Screening method for neutral site in corynebacterium glutamicum and application
By screening and identifying neutral sites in Corynebacterium glutamicum ATCC 13032, the problem of limited integration site selection in gene editing was solved, achieving efficient and stable gene expression and metabolic network optimization, and improving the synthesis efficiency of target products.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- JIANGNAN UNIV
- Filing Date
- 2026-04-15
- Publication Date
- 2026-07-24
AI Technical Summary
Existing gene editing technologies for Corynebacterium glutamicum suffer from problems such as low editing efficiency, significant impact on the physiological functions of the host bacteria, and limited selection of integration sites. In particular, it is difficult to achieve efficient and stable genome integration in high GC environments.
Neutral sites in the genome of Corynebacterium glutamicum ATCC 13032 were screened and identified. Nucleic acid cassettes containing 5' and 3' homologous arm sequences were designed to integrate exogenous genes, ensuring that the gene editing process did not affect the normal physiological function of the host bacterium. An expression vector was constructed through PCR amplification and homologous recombination to achieve efficient and stable gene expression.
It provides efficient and widely applicable genome integration sites, ensuring that the stable expression of exogenous genes does not affect the normal physiological metabolism of the host bacteria, improving the synthesis efficiency of target products, and expanding the application potential of metabolic network optimization.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, and in particular to a method and application for screening neutral sites in Corynebacterium glutamicum. Background Technology
[0002] Corynebacterium glutamicum model strain C. glutamicum ATCC 13032 is a Gram-positive, facultative aerobic, non-spore-forming, non-flagellated high-GC actinomycete, named for its ability to secrete excessive amounts of glutamate in a hyperosmolar environment. With its thick cell wall, low permeability barrier, and "GRAS" safety certification and mature genetic system, it has become the industrial microbial chassis, widely used in the large-scale fermentation production of amino acids, organic acids, bio-based monomers, and fuels.
[0003] The existing gene editing technologies of Corynebacterium glutamicum have formed a three-tiered system of "traditional recombination - CRISPR / Cas - base editing": (1) Single and double crossover homologous recombination mediated by suicide plasmids or temperature-sensitive plasmids is still the most basic "knockout / insertion" method. The technical threshold is low, but the editing efficiency is only 0.1-1%, and there are inherent defects such as sacB reverse screening inactivation, high false positives, and difficulty in integrating large fragments; (2) Cre / loxP or Flp / FRT site-specific recombination systems can achieve scarless deletion with an efficiency of 30-50%, but require pre-setting recognition sites in the genome, which is cumbersome; (3) CRISPR / Cas9 and CRISPR-Cpf1 (Cas12a) systems can improve the precise knockout / insertion efficiency to 70-95% by introducing programmable nucleases and single-stranded or double-stranded donor DNA, and can delete 50 kb or insert 7.5 kb fragments at one time. However, Cas9 has obvious toxicity to high GC Corynebacterium glutamicum, while Cas12a is limited by TTTN. PAM, and the efficiency drops sharply when editing multiple sites simultaneously due to cytotoxicity or the lengthy structure of sgRNA; (4) The latest cytosine or adenine base editor (BE / AE) combined with tRNA-processing multiple sgRNA arrays can achieve more than 90% single base precise replacement and simultaneous editing of three genes without DSB and donor DNA, but the efficiency of more than three targets is still less than 35%, and multiple sgRNAs have bottlenecks such as repetitive sequence interference and cumbersome target replacement.
[0004] Due to the Corynebacterium glutamicum model strain C. glutamicum ATCC 13032 is an important strain for producing various bioproducts; therefore, when modifying it using genetic engineering techniques to increase the yield of target products, screening suitable gene integration sites is crucial. Traditional integration sites often face limitations such as affecting the physiological function of the host bacterium, unstable expression, or insufficient regulatory precision. Neutral integration sites, due to their characteristic of not significantly interfering with host growth and metabolism after integrating foreign genes, have become an effective approach to solving these problems. Currently... C. glutamicum The number of validated neutral sites in ATCC 13032 is limited. Therefore, systematically screening for neutral sites with high stability and high editing efficiency, and expanding their application in gene editing, is of great value to the scientific research and industrial transformation of this strain. Summary of the Invention
[0005] This invention aims to solve a key problem in the field of gene editing: how to provide efficient and widely applicable genome integration sites that can ensure the stable expression of foreign genes without interfering with the normal physiological and metabolic activities of the host bacteria.
[0006] The primary objective of this invention is to screen and identify Corynebacterium glutamicum. C. glutamicum The neutral site in the ATCC 13032 genome provides a reliable integration site for the genetic modification of this strain, ensuring that the gene editing process does not affect the normal physiological functions of the host, while maintaining the efficient expression of the exogenous gene.
[0007] A further objective of this invention is to obtain a universally applicable neutral site and to investigate its potential applications in metabolic engineering, including optimizing the metabolic network of the host bacterium and improving the synthesis efficiency of the target product.
[0008] This invention provides a nucleic acid cassette integrated into the genome of Corynebacterium glutamicum, comprising: a 5' homologous arm sequence and a 3' homologous arm sequence at a neutral site, and a target protein gene, wherein the target protein gene is located in the region between the 5' homologous arm sequence and the 3' homologous arm sequence; the 5' homologous arm sequence and the 3' homologous arm sequence at the neutral site are obtained by PCR amplification using the Corynebacterium glutamicum ATCC 13032 genome as a template and primers of the 5' homologous arm sequence and the 3' homologous arm sequence; The nucleotide sequences of the neutral sites are shown in any one of SEQ ID NO.1 to SEQ ID NO.5; The primer sequences for the 5' homologous arm sequence and 3' homologous arm sequence of the neutral site of the nucleotide sequence as described in SEQ ID NO. 1 are shown in SEQ ID NO. 6 to SEQ ID NO. 9, respectively. The primer sequences for the 5' homologous arm sequence and 3' homologous arm sequence of the neutral site of the nucleotide sequence as described in SEQ ID NO. 2 are shown in SEQ ID NO. 10 to SEQ ID NO. 13, respectively. The primer sequences for the 5' homologous arm sequence and the 3' homologous arm sequence of the neutral site of the nucleotide sequence, such as SEQ ID NO. 3, are shown in SEQ ID NO. 14 to SEQ ID NO. 17, respectively. The primer sequences for the 5' homologous arm sequence and 3' homologous arm sequence of the neutral site of the nucleotide sequence, such as SEQ ID NO. 4, are shown in SEQ ID NO. 18 to SEQ ID NO. 21, respectively. The primer sequences for the 5' homologous arm sequence and the 3' homologous arm sequence of the neutral site of the nucleotide sequence as described in SEQ ID NO. 5 are shown in SEQ ID NO. 22 to SEQ ID NO. 25, respectively. In one embodiment of the present invention, the Corynebacterium glutamicum is Corynebacterium glutamicum ATCC 13032.
[0009] The present invention provides an expression vector for expressing a target protein in Corynebacterium glutamicum, the expression vector containing the nucleic acid cassette as described in claim 1.
[0010] In one embodiment of the present invention, the Corynebacterium glutamicum is Corynebacterium glutamicum ATCC 13032.
[0011] In one embodiment of the present invention, the expression vector further includes a promoter sequence located upstream of the coding gene of the protein, the promoter controlling the expression of the protein.
[0012] In one embodiment of the present invention, the promoter includes, but is not limited to, P. tuf P gapA P trc Promoter.
[0013] The present invention also provides a method for improving the gene editing efficiency of Corynebacterium glutamicum, wherein the method involves integrating the coding gene of the target protein or polypeptide into a neutral site; the nucleotide sequence of the neutral site is shown in any one of SEQ ID NO.1 to SEQ ID NO.5.
[0014] In one embodiment of the present invention, the Corynebacterium glutamicum is Corynebacterium glutamicum ATCC 13032.
[0015] The present invention also provides a genetically engineered bacterium, which is obtained by introducing the above expression vector into Corynebacterium glutamicum ATCC 13032 as a chassis cell.
[0016] The present invention also provides an application of a neutral site in the expression of a target protein or polypeptide in Corynebacterium glutamicum, wherein the application involves integrating the coding gene of the target protein or polypeptide into the neutral site; the nucleotide sequence of the neutral site is shown in any one of SEQ ID NO.1 to SEQ ID NO.5.
[0017] In one embodiment of the present invention, the Corynebacterium glutamicum is Corynebacterium glutamicum ATCC 13032.
[0018] The present invention also provides a recombinant Corynebacterium glutamicum that expresses a target protein at a specific site. The recombinant Corynebacterium glutamicum is obtained by transferring the above expression vector, the sgRNA plasmid corresponding to the target protein sequence, and the Cas9 plasmid into Corynebacterium glutamicum.
[0019] In one embodiment of the present invention, the Corynebacterium glutamicum is Corynebacterium glutamicum ATCC 13032.
[0020] The present invention also provides a method for expressing a target protein or polypeptide in Corynebacterium glutamicum ATCC 13032, wherein the method comprises integrating the target protein into a neutral site on the genome of Corynebacterium glutamicum ATCC 13032; The nucleotide sequences of the neutral sites are shown in any one of SEQ ID NO.1 to SEQ ID NO.5.
[0021] The present invention also provides the application of the above-mentioned nucleotide sequences as any of the presumed proteins shown in SEQ ID NO.1 to SEQ ID NO.5 as neutral sites.
[0022] This invention also provides a method for constructing recombinant Corynebacterium glutamicum expressing a target protein, the method comprising the following steps: (1) Construct neutral site knockout plasmids pk18-ΔRS13740, pk18-ΔRS13750, pk18-ΔRS00195, pk18-ΔRS00280 or pk18-ΔRS00320: Using the ATCC 13032 genome as a template, design upstream and downstream homologous arm primers for knockout. The primer sequences are shown in SEQ ID NO.6~SEQ ID NO.25. Amplify the upstream and downstream recombinant fragments of the neutral site knockout fragment; Primers were designed based on the sequence information of plasmid pK18mobsacB. Using plasmid pK18mobsacB as a template, linearized vector fragments were obtained by reverse PCR amplification. After the above fragments were recovered, they were recombined and ligated to prepare pk18-ΔRS13740, pk18-ΔRS13750, pk18-ΔRS00195, pk18-ΔRS00280 or pk18-ΔRS00320 respectively. (2) Using plasmids pk18-ΔRS13740, pk18-ΔRS13750, pk18-ΔRS00195, pk18-ΔRS00280 or pk18-ΔRS00320 as templates, primers for the target gene were designed, and linearized vector fragments were obtained by reverse PCR amplification. Plasmids for overexpressing the target gene were constructed by homologous recombination. (3) The plasmid obtained in step (2) is introduced into Corynebacterium glutamicum ATCC 13032 or modified Corynebacterium glutamicum ATCC 13032 to construct a strain in which the target gene is inserted at a neutral site. The nucleotide sequences of the neutral sites are shown in any one of SEQ ID NO.1 to SEQ ID NO.5.
[0023] Beneficial effects The neutral site of Corynebacterium glutamicum identified in this invention has the following important characteristics: when performing gene editing operations (such as gene knockout or knock-in), this site can ensure that the normal physiological metabolism of the strain is not affected, and can maintain the efficient and stable expression of exogenous genes; at the same time, it exhibits excellent editing efficiency, providing an ideal target site with good universality and high reliability for the genetic modification of Corynebacterium glutamicum.
[0024] In practical applications, this neutral site can serve as an ideal integration site for key genes in the threonine biosynthesis pathway. Experimental data show that integrating multiple key genes in the threonine biosynthesis pathway into this neutral site significantly increases threonine production. Furthermore, the neutral site screening strategy can effectively identify genes with unknown functions in the genome, elucidating their potential regulatory mechanisms in bacterial growth and threonine synthesis, thus providing new target selections for subsequent metabolic network optimization and strain modification. Detailed Implementation
[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.
[0026] The strains involved in the following examples: Corynebacterium glutamicum C. glutamicum ATCC 13032, purchased from the American Type Culture Collection (ATCC), strain number 13032.
[0027] The culture media involved in the following examples: LBGB liquid medium for culturing Corynebacterium glutamicum: 10 g / L NaCl, 5 g / L yeast extract, 10 g / L peptone, 5 g / L glucose, 18.5 g / L brain and heart extract; brain and heart broth 38.5 g / L; LBGB solid medium for culturing Corynebacterium glutamicum: 10 g / L NaCl, 5 g / L yeast extract, 10 g / L peptone, 5 g / L glucose, 18.5 g / L brain and heart extract; brain and heart broth 38.5 g / L; 10 g / L agar powder; LBHIS recovery medium for Corynebacterium glutamicum after electroporation: 5 g / L NaCl, 2.5 g / L yeast extract, 5 g / L peptone, 18.5 g / L brain heart extract, 91 g / L D-sorbitol. Epo medium for competent cells of Corynebacterium glutamicum: 10 g / L NaCl, 5 g / L yeast extract, 10 g / L peptone, 1 g / L Tween 80, 25 g / L glycine, 0.4 g / L isoniazid; Slant culture medium: urea 3 g / L, K2HPO4·3H2O 1 g / L, MgSO4·7H2O 0.1 g / L, yeast extract (OXOID) 5 g / L, soybean peptone (Solepro) 9 g / L, biotin 10 μg / L, vitamin B1 0.1 mg / L, agar powder 15 g / L, glucose 10 g / L, succinic acid 0.5 g / L, MOPS 20 g / L, pH adjusted to 7.20 with NaOH.
[0028] Seed culture medium: urea 3 g / L, K2HPO4·3H2O 1 g / L, MgSO4·7H2O 0.1 g / L, yeast extract powder 5 g / L, soybean peptone 9 g / L, biotin 10 μg / L, vitamin B1 0.1 mg / L, agar powder 15 g / L, glucose 10 g / L, MOPS 20 g / L, pH adjusted to 7.20 with NaOH.
[0029] Fermentation medium: (NH4)2SO4 15 g / L, KH2PO4 6 g / L, MgSO4·7H2O 0.5 g / L, yeast extract powder 3 g / L, FeSO4·7H2O 0.1 g / L, MnSO4·H2O 0.05 g / L, biotin 1 mg / L, vitamin B1 1 mg / L, antifoaming agent 0.07 mL / L, glucose 50 g / L, soybean peptone 4 g / L, sodium chloride NaOH 4 g / L, pH adjusted to 7.0 with ammonia.
[0030] The detection methods involved in the following embodiments: Knockout site growth assay: The bacterial culture was appropriately diluted, and the OD was measured using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 600 nm. 600 value.
[0031] Knock-in site fluorescence assay: The bacterial culture was appropriately diluted, and the red fluorescence value was measured using an enzyme-linked immunosorbent assay (ELISA) reader at wavelengths of 588 nm and 645 nm.
[0032] Determination of threonine products: Fermentation broth pretreatment: After centrifuging the fermentation broth at 12000 rpm for 10 min, take the supernatant and filter it through a 0.22 μm filter membrane.
[0033] High-performance liquid chromatography (HPLC) was used to determine the L-threonine content in the fermentation broth: Mobile phase A was 0.01 mol / L KH₂PO₄ solution, adjusted to pH 5.3 with KOH; Mobile phase B was a mixture of acetonitrile, methanol, and mobile phase A in a volume ratio of 5:3:1, adjusted to pH 5.3 with acetic acid. An Aglient ZORBAX SB-Aq column (250 × 4.6 mm, 5 µm) was used. Pre-column online derivatization was employed with OPA as the derivatizing agent. Gradient elution was performed at a column temperature of 35 ℃ and a flow rate of 1.0 mL / min. The detection wavelength was UV 254 nm; FLD: excitation wavelength 330 nm / emission wavelength 465 nm.
[0034] The editing efficiency detection and calculation methods involved in the construction of neutral site knockout strains in the following examples are as follows: .
[0035] Example 1: Screening for neutral sites that enable gene editing This invention analyzes the published Corynebacterium glutamicum. C. glutamicum Five neutral protein gene loci were identified in the ATCC 13032 genome: CGL_RS13740, CGL_RS13750, CGL_RS00195, CGL_RS00280, and CGL_RS00320. The sequences described in this example are shown in Table 1. Table 1: Neutral site sequences
[0036] Example 2: Evaluation of the efficiency of genome integration at neutral sites The specific steps are as follows: (1) Construction of knockout plasmid According to the neutral site in Table 1 of Example 1, taking the neutral site CGL_RS13740 as an example: (1) Select the genome of Corynebacterium glutamicum model strain ATCC 13032 as a template, and design upstream and downstream homologous arm primers 13740 for this neutral site. Up-F / 13740-Up-R and 13740 Down-F / 13740-Down-R, and amplify the upstream and downstream homologous arm fragments of the neutral site by PCR; (2) Design primers pK18 based on the sequence information of plasmid pK18mobsacB. 13740-F / pK18 Using plasmid pK18mobsacB as a template, a linearized vector fragment was obtained by reverse PCR amplification of 13740-R. The three fragments were recovered and ligated via homologous recombination. The transformants obtained after chemical transformation were then amplified, and plasmid extraction yielded the putative neutral site knockout plasmid pk18-ΔRS13740. Primers used in this example are shown in Table 2.
[0037] Table 2: Primers for constructing neutral site knockout plasmids
[0038] Using the above methods and primers, neutral site knockout plasmids were prepared as follows: pk18-ΔRS13740, pk18-ΔRS13750, pk18-ΔRS00195, pk18-ΔRS00280 and pk18-ΔRS00320.
[0039] (2) Prepared using methods reported in the literature C. glutamicum ATCC 13032 competent cells (Biotechnology Letters, 2015, 37: 2445) 52.) The 13032 competent cells obtained above were electroporated with 10 μg of plasmids pk18-ΔRS13740, pk18-ΔRS13750, pk18-ΔRS00195, pk18-ΔRS00280, or pk18-ΔRS00320, respectively. 1 mL of preheated LBHIS medium (46 ℃) was added, and the cells were incubated at 46 ℃ for 6 min, then at 30 ℃ for 6 h. The transformed cells were then plated onto LBGB solid medium containing 25 μg / mL kanamycin and cultured at 30 ℃ for 2 days to obtain transformants for the first homologous recombination. Correct transformants were transferred to LBGB medium and cultured at 30 ℃ for 2–6 h, followed by screening on LBGB medium supplemented with 150 g / L sucrose.
[0040] Taking the neutral site CGL_RS13740 as an example, the transformants selected by sucrose were sequenced and verified using the corresponding primers in Table 3, resulting in the neutral site knockout strain Cgl13032-Δ13740. The primers used in this example are shown in Table 3.
[0041] Table 3: Primers required for sequencing validation
[0042] The knockout strains were prepared according to the above method and the primers in Table 3: Strains with the CGL_RS13740 gene fragment knocked out C. glutamicum ATCC 13032ΔCGL_RS13740, named Cgl13032-Δ13740; Strains with the CGL_RS13750 gene fragment knocked out C. glutamicum ATCC 13032ΔCGL_RS13750, named Cgl13032-Δ13750; Strains with the CGL_RS00195 gene segment knocked out C. glutamicum ATCC 13032ΔCGL_RS00195, named Cgl13032-Δ00195; Strains with the CGL_RS00280 gene fragment knocked out C. glutamicum ATCC 13032ΔCGL_RS00280, named Cgl13032-Δ00280; Strains with the CGL_RS00320 gene fragment knocked out C. glutamicum ATCC 13032ΔCGL_RS00320, named Cgl13032-Δ00320.
[0043] The editing efficiency during the construction of neutral site knockout strains is shown in Table 4. Among them, strains Cgl13032-Δ13740, Cgl13032-Δ13750, Cgl13032-Δ00195, and Cgl13032-Δ00280 had higher editing success rates during construction, indicating that the CGL_RS13740, CGL_RS13750, CGL_RS00195, and CGL_RS00280 sites have good editability.
[0044] Table 4: Success rate of constructing neutral site knockout strains (editing efficiency)
[0045] (3) To identify the hypothetical neutral sites, the constructed neutral site knockout strains Cgl13032-Δ13740, Cgl13032-Δ13750, Cgl13032-Δ00195, Cgl13032-Δ00280 and Cgl13032-Δ00320 were evaluated for growth, while wild-type strains were used as a comparison. C. glutamicum ATCC 13032 (hereinafter referred to as strain Cgl13032) was used as a control. First, the control strain and the knockout strains Cgl13032-Δ13740, Cgl13032-Δ13750, Cgl13032-Δ00195, Cgl13032-Δ00280, and Cgl13032-Δ00320 were inoculated into LBGB growth medium and cultured at 30°C for 12 hours. The resulting culture was used as a seed culture and inoculated into 96-well plates containing 800 μL of LBGB medium per well. Initial OD... 600 The control value was approximately 0.2. The plates were incubated at 30°C for 24 hours, with OD measured every 4 hours. 600 The growth results are shown in Table 5.
[0046] Table 5: Growth of neutral site knockout strains at different time points (OD) 600 )
[0047] The results show: Among them, the growth of strains Cgl13032-Δ13740, Cgl13032-Δ13750, Cgl13032-Δ00195, Cgl13032-Δ00280, and Cgl13032-Δ00320 was not significantly different from that of the wild-type strain Cgl13032, indicating that the knockout of these neutral gene sites had no effect on the growth of the strains. That is, these five sites are all usable neutral sites. Among them, the editing efficiency of CGL_RS13740, CGL_RS13750, CGL_RS00195, and CGL_RS00280 sites is higher, and therefore they are more suitable as neutral sites for gene knockout.
[0048] Example 3: Testing and application of neutral site knock-in strains To verify the availability of neutral sites, three currently disclosed strong promoters were selected for evaluation: P tuf P gapA P trc ,use mKate Red fluorescent protein is used as a reporter gene to characterize the effect of the knock-in gene on the neutral site. The pECXK99E-P involved... tuf -mKate、pECXK99E-P gapA-mKate and pECXK99E-P trc The method for constructing -mKate is as follows: Chemically synthesized P tuf promoter and mKate The red fluorescent protein gene fragment was ligated and cloned into the pECXK99E vector to construct the recombinant vector pECXK99E-P. tuf - mKate ; chemically synthesized P gapA promoter and mKate The red fluorescent protein gene fragment was ligated and cloned into the pECXK99E vector to construct the recombinant vector pECXK99E-P. gapA - mKate ; chemically synthesized P trc promoter and mKate The red fluorescent protein gene fragment was ligated and cloned into the pECXK99E vector to construct the recombinant vector pECXK99E-P. trc - mKate .
[0049] The specific steps are as follows: (1) Construction of strains P tuf P gapA P trc and mKate The nucleotide sequence of red fluorescent protein is shown in Table 6. The neutral site CGL_RS13740 was knocked out, and the strong promoter P was knocked in. tuf and mKate Taking red fluorescent protein as an example, using plasmid pk18-ΔRS13740 as a template (prepared in Example 2), primers pk18-13740-tuf-F / pk18-13740-tuf-R (or pk18-13740-gapA-F / pk18-13740-gapA-R, or pk18-13740-trc-F / pk18-13740-trc-R) were designed, and linearized pk18 vector fragments were obtained by reverse PCR amplification; using plasmid pECXK99E-P tuf - mKate (or pECXK99E-P) gapA - mKate or pECXK99E-P trc - mKate Using 13740-tuf-F / 13740-tuf-R as a template, primers were designed to amplify P. tuf Promoter fragment (design primers 13740-gapA-F / 13740-gapA-R to amplify P) gapAPromoter fragment, primers 13740-trc-F / 13740-trc-R were designed to amplify P trc (Promoter fragment), design primers 13740-tuf-mkate-F / 13740-tuf-mkate-R (or 13740-gapA-mkate-F / 13740-gapA-mkate-R, or 13740-trc-mkate-F / 13740-trc-mkate-R) for amplification. mKate Red fluorescent protein fragments, homologous recombination of the above three fragments, transformation of the transformed individuals obtained after chemical transformation, amplification, and plasmid extraction yield neutral site knock-in P. tuf and mKate The recombinant plasmid pk18-ΔRS13740::P for red fluorescent protein tuf - mKate Using the same operation, construct the input P. gapA and mKate P trc and mKate plasmid pk18-ΔRS13740::P gapA - mKate and pk18-ΔRS13740::P trc - mKate Sequencing verification was performed using cg13740-F / cg13740-R (cg13750-F / cg13750-R, cg00195-F / cg00195-R, cg00280-F / cg00280-R, cg00320-F / cg00320-R). After successful verification, the cells were electroporated into *Corynebacterium glutamicum* ATCC13032 competent cells. After two rounds of homologous recombination selection, the neutral site knock-in strain cgl13032-Δ13740::P was successfully performed. tuf - mKate cgl13032-Δ13740:: P gapA - mKate cgl13032-Δ13740:: P trc - mKate .
[0050] Knock-in strains were prepared separately: C.glutamicum ATCC 13032 / Δ13740:: P tuf - mKate The strain was named Cg13740-tuf- mKate ; C. glutamicum ATCC 13032 / Δ13740:: P gapA - mKate The strain was named Cg13740-gapA- mKate ; C. glutamicum ATCC 13032 / Δ13740:: P trc - mKate The strain was named Cg13740-trc- mKate ; C. glutamicum ATCC 13032 / Δ13750:: P tuf - mKate The strain was named Cg13750-tuf- mKate ; C. glutamicum ATCC 13032 / Δ13750:: P gapA - mKate The strain was named Cg13750-gapA- mKate ; C. glutamicum ATCC 13032 / Δ13750:: P trc - mKate The strain was named Cg13750-trc- mKate ; C. glutamicum ATCC 13032 / Δ00195:: P tuf - mKate The strain was named Cg00195-tuf- mKate ; C. glutamicum ATCC 13032 / Δ00195:: P gapA - mKate The strain was named Cg00195-gapA- mKate ; C. glutamicum ATCC 13032 / Δ00195:: P trc - mKate The strain was named Cg00195-trc- mKate ; C. glutamicum ATCC 13032 / Δ00280:: P tuf - mKate The strain was named Cg00280-tuf- mKate ; C. glutamicum ATCC 13032 / Δ00280:: P gapA - mKate The strain was named Cg00280-gapA- mKate ; C. glutamicum ATCC 13032 / Δ00280:: P trc - mKate The strain was named Cg00280-trc- mKate ; C. glutamicum ATCC 13032 / Δ00320:: P tuf - mKate The strain was named Cg00320-tuf- mKate ; C. glutamicum ATCC 13032 / Δ00320:: P gapA - mKate The strain was named Cg00320-gapA- mKate ; C. glutamicum ATCC 13032 / Δ00320:: P trc - mKate The strain was named Cg00320-trc- mKate .
[0051] The sequences described in this embodiment are shown in Table 6, and the primers used in this embodiment are shown in Table 7.
[0052] Table 6: Sequence Information
[0053] Table 7: Primers for constructing strains with neutral site knock-in
[0054] (2) The neutral site knock-in strain constructed in step (1) was subjected to fluorescence measurement, with the wild-type strain ATCC13032 as a control. First, the strain was inoculated into LBGB growth medium and cultured at 30°C for 12 hours. The prepared culture was then used as seed culture in a 96-well plate containing 800 μL of LBGB medium per well. Initial OD... 600 The control value was approximately 0.2. The plates were incubated at 30°C for 24 hours, and the final fluorescence value was measured. The unit fluorescence results are shown in Table 8.
[0055] Table 8: Fluorescence assay data of knock-in strains
[0056] The results show: The three different promoters showed the same fluorescence trend at sites CGL_RS13740, CGL_RS13750, CGL_RS00195, CGL_RS00280, and CGL_RS00320, with promoter intensities of P. tuf >P gapA >P trc Furthermore, the fluorescence data of all knock-in strains were higher than those of the control strain Cg13032, indicating that the five sites CGL_RS13740, CGL_RS13750, CGL_RS00195, CGL_RS00280, and CGL_RS00320 are stable, efficient gene integration sites that do not affect the normal physiological function of the host bacteria, and are relatively stable neutral sites in Corynebacterium glutamicum ATCC 13032.
[0057] Example 4: Application of neutral sites in the fermentation of threonine-producing strains L-Threonine is an essential amino acid, widely used in feed additives, food additives, and pharmaceutical intermediates. In Corynebacterium glutamicum, the synthesis of L-Threonine begins with aspartic acid, followed by aspartate kinase (…). lysC Encoding), aspartate-β-semialdehyde dehydrogenase ( asd Encoding), homoserine dehydrogenase ( man Encoding), homoserine kinase ( thrB Encoding) and threonine synthase ( thrC The process involves multiple enzymatic reactions, including coding. Previous studies have reported that knocking out… lysA (Encoding diaminopimelic acid decarboxylase) metX (Encoding homoserine O-acetyltransferase) and ilva (Encoding threonine dehydratase) can block or weaken the production of L-lysine, L-methionine, and L-isoleucine, focusing carbon flux on L-threonine synthesis and thus increasing L-threonine accumulation. Furthermore, overexpression of key genes... lysC , man and thrB It has been proven that it can further enhance the carbon flow in the L-threonine main pathway, and has a significant effect on increasing the yield of L-threonine.
[0058] The specific steps are as follows: 1. Construction of the production strain Cg-Thr (1) Using the genome of Corynebacterium glutamicum strain ATCC 13032 as a template, a knockout design was created. lysA upstream and downstream homologous arm primers lysA Up-F / lysA-Up-R and lysA Down-F / lysA-Down-R, and knocked out by PCR amplification. lysA The upstream and downstream homologous arm fragments; primers pK18 were designed based on the sequence information of plasmid pK18mobsacB. lysA-F / pK18 Using plasmid pK18mobsacB as a template, linearized vector fragments were obtained by reverse PCR amplification of lysA-R. The three fragments were then recovered and homologous recombination ligated. The transformants obtained after chemical transformation were then amplified, and the knockout plasmid pk18-Δ was extracted to obtain the knockout plasmid. lysA The correct knockout plasmid pk18 will be verified. Δ lysA Electroporation was performed into *Corynebacterium glutamicum* ATCC 13032 competent cells. The strain was obtained through two rounds of homologous recombination screening (first round screening for resistant clones, second round reverse selection on sucrose-containing plates). C.glutamicum ATCC 13032Δ lysA It was named Cg-Thr-1.
[0059] (2) Using the genome of Corynebacterium glutamicum strain ATCC 13032 as a template, a knockout design was created. metX upstream and downstream homologous arm primers metX Up-F / metX-Up-R and metX Down-F / metX-Down-R, and knocked out via PCR amplification. metX The upstream and downstream homologous arm fragments; primers pK18 were designed based on the sequence information of plasmid pK18mobsacB. metX-F / pK18 metX-R, using plasmid pK18mobsacB as a template, was used to obtain a linearized vector fragment via reverse PCR amplification. The three fragments were then recovered and ligated via homologous recombination. The transformants obtained after chemical transformation were then amplified, and the knockout plasmid pk18-Δ was extracted to obtain the knockout plasmid. metX The correct knockout plasmid pk18 will be verified. Δ metX Electroporation was performed into *Corynebacterium glutamicum* Cg-Thr-1 competent cells. The strain was obtained through two rounds of homologous recombination screening (first round screening for resistant clones, second round reverse selection on sucrose-containing plates). C.glutamicum ATCC 13032Δ lysA Δ metX It was named Cg-Thr-2.
[0060] (3) Using the genome of Corynebacterium glutamicum model strain ATCC 13032 as a template, a knockout design was created. ilva upstream and downstream homologous arm primers ilvA Up-F / ilvA-Up-R and ilvA Down-F / ilvA-Down-R, and knocked out by PCR amplification. ilva The upstream and downstream homologous arm fragments; primers pK18 were designed based on the sequence information of plasmid pK18mobsacB. ilvA-F / pK18 Using plasmid pK18mobsacB as a template, linearized vector fragments were obtained by reverse PCR amplification of ilvA-R. The three fragments were then recovered and homologous recombination ligated. The transformants obtained after chemical transformation were then amplified, and the knockout plasmid pk18-Δ was extracted to obtain the knockout plasmid. ilva The correct knockout plasmid pk18 will be verified. Δ ilva Electroporation was performed into *Corynebacterium glutamicum* Cg-Thr-2 competent cells. The strain was obtained through two rounds of homologous recombination screening (first round screening for resistant clones, second round reverse selection on sucrose-containing plates). C. glutamicum ATCC 13032Δ lysA Δ metX Δ ilva It was named Cg-Thr-3.
[0061] (4) Based on the neutral sites in Table 1 of Example 1, taking CGL_RS13740 as an example, using the pK18-ΔRS13740 plasmid constructed in Example 2 as a template, primers RpK18-13740-F / RpK18-13740-R were designed, and the pK18-ΔRS13740 plasmid was linearized by reverse PCR; using the genome of Corynebacterium glutamicum model strain ATCC 13032 as a template, primers 13740-lysC-F / 13740-lysC-R were designed, P tac Promoter design in pK18 reverse amplification downstream primers and lysC The upstream primer was obtained by PCR. lysC Gene fragment, primers designed: 13740-hom-F / 13740-hom-R, P tac Promoter design in lysC Downstream primers and man The upstream primer was obtained by PCR. man Gene fragment, primers designed 13740-thrB-F / 13740-thrB-R, P tac Promoter design in man Downstream primers and thrB The upstream primer was obtained by PCR. thrBGene fragments. The above four fragments were recovered and homologous recombination ligated. The transformants obtained after chemical transformation were then amplified, and the plasmid was extracted to obtain the recombinant plasmid pk18-ΔRS13740::P. tac - lysC - P tac - man - P tac - thrB The recombinant plasmid pk18-ΔRS13740::P was verified to be correct. tac - lysC - P tac - man - P tac - thrB Electroporation was performed into *Corynebacterium glutamicum* Cg-Thr-3 competent cells. The strain was obtained through two rounds of homologous recombination screening (first round screening for resistant clones, second round reverse selection on sucrose-containing plates). C. glutamicum ATCC 13032Δ lysA Δ metX Δ ilva ΔRS13740:: P tac - lysC - P tac - man - P tac - thrB It was named Cg-Thr-4.1.
[0062] Following the same procedure, the following strains were constructed: inserted into the neutral site CGL_RS13750. lysC - man - thrB Gene-constructed strains C.glutamicum ATCC 13032Δ lysA Δ metX Δ ilva ΔRS13750::P tac - lysC - P tac - man - P tac - thrB It was named Cg-Thr-4.2; Insert at neutral site CGL_RS00195 lysC - man - thrB Gene-constructed strains C.glutamicum ATCC13032Δ lysA Δ metX Δ ilva ΔRS00195:: P tac - lysC - Ptac - man - P tac - thrB It was named Cg-Thr-4.3; Insert at neutral site CGL_RS00285 lysC - man - thrB Gene-constructed strains C.glutamicum ATCC13032Δ lysA Δ metX Δ ilva ΔRS00285:: P tac - lysC - P tac - man - P tac - thrB It was named Cg-Thr-4.4; Insert at neutral site CGL_RS00320 lysC - man - thrB Gene-constructed strains C. glutamicum ATCC13032Δ lysA Δ metX Δ ilvA ΔRS00320:: P tac - lysC - P tac - hom - P tac - thrB It was named Cg-Thr-4.5.
[0063] Additionally, following the above procedure, insert the following into the reported neutral site CGL_RS03085 (in the patent application document with publication number CN121271966A). lysC - hom - thrB Gene-constructed strains C.glutamicum ATCC 13032Δ lysA Δ metX Δ ilvA ΔRS03085:: P tac - lysC - P tac - hom - P tac - thrB It was named Cg-Thr-4.0.
[0064] The sequences described in this embodiment are shown in Table 9, and the primers used in this embodiment are shown in Table 10.
[0065] Table 9: Sequence Information
[0066] Table 10: Primers for constructing threonine-producing strains
[0067] 2. Fermentation validation of the production strain Cg-Thr The Cg-Thr-3, Cg-Thr-4.0, Cg-Thr-4.1, Cg-Thr-4.2, Cg-Thr-4.3, Cg-Thr-4.4, and Cg-Thr-4.5 production strains obtained in step 1 were inoculated into test tubes containing 5 mL of LBGB medium and cultured at 30℃ and 220 rpm for 12–18 h. Then, 0.1–0.2 mL of the activated bacterial culture was transferred to a 500 mL Erlenmeyer flask containing 30 mL of seed culture medium and cultured at 30℃ and 220 rpm for 8–10 h until the OD of the seed culture was reached. 600 The value reached 30±3. Then, 3 mL of seed culture was inoculated into a 500 mL Erlenmeyer flask containing 30 mL of Cg-Thr fermentation medium. Each strain required 3 replicates, and fermentation was carried out at 30℃ and 220 rpm for 48 h. At 0 h, 10 h, 12 h, 14 h, 17 h, and 20 h of fermentation, 0.4 mL, 0.24 mL, 0.24 mL, 0.24 mL, and 0.24 mL of 300 g / L urea were added, respectively. The L-threonine yield was measured. The fermentation test results are shown in Table 11.
[0068] Table 11: Fermentation results of L-threonine
[0069] The results showed that overexpression occurred at different neutral sites. lysC , hom and thrB All of these methods can increase L-threonine accumulation, and the integration effects at neutral sites CGL_RS13740, CGL_RS13750, CGL_RS00195, CGL_RS00280, and CGL_RS00320 are superior to the previously reported neutral site CGL_RS03085. Among these, integration at neutral sites CGL_RS13740, CGL_RS13750, CGL_RS00195, and CGL_RS00280 is particularly effective. lysC - hom - thrBThe strains (Cg-Thr-4.1~4.4) with high gene yields indicate that these sites are more suitable for efficient expression and stable inheritance of exogenous genes, possessing excellent versatility and stability, and are applicable to the metabolic engineering of Corynebacterium glutamicum.
[0070] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.
Claims
1. A nucleic acid cassette integrated into the genome of Corynebacterium glutamicum, characterized in that, It comprises: the 5' homologous arm sequence and the 3' homologous arm sequence of the neutral site, and the target protein gene, wherein the target protein gene is located in the middle region of the 5' homologous arm sequence and the 3' homologous arm sequence; the 5' homologous arm sequence and the 3' homologous arm sequence of the neutral site are obtained by PCR amplification using the Corynebacterium glutamicum ATCC13032 genome as a template and primers of the 5' homologous arm sequence and the 3' homologous arm sequence; The nucleotide sequences of the neutral sites are shown in any one of SEQ ID NO.1 to SEQ ID NO.5; The primer sequences for the 5' homologous arm sequence and 3' homologous arm sequence of the neutral site of the nucleotide sequence as described in SEQ ID NO. 1 are shown in SEQ ID NO. 6 to SEQ ID NO. 9, respectively. The primer sequences for the 5' homologous arm sequence and 3' homologous arm sequence of the neutral site of the nucleotide sequence as described in SEQ ID NO. 2 are shown in SEQ ID NO. 10 to SEQ ID NO. 13, respectively. The primer sequences for the 5' homologous arm sequence and the 3' homologous arm sequence of the neutral site of the nucleotide sequence, such as SEQ ID NO. 3, are shown in SEQ ID NO. 14 to SEQ ID NO. 17, respectively. The primer sequences for the 5' homologous arm sequence and 3' homologous arm sequence of the neutral site of the nucleotide sequence, such as SEQ ID NO. 4, are shown in SEQ ID NO. 18 to SEQ ID NO. 21, respectively. The primer sequences for the 5' homologous arm sequence and the 3' homologous arm sequence of the neutral site of the nucleotide sequence as described in SEQ ID NO. 5 are shown in SEQ ID NO. 22 to SEQ ID NO. 25, respectively. Preferably, the Corynebacterium glutamicum is Corynebacterium glutamicum ATCC 13032.
2. An expression vector for expressing a target protein in Corynebacterium glutamicum, characterized in that, The expression vector contains the nucleic acid cassette according to claim 1; Preferably, the Corynebacterium glutamicum is Corynebacterium glutamicum ATCC 13032.
3. The expression vector according to claim 2, characterized in that, The expression vector also includes a promoter sequence located upstream of the coding gene of the protein, and the promoter controls the expression of the protein; Preferably, the promoter includes, but is not limited to, P tuf P gapA P trc Promoter.
4. A method for improving the gene editing efficiency of Corynebacterium glutamicum, characterized in that, The method involves integrating the gene encoding the target protein or polypeptide into a neutral site; the nucleotide sequence of the neutral site is shown in any one of SEQ ID NO.1 to SEQ ID NO.
5. Preferably, the Corynebacterium glutamicum is Corynebacterium glutamicum ATCC 13032.
5. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria are obtained by introducing the expression vector described in claim 2 or 3 into Corynebacterium glutamicum ATCC 13032 as the chassis cell.
6. The application of a neutral site in the expression of a target protein or polypeptide in Corynebacterium glutamicum, characterized in that, The application involves integrating the gene encoding the target protein or polypeptide into the neutral site; the nucleotide sequence of the neutral site is shown in any one of SEQ ID NO.1 to SEQ ID NO.5; Preferably, the Corynebacterium glutamicum is Corynebacterium glutamicum ATCC 13032.
7. A recombinant Corynebacterium glutamicum that expresses a target protein through site-specific integration, characterized in that, The recombinant Corynebacterium glutamicum is obtained by transferring the expression vector described in claim 2 or 3, the sgRNA plasmid corresponding to the target protein sequence, and the Cas9 plasmid into Corynebacterium glutamicum. Preferably, the Corynebacterium glutamicum is Corynebacterium glutamicum ATCC 13032.
8. A method for expressing a target protein or polypeptide in Corynebacterium glutamicum ATCC 13032, characterized in that, The method involves integrating the target protein into a neutral site on the genome of Corynebacterium glutamicum ATCC 13032; The nucleotide sequences of the neutral sites are shown in any one of SEQ ID NO.1 to SEQ ID NO.
5.
9. The use of a hypothetical protein with a nucleotide sequence as shown in any of SEQ ID NO.1 to SEQ ID NO.5 as a neutral site.
10. A method for constructing recombinant Corynebacterium glutamicum expressing a target protein, characterized in that, The method includes the following steps: (1) Construct neutral site knockout plasmids pk18-ΔRS13740, pk18-ΔRS13750, pk18-ΔRS00195, pk18-ΔRS00280 or pk18-ΔRS00320: Using the ATCC 13032 genome as a template, design upstream and downstream homologous arm primers for knockout. The primer sequences are shown in SEQ ID NO.6~SEQ ID NO.
25. Amplify the upstream and downstream recombinant fragments of the neutral site knockout fragment; Primers were designed based on the sequence information of plasmid pK18mobsacB. Using plasmid pK18mobsacB as a template, linearized vector fragments were obtained by reverse PCR amplification. After the above fragments were recovered, they were recombined and ligated to prepare pk18-ΔRS13740, pk18-ΔRS13750, pk18-ΔRS00195, pk18-ΔRS00280 or pk18-ΔRS00320 respectively. (2) Using plasmids pk18-ΔRS13740, pk18-ΔRS13750, pk18-ΔRS00195, pk18-ΔRS00280 or pk18-ΔRS00320 as templates, primers for the target gene were designed, and linearized vector fragments were obtained by reverse PCR amplification. Plasmids for overexpressing the target gene were constructed by homologous recombination. (3) The plasmid obtained in step (2) is introduced into Corynebacterium glutamicum ATCC 13032 or modified Corynebacterium glutamicum ATCC 13032 to construct a strain in which the target gene is inserted at a neutral site. The nucleotide sequences of the neutral sites are shown in any one of SEQ ID NO.1 to SEQ ID NO.5.