5-aminolevulinic acid production related transcription factor Cgl2988 based on network model prediction and application

Abstract

The invention discloses a transcription factor Cgl2988 related to production of 5-aminolevulinic acid based on network model prediction and application, the amino acid sequence of the transcription factor Cgl2988 is as shown in SEQ ID NO.1. The transcription factor Cgl2988 related to production of 5-ALA is simulated and predicted based on a metabolic network model system mcCGL, and experiments prove that the transcription factor Cgl2988 related to production of 5-ALA has the advantages that the transcription factor Cgl2988 related to production of 5-ALA can be used for preparing the transcription factor Cgl2988 related to production of 5-ALA; the expression strain Cgh3-low-cgl2988 of the transcription factor gene cgl2988 in the down-regulated corynebacterium glutamicum is subjected to shake flask fermentation, and the yield of 5-ALA is 5.52 + / -0.19 g / L and is increased by about 1.64 times compared with that of a control strain; the expression strain Cgh3-pX-cgl2988 of the transcription factor gene cgl2988 in the up-regulated corynebacterium glutamicum is subjected to shake flask fermentation, and the yield of the 5-ALA is 5.76 + / -0.11 g / L, which is about 2.84 times higher than that of a control strain.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology and applications, and particularly relates to a transcription factor Cgl2988 related to 5-aminolevulinic acid production based on network model prediction and its applications. Background Art

[0002] 5-aminolevulinic acid (5-ALA), as a key precursor of heme, chlorophyll and vitamin B12, has important application values in the fields of photodynamic therapy, agricultural biostimulants and microbial manufacturing. [1,2] At present, the production of 5-ALA based on Corynebacterium glutamicum mainly relies on metabolic engineering strategies, such as directed evolution of 5-ALA synthase, perturbation of side branches or downstream pathways, or dynamic regulated expression of key genes. [3] However, so far, the research on transcription factors specific to the 5-ALA synthesis pathway is still blank.

[0003] In recent years, network model-driven synthetic biology strategies have provided new ideas for microbial metabolic regulation. By integrating genomic, transcriptomic and metabolomic data to construct a metabolic network model, potential transcription factors and their target genes can be systematically predicted, breaking through the limitations of the traditional "trial and error method". For example, in 2017, Kim [4] et al. used the Escherichia coli metabolic network model iJO1366 as the chassis, integrated 3672 transcriptional regulatory relationships obtained from the RegulonDB database, constructed an Escherichia coli transcriptional regulatory metabolic network model, and used the BeReTa algorithm to predict transcription factors related to the synthesis of various products in Escherichia coli, such as the transcription factors ArgR, TdcA, ArgP and Rob for synthesizing arginine. In 2019, Shen [5] et al. used the Saccharomyces cerevisiae metabolic network model Yeast 7.6 as the chassis model, combined the transcriptional regulatory network and the metabolic network model using the IDREAM method, and used the OptRAM algorithm to predict genes and transcription factors related to high-yield succinic acid. The upregulation of the transcription factors PHO4 and PDR1 significantly increased the succinic acid yield.

[0004] Based on the above case demonstrations, combined with the deficiencies in the current 5-ALA production, using network model prediction to screen transcription factors related to high-yield 5-ALA can accurately analyze the metabolic regulation mechanism and guide the transformation of targeted strains, significantly improving the 5-ALA biosynthesis efficiency, and providing key targets and theoretical supports for large-scale industrial production and metabolic engineering optimization. However, there is currently no report on the transcription factor Cgl2988 related to 5-aminolevulinic acid production based on network model prediction. Summary of the Invention

[0005] The object of the present invention is to overcome the deficiencies of the prior art and provide a transcription factor Cgl2988 related to the production of 5-aminolevulinic acid based on network model prediction and its application.

[0006] The second object of the present invention is to provide a gene encoding the above transcription factor Cgl2988.

[0007] The third object of the present invention is to provide a pCRISPRi-low-cgl2988 plasmid or a pX-cgl2988 plasmid containing the above gene;

[0008] The fourth object of the present invention is to provide a recombinant bacterium containing the above pCRISPRi-low-cgl2988 plasmid or a recombinant bacterium containing the above pX-cgl2988 plasmid.

[0009] The fifth object of the present invention is to provide the application of the above recombinant bacterium in fermentation to obtain the fermentation product 5-aminolevulinic acid.

[0010] The technical solution of the present invention is outlined as follows:

[0011] A transcription factor Cgl2988 related to the production of 5-aminolevulinic acid based on network model prediction, and the amino acid sequence of the transcription factor Cgl2988 is shown as SEQ ID NO.1.

[0012] A gene encoding the above transcription factor Cgl2988, and the nucleotide sequence of the gene is shown as SEQ ID NO.2.

[0013] A pCRISPRi-low-cgl2988 plasmid or a pX-cgl2988 plasmid containing the above gene;

[0014] The pCRISPRi-low-cgl2988 plasmid is constructed in the following manner:

[0015] Using pdCas9gRNA as a template, and l-cgl2988-F1 and cas-R1 as upstream and downstream primers, perform PCR amplification to obtain a linearized vector fragment 1;

[0016] Using pdCas9gRNA as a template, and ori-F and CRISPRi-R2 as upstream and downstream primers, perform PCR amplification to obtain a linearized vector fragment 2;

[0017] Connect the fragment 1 and the fragment 2, and verify by colony PCR to obtain the pCRISPRi-low-cgl2988 plasmid;

[0018] The nucleotide sequence of the l-cgl2988-F1 is shown as SEQ ID NO.3;

[0019] The nucleotide sequence of the cas-R1 is shown as SEQ ID NO.4;

[0020] The nucleotide sequence of the ori-F is shown as SEQ ID NO.5;

[0021] The nucleotide sequence of the CRISPRi-R2 is shown as SEQ ID NO.6;

[0022] The pX-cgl2988 plasmid was constructed in the following manner:

[0023] Using pXMJ19 as a template and pX-cgl2988-F and pX-cgl2988-R as upstream and downstream primers, PCR amplification was performed to obtain a linearized vector fragment 3;

[0024] Using the Corynebacterium glutamicum ATCC 13032 genome as a template and cgl2988-F and cgl2988-R as upstream and downstream primers, PCR amplification was performed to obtain a linearized vector fragment 4;

[0025] The fragment 3 and fragment 4 were ligated and verified by colony PCR to obtain the pX-cgl2988 plasmid;

[0026] The nucleotide sequence of the pX-cgl2988-F is shown as SEQ ID NO.7;

[0027] The nucleotide sequence of the pX-cgl2988-R is shown as SEQ ID NO.8;

[0028] The nucleotide sequence of the cgl2988-F is shown as SEQ ID NO.9;

[0029] The nucleotide sequence of the cgl2988-R is shown as SEQ ID NO.10.

[0030] A recombinant bacterium containing the pCRISPRi-low-cgl2988 plasmid or a recombinant bacterium containing the pX-cgl2988 plasmid,

[0031] The recombinant bacterium containing the pCRISPRi-low-cgl2988 plasmid was constructed in the following manner:

[0032] The pCRISPRi-low-cgl2988 plasmid was integrated into the Corynebacterium glutamicum recombinant strain Cgh3 by electrotransformation and verified by colony PCR to obtain the strain Cgh3-low-cgl2988 with down-regulated expression of the transcription factor gene cgl2988 in Corynebacterium glutamicum;

[0033] The recombinant bacterium containing the pX-cgl2988 plasmid was constructed in the following manner:

[0034] The pX-cgl2988 plasmid was integrated into the Corynebacterium glutamicum recombinant strain Cgh3 by electrotransformation and verified by colony PCR to obtain the strain Cgh3-pX-cgl2988 that up-regulates the expression of the transcription factor gene cgl2988 in Corynebacterium glutamicum.

[0035] Application of the above recombinant bacterium in the fermentation production of 5-aminolevulinic acid.

[0036] Advantages of the present invention:

[0037] For the first time, the present invention simulates and predicts the transcription factor Cgl2988 related to 5-ALA production based on the metabolic network model system mcCGL. Experiments have proved that the strain Cgh3-low-cgl2988 with the down-regulated expression of the transcription factor gene cgl2988 in Corynebacterium glutamicum was fermented in a shake flask, and the 5-ALA yield was 5.52 ± 0.19 g / L, which was about 1.64 times higher than that of the control strain Cgh3-pXi; the strain Cgh3-pX-cgl2988 with the up-regulated expression of the transcription factor gene cgl2988 in Corynebacterium glutamicum was fermented in a shake flask, and the 5-ALA yield was 5.76 ± 0.11 g / L, which was about 2.84 times higher than that of the control strain Cgh3-pX. Description of the drawings

[0038] Figure 1 It is the plasmid map of pCRISPRi-low-cgl2988.

[0039] Figure 2 It is the plasmid map of pX-cgl2988.

[0040] Figure 3 It is the plasmid map of pD-sucCD.

[0041] Figure 4 It is the 5-ALA yield graph of the strains Cgh3-low-cgl2988 and Cgh3-pX-cgl2988 under shake flask conditions. Detailed implementation manners

[0042] The following further illustrates the present invention with specific embodiments. The following embodiments are intended to enable those skilled in the art to better understand the present invention, but do not impose any limitations on the present invention.

[0043] The original strain Corynebacterium glutamicum ATCC13032 used in the present invention was sourced from ATCC (The Global Bioresource Center, http: / / www.atcc.org / ) and purchased in October 2012;

[0044] The starting strain used in the present invention is the recombinant strain of Corynebacterium glutamicum CGL11-RM-BEI, and the construction method of CGL11-RM-BEI has been described in detail in the authorized patent "Mutant of Corynebacterium glutamicum Transcriptional Regulator IpsA and Its Application" (Patent No.: ZL202010333584.X).

[0045] The genotype of the recombinant strain of Corynebacterium glutamicum CGL11-RM-BEI is C.glutamicum ATCC13032ΔldhAΔackA-ptaΔpqoΔcat Ppyc::Psod Pppc::Psod Ptal::Psod Ptkt::Psod ParaE::Ptuf(del 21bp)PackA-pta::Psod ldhA::araE ackA-pta::xylAB:C131T NCgl2538:C331T.

[0046] The integration vectors pD-aceA::hemA C132A 、pD-catA::hemA C132A 、pD-pqo::hemA C132A The specific operation methods for the construction, such as the primers used, have been described in detail in the patent "Engineering Bacteria of Corynebacterium glutamicum for Producing 5-Aminolevulinic Acid and Its Construction Method" (Application No.: CN202410677943.1).

[0047] The construction method of the strong promoter Psod-sx involved in the construction of Cgh3 has been described in detail in the published patent "A High-Strength Promoter Psod-sx of Corynebacterium glutamicum and Its Application" (Patent No.: CN202010351748.1).

[0048] The construction method of the hemA C132A gene involved in the construction of Cgh3 has been described in detail in the authorized patent "Mutant of 5-Aminolevulinic Acid Synthase of Rhodopseudomonas palustris and Its Application" (Application No.: CN202210142094.0).

[0049] The genotype of Cgh3 is C.glutamicum ATCC13032△ldhA△pqo::Psod-sx hemAC132A △cat::Psod-sx hemA C132A △pta△ackA Psod pyc Psod ppc,△aceA::Psod-sx hemA C132A ,Psod tal Psod tkt,Ptuf araE(del 21bp),ackA-pta::Psod xylAB:C131T,NCgl2538:C311T。

[0050] For the specific operation methods of the scarless operation technology of Corynebacterium glutamicum and the construction of the tool vector pD-sacB involved in the present invention, reference can be made to the authorized patent (Application No.: CN201710215459.7, Corynebacterium glutamicum strain for high-yield chiral D-(-)-acetoin and its construction and application).

[0051] The preparation of pdCas9gRNA can be referred to Reference 6.

[0052] The 5-aminolevulinic acid standard product was purchased from sigma company (http: / / www.sigmaaldrich.com / sigmaaldrich).

[0053] The molecular biology reagents such as restriction endonucleases and DNA ligases used were purchased from Thermo company (http: / / www.thermoscientificbio.com / fermentas), and other biochemical reagents used were purchased from Sangon Biotech (Shanghai)

[0054] Co., Ltd. ([[]] http: / / www.sangon.com / ).

[0055] E. coli DH5α competent cells were prepared by the conventional CaCl 2 method;

[0056] LB liquid medium: yeast extract powder 5 g / L, tryptone 10 g / L, NaCl 10 g / L, and 2% agar powder was added to the LB solid medium.

[0057] CGIII medium: yeast extract powder 10 g / L, tryptone 10 g / L, MOPS 21 g / L, NaCl 2.5 g / L, and the pH was adjusted to 7 with 5 M NaOH aqueous solution.

[0058] BHIS liquid medium: brain heart infusion broth powder 74 g / L. 2% agar powder was added to the BHIS solid medium.

[0059] The antibiotic concentrations were: kanamycin 40 μg / mL, chloramphenicol 10 μg / mL.

[0060] Detection method of 5-ALA: Take 250 μL of 5-aminolevulinic acid standard solution (final concentrations of 1, 2, 4, 6, 8 mg / L), or diluted fermentation broth, or the reaction solution after the termination of enzyme activity assay, add 125 μL of sodium acetate buffer with pH = 4.6, then add 62.5 μL of acetylacetone, incubate in a metal bath at 100 °C for 15 min, after cooling to room temperature, add 440 μL of freshly prepared Modified Ehrlich's reagent (0.2 g of p-dimethylaminobenzaldehyde, 1 mL of glacial acetic acid, 1 mL of perchloric acid, made up to 10 mL with glacial acetic acid), mix well, measure the absorbance value of the reaction solution at a wavelength of 554 nm after reacting at room temperature for 20 min. Calculate the content of 5-ALA in the fermentation broth or the reaction solution after the termination of enzyme activity assay using the standard curve obtained from the determination of 5-aminolevulinic acid standard product.

[0061] Example 1 Transcription factor Cgl2988 related to 5-aminolevulinic acid production predicted based on a network model

[0062] Based on the Corynebacterium glutamicum multi-constraint network model mcCGL [7] , use the BeReTa algorithm to predict the targets of transcription factors related to 5-ALA synthesis on the multi-constraint network model in ETGEMs2.0 format. Utilize the integrated transcriptome data and transcriptional regulatory relationships of Corynebacterium glutamicum, and by maximizing the fluxes of biomass synthesis reaction and 5-ALA exchange reaction respectively as the objectives of model calculation, simulate and predict the transcription factors related to 5-ALA synthesis. Read the Corynebacterium glutamicum regulatory relationship file in DataFrame format, and use it together with the multi-constraint network model as parameters to input into the BeReTa_use function, and run BeReTa to obtain the result matrix. Screen the transcription factors with P < 0.05, number of valid genes / reactions ≥ 2, and proportion of valid genes > 0.1 in the matrix as the transcription factors related to 5-ALA synthesis. Finally, it is found that the transcription factor Cgl2988 (the amino acid sequence of transcription factor Cgl2988 is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding transcription factor Cgl2988 is shown in SEQ ID NO.2) is highly related to 5-ALA production, and the correlation coefficient is -1.511.

[0063] Example 2 Construction of gene expression vector

[0064] (1) Construction of pCRISPRi-low-cgl2988 plasmid

[0065] Using pdCas9gRNA as a template and l-cgl2988-F1 (SEQ ID NO.3) and cas-R1 (SEQ ID NO.4) as upstream and downstream primers, perform PCR amplification to obtain linearized vector fragment 1;

[0066] Using pdCas9gRNA as a template and ori-F (SEQ ID NO.5) and CRISPRi-R2 (SEQ ID NO.6) as upstream and downstream primers, perform PCR amplification to obtain linearized vector fragment 2.

[0067] Use seamless cloning technology to ligate fragment 1 and fragment 2, and verify by colony PCR to obtain the pCRISPRi-low-cgl2988 plasmid, the map of which is as Figure 1 shown.

[0068] PCR amplification system: 2 μL of template, 2 μL each of upstream and downstream primers, 1 μL of dNTP, 25 μL of phanta Buffer, 17 μL of sterilized double-distilled water, 1 μL of DNA polymerase, with a total volume of 50 μL.

[0069] The PCR reaction conditions are: 95°C for 3 min, 30 cycles (95°C for 20 s, 58°C for 20 s, 72°C for 1 min), 72°C for 10 min, 4°C for 10 min. Use a gel extraction kit to recover and purify the PCR product.

[0070] (2) Construction of pX-cgl2988 plasmid

[0071] Using pXMJ19 (commercial) as a template and pX-cgl2988-F (SEQ ID NO.7) and pX-cgl2988-R (SEQ ID NO.8) as upstream and downstream primers, perform PCR amplification to obtain linearized vector fragment 3;

[0072] Using the Corynebacterium glutamicum ATCC13032 genome as a template and cgl2988-F (SEQ ID NO.9) and cgl2988-R (SEQ ID NO.10) as upstream and downstream primers, perform PCR amplification to obtain linearized vector fragment 4.

[0073] Use seamless cloning technology to ligate fragment 3 and fragment 4, and verify by colony PCR. The obtained pX-cgl2988 plasmid, the map of which is as Figure 2 shown.

[0074] PCR amplification system: 2 μL of template, 2 μL each of upstream and downstream primers, 1 μL of dNTP, 25 μL of phanta Buffer, 17 μL of sterilized double-distilled water, 1 μL of DNA polymerase, with a total volume of 50 μL.

[0075] The PCR reaction conditions were as follows: 95°C for 3 min, 30 cycles (95°C for 20 s, 58°C for 20 s, 72°C for 1 min), 72°C for 10 min, 4°C for 10 min. The PCR products were recovered and purified using a gel extraction kit.

[0076] Example 3 Construction of strain Cgh3

[0077] 1 Construction of strain Cgh

[0078] (1) Construction of the sucCD knockout plasmid pD-sucCD

[0079] Using pD-sacB (commercial) as the template and up-sucCD-F (SEQ ID NO.11), up-sucCD-R (SEQ ID NO.12) as the upstream and downstream primers, PCR amplification was performed to obtain the upstream fragment;

[0080] Using pD-sacB as the template and down-sucCD-F (SEQ ID NO.13), down-sucCD-R (SEQ ID NO.14) as the upstream and downstream primers, PCR amplification was performed to obtain the downstream fragment;

[0081] Using pD-sacB as the template and pD-F (SEQ ID NO.15), pD-R (SEQ ID NO.16) as the upstream and downstream primers, PCR amplification was performed to obtain the pD vector fragment;

[0082] After recovering and purifying the upstream fragment, downstream fragment, and pD vector fragment, the upstream fragment, downstream fragment, and pD vector fragment were ligated using the 2X MultiF Seamless Assembly Mix reagent by molecular cloning methods and transformed into Escherichia coli DH5α competent cells. The obtained transformants were verified by colony PCR using the primer pair test-pD-F (SEQ ID NO.17) / test-pD-R (SEQ ID NO.18). After correct sequencing verification, the pD-sucCD plasmid was obtained. The plasmid map of pD-sucCD is as Figure 3 shown.

[0083] (2) Construction of strain Cgh

[0084] The pD-sucCD plasmid was introduced into the Corynebacterium glutamicum recombinant strain CGL11-RM-BEI by electrotransformation. According to the scarless operation method described in the authorized patent CN201710215459.7, the sucCD gene in the genome of CGL11-RM-BEI was knocked out to obtain the strain Cgh.

[0085] Construction of strain Cgh3

[0086] The integration vectors pD-aceA::hemA C132A , pD-catA::hemA C132A , pD-pqo::hemA C132A were successively introduced into the Corynebacterium glutamicum recombinant strain Cgh by electrotransformation. According to the scarless operation method described in the authorized patent CN201710215459.7, 3 copies of the hemA C132A gene were integrated at the aceA, catA, and pqo loci on the chromosome of the Corynebacterium glutamicum recombinant strain Cgh to obtain the strain Cgh3.

[0087] Example 4 Construction and application of the recombinant bacterium containing the pCRISPRi-low-cgl2988 plasmid or the recombinant bacterium containing the pX-cgl2988 plasmid

[0088] (1) Construction of the recombinant bacterium containing the pCRISPRi-low-cgl2988 plasmid or the recombinant bacterium containing the pX-cgl2988 plasmid

[0089] The plasmids pCRISPRi-low-cgl2988 and pX-cgl2988 with correct sequencing results were respectively integrated into Corynebacterium glutamicum Cgh3 in the form of electrotransformation and evenly spread on a BHIS solid plate with chloramphenicol resistance (final concentration 10 μg / mL). Single colonies were separately picked and subjected to PCR verification with the following primers test-pX-1 (SEQ ID NO.19) / test-pX-2 (SEQ ID NO.20). Those with correct sequencing were successfully inserted with the plasmid, and the strain Cgh3-low-cgl2988 capable of downregulating the expression of the transcription factor gene cgl2988 and the strain Cgh3-pX-cgl2988 capable of upregulating the expression of the transcription factor gene cgl2988 were obtained.

[0090] The pdCas9gRNA plasmid was integrated into the Corynebacterium glutamicum recombinant strain Cgh3 in the form of electrotransformation to obtain the strain Cgh3-pXi; the pXMJ19 plasmid was integrated into the Corynebacterium glutamicum recombinant strain Cgh3 in the form of electrotransformation to obtain the strain Cgh3-pX as a control strain.

[0091] (2) Shake flask fermentation of the production strain

[0092] Perform shake flask fermentation on strain Cgh3-low-cgl2988 or Cgh3-pX-cgl2988.

[0093] First, streak Cgh3-low-cgl2988 and Cgh3-pX-cgl2988 on BHIS solid medium and place them in an incubator at 30 °C for about 18 h. Pick single colonies on the plate and inoculate them into 5 mL of BHIS liquid medium. Culture at 30 °C and 220 rpm for about 12 h, then take 1 mL and transfer it to CGIII medium for continued culture for 12 h. Use CGIII medium with an initial OD 600 of about 0.5 and a final glucose concentration of 30 g / L. Place it in a constant temperature shaker at 30 °C and 220 rpm and shake culture for 4 h until the OD 600 is about 5.0. Then add 7.5 g / L of the precursor glycine. Measure its 5-ALA production during the culture period (see Figure 4 ). After culturing for 72 h, perform shake flask fermentation on the strain Cgh3-low-cgl2988 with the expression of the transcription factor gene cgl2988 in Corynebacterium glutamicum down-regulated. The 5-ALA production is 5.52 ± 0.19 g / L, which is about 1.64 times higher than that of the control strain Cgh3-pXi; perform shake flask fermentation on the strain Cgh3-pX-cgl2988 with the expression of the transcription factor gene cgl2988 in Corynebacterium glutamicum up-regulated. The 5-ALA production is 5.76 ± 0.11 g / L, which is about 2.84 times higher than that of the control strain Cgh3-pX.

[0094] References

[0095] 1. Szeimies RM, Methyl aminolevulinate-photodynamic therapy for basal cell carcinoma. Dermatol Clin, 2007, 25: 89-94.

[0096] 2. Inoue K, 5-Aminolevulinic acid-mediated photodynamic therapy for bladder cancer. Int J Urol, 2017, 24: 97-101.

[0097] 3. Shih IT, Yi YC, and Ng IS. Plasmid-free system and modular design for efficient 5-aminolevulinic acid production by engineered Escherichia coli. Appl Biochem Biotechnol, 2021, 193:2858-2871.

[0098] 4. Kim M, Sun G, Lee DY, et al. BeReTa: a systematic method for identifying target transcriptional regulators to enhance microbial production of chemicals. Bioinformatics, 2017, 33:87-94.

[0099] 5. Shen F, Sun R, Yao J, et al. OptRAM: In-silico strain design via integrative regulatory-metabolic network modeling. PLoS Comput Biol, 2019, 15: e1006835.

[0100] 6. Liu J., Liu M., Shi T., Sun G., Gao N., Zhao X., Guo X., Ni X., Yuan Q., Feng J., Liu Z., Guo Y., Chen J., Wang Y., Zheng P., Sun J. CRISPR-assisted rational flux-tuning and arrayed CRISPRi screening of an L-proline exporter for L-proline hyperproduction. Nat Commun, 2022, 13:891.

[0101] 7.Zhao J,Sun X,Mao Z,et al,Independent component analysis of Corynebacterium glutamicum transcriptomes reveals its transcriptional regulatory network.Microbiol Res,2023,276:127485.

Claims

1. The 5-aminolevulinic acid production-related transcription factor Cgl2988 predicted based on the network model is characterized by The amino acid sequence of the transcription factor Cgl2988 is shown in SEQ ID NO.

1.

2. A gene encoding the transcription factor Cgl2988 according to claim 1, wherein the nucleotide sequence of the gene is shown in SEQ ID NO.

2.

3. pCRISPRi-low-cgl2988 plasmid or pX-cgl2988 plasmid comprising the gene of claim 2; The pCRISPRi-low-cgl2988 plasmid was constructed in the following manner: Using pdCas9gRNA as a template and l-cgl2988-F1 and cas-R1 as upstream and downstream primers, PCR amplification was performed to obtain linearized vector fragment 1; Using pdCas9gRNA as a template and ori-F and CRISPRi-R2 as upstream and downstream primers, PCR amplification was performed to obtain linearized vector fragment 2; The fragment 1 and the fragment 2 were connected and verified by colony PCR to obtain the pCRISPRi-low-cgl2988 plasmid; The nucleotide sequence of l-cgl2988-F1 is shown in SEQ ID NO.3; The nucleotide sequence of cas-R1 is shown in SEQ ID NO.4; The nucleotide sequence of ori-F is shown in SEQ ID NO.5; The nucleotide sequence of the CRISPRi-R2 is shown in SEQ ID NO.6; The pX-cgl2988 plasmid was constructed in the following manner: Using pXMJ19 as a template and pX-cgl2988-F and pX-cgl2988-R as upstream and downstream primers, PCR amplification was performed to obtain linearized vector fragment 3; Using the genome of Corynebacterium glutamicum ATCC 13032 as a template and cgl2988-F and cgl2988-R as upstream and downstream primers, PCR amplification was performed to obtain a linearized vector fragment 4; The fragment 3 and the fragment 4 were connected and verified by colony PCR to obtain the pX-cgl2988 plasmid; The nucleotide sequence of pX-cgl2988-F is shown in SEQ ID NO.7; The nucleotide sequence of pX-cgl2988-R is shown in SEQ ID NO.8; The nucleotide sequence of cgl2988-F is shown in SEQ ID NO.9; The nucleotide sequence of cgl2988-R is shown in SEQ ID NO.

10.

4. A recombinant bacterium comprising the pCRISPRi-low-cgl2988 plasmid or a recombinant bacterium comprising the pX-cgl2988 plasmid, characterized in that The recombinant bacteria containing the pCRISPRi-low-cgl2988 plasmid were constructed in the following manner: The pCRISPRi-low-cgl2988 plasmid was integrated into the recombinant strain Cgh3 of Corynebacterium glutamicum by electroporation, and verified by colony PCR to obtain the expression strain Cgh3-low-cgl2988 that down-regulated the transcription factor gene cgl2988 in Corynebacterium glutamicum; The recombinant bacteria containing the pX-cgl2988 plasmid were constructed in the following manner: The pX-cgl2988 plasmid was integrated into the recombinant strain Cgh3 of Corynebacterium glutamicum by electroporation and verified by colony PCR to obtain the expression strain Cgh3-pX-cgl2988 which up-regulated the transcription factor gene cgl2988 in Corynebacterium glutamicum.

5. Use of the recombinant bacteria according to claim 4 in fermentation production of 5-aminolevulinic acid.

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