Recombinant plant lactobacillus of overexpression adenosine triphosphate binding cassette transporter protein gene and application of recombinant plant lactobacillus

Abstract

The invention discloses a recombinant plant lactobacillus for overexpressing an adenosine triphosphate binding cassette transporter protein gene and application of the recombinant plant lactobacillus. Belongs to the technical field of bioengineering. The invention aims to improve the cold-resistant growth capability of the plant lactobacillus, increase the capability of producing lactic acid and degrading nitrite, and improve the integrity and permeability of the plant lactobacillus cells. The invention provides a recombinant lactobacillus plantarum, which is characterized in that the lactobacillus plantarum is used as an original strain, and a nucleic acid molecule as shown in SEQ ID NO.1 is overexpressed. The method is used for fermenting products in food industry.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and particularly relates to a recombinant Lactobacillus plantarum over-expressing an adenosine triphosphate-binding cassette transporter protein gene and an application thereof. Background Art

[0002] Low-temperature fermented foods are of high quality, with a softer, more delicious taste and no odor. However, microorganisms in the fermentation system are often affected by low-temperature stress, which reduces the fermentation performance of the microorganisms and thus affects the product quality. Therefore, the present invention provides a method for constructing a recombinant Lactobacillus plantarum that overexpresses an ATP-binding cassette transporter protein gene, has good cold tolerance in low-temperature environments, and is used for low-temperature fermentation to produce acid and degrade nitrite, in order to improve the flavor and quality of low-temperature fermented foods such as sauerkraut, while shortening the fermentation time.

[0003] In the food industry, many fermented products (such as yogurt, fermented milk, and kimchi) require storage and transportation at low temperatures to extend shelf life and inhibit the growth of harmful microorganisms. If Lactobacillus plantarum possesses good cold tolerance, it can remain active at low temperatures, helping to maintain the fermentation characteristics, flavor, and safety of the product. Cold-tolerant Lactobacillus plantarum can be applied to a wider range of fermentation or health food production processes requiring low temperatures, such as refrigerated fermented foods and probiotic preparations stored at low temperatures, expanding its industrial application potential.

[0004] The accumulation of lactic acid by Lactobacillus plantarum during fermentation not only lowers pH but also increases lactic acid production, helping to improve the quality and consistency of fermented foods. Furthermore, microbial fermentation can efficiently produce lactic acid, an industrial chemical, thereby reducing environmental pollution and resource waste, facilitating industrial applications.

[0005] An intact cell membrane and cell wall ensure the stability of the intracellular environment, enabling the proper functioning of various organelles and ensuring the smooth progress of basic life activities such as metabolism and reproduction. Maintaining normal cellular physiological functions is crucial for enhancing Lactobacillus plantarum's growth and metabolic capacity in low-temperature environments and for unleashing its fermentation properties and functions. For example, an intact cell membrane maintains appropriate intracellular ion concentrations and pH values, creating optimal conditions for enzyme activity and ensuring the proper functioning of processes such as energy metabolism and biosynthesis. Lactobacillus plantarum possessing these capabilities is urgently needed. Summary of the Invention

[0006] The purpose of the invention is to improve the cold-resistant growth ability of Lactobacillus plantarum, increase the ability to produce lactic acid and degrade nitrite as well as the integrity and permeability of Lactobacillus plantarum cells.

[0007] The present invention provides a recombinant Lactobacillus plantarum (Lactiplantibacillus plantarum ), using Lactobacillus plantarum SC-MDJ as the starting strain, overexpressing the gene shown in SEQ ID NO.1; the Lactobacillus plantarum SC-MDJ, with the deposit number CGMCC NO.28112, is deposited in the China General Microorganism Collection Center on August 7, 2023, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] The present invention provides a microbial preparation containing the recombinant Lactobacillus plantarum SC-MDJ.

[0009] The present invention provides a method for cultivating cold-resistant Lactobacillus plantarum or a method for improving the cold-resistant ability of Lactobacillus plantarum. The gene shown in SEQ ID NO.1 is overexpressed in Lactobacillus plantarum SC-MDJ.

[0010] The present invention provides a breeding method for improving the lactic acid yield of Lactobacillus plantarum, and overexpresses the gene shown in SEQ ID NO.1 in Lactobacillus plantarum SC-MDJ.

[0011] A breeding method for improving the nitrite degradation ability of Lactobacillus plantarum, comprising overexpressing the gene shown in SEQ ID NO. 1 in Lactobacillus plantarum SC-MDJ.

[0012] The present invention provides an application of the recombinant Lactobacillus plantarum in cold-resistant fermentation, lactic acid-producing fermentation or nitrite-degrading fermentation.

[0013] Beneficial effect: Vaccination with overexpression of ATP-binding cassette transporter gene L. plantarum Fermentation of SC-MDJ-NC8_RS00340 under low temperature conditions can increase the OD 600nm The fluorescence intensity of the cell membrane decreased to 1.884, a 32.68% increase compared to the control group. The pH of the fermentation system was also reduced to 4.47, a 10.24% decrease compared to the control group. Furthermore, the integrity of the cell membrane was inversely proportional to the fluorescence intensity. When the fluorescence intensity decreased to 135.35, the integrity of the cell membrane increased by 74.39% compared to the control group. The permeability of the cell membrane was directly proportional to the conductivity. When the conductivity increased to 65.10%, the permeability of the cell membrane increased by 181% compared to the control group. Inoculation of cells overexpressing the ATP-binding cassette transporter gene L. plantarum Fermentation of SC-MDJ-NC8_RS00340 at 15 °C increased the total acid content in the fermentation system to 15.70 mg / mL, while reducing the sodium nitrite content to 0.013 mg / mL, and the nitrite degradation rate reached 98.70%. Inoculation of cells overexpressing the ATP-binding cassette transporter gene L. plantarum Fermentation of SC-MDJ-NC8_RS00340 at 15°C increased the lactic acid content in the fermentation system to 11.50 mg / mL, a 39.73% increase compared to the control. This increase in lactic acid content can enhance the unique sour taste of foods, thereby promoting the development of a good flavor.

[0014] [Biological preservation information]: Lactobacillus plantarum is named Lactobacillus plantarum SC-MDJ, the preservation number is CGMCCNO.28112, and it is deposited in the China General Microorganism Collection Center. The preservation date is August 7, 2023. The preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The construction process of the ATP-binding cassette transporter protein gene overexpression plasmid; Figure 2 Overexpression of ATP-binding cassette transporter gene L. plantarum PCR identification results; Figure 3 This is the result graph of the relative expression level of NC8_RS00340 gene mRNA. DETAILED DESCRIPTION

[0016] MRS broth medium: peptone 10.0 g, beef extract powder 8.0 g, yeast extract powder 4.0 g, glucose 20.0 g, dipotassium hydrogen phosphate 2.0 g, diammonium hydrogen citrate 2.0 g, sodium acetate 5.0 g, magnesium sulfate 0.2 g, manganese sulfate 0.04 g, Tween 80 1.0 g, distilled water 1000 mL, pH = 5.7 ± 0.2.

[0017] LB broth medium: 10.0 g tryptone, 5.0 g yeast extract powder, 10.0 g sodium chloride, 1000 mL distilled water, pH = 7.0 ± 0.1.

[0018] Example 1. Method for constructing recombinant Lactobacillus plantarum (1) Extraction of genomic DNA and acquisition of ATP-binding cassette transporter protein genes Lactobacillus plantarum ( Lactiplantibacillus plantarumSC-MDJ was inoculated into MRS broth at a 2% inoculum volume and cultured at 30 °C, 200 rpm until the logarithmic growth phase and passaged twice. Then, genomic DNA of the strain was extracted using a bacterial genomic DNA extraction kit. L. plantarum Reference genes NC8_RS00340 Sequence design primers were used for PCR amplification. After the PCR product was detected by 1% agarose gel electrophoresis, the target gene fragment was cut out and recovered using a spin column type ordinary agarose gel DNA recovery kit and stored in a -20°C refrigerator until use. NC8_RS00340

[0019] Table 1 Primers and sequences used

[0020] Table 2 PCR amplification reaction system

[0021] Table 3 PCR amplification reaction program

[0022] (2) Plasmid extraction and linearization Escherichia coli ( Escherichia coli ) was inoculated into LB broth containing erythromycin at a final concentration of 600 μg / mL at a 2% inoculum volume, cultured at 37 °C, 200 rpm until the logarithmic growth phase and passaged twice, and then the plasmid was extracted using a plasmid extraction kit. Xba I and Hind The pMG36e plasmid was double-digested with the restriction endonuclease III. The digestion reaction was carried out in a 37°C water bath for 20 min. After completion of the reaction, the fragment was detected by 1% agarose gel electrophoresis. The target fragment was recovered using a spin column-based agarose gel DNA recovery kit and stored at -20°C until use. The double-digestion reaction system is shown in Table 4.

[0023] Table 4 Double enzyme digestion reaction system

[0024] (3) Ligation and transformation of target gene and plasmid The purified target gene was ligated with the linearized pMG36e plasmid using a one-step cloning kit, reacted in a 37°C water bath for 30 min, and immediately placed on ice after the reaction. E. coli DH5α competent cells were placed on ice for slowing down. 100 μL competent cells were gently mixed with 10 μL plasmid and placed on ice for 30 min. The ligated plasmid was transformed into E. coli Finally, the DH5α competent cells were plated on LB agar medium containing 600 µg / mL erythromycin. After colonies grew on the plate, transformants were screened and verified.

[0025] (4) Screening and verification of E. coli transformants After picking individual colonies with a sterile pipette tip, transformants were verified by PCR using primers pMG36e-F and pMG36e-R. Correctly identified colonies were inoculated into LB broth containing a final concentration of 600 µg / mL erythromycin and cultured at 37°C, 200 rpm, until the logarithmic growth phase. The bacterial suspension was aspirated and washed twice with sterile saline (0.85% NaCl, w / v) before DNA sequencing. The E. coli transformant suspension was stored in a glycerol freezer at -80°C, completing the construction of the recombinant plasmid pMG36e-NC8_RS00340. The PCR verification reaction system for transformants is shown in Table 5, and the amplification reaction procedure is shown in Table 6.

[0026] Table 5 Transformant PCR verification reaction system

[0027] Table 6 PCR amplification reaction program

[0028] (5) Extraction and transformation of recombinant plasmid The recombinant plasmid pMG36e-NC8_RS00340 was E. coli DH5α strain was inoculated into LB broth with a final concentration of 600 μg / mL erythromycin at a 2% inoculum volume and cultured at 37 °C, 200 rpm until the logarithmic growth phase. The recombinant plasmid was extracted using a plasmid extraction kit. 10 μL of plasmid was mixed with 100 μL of L. plantarum Gently mix the SC-MDJ competent cells and incubate on ice for 5 minutes. Then, use an electroporator to transform the plasmid into the competent cells. Using a 1 mm gap cuvette, adjust the voltage to 1.25 kV and perform two consecutive electroporations. Immediately add 890 μL of pre-chilled MRS broth and incubate at 30°C for 2.5 hours. After completion, concentrate the culture solution by centrifugation and spread it on MRS agar containing 100 μg / mL erythromycin. Incubate at 30°C until colonies emerge for screening and verification.

[0029] (6) Screening and verification of gene overexpression strains A single colony was inoculated into MRS broth containing a final concentration of 10 μg / mL erythromycin using a sterile pipette tip and cultured at 30 °C, 200 rpm until the logarithmic growth phase. The bacterial liquid was aspirated and washed twice with sterile saline (0.85% NaCl, w / v). PCR verification was performed using pMG36e-F and pMG36e-R primers. The verified bacterial liquid was again inoculated into MRS broth containing a final concentration of 10 μg / mL erythromycin and cultured at 30 °C, 200 rpm until the logarithmic growth phase. The bacterial liquid was stored in a -80 °C freezer by glycerol preservation. L. plants The construction is completed and named L. plantarum SC-MDJ-NC8_RS00340. The PCR verification reaction system is the same as in Table 5, and the amplification reaction procedure is the same as in Table 6.

[0030] Figure 1 The following is the process for constructing the overexpression plasmid of ATP-binding cassette transporter protein gene. Figure 2 Overexpression of ATP-binding cassette transporter gene L. plantarum The PCR identification results were obtained. The successfully constructed gene recombinant plasmid pMG36e-NC8_RS00340 was extracted from E. coli DH5α was extracted and transformed into L. plantarum In SC-MDJ, single colonies were picked from erythromycin-resistant MRS agar for PCR verification of transformants to check whether the recombinant plasmid was successfully transformed into L. plantarum SC-MDJ was used to obtain an ATP-binding cassette transporter gene overexpression strain. The results of 1% agarose gel electrophoresis were as follows: Figure 2 As shown, there is a single bright band at about 2505 bp, which is of good quality, indicating that the recombinant plasmid pMG36e-NC8_RS00340 was successfully transformed into L. plants SC-MDJ, ATP-binding cassette transporter gene overexpression strain L. plantarum SC-MDJ-NC8_RS00340 is built. In addition, L. plantarum SC-MDJ-NC8_RS00340 NC8_RS00340 The relative expression of gene mRNA increased by 26.41 times, which also shows that the gene was successfully overexpressed. Figure 3 shown.

[0031] Example 2. Improvement L. plantarum Determination of cold-resistant growth ability of SC-MDJ-NC8_RS00340 (1) Preparation of starter culture Overexpression strains L. plantarum SC-MDJ-NC8_RS00340 (experimental group) and control strains L. plants SC-MDJ were inoculated into MRS broth containing erythromycin at a final concentration of 10 μg / mL at a 2% inoculum volume, cultured at 30 °C, 200 rpm until the logarithmic growth phase, subcultured twice, and the cultures were collected.

[0032] (2) Establishment of fermentation system The culture was inoculated into MRS broth containing erythromycin at a final concentration of 10 µg / mL to inoculate L. plants The SC-MDJ fermentation system was used as the control group and inoculated L. plantarum The fermentation system of SC-MDJ-NC8_RS00340 was used as the experimental group and fermented at 15 °C and 200 rpm for 6 days.

[0033] (3) Determination of fermentation culture growth, pH, and cell membrane characteristics After the culture was collected, the OD was measured using a spectrophotometer. 600nm To evaluate the growth ability of the strain, the pH of the fermentation broth was measured using a pH meter to evaluate the acid production ability of the strain. The strain (OD 600nm =0.8) and the cell membrane integrity (fluorescence intensity) of the strains were measured using a conductivity meter (representing cell membrane permeability) to evaluate the cell membrane properties.

[0034] Table 7 L. plantarum OD of SC-MDJ-NC8_RS00340 600nm , pH, cell membrane integrity and permeability

[0035] Note: Different lowercase letters in the vertical row indicate significant differences among treatments ( P <0.05).

[0036] Table 7 is L. plantarum OD of SC-MDJ-NC8_RS00340 600nm , pH, cell membrane integrity and permeability test results. The test results showed that the cells overexpressing the ATP-binding cassette transporter gene L. plants Fermentation of SC-MDJ-NC8_RS00340 under low temperature conditions can increase the OD 600nmIt increased to 1.884, an increase of 32.68% compared with the control group. At the same time, the pH of the fermentation system was reduced to 4.47, a decrease of 10.24% compared with the control group. In addition, the integrity of the cell membrane was inversely proportional to the fluorescence intensity. When the fluorescence intensity decreased to 135.35, the integrity of the cell membrane increased by 74.39% compared with the control group. The permeability of the cell membrane was positively correlated with the conductivity. When the conductivity increased to 65.10%, the permeability of the cell membrane increased by 181% compared with the control group. Therefore, the above results show that overexpression of the ATP-binding cassette transporter protein gene can significantly improve L. plantarum SC-MDJ's ability to grow, produce acid, and possess cell membrane properties in low-temperature environments endows the fermentation agent with unique physiological functions, providing favorable conditions for the strain to restore normal physiological metabolism. This not only helps to increase the fermentation rate of low-temperature fermented vegetable products such as sauerkraut, but also improves the flavor, quality, and safety of the products, and reduces resource waste in the food production process.

[0037] Example 3. L. plantarum Determination of total acid and nitrite degradation levels in fermentation of SC-MDJ-NC8_RS00340 L. plantarum Determination of total acid produced by fermentation of SC-MDJ-NC8_RS00340: (1) Establishment of fermentation system 1 mg / mL sodium nitrite was added to MRS broth containing erythromycin at a final concentration of 10 μg / mL, and the culture medium without inoculation of starter culture was used as a control. L. plantarum SC-MDJ-NC8_RS00340 was the inoculated starter group, and fermentation was carried out at 15 °C and 200 rpm for 4 days to determine the total acid content in the fermentation system.

[0038] (2) Determination of total acid content Pipette 10.0 mL of fermentation broth and add purified water to a volume of 100 mL. Filter impurities through gauze. Collect 50 mL of filtrate and titrate with 0.1 mol / L NaOH. Simultaneously, add two drops of 10 g / L phenolphthalein indicator. Record the volume of NaOH solution when the solution reaches a slightly reddish color (V1) and the volume of NaOH solution consumed by replacing the sample solution with purified water (V2).

[0039] (3) The total acid content (mg / mL) is calculated as follows:

[0040] Note: c: concentration of sodium hydroxide standard titrant, in moles per liter (mol / L); V1: volume of sodium hydroxide standard titrant consumed in titrating the test solution, in milliliters (mL); V2: volume of sodium hydroxide standard titrant consumed in the blank test, in milliliters (mL); k: acid conversion factor (calculated based on lactic acid, 0.090); F: dilution factor of the solution; m: mass of the sample, in milliliters (mL); 1000: conversion factor.

[0041] L. plantarum Determination of nitrite degradation by fermentation of SC-MDJ-NC8_RS00340: (1) Establishment of fermentation system 1 mg / mL sodium nitrite was added to MRS broth containing erythromycin at a final concentration of 10 μg / mL, and the culture medium without inoculation of starter culture was used as a control. L. plantarum SC-MDJ-NC8_RS00340 was the inoculated starter group. Fermentation was carried out at 15 °C and 200 rpm for 4 days, and the sodium nitrite content in the fermentation system was measured.

[0042] (2) Determination of nitrite content Nitrite content was determined using a nitrite content assay kit for soil and water (Greis, Suzhou, China).

[0043] (3) The calculation formula of nitrite degradation rate is as follows:

[0044] Note: X1 is the sodium nitrite content in the control culture medium, in milligrams per milliliter (mg / mL); X2 is the sodium nitrite content in the inoculated starter culture medium, in milligrams per milliliter (mg / mL).

[0045] Table 8 L. plantarum Total acid and sodium nitrite content in SC-MDJ-NC8_RS00340 fermentation system

[0046] Note: Different lowercase letters in the vertical row indicate significant differences between samples ( P <0.05).

[0047] Table 8 is L. plantarum The results of total acid and sodium nitrite content determination in SC-MDJ-NC8_RS00340 fermentation system. The test results showed that the inoculated L. plantarumFermentation of SC-MDJ-NC8_RS00340 at 15°C increased the total acid content in the fermentation system to 15.70 mg / mL, while reducing the sodium nitrite content to 0.013 mg / mL, and achieving a nitrite degradation rate of 98.70%. The increase in total acid content can enhance the taste and flavor of food, thereby promoting the formation of a good flavor in food. L. plants The SC-MDJ-NC8_RS00340 starter culture also enhances the flavor of fermented foods. Compared with medium-temperature fermentation, it can significantly improve the acid production and nitrite degradation capabilities of the starter culture under lower temperature conditions. This not only helps to improve the quality and safety of low-temperature fermented vegetable foods such as sauerkraut, but also greatly increases the fermentation speed of the product and reduces resource waste in the production process.

[0048] Example 4. L. plantarum Lactate production capacity assay of SC-MDJ-NC8_RS00340 (1) Preparation of starter culture Overexpression strains L. plantarum SC-MDJ-NC8_RS00340 and control strains L. plantarum SC-MDJ were inoculated into MRS broth containing erythromycin at a final concentration of 10 μg / mL at a 2% inoculum volume, cultured at 30 °C, 200 rpm until the logarithmic growth phase, subcultured twice, and the cultures were collected.

[0049] (2) Establishment of fermentation system The culture was inoculated into MRS broth containing erythromycin at a final concentration of 10 µg / mL to inoculate L. plants The SC-MDJ fermentation system was used as the control group and inoculated L. plantarum The fermentation system of SC-MDJ-NC8_RS00340 was used as the experimental group and fermented at 15 °C and 200 rpm for 6 days.

[0050] (3) Determination of lactic acid production capacity After the fermentation broth was collected, the lactic acid content in the fermentation broth was determined using high performance liquid chromatography to evaluate the lactic acid production ability of the strain under low temperature environment.

[0051] Table 9 L. plantarum Lactic acid production capacity of SC-MDJ-NC8_RS00340

[0052] Note: Different lowercase letters in the vertical row indicate significant differences among treatments ( P <0.05).

[0053] Table 9 is L. plantarum The results of the lactic acid content determination in the SC-MDJ-NC8_RS00340 fermentation system showed that the inoculated L. plantarum Fermentation of SC-MDJ-NC8_RS00340 at 15 °C increased the lactic acid content in the fermentation system to 11.50 mg / mL, a 39.73% increase compared to the control group. The increase in lactic acid content can increase the unique sour taste of food, thereby promoting the formation of good food flavor. L. plantarum The SC-MDJ-NC8_RS00340 starter culture also enhances the flavor of fermented foods, helping to improve the quality and safety of low-temperature fermented vegetable foods like sauerkraut and promoting product maturation. Furthermore, since microbial fermentation can efficiently produce lactic acid, an industrial chemical, while also reducing environmental pollution and resource waste, it is promising for industrial applications.

Claims

1. A recombinant Lactobacillus plantarum ( Lactiplantibacillus plantarum ), characterized in that, Lactobacillus plantarum SC-MDJ was used as the starting strain to overexpress the gene shown in SEQ ID NO.1; the Lactobacillus plantarum SC-MDJ had a deposit number of CGMCC NO.28112 and was deposited in the China General Microorganism Collection Center on August 7, 2023. The deposit address was the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. A microbial preparation containing the recombinant Lactobacillus plantarum according to claim 1.

3. A breeding method for improving the cold tolerance of Lactobacillus plantarum, characterized in that: The gene shown in SEQ ID NO. 1 was overexpressed in Lactobacillus plantarum SC-MDJ.

4. A breeding method for increasing lactic acid production of Lactobacillus plantarum, characterized in that: The gene shown in SEQ ID NO. 1 was overexpressed in Lactobacillus plantarum SC-MDJ.

5. A breeding method for improving the ability of Lactobacillus plantarum to degrade nitrite, characterized in that: The gene shown in SEQ ID NO. 1 was overexpressed in Lactobacillus plantarum SC-MDJ.

6. Use of the recombinant plant lactobacillus according to claim 1 in cold-resistant fermentation, lactic acid-producing fermentation or nitrite-degrading fermentation.

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