. The resulting plasmid was named pK18-Pzwf-pfk(14067).

[0296] 2.3.10 Construction of pK18-PrecA-pfk(14067) plasmid

[0297] 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 the upstream and downstream homology arms UP and DN 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 (Tiangen) and stored at -20°C for later use.

[0298] 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 (TransGen Biotech) were transformed. Kanamycin-resistant clones were selected and identified using the primer pair P81 / P86. The correct band size was 1765 bp. Sequencing (GENEWIZ) confirmed the correct insertion of the fragment using the primers P81 / P86 / PE889-PrecA-F. The resulting plasmid was named pK18-PrecA-pfk(14067).

[0299] 2.3.11 Construction of pK18-pfk-E171D(14067) plasmid

[0300] 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). Using PE892-pfk-E171K-UP-1F / PE893-pfk-E171K-UP-1R and PE897-pfk-E171D-DN-2F / P14-18 as primer pairs, upstream and downstream homology arms UP and DN 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 until further use.

[0301] pK18mobsacB was digested with XbaI / HindIII restriction endonucleases. The amplified UP and DN fragments were mixed with the double-digested vector in the ClonExpress MultiS One-Step Cloning Kit (Novagen Biotech Co., Ltd.) in the appropriate ratios. After pipetting and mixing, the mixture was 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) confirmed the correct insertion of the fragment using the primers P81 / P86. The resulting plasmid was named pK18-pfk-E171D (14067).

[0302] Example 3: Construction of strains overexpressing xfp from different sources and their glutamate conversion rates

[0303] 3.1 WJ0140-pXMJ19-PcspB-Ll-xfp

[0304] Prepare WJ0140 competent cells according to the Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), recombinant plasmid pXMJ19-PcspB-L1-xfp is changed into Corynebacterium glutamicum WJ0140 competent cells with electrotransformation method, and recombinants are screened on the BHI substratum containing 5mg / L chloramphenicol. By PCR amplification of target sequence (PE813-L1-XFP-1F / PE814-L1-XFP-2R), nucleotide sequencing analysis, obtain importing the plasmid recombinant microorganism strain containing the target gene.

[0305] 3.2 WJ0140-pXMJ19-PcspB-Pa-xfp

[0306] 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 transferred into Corynebacterium glutamicum WJ0140 competent cells using electroporation. Recombinants were screened on BHI medium containing 5 mg / L chloramphenicol. The target sequence (PE817-Pa-XFP-1F / PE818-Pa-XFP-2R) was amplified by PCR and analyzed by nucleotide sequencing to obtain a recombinant microbial strain containing the plasmid containing the target gene.

[0307] 3.3 WJ0140-pXMJ19-PcspB-Bb-xfp

[0308] WJ0140 competent cells were prepared according to the classical method for Corynebacterium glutamicum (C.glutamicum Handbook, Chapter 23). The recombinant plasmid pXMJ19-PcspB-Bb-xfp was transferred into Corynebacterium glutamicum WJ0140 competent cells using electroporation. Recombinants were screened on a BHI medium supplemented with 5 mg / L chloramphenicol. The target sequence (PE823-Bb-XFP-1F / PE824-Bb-XFP-2R) was amplified by PCR and analyzed by nucleotide sequencing to obtain a recombinant microbial strain containing the plasmid containing the target gene.

[0309] 3.4 WJ0140-pXMJ19-PcspB-Bm-xfp

[0310] Prepare WJ0140 competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), recombinant plasmid pXMJ19-PcspB-Bm-xfp is changed into Corynebacterium glutamicum WJ0140 competent cell with electrotransformation method, on the BHI substratum of the chloramphenicol that contains 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 importing the plasmid recombinant microorganism strain that contains the target gene.

[0311] 3.5 WJ0140-pXMJ19-PcspB-Lm-xfp

[0312] 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 BHI medium containing 5 mg / L chloramphenicol. The target sequence (PE815-Lm-XFP-1F / PE816-Lm-XFP-2R) was amplified by PCR and analyzed by nucleotide sequencing to obtain a recombinant microbial strain containing the plasmid containing the target gene.

[0313] 3.6 WJ0140-pXMJ19-PcspB-Bab-xfp

[0314] WJ0140 competent cells were prepared according to the classical method for Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23). The recombinant plasmid pXMJ19-PcspB-Bab-xfp was transferred into Corynebacterium glutamicum WJ0140 competent cells by electroporation, and recombinants were screened on BHI medium containing 5 mg / L chloramphenicol. The target sequence (PE807-Bm-XFP-1F / PE810-Bm-XFP-2R) was amplified by PCR and analyzed by nucleotide sequencing to obtain a recombinant microbial strain containing the plasmid containing the target gene.

[0315] 3.7 WJ0140-pXMJ19-PcspB-An-xfp

[0316] 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 transferred into Corynebacterium glutamicum WJ0140 competent cells by electroporation, and recombinants were screened on BHI medium containing 5 mg / L chloramphenicol. The target sequence (PE819-An-XFP-1F / PE820-An-XFP-2R) was amplified by PCR and analyzed by nucleotide sequencing to obtain a recombinant microbial strain containing the plasmid containing the target gene.

[0317] 3.8 WJ0140-pXMJ19-PcspB-Ban-xfp

[0318] WJ0140 competent cells were prepared according to the classical method for Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23). The recombinant plasmid pXMJ19-PcspB-Ban-xfp was transferred into Corynebacterium glutamicum WJ0140 competent cells using electroporation. Recombinants were screened on BHI medium containing 5 mg / L chloramphenicol. The target sequence (PE821-Ban-XFP-1F / PE822-Ban-XFP-2R) was amplified by PCR and analyzed by nucleotide sequencing to obtain a recombinant microbial strain containing the plasmid containing the target gene.

[0319] 3.9 WJ0140-pXMJ19-PcspB-Bl-xfp

[0320] WJ0140 competent cells were prepared according to the Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23). The recombinant plasmid pXMJ19-PcspB-Bl-xfp was transferred into Corynebacterium glutamicum WJ0140 competent cells with electroporation, and recombinants were screened on BHI medium containing 5 mg / L chloramphenicol. The target sequence (PE811-Bl-XFP-1F / PE812-Bl-XFP-2R) was amplified by PCR and analyzed by nucleotide sequencing to obtain a recombinant microbial strain containing the plasmid containing the target gene.

[0321] 3.10 WJ0140-pXMJ19 No-load

[0322] WJ0140 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C.glutamicum Handbook, Chapter 23). Empty plasmid pXMJ19 (GenBank: AJ133195.1) was transformed into Corynebacterium glutamicum WJ0140 competent cells with electroporation and screened on a BHI culture medium containing 5 mg / L chloramphenicol. The target sequence (PE911-Lm-28a-F / PE912-Lm-28a-R) was amplified by PCR and analyzed by nucleotide sequencing to obtain a recombinant microbial strain that had imported the empty plasmid.

[0323] 3.11 Shake flask validation

[0324] 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.

[0325] 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.

[0326] Table 6. Conversion rate of glutamate in strains with different sources of xfp

[0327] Example 4: Construction of xfp multi-copy strains and their glutamate conversion efficiency starting from WJ0140

[0328] 4.1 WJ0140-alaT-Ptuf-Bl_xfp(SME762a)xfp one copy

[0329] 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 and cultured 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 sieving 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 / PE1024-alaT-ID-R. The correct size was 4054 bp. The recombinant strain with the correct identified band size was sent to Genewise for sequencing. Subsequently, nucleotide sequencing analysis was performed, and the recombinant microbial strain with the correct sequence was named SME762a.

[0330] 4.2 SME762a-cg3364-Ptuf-Bl_xfp (SME768a) xfp Second Copy

[0331] 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 BHI substratum that contains the kanamycin of 15mg / L, screen a recombinant.The 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, and 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.The secondary identification primer is PE1123-ID-F / PE1124-ID-R. The recombinant bacteria with the correct identification band size were sent to GenWeiZi for sequencing, followed by nucleotide sequencing analysis, and the recombinant microbial strain with the correct sequencing was named SME768a.

[0332] 4.3 SME768a-cg2564-Ptuf-Bl_xfp (SME777a) xfp three copies

[0333] 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 BHI substratum that contains the kanamycin of 15mg / L, screen a recombinant.The 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, and 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 GenWeichi for sequencing, followed by nucleotide sequencing analysis, and the recombinant microbial strain with the correct sequencing was named SME777a.

[0334] 4.4 SME777a-cg3125-Ptuf-Bl_xfp(SME778a)xfp Four copies

[0335] 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 BHI substratum that contains the kanamycin of 15mg / L, screen a recombinant.The 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, and 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.The 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.

[0336] 4.5 SME778a-cg3384-Ptuf-Bl_xfp(SME783a)xfp five copies

[0337] 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 BHI substratum that contains the kanamycin of 15mg / L, screen a recombinant.The 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, and 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 GenWeichi for sequencing, followed by nucleotide sequencing analysis, and the recombinant microbial strain with the correct sequencing was named SME783a.

[0338] 4.6 SME783a-BBD29_07270-Ptuf-Bl_xfp (SME787a) xfp six copies

[0339] Prepare SME783a competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), recombinant plasmid pK18-BBD29-07270-Ptuf-Bl_xfp is changed in Corynebacterium glutamicum SME783a competent cell with the electrotransformation method, on the BHI substratum 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 without, 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 GeneWeiZ for sequencing, followed by nucleotide sequencing analysis. The recombinant microbial strain with the correct sequence was named SME787a.

[0340] 4.7 SME787a-cg0928-Ptuf-Bl_xfp(SME789a)xfp Seven Copies

[0341] Prepare SME787a competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), recombinant plasmid pK18-cg0928-Ptuf-Bl_xfp (13869) is changed in Corynebacterium glutamicum SME787a competent cell with the electrotransformation method, on the BHI substratum that contains the kanamycin of 15mg / L, screen a recombinant.Once 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 the anti-BHI substratum without, 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 / PE1132-ID-R. Recombinant strains with the correct identification band size were sent to GeneWeiZ for sequencing, followed by nucleotide sequencing analysis. The recombinant strain with the correct sequence was named SME789a.

[0342] 4.8 SME789a-cg2337-Ptuf-Bl_xfp(SME870a)xfp eight copies

[0343] Prepare SME789a competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), recombinant plasmid pK18-cg2337-Ptuf-Bl_xfp (building among this patent 6.1.1) is changed in the Corynebacterium glutamicum SME789a competent cell with the electrotransformation method, on the BHI substratum 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 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, reorganization takes place second time in transformant, and by homologous recombination exchange, carrier sequence is removed from genome. The bacterial solution was diluted 100-1000 times and then plated on 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 PE1367-ID-F / PE1368-ID-R, and the band size was 4101bp. The recombinant bacteria with the correct identification band size were sent to GenWeiZ for sequencing using the sequencing primers PE1367-ID-F / PE1368-ID-R / PE962-Bl-check-F1 / PE963-Bl-check-F2 / PE1349-xfp-F / PE1350-xfp-R. Subsequently, nucleotide sequencing analysis was performed, and the recombinant microbial strain with the correct sequence was named SME870a.

[0344] 4.9 SME870a-BBD29-07210-Ptuf-Bl_xfp(SME879a)xfp nine copies

[0345] Prepare SME870a competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), recombinant plasmid pK18-BBD29-07210-Ptuf-Bl_xfp (building among this patent 6.1.3) is changed in the Corynebacterium glutamicum SME870a competent cell with the electrotransformation method, on the BHI substratum 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 PE1389-7210-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, reorganization takes place second time in transformant, and by homologous recombination exchange, carrier sequence is removed from genome. The bacterial solution was diluted 100-1000 times and then plated on 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 PE1419-ID-F / PE1420-ID-R, and the band size was 4101bp. The recombinant bacteria with the correct identification band size were sent to GenWeiZ for sequencing using the sequencing primers PE1419-ID-F / PE1420-ID-R / PE962-Bl-check-F1 / PE963-Bl-check-F2 / PE1393-xfp-F / PE1394-xfp-R. Subsequently, nucleotide sequencing analysis was performed, and the recombinant microbial strain with the correct sequence was named SME879a.

[0346] 4.10 SME879a-odhA-Ptuf-Bl_xfp(SME886a)xfp ten copies

[0347] 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 BHI substratum 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 strains with the correct identification band size were sent to GeneWeiZ for sequencing, followed by nucleotide sequencing analysis. The recombinant strain with the correct sequence was named SME886a.

[0348] 4.11 Shake flask validation

[0349] 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%.

[0350] Table 7. Conversion rate of glutamate in strains overexpressing xfp

[0351] Example 5: Construction of xfp multi-copy strains and their glutamate conversion efficiency starting from WJ0140

[0352] 5.1 WJ0140 overlay odhA-Ptuf-Bl_xfp, xfp one copy

[0353] Prepare WJ0140 competent cell 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 cell with electrotransformation method, on the BHI substratum 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 GeneWeiZ for sequencing, followed by nucleotide sequencing analysis.

[0354] 5.2 Further superposition of alaT-Ptuf-Bl_xfp, two copies of xfp

[0355] According to the Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), prepare the correct recombinant competent cells of the sequencing obtained in section 5.1, further recombinant plasmid pK18-alaT-Ptuf-Bl_xfp is changed into above-mentioned Corynebacterium glutamicum with electrotransformation method, and on the BHI substratum containing the kanamycin of 15mg / L, screen a recombinant. The primary 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 the non-anti-BHI substratum, and 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, be coated on the solid BHIS substratum that contains 10% sucrose and cultivate 36 hours. The secondary identification primer is PE1023-alaT-ID-F / PE1024-alaT-ID-R. The recombinant bacteria with the correct identification band size were sent to GeneWeichi for sequencing.

[0356] 5.3 Further stacking of odhA-PcspB-Bl_xfp, three copies of xfp

[0357] According to the Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), prepare the correct recombinant bacteria competent cell of sequencing that part obtains in 5.2, further recombinant plasmid pK18-odhA-PcspB-Bl_xfp is changed in the above-mentioned Corynebacterium glutamicum with the electrotransformation method, on the BHI substratum that contains the kanamycin of 15mg / L, screen a recombinant.The primary identification primer is P86 / PE617-UP-1F, PE624-DN-4R / 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.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 primer is PE625-ID-F / PE626-ID-R. The recombinant bacteria with the correct identification band size were sent to GeneWeichi for sequencing, followed by nucleotide sequencing analysis.

[0358] 5.4 Further superimpose cg3384-Ptuf-Bl_xfp, four copies of xfp

[0359] Prepare the correct recombinant bacteria competent cells of the sequencing obtained in 5.3 part according to the Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), further recombinant plasmid pK18-cg3384-Ptuf-Bl_xfp is changed into above-mentioned competent cells with electrotransformation method, and on the BHI substratum containing the kanamycin of 15mg / L, screen a recombinant. The 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 the non-anti-BHI substratum, and 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, be coated on the solid BHIS substratum that contains 10% sucrose and cultivate 36 hours. The secondary identification primer is PE1107-ID-F / PE1108-ID-R. The recombinant bacteria with the correct identification band size were sent to GeneWeichi for sequencing, followed by nucleotide sequencing analysis.

[0360] 5.5 Further superimpose cg2564-Ptuf-Bl_xfp, xfp five copies

[0361] Prepare the correct recombinant bacteria competent cells of the sequencing obtained in section 5.4 according to the Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), further the recombinant plasmid pK18-cg2564-Ptuf-Bl_xfp is transferred into the competent cells with the electroporation method, and the recombinant is screened on the BHI substratum containing 15mg / L of kanamycin. The primary identification primers are P86 / PE1351-2564-UP-1F and PE1354-2564-DN-2R / P81. The recombinant obtained by screening is inoculated into the non-anti-BHI substratum and cultivated for 12-14 hours. The cultivation temperature is 30 ℃, and the culture is shaken at 220rpm on a rotary shaker. After the bacterial liquid is diluted 100-1000 times, it is coated on the solid BHIS substratum containing 10% sucrose and cultivated for 36 hours. The secondary identification primers are PE1369-ID-F / PE1370-ID-R. The recombinant bacteria with the correct identification band size were sent to GeneWeichi for sequencing, followed by nucleotide sequencing analysis.

[0362] 5.6 Further superimpose cg2211-Ptuf-Bl_xfp, six copies of xfp

[0363] According to the Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), prepare the correct recombinant competent cell of the sequencing that part obtains in 5.5, recombinant plasmid pK18-cg2211-Ptuf-Bl_xfp is changed in the above-mentioned Corynebacterium glutamicum with the electrotransformation method, on the BHI substratum that contains the kanamycin of 15mg / L, screen a recombinant.The primary identification primer is P86 / PE1339-2211-UP-1F, PE1342-2211-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.Bacterium liquid is diluted 100-1000 times and is coated on the solid BHIS substratum that contains 10% sucrose and cultivated 36 hours.The secondary identification primer is PE1365-ID-F / PE1366-ID-R. The recombinant bacteria with the correct identification band size were sent to GeneWeichi for sequencing, followed by nucleotide sequencing analysis.

[0364] 5.7 Further superimpose cg3364-Ptuf-Bl_xfp, xfp seven copies

[0365] According to the Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), prepare the correct recombinant bacteria competent cell of sequencing that part obtains in 5.6, recombinant plasmid pK18-cg3364-Ptuf-Bl_xfp is changed in the above-mentioned Corynebacterium glutamicum with the electrotransformation method, on the BHI substratum that contains the kanamycin of 15mg / L, screen a recombinant.The 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, and cultivation temperature is 30 ℃, rotary shaker 220rpm shaking culture.Bacterium liquid is diluted 100-1000 times and is coated on the solid BHIS substratum that contains 10% sucrose and cultivated 36 hours.The secondary identification primer is PE1123-ID-F / PE1124-ID-R. The recombinant bacteria with the correct identification band size were sent to GeneWeichi for sequencing, followed by nucleotide sequencing analysis.

[0366] 5.8 Further superimpose cg1512-Ptuf-Bl_xfp, xfp eight copies

[0367] According to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), prepare the correct recombinant bacteria competent cell of the order-checking that 5.7 part obtains, recombinant plasmid pK18-cg1512-Ptuf-Bl_xfp (this patent 6.1.2 makes up) is changed in the above-mentioned Corynebacterium glutamicum with the electrotransformation method, on the BHI substratum 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 PE1133-1512-UP-1F / P86 and P81 / PE1136-1512-DN-2R.The recombinant that will sieve 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, 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 for 36 hours. Strains grown on sucrose medium do not carry the inserted vector sequence in their genomes. 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 GenWeiZ for sequencing, followed by nucleotide sequencing analysis.

[0368] 5.9 Further superimpose cg3125-Ptuf-Bl_xfp, xfp nine copies

[0369] According to the Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), prepare the correct recombinant bacteria competent cell of the sequencing that part obtains in 5.8, recombinant plasmid pK18-cg3125-Ptuf-Bl_xfp is changed into above-mentioned Corynebacterium glutamicum competent cell with the electrotransformation method, on the BHI substratum of the kanamycin that contains 15mg / L, screen a recombinant.The 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, and cultivation temperature is 30 ℃, rotary shaking table 220rpm shaking culture.Bacterium liquid is diluted 100-1000 times and is coated on the solid BHIS substratum that contains 10% sucrose and cultivated 36 hours.The secondary identification primer is PE1371-ID-F / PE1372-ID-R. The recombinant bacteria with the correct identification band size were sent to GeneWeichi for sequencing, followed by nucleotide sequencing analysis.

[0370] 5.10 further superimpose cg0928-Ptuf-Bl_xfp, xfp ten copies

[0371] According to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), prepare the correct recombinant bacteria competent cell of the order-checking that 5.9 part obtains, recombinant plasmid pK18-cg0928-Ptuf-Bl_xfp (13869) is changed in the above-mentioned Corynebacterium glutamicum competent cell with the electrotransformation method, on the BHI substratum 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 P86, PE1125-0928-UP-1F / PE1128-0928-DN-2R, P81.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, reorganization takes place the 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 for 36 hours. Strains grown on sucrose medium do not carry the inserted vector sequence in their genome. The secondary identification primers were PE1131-ID-F / PE1132-ID-R, and the band size was 4101 bp. Recombinant strains with the correct identification band size were sent to GeneWeichi for sequencing, followed by nucleotide sequencing analysis.

[0372] 5.11 Shake flask validation

[0373] 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 WJ0140, 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.

[0374] Table 8. Conversion rate of glutamate in strains overexpressing xfp

[0375] Example 6: Construction of xfp multi-copy strains and their glutamate conversion efficiency starting from WJ0140

[0376] 6.1 Plasmid construction

[0377] 6.1.1 Construction of pK18-cg2337-Ptuf-Bl_xfp plasmid

[0378] The upstream homology arm UP was amplified using primers PE1345-2337-UP-1F / PE1346-2337-UP-1R from the genome of Corynebacterium glutamicum ATCC13869. The downstream homology arm DN was amplified using primers PE1347-2337-DN-2F / PE1348-2337-DN-2R. The pk18-odhA-Ptuf-BL_xfp plasmid (constructed in 2.1.3 of this patent) was used as a template, and primers PE1349-xfp-F / PE1350-xfp-R were used to amplify Ptuf_xfp. The PCR procedure 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 / 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 / PE1349-xfp-F / PE1350-xfp-R. The resulting plasmid was named pK18-cg2337-Ptuf-Bl_xfp.

[0379] 6.1.2 Construction of pK18-cg1512-Ptuf-Bl_xfp plasmid

[0380] The upstream homology arms UP and DN were amplified using the C. glutamicum ATCC13869 genome as a template 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. The tuf_xfp gene was amplified using the pk18-odhA-Ptuf-BL_xfp plasmid as a template using 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 / 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 / PE1137-xfp-F / PE1138-xfp-R. The resulting plasmid was named pK18-cg1512-Ptuf-Bl_xfp.

[0381] 6.1.3 Construction of pK18-BBD29-07210-Ptuf-Bl_xfp Plasmid

[0382] The genome of Corynebacterium glutamicum ATCC13869 was used as a template, and the upstream homology arms UP and DN were amplified using primers PE1389-7210-UP-1F / PE1390-7210-UP-1R. The downstream homology arms UP and DN were amplified using primers PE1391-7210-DN-2F / PE1392-7210-DN-2R. The pK18-cg0928-Ptuf-Bl_xfp(13869) plasmid was used as a template, and tuf_xfp was amplified using 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 / 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 / PE1393-xfp-F / PE1394-xfp-R. The resulting plasmid was named pK18-BBD29-07210-Ptuf-Bl_xfp.

[0383] 6.2 Strain Construction

[0384] 6.2.1 SME777a superimposed with pK18-odhA-Ptuf-Bl_xfp, four copies of xfp (SME872a)

[0385] According to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), the SME777a competent cell that embodiment 4.3 part makes, recombinant plasmid pK18-odhA-Ptuf-Bl_xfp is changed in Corynebacterium glutamicum with electrotransformation method, on the BHI substratum of the kanamycin that contains 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, transformant second time reorganization takes place, and by homologous recombination exchange, carrier sequence is removed from genome. The bacterial solution was diluted 100-1000 times and plated on 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 4017bp. The recombinant strain with the correct identification band size was sent to GenWeiZ for sequencing using the sequencing primers PE962-Bl-check-F1 / PE963-Bl-check-F2 / PE1009-odhA-BL-DN-R / PE1006-odhA-BL-UP-F / PE1010-odhA-tuf-F. Subsequently, nucleotide sequencing analysis was performed, and the strain with the correct sequence was named SME872a.

[0386] 6.2.2 SME872a superimposed with pK18-cg0928-Ptuf-Bl_xfp, five copies of xfp (SME874a)

[0387] Prepare SME872a competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), further recombinant plasmid pK18-cg0928-Ptuf-Bl_xfp (13869) is changed in Corynebacterium glutamicum with electrotransformation method, on the BHI substratum 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 P86, PE1125-0928-UP-1F / PE1128-0928-DN-2R, P81.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, transformant second time reorganization takes place, exchanges the carrier sequence from genome, and is removed. The bacterial solution was diluted 100-1000 times and then spread on 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 PE1131-ID-F / PE1132-ID-R, and the band size was 4101bp. The recombinant bacteria with the correct identification band size were sent to GenWeiZ for sequencing using the sequencing primers PE1131-ID-F / PE1132-ID-R / PE962-Bl-check-F1 / PE963-Bl-check-F2 / PE1129-xfp-F / PE1130-xfp-R. Subsequently, nucleotide sequencing analysis was performed, and the strain with the correct sequence was named SME874a.

[0388] 6.2.3 SME874a superimposed with pK18-cg2337-Ptuf-Bl_xfp, six copies of xfp (SME878a)

[0389] Prepare SME874a competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), further recombinant plasmid pK18-cg2337-Ptuf-Bl_xfp is changed in Corynebacterium glutamicum with electrotransformation method, on the BHI substratum 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 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, and by homologous recombination exchange, carrier sequence is removed from genome. The bacterial solution was diluted 100-1000 times and then plated on 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 PE1367-ID-F / PE1368-ID-R, and the band size was 4101bp. The recombinant strain with the correct identification band size was sent to GenWeiZ for sequencing using the sequencing primers PE1367-ID-F / PE1368-ID-R / PE962-Bl-check-F1 / PE963-Bl-check-F2 / PE1349-xfp-F / PE1350-xfp-R. Subsequently, nucleotide sequencing analysis was performed, and the strain with the correct sequence was named SME878a.

[0390] 6.2.4 SME878a superimposed with pK18-cg1512-Ptuf-Bl_xfp, seven copies of xfp (SME885)

[0391] Prepare SME878a competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), further recombinant plasmid pK18-cg1512-Ptuf-Bl_xfp is changed in Corynebacterium glutamicum with electrotransformation method, on the BHI substratum 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 PPE1133-1512-UP-1F / P86 and P81 / PE1136-1512-DN-2R.The recombinant that sieves is inoculated into and does not have in anti-YB substratum and cultivates 12-14h, 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 solution was diluted 100-1000 times and then spread on 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 4101bp. The recombinant bacteria with the correct identification band size were sent to GenWeiZ for sequencing using the sequencing primers PE1371-ID-F / PE1372-ID-R / PE962-Bl-check-F1 / PE963-Bl-check-F2 / PE1137-xfp-F / PE1138-xfp-R. Subsequently, nucleotide sequencing analysis was performed, and the strain with the correct sequence was named SME885.

[0392] 6.2.5 SME778a superimposed with pK18-cg0928-Ptuf-Bl_xfp, five copies of xfp (SME869a)

[0393] Prepare SME778a competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), prepare 5.4 part xfp four copies competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), recombinant plasmid pK18-cg0928-Ptuf-Bl_xfp (13869) is changed in Corynebacterium glutamicum with electrotransformation method, on the BHI substratum 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, cultivation temperature is 30 ℃, rotary shaking table 220rpm shaking culture. 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 spread 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 PE1131-ID-F and PE1132-ID-R, and the band size was 4101bp. The recombinant bacteria with the correct identification band size were sent to Jinweizhi Company for sequencing. The sequencing primers were PE1131-ID-F / PE1132-ID-R, PE962-Bl-check-F1 / PE963-Bl-check-F2 / PE1129-xfp-F / PE1130-xfp-R. Subsequently, nucleotide sequencing analysis was performed, and the strain with the correct sequencing was named SME869a.

[0394] 6.2.6 SME869a superimposed with pK18-BBD29-07210-Ptuf-Bl_xfp, six copies of xfp (SME877a)

[0395] Prepare SME869a competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), further recombinant plasmid pK18-BBD29-07210-Ptuf-Bl_xfp is changed in Corynebacterium glutamicum with electrotransformation method, on the BHI substratum 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 PE1389-7210-UP-1F / P86 and P81 / PE1392-7210-DN-2R.The recombinant that sieves is inoculated into and does not 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, and by homologous recombination exchange, carrier sequence is removed from genome. The bacterial solution was diluted 100-1000 times and then plated on 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 PE1419-ID-F / PE1420-ID-R, and the band size was 4101bp. The recombinant strain with the correct identification band size was sent to GenWeiZ for sequencing using the sequencing primers PE1419-ID-F / PE1420-ID-R / PE962-Bl-check-F1 / PE963-Bl-check-F2 / PE1393-xfp-F / PE1394-xfp-R. Subsequently, nucleotide sequencing analysis was performed, and the strain with the correct sequence was named SME877a.

[0396] 6.2.7 SME877a superimposed with seven copies of pK18-odhA-Ptuf-Bl_xfp (SME882)

[0397] Prepare SME877a competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), further recombinant plasmid pK18-odhA-Ptuf-Bl_xfp is changed in Corynebacterium glutamicum with electrotransformation method, on the BHI substratum of the kanamycin that contains 15mg / L, screen a recombinant, wherein target gene is inserted in karyomit(e) due to homologous recombination.Once identification primer is P81, PE1009-odhA-BL-DN-R / PE1006-odhA-BL-UP-F, P86.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, transformant second time reorganization takes place, exchanges carrier sequence from genome, is removed by homologous recombination. The bacterial solution was diluted 100-1000 times and then spread on 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 4017bp. The recombinant bacteria with the correct identification band size were sent to GenWeiZ for sequencing using the sequencing primers PE962-Bl-check-F1 / PE963-Bl-check-F2 / PE1009-odhA-BL-DN-R / PE1006-odhA-BL-UP-F / PE1010-odhA-tuf-F. Subsequently, nucleotide sequencing analysis was performed, and the strain with the correct sequence was named SME882.

[0398] 6.3 Shake flask validation

[0399] We performed shake flask fermentation on the phosphoketolase-expressing strain to verify its glutamate production performance. For detailed shake flask fermentation methods, refer to the shake flask fermentation method in Section 1.1.3. The fermentation results are shown in the table below.

[0400] Table 9. Glutamate content detection of recombinant strains

[0401] The above shake flask results show that whether randomly stacking from three copies of WJ0140-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.

[0402] Example 7: Construction of xfp multi-copy strains and their glutamate conversion efficiency

[0403] 7.1 Starting from ATCC13032, xfp multiple copies

[0404] 7.1.1 Construction of MH13032-10-alaT-Ptuf-Bl_xfp Strain (MH13032-11)

[0405] 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 sieves is inoculated into and cultivated 12-14 hour in no anti-BHI substratum, and cultivation temperature is 30 ℃, rotary shaker 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 PE1023-alaT-ID-F / 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.

[0406] 7.1.2 Construction of MH13032-11-cg3364-Ptuf-Bl_xfp strain (MH13032-12)

[0407] Prepare MH13032-11 competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), recombinant plasmid pK18-cg3364-Ptuf-Bl_xfp (13032) is changed into Corynebacterium glutamicum MH13032-11 competent cell with the electrotransformation method, on the BHI substratum 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 P13-11 / 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.

[0408] 7.1.3 Construction of MH13032-12-cg2564-Ptuf-Bl_xfp strain (MH13032-13)

[0409] Prepare MH13032-12 competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), recombinant plasmid pK18-cg2564-Ptuf-Bl_xfp (13032) is changed into Corynebacterium glutamicum MH13032-12 competent cell with the electrotransformation method, on the BHI substratum 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 / 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.

[0410] 7.1.4 Construction of MH13032-13-cg3125-Ptuf-Bl_xfp strain (MH13032-14)

[0411] Prepare MH13032-13 competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), recombinant plasmid pK18-cg3125-Ptuf-Bl_xfp (13032) is changed into Corynebacterium glutamicum MH13032-13 competent cell with the electrotransformation method, on the BHI substratum 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 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 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.

[0412] 7.1.5 Construction of MH13032-14-cg3384-Ptuf-Bl_xfp strain (MH13032-15)

[0413] Prepare MH13032-14 competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), recombinant plasmid pK18-cg3384-Ptuf-Bl_xfp (13032) is changed into Corynebacterium glutamicum MH13032-14 competent cell with the electrotransformation method, on the BHI substratum 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, 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.

[0414] 7.1.6 Construction of MH13032-15-BBD29_7270-Ptuf-Bl_xfp strain (MH13032-16)

[0415] Prepare MH13032-15 competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 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 BHI substratum 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 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 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.

[0416] 7.1.7 Construction of MH13032-16-cg0928-Ptuf-Bl_xfp strain (MH13032-17)

[0417] Prepare MH13032-16 competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), recombinant plasmid pK18-cg0928-Ptuf-Bl_xfp (13032) is changed into Corynebacterium glutamicum MH13032-16 competent cell with the electrotransformation method, on the BHI substratum 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 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 / 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.

[0418] 7.1.8 13032 XFP multi-copy strain shake flask verification

[0419] 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.

[0420] 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.

[0421] Table 10. Glutamate content detection of recombinant strains

[0422] 7.2 Starting from ATCC14067, xfp multiple copies

[0423] 7.2.1 Construction of MH14067-10-alaT-Ptuf-Bl_xfp Strain (MH14067-11)

[0424] 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.Bacterium liquid is diluted 100-1000 doubly 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 / 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.

[0425] 7.2.2 Construction of MH14067-11-cg3364-Ptuf-Bl_xfp strain (MH14067-12)

[0426] Prepare MH14067-11 competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), recombinant plasmid pK18-cg3364-Ptuf-Bl_xfp (14067) is changed into Corynebacterium glutamicum MH14067-11 competent cell with the electrotransformation method, on the BHI substratum 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 / 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.

[0427] 7.2.3 Construction of MH14067-12-cg2564-Ptuf-Bl_xfp strain (MH14067-13)

[0428] Prepare MH14067-12 competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), recombinant plasmid pK18-cg2564-Ptuf-Bl_xfp (14067) is changed into Corynebacterium glutamicum MH14067-12 competent cell with the electrotransformation method, on the BHI substratum 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 / 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.

[0429] 7.2.4 Construction of MH14067-13-cg3125-Ptuf-Bl_xfp strain (MH14067-14)

[0430] 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, and on the BHI substratum containing 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.

[0431] 7.2.5 Construction of MH14067-14-cg3384-Ptuf-Bl_xfp strain (MH14067-15)

[0432] Prepare MH14067-14 competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), recombinant plasmid pK18-cg3384-Ptuf-Bl_xfp (14067) is changed into Corynebacterium glutamicum MH14067-14 competent cell with the electrotransformation method, on the BHI substratum 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, 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 MH14067-15.

[0433] 7.2.6 Construction of MH14067-15-BBD29_7270-Ptuf-Bl_xfp strain (MH14067-16)

[0434] Prepare MH14067-15 competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 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 BHI substratum 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 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 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 MH14067-16.

[0435] 7.2.7 Construction of MH14067-16-cg0928-Ptuf-Bl_xfp strain (MH14067-17)

[0436] Prepare MH14067-16 competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), recombinant plasmid pK18-cg0928-Ptuf-Bl_xfp (14067) is changed into Corynebacterium glutamicum MH14067-16 competent cell with the electrotransformation method, on the BHI substratum 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 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 / 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.

[0437] 7.2.8 ATCC14067-based xfp multi-copy strain shake flask verification

[0438] 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.

[0439] 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.

[0440] Table 11. Glutamate content detection of recombinant strains

[0441] Example 8: Construction of the WJ0140 route xfp six-copy-weakened pfk strain and its glutamate conversion rate

[0442] 8.1 WJ0140 route xfp six copies of bacteria SME787 starting, weakening pfk

[0443] 8.1.1 SME787 overlay pfk-A1T (SME803)

[0444] SME787 competent cells were prepared according to the classical method for Corynebacterium glutamicum (C.glutamicum Handbook, Chapter 23). The recombinant plasmid pK18-pfk-A1T was electroporated into Corynebacterium glutamicum and recombinants were screened on a BHI medium supplemented with 15 mg / L of kanamycin. The primary identification primers were P86 / PE878-pfk-TTG-UP-1F and PE881-pfk-UP-2R / P81. The recombinants were inoculated into a non-antibiotic 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 times and then plated on a solid BHIS medium supplemented with 10% sucrose and cultured for 36 hours. The secondary identification primers were PE898-A1T-ID-F / PE899-A1T-ID-R. The recombinant bacteria with the correct identification band size were sent to GenWeiZi for sequencing, followed by nucleotide sequencing analysis, and the strain with the correct sequencing was named SME803.

[0445] 8.1.2 SME787 overlaying PrecA-pfk (SME804)

[0446] Prepare SME787 competent cells according to the classical method of Corynebacterium glutamicum (C.glutamicum Handbook, Chapter 23). Recombinant plasmid pK18-PrecA-pfk is transferred into Corynebacterium glutamicum with the electroporation method. Recombinants are screened once on the BHI culture medium containing 15mg / L of kanamycin. The primary identification primers are P86 / PE882-pfk-UP-1F and PE885-pfk-DN-2R / P81. The recombinants sieved are inoculated into the non-anti-BHI culture medium and cultivated for 12-14 hours. The cultivation temperature is 30 ℃ with a rotary shaker at 220rpm. After the bacterial liquid is diluted 100-1000 times, it is coated on the solid BHIS culture medium containing 10% sucrose and cultivated for 36 hours. The secondary identification primers are PE891-PrecA-check-F / PE899-A1T-ID-R. The recombinant bacteria with the correct identification band size were sent to GenWeiZi for sequencing, followed by nucleotide sequencing analysis, and the strain with the correct sequencing was named SME804.

[0447] 8.1.3 SME787 overlay pfk-E171D (SME805)

[0448] Prepare SME787 competent cells according to the classical method of Corynebacterium glutamicum (C.glutamicum Handbook, Chapter 23). Recombinant plasmid pK18-pfk-E171D is transferred into Corynebacterium glutamicum with electroporation method. Recombinants are screened once on the BHI culture medium containing 15mg / L kanamycin. The primary identification primers are P86 / PE892-pfk-E171K-UP-1F and PE895-pfk-E171K-DN-2R / P81. The recombinants sieved are inoculated into the non-anti-BHI culture medium and cultivated for 12-14 hours. The cultivation temperature is 30 ℃ with a rotary shaker at 220rpm. After the bacterial liquid is diluted 100-1000 times, it is coated on the solid BHIS culture medium containing 10% sucrose and cultivated for 36 hours. The secondary identification primers are PE946-E171-ID-F / PE947-E171-ID-R. The recombinant bacteria with the correct identification band size were sent to GenWeiZi for sequencing, followed by nucleotide sequencing analysis, and the strain with the correct sequencing was named SME805.

[0449] 8.1.4 SME787 overlay pK18-Pzwf-pfk (SME806)

[0450] Prepare SME787 competent cells according to the classical method of Corynebacterium glutamicum (C.glutamicum Handbook, Chapter 23). Recombinant plasmid pK18-Pzwf-pfk is transferred into Corynebacterium glutamicum with electroporation method. Recombinants are screened once on BHI culture medium containing 15mg / L of kanamycin. The primary identification primers are P86 / PE882-pfk-UP-1F and PE885-pfk-DN-2R / P81. The recombinants sieved are inoculated into non-antibiotic BHI culture medium and cultured for 12-14 hours at 30°C on a rotary shaker at 220rpm. After the bacterial solution is diluted 100-1000 times, it is coated on a solid BHIS culture medium containing 10% sucrose and cultured for 36 hours. The secondary identification primers are PE888-zwf-check-F / PE899-A1T-ID-R. The recombinant bacteria with the correct identification band size were sent to GenWeiZi for sequencing, followed by nucleotide sequencing analysis, and the strain with the correct sequencing was named SME806.

[0451] 8.1.5 Shake flask validation

[0452] 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.

[0453] 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.

[0454] Table 12. Conversion rate of glutamate in pfk-modified strains

[0455] 8.2 Starting from the six-copy xfp bacteria (the recombinant competent cells with correct sequencing obtained in section 5.6), weaken the pfk

[0456] 8.2.1 Superposition of Pzwf-pfk

[0457] According to the classical method of Corynebacterium glutamicum (C.glutamicum Handbook, Chapter 23), the correct xfp six-copy recombinant bacteria competent cells obtained in section 5.6 were prepared. The recombinant plasmid pK18-Pzwf-pfk was transferred into the above-mentioned Corynebacterium glutamicum by electroporation. The recombinants were screened once on a BHI medium containing 15mg / L of kanamycin. The primary identification primers were P86 / PE882-pfk-UP-1F and PE885-pfk-DN-2R / P81. The recombinants screened were inoculated into the non-antibiotic BHI 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 medium containing 10% sucrose and cultured for 36 hours. The secondary identification primers were PE888-zwf-check-F / PE899-A1T-ID-R. The recombinant bacteria with the correct identification band size were sent to GeneWeichi for sequencing, followed by nucleotide sequencing analysis.

[0458] 8.2.2 Superimposing PrecA-pfk

[0459] According to the classical method of Corynebacterium glutamicum (C.glutamicum Handbook, Chapter 23), prepare the correct xfp six-copy recombinant bacteria competent cells of the sequencing obtained in section 5.6. The recombinant plasmid pK18-PrecA-pfk was transferred into the above-mentioned Corynebacterium glutamicum with the electroporation method. The recombinant was screened once on the BHI medium containing 15mg / L of kanamycin. The primary identification primers were P86 / PE882-pfk-UP-1F and PE885-pfk-DN-2R / P81. The recombinant was inoculated into the non-anti-BHI 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 medium containing 10% sucrose and cultivated for 36 hours. The secondary identification primers were PE891-PrecA-check-F / PE899-A1T-ID-R. The recombinant bacteria with the correct identification band size were sent to GeneWeichi for sequencing, followed by nucleotide sequencing analysis.

[0460] 8.2.3 Overlaying pfk-E171K

[0461] According to the Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), prepare the correct xfp six-copy recombinant bacteria competent cells of the sequencing that part obtains in 5.6, recombinant plasmid pK18-pfk-E171K is changed into above-mentioned Corynebacterium glutamicum with the electrotransformation method, and on the BHI substratum 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 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 PE946-E171-ID-F / PE947-E171-ID-R. Recombinant bacteria with the correct identification band size were sent to GeneWeiZ for sequencing and subsequent nucleotide sequencing analysis.

[0462] 8.2.4 Shake flask validation

[0463] 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.

[0464] 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.

[0465] Table 13. Conversion rate of glutamate in pfk-modified strains

[0466] 8.3 Starting from ATCC13032, weakening pfk

[0467] 8.3.1 Construction of MH13032-17-pfk-A1T strain (MH13032-18)

[0468] 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, on the BHI substratum 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 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 / 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.

[0469] 8.3.2 Construction of MH13032-18-PrecA-pfk strain (MH13032-19)

[0470] Prepare MH13032-18 competent cells according to 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 BHI substratum 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 the non-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 / 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.

[0471] 8.3.3 Construction of MH13032-19-pfk-E171D strain (MH13032-20)

[0472] 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 BHI substratum 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 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 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.

[0473] 8.3.4 Construction of MH13032-20-Pzwf-pfk Strain (MH13032-21)

[0474] 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 BHI substratum 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 the non-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 PE888-zwf-check-F / 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.

[0475] 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%.

[0476] Table 14. Glutamate content detection of recombinant strains

[0477] 8.4 Starting from ATCC14067, weakening pfk

[0478] 8.4.1 Construction of MH14067-17-pfk-A1T Strain (MH14067-18)

[0479] 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 BHI substratum 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 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 / 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.

[0480] 8.4.2 Construction of MH14067-18-PrecA-pfk strain (MH14067-19)

[0481] Prepare MH14067-18 competent cell according to Corynebacterium glutamicum classical method (C.glutamicum Handbook, Chapter 23), recombinant plasmid pK18-PrecA-pfk (14067) is changed into Corynebacterium glutamicum MH14067-18 competent cell with the electrotransformation method, on the BHI substratum that contains the kanamycin of 15mg / L, screen a recombinant.One time 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 the non-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 / 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.

[0482] 8.4.3 Construction of MH14067-19-pfk-E171D strain (MH14067-20)

[0483] Prepare MH14067-19 competent cells according to the classical method of Corynebacterium glutamicum (C.glutamicum Handbook, Chapter 23). Recombinant plasmid pK18-pfk-E171D (14067) was transferred into Corynebacterium glutamicum MH14067-19 competent cells with the electroporation method. The recombinant was screened on the BHI substratum containing 15mg / L of kanamycin. The primary identification primers were P86 / PE892-pfk-E171K-UP-1F and P14-18 / P81. The recombinant obtained by screening was inoculated into the non-anti-BHI substratum 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 the solid BHIS substratum containing 10% sucrose and cultivated for 36 hours. The secondary identification primer was 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.

[0484] 8.4.4 Construction of MH14067-20-Pzwf-pfk strain (MH14067-21)

[0485] 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 BHI substratum 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 the non-anti-BHI substratum, and the cultivation temperature is 30 ℃, rotary shaking table 220rpm shaking culture. Bacterial liquid is diluted 100-1000 times and is coated on the solid BHIS substratum that contains 10% sucrose and cultivated 36 hours. The secondary identification primers were PE888-zwf-check-F / 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.

[0486] 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%.

[0487] Table 15. Glutamate content detection of recombinant strains

[0488] Example 9: Construction of xfp multi-copy strains and their glutamate conversion efficiency based on WJ0138-7

[0489] We performed multi-copy genomic expression of xfp based on WJ0138-7 (gdh / odhA / yggB combination). We completed the transformation of 7 copies of Bl-xfp and found that the strain with the sixth copy of xfp had the best performance. The optional insertion sites for 7-copy xfp transformation are 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. The order of exemplary 7-copy xfp insertion sites is shown in Table 16, but is not limited to these 7 insertion sites:

[0490] Table 16. Exemplary xfp insertion sites

[0491] Other approaches to reduce odhA activity included complete deletion of the odhA gene sequence, truncation of odhA with simultaneous repair of the frameshift mutation to wild type, and other truncated forms of odhA that reduced activity by more than 10%. Other approaches to enhance gdh activity included enhancing gdh with a strong promoter. All of these substitutions yielded similar results.

[0492] 9.1 Construction of WJ0138-7-odhA-Ptuf-Bl_xfp strain (WJ0138-10)

[0493] WJ0138-7 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C.glutamicum Handbook, Chapter 23). The recombinant plasmid pK18-odhA-Ptuf-Bl_xfp was transferred into the competent cells using electroporation and cultured for 48 hours on a BHI medium plate containing 15 mg / L kanamycin. The recombinant identification primers were P81 / PE1009-odhA-BL-DN-R and PE1006-odhA-BL-UP-F / P86. The recombinant was inoculated into the non-antibiotic 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 times and then plated on a solid BHIS medium containing 10% sucrose and cultured for 36 hours. The primers for secondary recombinant identification were PE1009-odhA-BL-DN-R / PE1006-odhA-BL-UP-F, yielding a band size of 4017 bp. Recombinant strains with the correct identified band size were sent to GENEWIZ for sequencing. Subsequently, nucleotide sequencing analysis was performed, and the recombinant strain with the correct sequence was named WJ0138-10.

[0494] 9.2 Construction of WJ0138-10-alaT-Ptuf-Bl_xfp strain (WJ0138-11)

[0495] WJ0138-10 competent cells were prepared according to the classical method for Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23). The recombinant plasmid pK18-alaT-Ptuf-Bl_xfp was electroporated into the competent cells and cultured on BHI medium plates containing 15 mg / L kanamycin for 48 hours. The primary recombinant 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 times and then plated on solid BHIS medium containing 10% sucrose and cultured for 36 hours. The secondary recombinant identification primers were PE1023-alaT-ID-F / PE1024-alaT-ID-R. The correct size was 4054 bp, and the recombinant strain with the correct identified band size was sent to GENEWIZ for sequencing. Subsequently, nucleotide sequencing analysis was performed, and the recombinant microbial strain with the correct sequence was named WJ0138-11.

[0496] 9.3 Construction of WJ0138-11-odhA-PcspB-Bl_xfp strain (WJ0138-12)

[0497] Prepare WJ0138-11 competent cells according to the classical method of Corynebacterium glutamicum (C.glutamicum Handbook, Chapter 23). The recombinant plasmid pK18-odhA-PcspB-Bl_xfp was transferred into the competent cells with the electroporation method. The recombinants were screened on a BHI culture medium containing 15mg / L of kanamycin. The primers for primary recombinant identification were P86 / PE617-UP-1F and PE624-DN-4R / P81. The recombinants screened were inoculated into the non-antibiotic 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 primers for secondary recombinant identification were PE625-ID-F / PE626-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 sequencing was named WJ0138-12.

[0498] 9.4 Construction of WJ0138-12-cg3384-Ptuf-Bl_xfp strain (WJ0138-13)

[0499] Prepare WJ0138-12 competent cells according to the classical method of Corynebacterium glutamicum (C.glutamicum Handbook, Chapter 23). The recombinant plasmid pK18-cg3384-Ptuf-Bl_xfp was transferred into the competent cells with the electroporation method. The recombinants were screened on a BHI culture medium containing 15mg / L of kanamycin. The primers for primary recombinant identification were P86 / PE1101-3384-UP-1F and PE1104-3384-DN-2R / P81. The recombinants screened were inoculated into the non-antibiotic 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 primers for secondary recombinant identification were PE1107-ID-F / PE1108-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 sequencing was named WJ0138-13.

[0500] 9.5 Construction of WJ0138-13-cg2564-Ptuf-Bl_xfp strain (WJ0138-14)

[0501] Prepare WJ0138-13 competent cells according to the classical method of Corynebacterium glutamicum (C.glutamicum Handbook, Chapter 23). Recombinant plasmid pK18-cg2564-Ptuf-Bl_xfp is transferred into competent cells with electroporation method. On the BHI culture medium containing 15mg / L of kanamycin, a recombinant is screened. The primers for primary recombinant identification are P86 / PE1351-2564-UP-1F and PE1354-2564-DN-2R / P81. The recombinant obtained by screening is inoculated into the non-antibiotic BHI culture medium and cultivated for 12-14 hours. The cultivation 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 culture medium containing 10% sucrose and cultivated for 36 hours. The primers for secondary recombinant identification are PE1369-ID-F / PE1370-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 sequencing was named WJ0138-14.

[0502] 9.6 Construction of WJ0138-14-cg2211-Ptuf-Bl_xfp strain (WJ0138-15)

[0503] Prepare WJ0138-14 competent cells according to the classical method of Corynebacterium glutamicum (C.glutamicum Handbook, Chapter 23). Recombinant plasmid pK18-cg2211-Ptuf-Bl_xfp is transferred into competent cells with electroporation method. On the BHI culture medium containing 15mg / L of kanamycin, a recombinant is screened. The primers for primary recombinant identification are P86 / PE1339-2211-UP-1F and PE1342-2211-DN-2R / P81. The recombinant obtained by screening is inoculated into the non-anti-BHI culture medium and cultivated for 12-14 hours. The cultivation 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 culture medium containing 10% sucrose and cultivated for 36 hours. The primers for secondary recombinant identification are PE1365-ID-F / PE1366-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 sequencing was named WJ0138-15.

[0504] 9.7 Construction of WJ0138-15-cg3364-Ptuf-Bl_xfp strain (WJ0138-16)

[0505] Prepare WJ0138-15 competent cells according to the classical method of Corynebacterium glutamicum (C.glutamicum Handbook, Chapter 23). The recombinant plasmid pK18-cg3364-Ptuf-Bl_xfp was transferred into the competent cells with the electroporation method. The recombinants were screened on the BHI culture medium containing 15mg / L of kanamycin. The primers for primary recombinant identification were P86 / PE1117-3364-UP-1F and PE1120-3364-DN-2R / P81. The recombinants screened were inoculated into the non-antibiotic 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 the solid BHIS culture medium containing 10% sucrose and cultured for 36 hours. The primers for secondary recombinant identification were PE1123-ID-F / PE1124-ID-R. The recombinant bacteria with the correct identification band size were sent to GeneWeichi for sequencing, followed by nucleotide sequencing analysis. The recombinant microbial strain with the correct sequencing was named WJ0138-16.

[0506] 9.8 Shake flask validation

[0507] 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.

[0508] Table 17 shows the results of shake flask experiments using multiple copies of the Bifidobacterium longum phosphoketolase (XFP) superimposed on WJ0138-7. The results show that glutamate conversion efficiency increased overall with the addition of 1 to 7 copies of XFP, reaching a peak of 35.31% at 6 copies of XFP. While there was an improvement with 7 copies, glutamate conversion decreased compared to 6 copies.

[0509] Table 17. Glutamate content detection of recombinant strains

[0510] Table 18. Promoter transformation table

[0511] Table 19. Gene insertion sites

[0512] Incorporated by Reference

[0513] Each patent and scientific document mentioned herein is incorporated by reference in its entirety for all purposes.

[0514] Equivalence

[0515] 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-glutamate, 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, further comprising a modification to reduce odhA activity, a mutation in the yggB encoding protein to increase glutamate production, and / or a modification to enhance gdh activity. The modified bacterium according to claim 5 , wherein the modification for reducing odhA activity is selected from the group consisting of complete knockout or partial knockout of the odhA gene.

7. The modified bacterium according to claim 5, wherein the yggB encoding protein mutation is selected from the group consisting of yggB encoding protein A100T, A111V, L93A, L109A, a mutation in the region encoding amino acid residues 419-533 of the wild-type YggB protein, or a mutation in the region encoding the transmembrane domain of the wild-type YggB protein.

8. The modified bacterium according to claim 5, wherein the modification for enhancing gdh activity is promoter modification, preferably, the promoter modification comprises using a strong promoter or a DNA sequence at position -35 of the gdh transcription start site being TTGTCA and a DNA sequence at position -10 of the transcription start site being TATAAT.

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

10. The modified bacterium according to any one of claims 1 to 9, wherein the bacterium further comprises a modification that reduces phosphofructokinase activity.

11. The modified bacterium according to claim 10, 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.

12. The modified bacterium of claim 11, 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.

13. The modified bacterium of claim 11, 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.

14. The modified bacterium according to any one of claims 1 to 13, comprising more than one copy of the phosphoketolase gene, preferably three to ten copies, more preferably six copies.

15. The modified bacterium of any one of claims 1 to 14, 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.

16. The modified bacterium of any one of claims 1 to 15, wherein 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 at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, or 17; or the nucleic acid sequence encoding the phosphoketolase comprises a sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, or 18, or a nucleic acid sequence at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, or 18.

17. The modified bacterium of any one of claims 1 to 16, further comprising gltA -10M-C361Y 、suc M199I ach S372C 、tktA T234I 、putA A755V 、ds G171D , and / or nadA D312N .

18. Use of the modified bacterium according to any one of claims 1 to 17 in the fermentative production of L-glutamic acid.

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

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