Recombinant bacteria and use thereof
By knocking out the mmgD gene in Bacillus sicca YB-1631 and blocking the 2-methylcitric acid cycle, a recombinant strain was constructed, which solved the problem of low γ-PGA production in glutamate-dependent strains and achieved a significant increase in γ-PGA production, demonstrating potential for industrial application.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- INST OF PLANT PROTECTION HENAN ACAD OF AGRI SCI
- Filing Date
- 2026-01-30
- Publication Date
- 2026-06-05
AI Technical Summary
The low yield of γ-PGA by existing glutamate-dependent strains limits the cost-effectiveness of industrial-scale γ-PGA production.
By knocking out the mmgD gene in Bacillus sicca YB-1631, blocking the 2-methylcitric acid cycle, and increasing the metabolic flux of the γ-PGA synthesis pathway, a recombinant strain was constructed using CRISPR-Cas9 gene editing technology.
It significantly improved the yield of γ-PGA. Under the same conditions, the yield of γ-PGA by recombinant strain increased by 25.62%, breaking through the bottleneck of yield improvement of existing strains. It is easy to operate and has good prospects for industrial application.
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Figure CN122146546A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of genetic engineering technology and relates to a recombinant bacterium and its applications. Background Technology
[0002] γ-polyglutamic acid (γ-PGA) is a water-soluble polyamino acid produced by microbial fermentation in nature. Its structure is a high-molecular-weight polymer formed by the condensation of glutamic acid units through amide bonds between α-amino and γ-carboxyl groups. It was initially discovered in the outer capsule of Bacillus anthracis and later found in natto, a traditional Japanese food. γ-PGA possesses excellent water solubility, strong adsorption capacity, and biodegradability. Its degradation product is harmless glutamic acid, making it an excellent environmentally friendly polymer material. It can be used as a water-retaining agent, heavy metal ion adsorbent, flocculant, slow-release agent, and drug carrier, and has significant commercial and social value in cosmetics, environmental protection, food, pharmaceuticals, agriculture, and desertification control industries.
[0003] Currently, the industrial production of γ-PGA mainly relies on microbial fermentation. Compared with natto extraction, chemical synthesis, and enzyme synthesis, this method has advantages such as low cost, simple process, and high product purity, and has become the mainstream production technology.
[0004] Industrially used γ-PGA production strains are mostly from the genus *Bacillus*, and can be divided into two categories based on whether they depend on the glutamate precursor: dependent and non-dependent. Non-glutamate-dependent strains can produce γ-PGA using glutamate-free media. Although these strains can reduce raw material costs, they are rarely used for large-scale production due to their low yield and limited potential for improvement. Glutamate-dependent strains, while requiring the addition of exogenous glutamate, have relatively higher yields and are currently the main choice for industrial production.
[0005] Therefore, improving the γ-PGA yield of glutamate-dependent strains through strain modification is key to reducing production costs and promoting industrial development. Summary of the Invention
[0006] The 2-methylcitric acid cycle is an important pathway for the metabolism of branched-chain amino acids and odd-carbon fatty acids in microorganisms, and the 2-methylcitric acid synthase encoded by the mmgD gene is a key enzyme in this cycle. Existing research indicates that the 2-methylcitric acid cycle may compete with the synthesis of microbial secondary metabolites for metabolic flux, but the regulatory role of this gene in γ-PGA synthesis has not been reported. Based on this, this invention knocks out the mmgD gene in Bacillus sicca using genetic engineering, unexpectedly discovering that it significantly increases γ-PGA production, providing a new technical solution for the efficient industrial production of γ-PGA.
[0007] To achieve this technical objective, the present invention adopts the following technical solution:
[0008] The present invention provides a recombinant bacterium, which is obtained by knocking out the mmgD gene of Bacillus sicca YB-1631, and the nucleotide sequence of the mmgD gene is shown in SEQ ID NO:1.
[0009] In this invention, Bacillus sicca YB-1631 refers to Bacillus sicca YB1631 with accession number CGMCC No.25784. For details, please refer to patent CN116286504A.
[0010] Preferably, the mmgD gene encodes 2-methylcitrate synthase.
[0011] Preferably, the knockout is achieved through homologous recombination or CRISPR-Cas9 gene editing.
[0012] Preferably, the Bacillus sicca YB-1631 can increase the production of γ-polyglutamic acid.
[0013] This invention provides the application of recombinant bacteria in increasing the yield of γ-polyglutamic acid, comprising the following steps: inoculating the recombinant bacteria into LB medium for seed culture, then inoculating the seed liquid into fermentation medium for fermentation culture, collecting the fermentation product, and separating and purifying to obtain γ-polyglutamic acid.
[0014] This invention provides a method for producing γ-polyglutamic acid by fermentation, comprising the following steps: inoculating recombinant bacteria into LB medium for seed culture, then inoculating the seed culture into fermentation medium for fermentation culture, collecting the fermentation product, and separating and purifying to obtain γ-polyglutamic acid.
[0015] Preferably, the fermentation medium includes monosodium glutamate, sodium citrate, MgSO4·7H2O, glycerol, KH2PO4, (NH4)2SO4, MnSO4, CaCl2, and FeCl3·6H2O.
[0016] Preferably, the separation and purification method includes: centrifuging the fermentation product to obtain the supernatant, adding anhydrous ethanol to precipitate, adding ultrapure water to redissolve the precipitate, and then freeze-drying.
[0017] The present invention provides a method for increasing the production of γ-polyglutamic acid, comprising the step of knocking out the mmgD gene encoding 2-methylcitrate synthase in Bacillus sicca YB-1631.
[0018] This invention provides a method for constructing recombinant bacteria, comprising the following steps: Using a plasmid containing the chloramphenicol resistance gene as a template, the chloramphenicol resistance fragment with the nucleotide sequence shown in SEQ ID NO:2 was amplified; Using the genome of Bacillus sicca YB-1631 as a template, the upstream and downstream homologous arms of the mmgD gene were amplified. The upstream homologous arm, chloramphenicol resistance fragment, and downstream homologous arm were seamlessly ligated to obtain a recombinant fragment with the nucleotide sequence shown in SEQ ID NO:3; The recombinant fragment was introduced into Bacillus sicca YB-1631 competent cells via electroporation transformation. The recombinant bacteria were obtained after chloramphenicol resistance screening and PCR verification.
[0019] SEQ ID NO:1 is shown below:
[0020] SEQ ID NO:2 is shown below:
[0021] SEQ ID NO:3 is shown below:
[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: The recombinant strain (YB-1631-ΔmmgD) provided by this invention significantly improved γ-PGA production under the same shake-flask fermentation conditions. Specific experimental data showed that the recombinant strain (YB-1631-ΔmmgD) achieved a γ-PGA yield of 45.8 g / L, while the original strain YB-1631 yielded 36.46 g / L, representing a 25.62% increase and breaking through the bottleneck of yield improvement in existing strains.
[0023] The results of this invention show that knocking out the mmgD gene can effectively block the 2-methylcitric acid cycle, reduce metabolic flux diversion, and allow carbon and nitrogen sources to flow more efficiently to the γ-PGA synthesis pathway, thereby improving raw material utilization and achieving a significant increase in yield.
[0024] The method of this invention is simple to operate, has significant effects, and has good prospects for industrial application. Attached Figure Description
[0025] Figure 1 Electrophoresis image used to verify the PCR of recombinant strain YB-1631-ΔmmgD using primer pairs Primer7 and Primer8.
[0026] Figure 2 Electrophoresis image for PCR verification of recombinant strain YB-1631-ΔmmgD using primer pairs Primer9 and Primer10.
[0027] In the figure, M: 2000 Maker; 1-5: recombinant bacteria YB-1631-ΔmmgD to be verified. Detailed Implementation
[0028] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods and detection methods described in each embodiment are conventional methods; unless otherwise specified, the reagents and materials can be purchased commercially.
[0029] Example 1 Experimental Materials 1. Strains and plasmids Bacillus sicca YB-1631 (selected and preserved in the applicant's laboratory), Escherichia coli JM109 (purchased from Beijing Coolerbot Technology Co., Ltd.), and Escherichia coli JM109 carrying plasmid pAD43-25 (containing chloramphenicol resistance gene).
[0030] 2. Culture medium LB medium: NaCl 10 g / L, tryptone 10 g / L, yeast extract 5 g / L, solid medium with 2% agar powder, sterilized at 121 ℃ for 20 min.
[0031] Fermentation medium: sodium glutamate 86.7 g / L, sodium citrate 17.94 g / L, MgSO4·7H2O 2.11 g / L, glycerol 25 g / L, KH2PO4 1.4 g / L, (NH4)2SO4 14 g / L, MnSO4 0.075 g / L, CaCl2 0.1 g / L, FeCl3·6H2O 0.04 g / L, pH adjusted to 7.2, sterilized at 121 ℃ for 20 min.
[0032] LBS hypertonic medium: NaCl 10 g / L, tryptone 10 g / L, yeast extract 5 g / L, sorbitol 91.085 g / L, dispensed into 45 mL / 250 mL Erlenmeyer flasks, sterilized at 121 ℃ for 20 min.
[0033] Electrolysis buffer: sorbitol 91.085 g / L, mannitol 91.085 g / L, glycerol 100 mL / L, sterilized at 121 °C for 20 min.
[0034] Resuscitation medium: NaCl 10 g / L, tryptone 10 g / L, yeast extract 5 g / L, sorbitol 91.085 g / L, mannitol 69.22 g / L, sterilized at 121 ℃ for 20 min.
[0035] 3. Reagents 10% Glycine: Weigh 10 g, bring the volume to 100 mL using a volumetric flask, sterilize with a 0.22 μm sterile filter, aliquot and store at -20 ℃ for later use.
[0036] 10% DL-Threonine: Weigh 10 g, bring the volume to 100 mL using a volumetric flask, sterilize with a 0.22 μm sterile filter, aliquot and store at -20 ℃ for later use.
[0037] 4. Primers The primer sequences used in the experiment are shown in Table 1.
[0038] Table 1
[0039] Example 2 Construction of recombinant bacteria 1. Preparation of competent cells (1) Activate the Bacillus sicca YB-1631 glycerol tube strain (YB-1631-WT) on LB agar medium, pick a single colony and inoculate it into a 5 mL LB test tube, and incubate overnight at 37 ℃ and 180 r / min. (2) Take 400 μL of the shaken bacterial culture and add it to 45 mL of LBS hypertonic medium. Shake at 37 ℃ and 200 r / min until the OD reaches 0. 600 Add 11.25 mL of 10% glycine (final concentration 2%) and 5 mL of 10% DL-threonine (final concentration 1%) to a concentration of approximately 0.5. (3) Continue shaking and incubation, taking a sample every 10 minutes. When the OD... 600 Stop shaking when the temperature begins to drop; cool in an ice bath for 20 minutes, then centrifuge at 8000 r / min and 4 ℃ for 10 minutes to collect the bacterial cells; (4) Resuspend the bacterial cells in pre-cooled electroporation buffer, centrifuge at 8000 r / min and 4 ℃ for 5 min to collect the bacterial cells, repeat 4 times, and finally resuspend in 1 mL of electroporation buffer and dispense into 100 μL / 2 mL tubes for later use.
[0040] 2. Construction of recombinant fragments (1) Select Escherichia coli JM109 containing plasmid pAD43-25 grown on LB agar plates (LB agar medium) containing chloramphenicol (cm 5 μg / mL) resistance, and verify and amplify the nucleotide sequence as shown in SEQ ID NO:2 using Primer1 and Primer2 as primers with Takara PrimeSTAR® MaxDNA Polymerase. (2) Using the genome of Bacillus sicca YB-1631 as a template, the upstream homologous arm of the mmgD gene was amplified by Primer3 and Primer4, and the downstream homologous arm of the mmgD gene was amplified by Primer5 and Primer6. (3) The upstream homologous arm, chloramphenicol resistance fragment, and downstream homologous arm were seamlessly ligated to obtain a recombinant fragment with a nucleotide sequence as shown in SEQ ID NO:3; (4) The recombinant fragment was introduced into Bacillus sicca YB-1631 competent cells by electroporation, and the recombinant bacteria were obtained by chloramphenicol resistance screening and PCR verification.
[0041] 3. Electrostatic Conversion and Screening (1) 5 μL of recombinant fragment was introduced into 100 μL of Bacillus sicca YB-1631 competent cells by electroporation. After mixing, the cells were immediately transferred into a pre-cooled 1 mm electroporation cuvette (electroporation conditions: 1.8 KV, 4.5-6 ms). (2) Immediately after the electric shock, add 900 μL of recovery medium preheated to 37 °C, transfer to a centrifuge tube, and recover at 37 °C and 160 r / min for 5-8 h; (3) After recovery, centrifuge at 8000 r / min for 5 min to concentrate the bacterial solution to 200 μL, spread it on LB agar containing 5 μg / mL chloramphenicol, and incubate at 37 ℃ for 16 h; (4) Select a single colony and perform PCR verification using Primer7 and Primer8, Primer9 and Primer10. The strains that amplify the bands of 1402 bp and 1788 bp are positive recombinant bacteria and named YB-1631-ΔmmgD. Among them, each set of primers, Primer7 and Primer8, and Primer9 and Primer10, has one end located in the original genome of YB-1631 and the other end located in the cm resistance fragment.
[0042] The strains with the correct band positions were sequenced for verification, and the results are as follows: Figure 1 , Figure 2 As shown.
[0043] Figure 1 The target lane showed a clear 1402bp specific band, perfectly matching the expected amplified fragment length. This result indicates that the recombinant fragment containing the chloramphenicol resistance gene has been successfully integrated into the genome of Bacillus cereus YB-1631, and the downstream homologous arm of the mmgD gene has correctly recombinated with the resistance fragment, preliminarily verifying the successful construction of the mmgD gene knockout recombinant bacterium.
[0044] Figure 2 The target lane showed a clear 1788bp specific band, perfectly matching the expected amplified fragment length. This result indicates that the upstream homologous arm of the mmgD gene correctly recombinated with the resistance fragment. This result is consistent with... Figure 1 The evidence corroborates each other, confirming that the recombinant fragment has been successfully and precisely integrated into the target site of the Bacillus sicca YB-1631 genome through homologous recombination. The mmgD gene has been successfully knocked out, and no reverse integration or false positive clones were observed. This further confirms that the recombinant strain YB-1631-ΔmmgD was correctly constructed and can be used for subsequent γ-PGA fermentation experiments.
[0045] Figure 1 and Figure 2 The results of dual PCR verification were consistent, and the successful construction of the mmgD gene knockout recombinant strain (nucleotide sequence as shown in SEQ ID NO:1) was fully confirmed by different primer combinations. This provides a reliable strain basis for the attribution analysis of the increased γ-PGA yield in subsequent fermentation experiments, ensuring that the subsequent yield difference is only due to the knockout of the mmgD gene, rather than other unexpected mutations in the strain construction process.
[0046] Example 3: γ-PGA fermentation production 1. Seed culture The positive recombinant bacteria YB-1631-ΔmmgD was activated on LB solid medium to produce single colonies, which were then inoculated into 50 mL / 100 mL Erlenmeyer flasks of LB liquid medium and cultured at 37 ℃ and 180 r / min for 12 h to obtain seed culture.
[0047] 2. Fermentation culture Adjust seed solution to OD 600 =1, and then inoculate the seed liquid into the fermentation medium (60mL / 250mL Erlenmeyer flask) at an inoculation rate of 2%, and ferment at 37 ℃ and 210 r / min for 48 h to obtain the fermentation broth.
[0048] 3. Determination of γ-PGA content Adjust the pH of the fermentation broth to around 3, centrifuge at 8000 r / min for 30 min, and collect the supernatant. Add 4 times the volume of anhydrous ethanol to the supernatant and mix well. Let it stand overnight at 4 ℃, centrifuge at 6000 r / min for 15 min, and collect the precipitate. Redissolve the precipitate with ultrapure water, freeze dry at -80 ℃, and finally weigh the dried product to obtain the γ-PGA yield of the positive recombinant strain YB-1631-ΔmmgD.
[0049] It should be noted that the γ-PGA yield of YB-1631-WT can be obtained using the same method described above. The γ-PGA yield of YB-1631-WT was determined in parallel using the same method as the positive recombinant strain YB-1631-ΔmmgD, and the results are shown in Table 2.
[0050] Table 2 Yield of γ-PGA by shake-flask fermentation
[0051] Note: A total of 5 independent parallel shake-flask fermentation experiments were conducted. "represent P <0.0001 (Analyze the significance between the two groups of data using multiple t-tests in GraphPad software).
[0052] Table 2 shows that the average yield of γ-PGA in the positive recombinant strain YB-1631-ΔmmgD was 45.8 g / L, while the average yield of the original strain YB-1631-WT was 36.46 g / L. The yield of the positive recombinant strain was increased by 25.62%, indicating that knocking out the 2-methylcitrate synthase gene in the 2-methylcitrate cycle pathway can significantly increase the yield of γ-PGA.
[0053] The embodiments described above are some, but not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but rather to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art through related deductions and substitutions based on the inventive concept, without inventive effort, are within the scope of protection of the present invention.
Claims
1. A recombinant bacterium, characterized in that, The recombinant bacteria were obtained by knocking out the mmgD gene of Bacillus sicca YB-1631, and the nucleotide sequence of the mmgD gene is shown in SEQ ID NO:
1.
2. The recombinant bacteria according to claim 1, characterized in that, The mmgD gene encodes 2-methylcitrate synthase.
3. The recombinant bacteria according to claim 1, characterized in that, The knockout is achieved through homologous recombination.
4. The recombinant bacteria according to claim 1, characterized in that, The Bacillus sicca YB-1631 can increase the production of γ-polyglutamic acid.
5. The application of the recombinant bacteria according to any one of claims 1-4 in increasing the yield of γ-polyglutamic acid, characterized in that, Includes the following steps: The recombinant bacteria were inoculated into LB medium for seed culture, and then the seed culture was inoculated into fermentation medium for fermentation culture. The fermentation products were collected and purified to obtain γ-polyglutamic acid.
6. A method for producing γ-polyglutamic acid by fermentation, characterized in that, The process includes the following steps: inoculating the recombinant bacteria according to any one of claims 1-4 into LB medium for seed culture, then inoculating the seed culture into fermentation medium for fermentation culture, collecting the fermentation product, and separating and purifying γ-polyglutamic acid.
7. The application according to claim 5 or the method according to claim 6, characterized in that, The fermentation medium includes sodium glutamate, sodium citrate, MgSO4·7H2O, glycerol, KH2PO4, (NH4)2SO4, MnSO4, CaCl2, and FeCl3·6H2O.
8. The application according to claim 5 or the method according to claim 6, characterized in that, The separation and purification method includes: centrifuging the fermentation product to obtain the supernatant, adding 4 times the amount of anhydrous ethanol to precipitate, adding ultrapure water to redissolve the precipitate, and then freeze-drying.
9. A method for increasing the yield of γ-polyglutamic acid, characterized in that, This includes the step of knocking out the mmgD gene encoding 2-methylcitrate synthase in Bacillus sicca YB-1631.
10. A method for constructing the recombinant bacteria according to any one of claims 1-4, characterized in that, Includes the following steps: Using a plasmid containing the chloramphenicol resistance gene as a template, the chloramphenicol resistance fragment with the nucleotide sequence shown in SEQ ID NO:2 was amplified; Using the genome of Bacillus sicca YB-1631 as a template, the upstream and downstream homologous arms of the mmgD gene were amplified. The upstream homologous arm, chloramphenicol resistance fragment, and downstream homologous arm were seamlessly ligated to obtain a recombinant fragment with the nucleotide sequence shown in SEQ ID NO:3; The recombinant fragment was introduced into Bacillus sicca YB-1631 competent cells via electroporation transformation. The recombinant bacteria were obtained after chloramphenicol resistance screening and PCR verification.