CRISPR / Cas9 technology-based lactobacillus food-like double-plasmid multi-gene editing system and application thereof

Abstract

The invention discloses a lactobacillus foodstuff-like double-plasmid multi-gene editing system based on a CRISPR / Cas9 technology and application of the lactobacillus foodstuff-like double-plasmid multi-gene editing system. The lactobacillus food-like double-plasmid multi-gene editing system comprises: (1) a plasmid pCRI01, wherein the plasmid pCRI01 comprises the following elements: a Cas9 protein coding gene; a temperature sensitive replicon pSC101; the sgRNA expression module is used for targeting a second plasmid replicon and is regulated and controlled by an inducible promoter Pnis; ampicillin resistance is achieved; (2) a plasmid pCRI02 comprising the following elements: at least one sgRNA expression module driven by a P23 promoter; the lambda-Red recombinase system comprises an Exo gene, a Beta gene and a Gam gene; an erythromycin resistance marker gene; and a kanamycin resistance gene. After the multi-gene editing system provided by the invention is a small-size pCRI01 and pCRI02 double-plasmid system, the number of transformants after resistance screening is remarkably increased, and under the assistance of a lambda-Red recombinase system, the pCRI plasmid mediated gene editing efficiency is greatly improved.

Description

Technical Field

[0001] The present invention relates to a gene editing plasmid based on CRISPR / Cas9 technology and a method for gene editing of Lactobacillus paralimentarius, belonging to the fields of genetic engineering and microbial genetics. Background Art

[0002] Lactobacillus paralimentarius, as an important lactic acid bacterium, is deeply involved in the production process of traditional fermented foods such as sourdough, pickles and cheese. Its metabolites have a significant impact on food flavor, shelf life and functional properties. In recent years, with the development of synthetic biology and metabolic engineering, lactic acid bacteria have gradually become a research hotspot in the food industry and biomedical fields due to their food safety, probiotic characteristics and acid environment tolerance. Their metabolic regulation ability directly affects food quality. Therefore, developing a gene editing tool suitable for L. paralimentarius not only helps to reveal its metabolic regulation mechanism, but also provides strong technical support for improving food quality, extending shelf life and optimizing the production of functional fermented products.

[0003] With the development of genetic engineering technology, traditional gene editing techniques such as homologous recombination, transposon mutagenesis, and zinc finger nucleases (ZFNs) are gradually being replaced by emerging CRISPR / Cas9-based gene editing techniques due to their low efficiency, cumbersome procedures, and difficulty in editing multiple loci. Compared with traditional gene editing techniques, the application of CRISPR / Cas9 gene editing technology in lactic acid bacteria has higher efficiency, stronger specificity, and broader applicability. Traditional gene editing relies on inefficient homologous recombination, while CRISPR / Cas9 precisely cuts the target gene through sgRNA-guided Cas9 enzyme, greatly improving the editing efficiency. At the same time, it supports synchronous editing of multiple genes and is suitable for complex metabolic engineering transformation of lactic acid bacteria. In addition, CRISPR / Cas9 can achieve scarless mutations through a markerless homologous recombination repair mechanism (HDR), avoiding the residue of antibiotic resistance genes in traditional methods and improving the genetic stability of strains. Its high targeting reduces off-target effects and improves editing accuracy, while the experimental period is significantly shortened compared with traditional methods. In our previous study, the gene LpaMA was successfully knocked out in L. paralimentarius using the established gene editing plasmid pHSP02 in L. plantarum, with an efficiency of 75%; however, when performing multi-gene editing tasks, the number of transformants and gene editing efficiency decreased linearly with the increase of target genes. It is speculated that the reason is that the loading of the repair template makes the plasmid too large, affecting the transformation efficiency, and the reason for the decrease in editing efficiency is that the host cannot adapt to the repair pressure brought by multi-site DNA double-strand breaks caused by the multi-sgRNA-Cas9 protein complex. The effectiveness of existing gene editing tools is limited to a single locus, which severely restricts their application potential as cell factories.

[0004] The key strategies to achieve multi-gene editing based on CRISPR / Cas9 technology are to reduce plasmid size, optimize transformation efficiency, and introduce auxiliary mechanisms to improve homologous repair ability. Summary of the Invention

[0005] One of the objectives of the present invention is to provide a two-plasmid multi-gene editing system for Lactobacillus parafoodi based on CRISPR / Cas9 technology.

[0006] The technical solution to achieve the above invention objective is as follows: A two-plasmid multi-gene editing system for Lactobacillus parafoodi based on CRISPR / Cas9 technology, comprising:

[0007] (1) Plasmid pCRI01, containing the following elements: a Cas9 protein coding gene; a temperature-sensitive replicon pSC101; an sgRNA expression module targeting the second plasmid replicon, regulated by the inducible promoter Pnis; ampicillin resistance;

[0008] (2) Plasmid pCRI02, containing the following elements: at least one sgRNA expression module driven by the P23 promoter; the λ-Red recombinase system, containing the Exo, Beta, and Gam genes; an erythromycin resistance marker gene; and a kanamycin resistance gene.

[0009] In the dual-plasmid combined with the λ-Red recombination system of the present invention, the Exo, Beta, and Gam genes of the λ-Red recombination system are codon-optimized to adapt to the host expression environment of Lactobacillus paralimentarius, which is beneficial to improving the multi-gene editing efficiency of Lactobacillus paralimentarius.

[0010] The second object of the present invention is to provide a multi-gene editing method for the dual-plasmid multi-gene editing system of Lactobacillus paralimentarius based on the CRISPR / Cas9 technology described above.

[0011] The technical solution to achieve the above-mentioned invention object is as follows: A multi-gene editing method for the dual-plasmid multi-gene editing system of Lactobacillus paralimentarius based on the CRISPR / Cas9 technology, including the following steps:

[0012] (1) Electrotransform pCRI01 into the competent Lactobacillus paralimentarius strain by electrotransformation to obtain the competent Lactobacillus paralimentarius strain containing plasmid pCRI01;

[0013] (3) Construct a plasmid pCRI02 containing at least one sgRNA targeting the target gene;

[0014] (4) Construct a DNA homologous repair template for the target gene;

[0015] (5) Co-electrotransform the plasmid pCRI02 and the DNA homologous repair template for the target gene into the competent Lactobacillus paralimentarius containing plasmid pCRI01, incubate, concentrate the cell concentration, evenly coat it on an agar MRS plate containing ampicillin and erythromycin resistance, and statically culture it at 37°C to obtain transformants;

[0016] (6) Pick the transformants into a liquid MRS medium for culture, extract the genome and send it for sequencing to verify the editing results;

[0017] (7) Inoculate the transformants with correct editing results into a liquid MRS medium containing 10 ng / μL Nisin and culture them overnight at 40°C.

[0018] In step (2), the mass ratio of the plasmid pCRI02 to the DNA homologous repair template for the target gene is 1:2, and the volume ratio is 1:1.

[0019] In step (2), the sgRNA expression module of plasmid pCRI02 realizes tandem insertion of multiple sgRNAs through the BioBricks assembly method by using XbaⅠ and SpeⅠ restriction enzyme sites.

[0020] Specifically, using plasmid pCRI02 as a template, PCR is performed with the P23 promoter and the sgRNA module as upstream and downstream. The XbaⅠ restriction enzyme site is added to the 3' end primer of P23, and the SpeⅠ site and the gRNA of the target gene are added to the 5' end primer of the sgRNA module. The PCR product is a linearized pCRI02 plasmid; using pCRI02-1 as a template, PCR is performed with the sgRNA module and P23 as upstream and downstream. The SpeⅠ restriction enzyme site and the gRNA of the target gene are added to the 5' end primer of the sgRNA module, and the XbaⅠ restriction enzyme site is added to the 3' end primer of P23. Multiple PCRs are performed for the editing work of multiple sgRNAs. After all the above PCR products are purified and recovered by a DNA column, BioBricks assembly is carried out through the XbaⅠ-SpeⅠ restriction enzyme site to ensure the same direction of the target fragments.

[0021] The third object of the present invention provides a method for editing a Lactobacillus paralimentarius strain based on the CRISPR-Cas9 dual-plasmid system.

[0022] Specifically, for the method of editing a Lactobacillus paralimentarius strain based on the CRISPR-Cas9 dual-plasmid system, using Lactobacillus paralimentarius as the starting strain, the above multi-gene editing method is used to knockout the target gene to obtain an edited Lactobacillus paralimentarius strain.

[0023] The target gene is at least one of LpaMA, UgpA, and UlpRTF1.

[0024] Lactobacillus paralimentarius is preferably Lactobacillus paralimentarius LBM12001. The Lactobacillus paralimentarius LBM12001 was deposited at the Guangdong Provincial Microbiology Culture Collection Center on July 21, 2022, and received the deposit registration number GDMCC 62232. The deposit address is: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou.

[0025] Furthermore, the alanine at the 375th position and the serine at the 257th position of the gene LpaMA of Lactobacillus paralimentarius LBM12001 are respectively mutated to proline, and at least one of the genes UgpA and UlpRTF1 is knocked out.

[0026] The fourth object of the present invention provides an edited Lactobacillus paralimentarius strain.

[0027] The edited Lactobacillus parafoodi strain lacks at least one of LpaMA, UgpA, and UlpRTF1, and is obtained by knocking out the target gene using the gene editing method based on the CRISPR / Cas9 dual plasmid system described above.

[0028] Lactobacillus parafoodi is preferably Lactobacillus parafoodi LBM12001. Further, the alanine at position 375 and serine at position 257 in the amino acid sequence of the LpaMA gene of Lactobacillus parafoodi LBM12001 are respectively mutated to proline, and at least one of the genes UgpA and UlpRTF1 is knocked out.

[0029] The fifth object of the present invention provides the application of the edited Lactobacillus parafoodi strain.

[0030] Specifically, it relates to the application of the edited Lactobacillus parafoodi strain in food preparation; the application of the Lactobacillus parafoodi strain as a food ingredient; and the application of the edited Lactobacillus parafoodi strain as a bread improver.

[0031] In one embodiment, when the edited Lactobacillus parafoodi strain is used as a bread improver, it is added to the dough by the edited Lactobacillus parafoodi strain and maintained at 37°C for at least 90 minutes.

[0032] The present invention also provides a food composition comprising the above-mentioned edited Lactobacillus parafoodi strain.

[0033] The present invention also provides the use of the above-mentioned edited Lactobacillus parafoodi strain as a sourdough starter; and a sourdough comprising the above-mentioned edited Lactobacillus parafoodi strain.

[0034] In the present invention, the food is dough or a dough product, or batter or a batter product, a baked product, or a bakery or pastry shop product.

[0035] Preferably, the baked product of the present invention is a bakery or pastry shop product. Known bread or pastry products in the art include, but are not limited to, those selected from bread, mochi, soft rolls, bagels, doughnuts, Danish pastries, hamburger buns, pizza, pita bread, ciabatta, sponge cakes, cream cakes, pound cakes, muffins, cup cakes, steamed cakes, waffles, brownies, cake doughnuts, yeast-fermented doughnuts, baguettes, bread rolls, saltine crackers, sweet crackers, pie crusts, rusks, and / or other baked products. More preferably, the baked product is bread, baguette, and / or bread roll.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] 1. After splitting the large - volume plasmid pIBE01 into a two - plasmid system of small - volume pCRI01 and pCRI02, the number of transformants after resistance screening has been significantly increased. Thanks to the assistance of the λ - Red recombinase system, the gene editing efficiency mediated by the pCRI plasmid has been greatly improved. In addition, when performing single - gene editing tasks, the single - plasmid system takes 6 days. When multiple genes need to be edited, it takes (4 + 2N) days. When the two - plasmid editing system pCRI01 and pCRI02 performs single - gene editing, it takes 5 days. When performing multi - gene editing, to ensure the editing efficiency, the strategy should be adjusted according to the number of edited genes, and multiple rounds of editing are carried out in groups of 3 genes. Thus, it can be seen that gene editing mediated by the two - plasmid system greatly reduces the time required for large - scale genetic engineering transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is the structural diagram of the two - plasmid system of the present invention.

[0039] Figure 2 It is the schematic diagram of the principle of the two - plasmid system of the present invention.

[0040] Figure 3 It is the transformation rate of the two - plasmid system of the present invention.

[0041] Figure 4 It is the diagram of bread storage changes of the modification of A375P / S257P site with the superimposed silencing expression of UlpRTF1 transcriptional repressor in L.paralimentarius, wild - type L.paralimentarius - like food - grade lactic acid bacteria, and the blank control.

[0042] Note: Control represents ordinary wheat bread, WT - Sourdough represents sourdough wheat bread added with wild - type L.paralimentarius, and WHU - Sourdough represents sourdough wheat bread added with L.paralimentarius strain with the modification of A375P / S257P site and the superimposed silencing expression of UlpRTF1 transcriptional repressor.

[0043] Figure 5 It is the determination of the optimal pH of wild - type maltogenic amylase.

[0044] Figure 6 It is the determination of the optimal temperature and hydrolysis activity of wild - type, mutants A375P, S257P, and A375P / S257P.

[0045] Figure 7 It is the determination of the temperature stability of wild - type, mutants A375P, S257P, and A375P / S257P at 60 °C.

[0046] Figure 8 The half-lives (t 1 / 2 ) of wild-type and mutants A375P, S257P, and A375P / S257P were fitted at 60 °C. Detailed implementation manners

[0047] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0048] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0049] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0050] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the specification of the present invention, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0051] Regarding "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0052] The dual-plasmid gene editing system based on the CRISPR / Cas9 technology provided by the present invention, such as Figure 1As shown, it is possible to significantly reduce the experimental procedures and time required for multi-gene editing while maintaining a high editing efficiency. In the present invention, two plasmids were constructed, namely pCRI01 (8880 bp) and pCRI02 (6740 bp). Among them, pCRI01 is mainly used to express the Cas9 protein. To facilitate the elimination of the plasmid, a temperature-sensitive replicon of pSC101 was introduced. This replicon cannot replicate at 37°C, ensuring the convenient elimination of pCRI01. In addition, to facilitate the elimination of plasmid pCRI02, pCRI01 is equipped with an sgRNA targeting the replicon of pCRI02. This sgRNA is induced by the Pnis promoter. When the expression of sgRNA in pCRI02 ends, the sgRNA in pCRI01 is induced to express with a final concentration of 100 ng / μL of Nicin to cleave the replicon of pCRI02 and eliminate the plasmid; sgRNA is loaded on pCRI02 to direct the Cas9 protein to the target gene. In addition, a λ-Red recombinase system derived from λ phage optimized by species codons was introduced into pCRI02. λ-Red recombination is mediated by three proteins, Exo, Beta, and Gam. Exo is an exonuclease that can degrade exogenous double-stranded DNA along the 5'-3' direction to produce single-stranded DNA. Beta is a single-stranded DNA-binding protein that can protect single-stranded DNA and promote its homologous pairing with chromosomal DNA. Gam is an inhibitor that can prevent the bacterial own RecBCD enzyme from degrading exogenous DNA, which enables the homologous repair template not to be encoded on the plasmid, further improving the gene editing efficiency. To facilitate the loading of multiple sgRNAs, another version of pCRI02-1 was also constructed. In this version, the expression element of P23-sgRNAmodule is in the opposite direction to the original version, and the loading method of multiple sgRNAs adopts the classical BioBricks method. The schematic diagram of its principle is as shown in Figure 2 shown.

[0053] Specifically, taking plasmid pCRI02 as a template, PCR was performed with the P23 promoter and sgRNA module as the upstream and downstream. An XbaⅠ restriction site was added to the 3' end primer of P23, and a SpeⅠ site and the gRNA of the target gene were added to the 5' end primer of sgRNA module. The PCR product was a linearized pCRI02 plasmid; taking pCRI02-1 as a template, PCR was performed with sgRNAmodule and P23 as the upstream and downstream. A SpeⅠ restriction site and the gRNA of the target gene were added to the 5' end primer of sgRNAmodule, and an XbaⅠ restriction site was added to the 3' end primer of P23. Multiple PCRs were performed for the editing work of multiple sgRNAs. After all the above PCR products were purified and recovered by a DNA column, BioBricks assembly was carried out through the restriction sites XbaⅠ-SpeⅠ to ensure the consistent direction of the target fragments.

[0054] Example 1: Preparation of competent cells of L. paralimentarius

[0055] (1) The L. paralimentarius strain preserved in glycerol tube was streaked and activated on an agar MRS solid plate. After static culture at 37 °C, a single colony was picked and transferred into 20 mL of liquid MRS medium, and statically cultured at 37 °C for 24 h.

[0056] (2) 2 mL of the overnight culture was inoculated into 50 mL of MRS liquid medium (containing 0.4 mol·L -1 sucrose and 5 g·L -1 glycine), and cultured at 37 °C with a rotation speed of 200 r·min -1 until the OD 600 reached 0.3 - 0.4.

[0057] (3) The bacterial solution was transferred into a pre-cooled centrifuge tube and left standing on ice for 30 min.

[0058] (4) Centrifuged at 5000 r·min -1 for 10 min at 4 °C to collect the bacterial cells, and the supernatant was discarded.

[0059] (5) The electrotransformation buffer (containing 0.4 mol·L -1 sucrose and 5 g·L -1 glycine) was centrifuged at 5000 r·min -1 for 5 min at 4 °C to wash the bacterial cells twice. The supernatant was discarded, and the bacterial cells were collected into a pre-cooled centrifuge tube and left standing on ice for 10 min.

[0060] (6) The bacterial cells were resuspended with the electrotransformation buffer. Centrifuged at 5000 r·min -1 for 10 min at 4 °C, and the supernatant was discarded.

[0061] (7) The bacterial cells were resuspended again with 1 mL of the electrotransformation buffer, and aliquoted into pre-cooled 1.5 mL Eppendorf tubes at 100 μL per tube, and stored at -80 °C.

[0062] Example 2: Knockout of LpaMA gene

[0063] 1. Preparation of competent cells of L. paralimentarius strain containing plasmid pCRI01

[0064] (1) 10 μL of plasmid pCRI01 was pipetted into 100 μL of pre-thawed L. paralimentarius competent cells, and electrotransformation was carried out under the conditions of 2.5 kV and 5 ms. The electrotransformed bacterial solution was added with 900 μL of warm MRS medium and then recovered and cultured at 37 °C for 2 h.

[0065] (2) The bacterial solution after cultivation was placed in a 1.5 mL centrifuge tube and centrifuged at 8000 r / min for 1 min. 900 μL of the supernatant was discarded, and the remaining bacterial solution was evenly spread on an agar MRS plate containing ampicillin resistance and statically cultured at 37 °C for 48 h.

[0066] (3) The transformants on the agar plate were picked and the competent cells were prepared according to the method described in Example 1.

[0067] 2. Construction of pCRI02-ΔLpaMA plasmid

[0068] (1) The sgRNA of the corresponding gene was designed by GeniousPrime 8.0.1. The sgRNA was mounted to the corresponding position by whole plasmid PCR method.

[0069] (2) The whole plasmid PCR conditions were 98 °C for 1 minute, (98 °C for 30 seconds, 58 °C for 15 seconds, 72 °C for 3 minutes) × 30 cycles, 72 °C for 5 minutes, 16 °C for 5 minutes. Each PCR reaction tube contained 1 μL of forward and 1 μL of reverse primers (10 pM), 1 μL of template plasmid (10 ng), 25 μL of PrimeSTAR MAX DNA polymerase, and 22 μL of ultrapure water. The PCR products were analyzed by electrophoresis on an agarose gel, and the PCR products containing the correct gel electrophoresis bands were purified using a purification kit (Thermo Scientific).

[0070] Forward primer: 5’-GATGAAATCGGTATGGATGGGTTTTAGAGCTAGAAATAGCAAGT-3’;

[0071] Reverse primer: 5’-CCATCCATACCGATTTCATCAACATCATTGTCATTCATATTTTTCA-3’.

[0072] (3) The purified gene sample was digested with Dpn1 enzyme to remove the empty plasmid template. The enzyme digestion mixture was incubated at 37 °C for 1 h and purified again using a purification kit (Thermo Scientific). The purified product was transformed into Escherichia coli E.coli DH5α by chemical transformation method, and the bacterial solution was spread on an LB agar plate containing kanamycin resistance and statically cultured overnight at 37 °C.

[0073] (4) The transformants were picked and inoculated into a non-resistant LB liquid medium. After culturing for 8 h, the plasmid pCRI02-ΔLpaMA was extracted according to the instructions of the plasmid extraction kit (Vazyme).

[0074] (5) Determine the DNA concentration of the purified sample at 280 nm using a NanoDrop 2000c spectrophotometer (Thermo Scientific), and concentrate the plasmid concentration to 50 ng / μL using a vacuum concentrator dryer.

[0075] 3. Construction of the ΔLpaMA homologous repair template

[0076] (1) Select 500 bp of bases upstream and downstream of LpaMA as homologous arms. Using the L. paralimentarius genome as a template, 98°C for 1 minute, (98°C for 30 seconds, 58°C for 15 seconds, 72°C for 15 seconds) × 30 cycles, 72°C for 5 minutes, 16°C for 5 minutes. Each PCR reaction tube contains 1 μL of forward and 1 μL of reverse primers (10 pM), 1 μL of template, 25 μL of PrimeSTAR MAX DNA polymerase, and 22 μL of ultrapure water. Amplify the upstream and downstream homologous arms respectively, and purify the PCR amplification products using a purification kit (Thermo Scientific).

[0077] Forward primer for the upstream homologous arm: 5’-TCCCAATGGCTTTGGATGAA-3’;

[0078] Reverse primer for the upstream homologous arm: 5’-TGAAATAACCTCTCATTTTTAAAAATTTG-3’;

[0079] Forward primer for the downstream homologous arm: 5’-AAAAATGAGAGGTTATTTCAAGAGGTGCTTAATTTGCAAAAACA-3’;

[0080] Reverse primer for the downstream homologous arm: 5’-AGAGGTGAAGATAGTATTGACCAGTCTT-3’.

[0081] (2) The purified products of the upstream and downstream homologous arms were ligated by overlap extension PCR (overlap). After the ligation products were verified by gel electrophoresis and purified by PCR product purification kit, they were used as homologous repair templates. The overlap method is as follows: Dilute the upstream and downstream homologous arms to 10 ng / μL. The first round of PCR uses a 10 μL system, including 1 μL of the upstream fragment, 1 μL of the downstream fragment, 5 μL of PrimeSTAR MAX DNA polymerase, and 3 μL of ultrapure water. The reaction conditions are 98°C for 1 minute, (98°C for 30 seconds, 58°C for 15 seconds, 72°C for 30 seconds) × 10 cycles, 72°C for 5 minutes, and 16°C for 5 minutes. The second round of PCR uses a 50 μL system, including 1 μL of the upstream primer of the upstream homologous arm, 1 μL of the downstream primer of the downstream homologous arm, 1 μL of the first round of PCR product, 25 μL of PrimeSTAR MAX DNA polymerase, and 22 μL of production pure water. The reaction conditions are 98°C for 1 minute, (98°C for 30 seconds, 58°C for 15 seconds, 72°C for 30 seconds) × 30 cycles, 72°C for 5 minutes, and 16°C for 5 minutes. After the products were verified by gel electrophoresis, they were purified using a purification kit (Thermo Scientific).

[0082] (3) Use a NanoDrop 2000c spectrophotometer (Thermo Scientific) to measure the DNA concentration of the purified sample at 280 nm, and concentrate the plasmid concentration to 100 ng / μL using a vacuum concentrator dryer.

[0083] 4. Construction of L. paralimentarius LBM12001ΔlpaMA strain

[0084] (1) Pipette 10 μL of plasmid pCRI02-ΔlpaMA and 10 μL of ΔLpaMA homologous repair template into 100 μL of competent cells of L. paralimentarius strain containing plasmid pCRI01 that has been pre-thawed, and perform electroporation under the conditions of 2.5 kV and 5 ms. The electroporated bacterial solution was added with 900 μL of warm MRS medium and then incubated at 37°C for 2 h.

[0085] (2) After the incubation, the bacterial solution was placed in a 1.5 mL centrifuge tube and centrifuged at 8000 r / min for 1 min. Discard 900 μL of the supernatant, and evenly spread the remaining bacterial solution on an agar MRS plate containing erythromycin resistance, and incubate at 37°C for 48 h.

[0086] (3) Pick the transformants on the agar plate and inoculate them into antibiotic-free liquid MRS medium for 24 h. Extract the genomes of the transformants according to the method described in the genomic DNA extraction kit (Vazyme), and send the extracted genomes to Shanghai Sangon Biotech Co., Ltd. for sequencing verification ( Figure 3 ). The comparison results with the single plasmid editing tool are as Figure 3as shown

[0087] 5. Plasmid curing

[0088] The correct transformants were inoculated into liquid MRS medium containing 10 ng / μL Nisin and cultured overnight at 40 °C for 12 h.

[0089] The results showed that: in the single gene knockout experiment, the dual plasmid editing system produced 63 ± 11 transformants, which was significantly higher than the 24 ± 2 transformants produced by the single plasmid, and the positive rate of gene editing was maintained at 75%.

[0090] Example 3: Knockout of LpaMA and UgpA genes

[0091] The specific steps are as follows:

[0092] 1. Preparation of competent cells of L. paralimentarius strain containing pCRI01 plasmid

[0093] The preparation method was as described in Example 2.

[0094] 2. Construction of pCRI02-ΔLpaMAΔUgpA plasmid

[0095] (1) According to the method Figure 2 as shown. Using pCRI02 as a template, linearize plasmid pCRI02 with P23 and sgRNA module as the boundary. When designing primers, introduce an XbaⅠ restriction site at the 3' end of P23, and introduce a SpeⅠ restriction site and the gRNA corresponding to UgpA at the 5' end of sgRNA module; using pCRI02-1 as a template, PCR amplify the sgRNA module-P23 fragment. When designing primers, introduce a SpeⅠ restriction site and the gRNA corresponding to LpaMA at the 5' end of sgRNA module, and introduce an XbaⅠ restriction site at the 3' end of P23.

[0096] (2) The linearized vector pCRI02 and the sgRNA module-P23 fragment were ligated by T4 DNA ligase in the order of gRNA to form the vector pCRI02-ΔLpaMAΔUgpA, and the subsequent work was referred to the operation in Example 2.

[0097] 3. Construction of ΔLpaMA and ΔUgpA homologous repair templates

[0098] The construction method was similar to that described in Example 2. Concentrate the concentration of the repair template to 200 ng / μL.

[0099] 4. Construction of L. paralimentarius LBM12001ΔlpaMAΔUgpA strain

[0100] (1) Pipette 10 μL of plasmid pCRI02-ΔlpaMA, 5 μL of ΔLpaMA, and 5 μL of the ΔUgpA homologous repair template into 100 μL of pre-thawed competent cells of L. paralimentarius strain containing plasmid pCRI01. Perform electrotransformation under the conditions of 2.5 kV and 5 ms. Add the electrotransformed bacterial solution to 900 μL of warm MRS medium and incubate at 37 °C for 2 h for resuscitation culture.

[0101] (2) Transfer the bacterial solution after the culture to a 1.5 mL centrifuge tube and centrifuge at 8000 r / min for 1 min. Discard 900 μL of the supernatant, and evenly spread the remaining bacterial solution on an agar MRS plate containing erythromycin resistance. Incubate statically at 37 °C for 48 h.

[0102] (3) Pick the transformants on the agar plate and inoculate them into antibiotic-free liquid MRS medium for culture for 24 h. Extract the genomes of the transformants according to the method described in the genomic DNA extraction kit (Vazyme). Send the extracted genomes to Shanghai Sangon Biotech Co., Ltd. for sequencing to verify the results ( Figure 3 ). The comparison results with the single-plasmid editing tool are as Figure 3 shown.

[0103] 5. Plasmid curing

[0104] According to the method described in Example 2.

[0105] The results showed that in the experiment of knocking out the LpaMA and UgpA double genes, the single-plasmid editing system produced 7 ± 1 transformants, and the double-plasmid editing system produced 60 ± 7 transformants, almost 10 times the number of the single-plasmid; in terms of the gene editing positive rate, the double-plasmid editing system remained at 75%, while the single-plasmid editing system only reached 13% ± 12%.

[0106] Example 4: Knockout of LpaMA, UgpA, and UlpRTF1 triple genes

[0107] The specific steps are as follows:

[0108] 1. Preparation of competent cells of L. paralimentarius strain containing plasmid pCRI01

[0109] The preparation method is as described in Example 2.

[0110] 2. Construction of plasmid pCRI02-ΔLpaMAΔUgpAΔUlpRTF1

[0111] (1) According to Figure 2The method shown. Using pCRI02 as a template, linearize plasmid pCRI02 with P23 and the sgRNA module as the boundaries. When designing primers, introduce an XbaⅠ restriction site at the 3' end of P23 and a SpeⅠ restriction site and the gRNA corresponding to UlpRTF1 at the 5' end of the sgRNA module; using pCRI02-1 as a template, PCR amplify the sgRNA module-P23 fragment. When designing primers, introduce a SpeⅠ restriction site and the gRNA corresponding to LpaMA (UgpA) at the 5' end of the sgRNA module and an XbaⅠ restriction site at the 3' end of P23.

[0112] (2) The linearized vector pCRI02 and the sgRNA module-P23 fragment are ligated by T4 DNA ligase in the order of gRNA to form vector pCRI02-ΔLpaMAΔUgpAΔUlpRTF1, and the subsequent work refers to the operation in Example 2.

[0113] 3. Construction of the homologous repair templates for ΔLpaMA, ΔUgpA, and ΔUlpRTF1

[0114] The construction method is similar to the method described in Example 2. Concentrate the concentration of each repair template to 330 ng / μL

[0115] 4. Construction of the L. paralimentarius LBM12001ΔlpaMAΔUgpAΔUlpRTF1 strain

[0116] (1) Pipette 10 μL of plasmid pCRI02-ΔlpaMA, 3.3 μL of ΔLpaMA, 3.3 μL of ΔUgpA, and 3.3 μL of the ΔUlpRTF1 homologous repair template into 100 μL of the competent cells of the L. paralimentarius strain containing the pCRI01 plasmid that has been pre-thawed, and perform electrotransformation under the conditions of 2.5 kV and 5 ms. The electrotransformed bacterial solution is added to 900 μL of warm MRS medium and then incubated and cultured at 37°C for 2 h.

[0117] (2) Place the bacterial solution after the culture in a 1.5 mL centrifuge tube and centrifuge at 8000 r / min for 1 min. Discard 900 μL of the supernatant, and evenly spread the remaining bacterial solution on an agar MRS plate containing erythromycin resistance, and statically culture at 37°C for 48 h.

[0118] (3) Pick the transformants on the agar plate and inoculate them into antibiotic-free liquid MRS medium for culture for 24 h. Extract the genomes of the transformants according to the method described in the genomic DNA extraction kit (Vazyme), and send the extracted genomes to Shanghai Sangon Biotech Co., Ltd. for sequencing to verify the results. The comparison results with the single plasmid editing tool are as Figure 3 shown.

[0119] 5. Plasmid curing

[0120] According to the method described in Example 2.

[0121] The results showed that: in the experiment of knocking out the three genes of lpaMA, UgpA, and UlpRTF1, the dual-plasmid system still showed great superiority. In terms of transformation efficiency, the dual-plasmid system produced 57 ± 8 transformants, while the single-plasmid system had a transformation efficiency close to 0 due to the too large plasmid volume; in terms of editing efficiency, the dual-plasmid system achieved a positive mutation rate of 46% ± 7%, still maintaining at a relatively high level.

[0122] Example 5: Construction of a strain with modified LpaMA A375P / S257P sites in L.paralimentarius gene and silenced expression of the UlpRTF1 transcriptional repressor and its application in bread anti-aging

[0123] 1. Construction of engineering strains

[0124] The engineering strains refer to Example 4.

[0125] Modification of the LpaMA A375P / S257P sites in the L.paralimentarius gene:

[0126] The wild-type maltogenic amylase is derived from Lactobacillus paralimentarius.

[0127] The gene encoding maltogenic amylase was chemically synthesized on the vector pET28a and transformed into Escherichia coli BL21(DE3) to form the wild-type pET28a-LpaMA / E.coli BL21 of this maltogenic amylase. The NCBI accession number of the gene is No. WP_025085606.1. Using the whole plasmid PCR technique, site-directed mutagenesis was performed with the obtained recombinant plasmid pET28a-LpaMA as the template to obtain mutants A375P, S257P, and A375P / S257P.

[0128] Amino acid sequence:

[0129] MGNTPAIYHRPDSEFAYLYKDNLMHIRLRTARGDIKEVGLLHGDPYTLDTDHWQDQPTPMKRYLTTDLYDFWTIEVTEPFKRISYAFKVTGNDGITIFYGDHGVFSFEKSVYDSPDNYFRLPYFHEVDRFKAPEWVKKTVWYQIFPERFANGDKSNDPENTLPWGSKTPSATDFFGGDLQGVIDHLDHLTDLGVNGIYFCPIFKAKSNHKYDTIDYMEIDPAFGDKATFKKLVQECHKRGIKVMLDAVFNHMGDSSPQWLDVVKNGKNSKYADWFHINKFPVSYEDDGFDQAKNISYDTFAFTPHMPKLNTANPEVKEYLLKIAKYWIEEFDIDAWRLDVANEVDHEFWRDFRRACDSVKKDFYILGEVWHSSQPWLQGDQFSAVMNYAYTDSISKYLIKKQIPIEKMVSDINDQLMLYRDQTDQIQFNVLDSHDTARLLTETNDDKDLMKQVLAFTYLQPGVPCIYYGDEIGMDGGNDPECRKCMVWDKSQQDLNLYDFFKHLIAFRKDNQKVLSEGEMVWEKVCDDGLLILSRKLNGTEVKVILNAGDTPKQVTTNQSVVLSNLVTENNHELTVDPKGFALVK.

[0130] Nucleotide sequence:

[0131]

[0132] Among them, the primers used for the mutation A375P are as follows:

[0133] A375P-For: 5’-TCACAACCGTGGCTCCAAGGAGATCAGTTTAG-3’;

[0134] A375P-Rev: 5’-TGGAGCCACGGTTGTGAGGAGTGCCAAACTTCA-3’.

[0135] The primers used for the mutation S257P (A375P / S257P mutant) are as follows:

[0136] S257P-For: 5’-CAGTTCACCGCAATGGTTGGATGTCGTTAAAAAT-3’;

[0137] S257P-Rev: 5’-ACCATTGCGGTGAACTGTCACCCATATGATTGAAA-3’.

[0138] The PCR reaction was carried out in a 50 μL system, and the reaction conditions were pre-denaturation at 94 °C for 4 min; then 30 cycles were entered: denaturation at 98 °C for 10 s, annealing at 55 °C for 5 s, extension at 72 °C for 5 min; finally, extension at 72 °C for 10 min and incubation at 4 °C.

[0139] The PCR amplification products were detected by 1% agarose gel electrophoresis. After the detection, 0.5 μL of methylation template digestion enzyme (Dpn I) was added to 10 μL of the amplification products, and they were mixed by pipetting with a pipette tip and reacted at 37 °C for 1.5 h. The amplification products treated with Dpn I were transformed into Escherichia coli E. coli BL21(DE3), and the transformation products were spread on LB solid medium and cultured at 37 °C for 8 - 10 h. 20 transformants were picked from the LB solid medium, inoculated into LB liquid medium for culture, and the plasmid was extracted after culturing at 37 °C for 10 h. The plasmid was sequenced, and if the sequencing was correct, recombinant Escherichia coli containing the genes encoding the mutants A375P, S257P, and A375P / S257P was obtained.

[0140] (2) Inductive culture and protein purification of the maltogenic amylase mutant

[0141] Using the maltogenic amylase with the amino acid sequence shown in SEQ ID NO.1 as the wild type, the obtained recombinant Escherichia coli pET28a-LpaMA / E. coli BL21 and the recombinant Escherichia coli containing the genes encoding mutants A375P, S257P, and A375P / S257P were spread on LB solid medium and cultured at 37 °C for 8 - 10 h to obtain single colonies. Single colonies were picked and inoculated into LB liquid medium and cultured at 37 °C, 200 rpm for 6 - 8 h to obtain seed liquid. The seed liquid was inoculated into LB liquid medium at an inoculation amount of 2% (v / v) and cultured at 37 °C, 200 rpm for 2 - 3 h. Then, IPTG with a final concentration of 0.1 mmol / L was added to the fermentation broth, and the induction culture was continued at 17 °C, 200 rpm for 12 - 17 h to obtain the fermentation broth. The fermentation broth was centrifuged at 4 °C, 8000 rpm for 5 min, the supernatant was discarded, and the precipitate was washed twice with 9% normal saline to obtain the wet cells of the wild type, mutant A375P, S257P, and A375P / S257P.

[0142] The wet cells were resuspended in buffer A and ultrasonically disrupted, and then centrifuged at 10000 rpm, 4 °C for 30 min to obtain the crude enzyme solution. After filtering with a 0.22 um aqueous filter membrane, the sample was slowly loaded onto a Ni-NAT affinity chromatography column. After loading, it was first washed with buffer A and then eluted with a gradient of buffer B. The elution peak corresponding to 300 mmol / L imidazole was collected, and then the imidazole in the pure enzyme was removed with a desalting column to obtain the pure enzymes of the wild type, mutant A375P, S257P, and A375P / S257P.

[0143] (3) Hydrolytic activity of different maltogenic amylase mutants on starch

[0144] Soluble starch (10 mg / mL) was added to 50 mol / L citrate buffer (pH 4.0 - 6.0), phosphate buffer (pH 6.0 - 7.0), and Tris-HCl buffer (pH 7.0 - 9.0) respectively to obtain reaction systems. 500 ul of each reaction system was incubated at 40 - 70 °C for 3 min, and then 100 ul of the pure enzymes of the wild type, mutant A375P, S257P, and A375P / S257P obtained in Example 2 was added to start the reaction. The control group did not contain the enzyme solution, and other components were the same. The reaction was carried out at 40 - 70 °C for 10 min. 10 ul of the product was diluted 20 times and an equal volume of 200 ul DNS (3,5-dinitrosalicylic acid) was added to end the reaction. After a 5-min boiling water bath at 100 °C and then cooling to room temperature on ice, the absorbance change at 540 nm was recorded to obtain the hydrolytic activities of the wild type, mutant A375P, S257P, and A375P / S257P on starch.

[0145] Hydrolytic activity (U / mg) = Ew × k × V / t / N × L × Protein concentration of pure enzyme solution

[0146] Among them, Ew is the absorbance change value at 540 nm within 10 min; k is the reducing sugar standard curve coefficient, with the unit of μmol / mL; V is the volume of the enzyme activity assay reaction system, with the unit of mL, which is 0.6 here; t is the reaction time, with the unit of min, which is 10 here; N is the enzyme solution dilution factor; L is the volume of added enzyme solution, with the unit of mL, which is 0.1 here; the protein concentration of the enzyme solution is measured using the Bradford protein kit (For measuring protein concentration with the Bradford protein kit, please refer to the literature: Zhou-Pan XR, ESérée, Zhou XJ, et al. Involvement of Human Liver Cytochrome P450 3A in Vinblastine Metabolism: Drug Interactions1[J]. Cancer Research, 1993, 53(21): 5121-5126.), with the unit of mg / mL.

[0147] The results are as follows: The optimal working temperature and pH of the wild type are 50 °C and 5.0 respectively, and the maximum hydrolytic activity is 274.79 U·mg -1 ; The optimal working temperatures of mutants A375P, S257P, and A375P / S257P are 60, 60, and 65 °C respectively; the maximum hydrolytic activities are 425.89, 325.82, and 430.15 U·mg -1 .

[0148] (4) Temperature stability of different maltogenic amylase mutants

[0149] The wild type, mutants A375P, S257P, and A375P / S257P enzyme solutions obtained in Example 2 were incubated in a water bath at 60 °C for 60 min. Samples were taken at regular intervals, and the catalytic activities of the wild type, mutants A375P, S257P, and A375P / S257P of maltogenic amylase on starch were measured at 50 °C after incubation. Taking the activity before incubation as 100%, the relative activity was calculated by comparing with the remaining activity after incubation to investigate the temperature stability and half-life of the wild type, mutants A375P, S257P, and A375P / S257P of maltogenic amylase. The Origin software was used to perform a linear fit on the calculated residual activity and the corresponding incubation time to obtain the half-life (t 1 / 2 ).

[0150] The results were as follows: the residual relative activities of mutants A375P, S257P, and A375P / S257P were 70.63%, 47.59%, and 95.27% respectively, and the half-lives were 109, 56, and 227 min respectively; the residual relative activity and half-life of the wild type under the same conditions were 30.73% and 32 min respectively. It can be seen that the double-point mutant A375P / S257P further achieved excellent thermal stability on the basis of the improved thermal stability of the single-point mutants A375P and S257P.

[0151] (5) Catalytic activities of wild-type maltogenic amylase and double-point mutant

[0152] In a reaction system of phosphate buffer (50 mmol / L, pH 5.0) containing 0 - 30 mg / mL β-cyclodextrin, 100 μL of the pure enzymes of the wild type and the double-point mutant A375P / S257P obtained in Example 2 were added to start the reaction. The control group did not contain the enzyme solution, and other components were the same; the reaction was carried out at the optimal temperature for 10 min to obtain the hydrolysis activities of the wild type and the double-point mutant A375P / S257P towards β-cyclodextrin; Origin software was used to perform non-linear fitting on the calculated hydrolysis activities and the corresponding substrate concentrations to obtain the catalytic efficiency (k cat / K m ) of the enzyme.

[0153] The results were as follows: the catalytic efficiency (k cat / K m ) of the wild type towards β-cyclodextrin was 13.36 mM -1 ·s -1 ; the catalytic efficiency (kcat / Km) of the double-point mutant A375P / S257P towards β-cyclodextrin was 30.42 mM -1 ·s -1 .

[0154] 2. Preparation of sourdough

[0155] The engineered L. paralimentarius strain was activated for two generations and cultured to the stationary phase. 100 μL of the bacterial solution was inoculated into 5 mL of MRS medium and cultured at 37 °C for 16 h, centrifuged at 8000 r / min for 5 min, and washed twice with sterile normal saline to obtain bacterial sludge. The centrifuged bacterial sludge was resuspended in sterile water again and inoculated into wheat substrate for culture. The dough yield DY value was 250. The bacterial sludge obtained by centrifuging every 10 mL of MRS medium was inoculated into 100 g of sourdough and placed in a constant temperature and humidity incubator at 30 °C and 85% humidity for 24 h to obtain sourdough. The DY value is the mass relationship between the substrate powder and water, and the calculation formula is as follows:

[0156] DY value = (1 + m 水 / m 粉 )×100

[0157] where: m 粉 is the mass (g) of the matrix powder; m 水 is the mass (g) of water.

[0158] 3. Preparation of wheat bread and sourdough bread

[0159] Use a 300 g bread formula system: 300 g wheat flour, 4.5 g yeast, 18 g sugar, 3 g salt, 180 g water. The specific processing procedure of the bread is as follows:

[0160] (1) Weigh wheat flour, yeast, white granulated sugar, edible salt (and maltogenic amylase) according to the formula system, pour them into a mixing bowl and stir until the ingredients are evenly mixed, then pour in an appropriate amount of water, stir at low speed for 3 min, and stir at high speed for 5 min to complete the kneading of the dough;

[0161] (2) Take out the dough, knead it manually moderately, and cover it with plastic wrap for 5 min;

[0162] (3) Roughly cut the relaxed dough into pieces of about 90 g each, perform preliminary shaping and degassing operations, and then cover it with plastic wrap again and let it proof for the first time at room temperature (5 min);

[0163] (4) Roll out each small dough in turn, ensure that the air bubbles are exhausted, then roll it up and put it into a baking mold, and let it proof for the second time at 37 °C and a relative humidity of 85% (90 min);

[0164] (5) Bake the proofed bread at 170 °C for the upper fire and 210 °C for the lower fire for 21 min;

[0165] The formula of the sourdough bread is: 255 g flour, 112.5 g sourdough, 4.5 g yeast, 18 g sugar, 3 g salt, 112.5 g water. The specific processing procedure of the bread is similar to the above.

[0166] 4. Test on the baking anti-aging performance of the sourdough made by engineering-transformed L. paralimentarius

[0167] After baking, the bread was left to cool at room temperature for 2 hours, and then its hardness was measured by Texture Profile Analysis (TPA). The remaining bread was stored at 4°C. The hardness changes of the bread after storage for 1, 3, 5, and 7 days were measured to evaluate its anti-aging properties. Before measuring the hardness of the bread, the bread was placed at room temperature for 2 hours. The hardness changes of ordinary wheat bread, bread made with sourdough made with wild-type L. paralimentarius, and bread made with sourdough made with engineered L. paralimentarius are shown in the figure below. Figure 4 shown.

[0168] The results show:

[0169] After 7 days of storage at 4°C, the hardness of standard wheat bread was 2070.7g ± 92.5g, while that of the WT sourdough group was 1770.3g ± 31.4g. This decreased to 1208.7g ± 38.5g in the WHU sourdough group. This suggests that sourdough made from wild-type L. paralimentarius significantly inhibits staling of wheat bread, while sourdough made from genetically engineered L. paralimentarius exhibits even stronger anti-staling properties.

[0170] The maltogenic amylase gene, LpaMA, was modified for thermal stability. Pure enzyme baking experiments confirmed that the heat-resistant LpaMA mutant, A375P / S257P, exhibited higher enzyme activity and heat resistance, demonstrating superior anti-aging properties in bread baking applications. A transcriptional repressor of the LpaMA gene, UlpRTF1, was discovered and identified, and its transcriptional repression of LpaMA was verified by RT-qPCR. Using the aforementioned dual-plasmid gene editing system, a double-trait stacked mutant was rapidly constructed. Sourdough produced from this mutant exhibited superior anti-aging properties compared to the wild-type.

[0171] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A dual-plasmid multi-gene editing system of Lactobacillus alimentarius based on the CRISPR / Cas9 technology, characterized in that, Comprising: (1) Plasmid pCRI01, containing the following elements: a Cas9 protein coding gene; a temperature-sensitive replicon pSC101; an sgRNA expression module targeting the second plasmid replicon, regulated by an inducible promoter Pnis; ampicillin resistance; (2) Plasmid pCRI02, containing the following elements: at least one sgRNA expression module, driven by the P23 promoter; a λ-Red recombinase system, containing Exo, Beta, and Gam genes; an erythromycin resistance marker gene; a kanamycin resistance gene.

2. The multi-gene editing method of the multi-gene editing system of Lactobacillus alimentarius dual-plasmid based on the CRISPR / Cas9 technology according to claim 1, characterized in that, Including the following steps: (1) Electrotransform pCRI01 into competent Lactobacillus parafarraginis strains by electroporation to obtain competent Lactobacillus parafarraginis strains containing plasmid pCRI01; (3) Construct plasmid pCRI02 containing at least one sgRNA corresponding to the target gene; (4) Construct a DNA homologous repair template for the target gene; (5) Co-electroporate the plasmid pCRI02 and the DNA homologous repair template for the target gene into the competent Lactobacillus parafarraginis containing plasmid pCRI01, incubate, concentrate the cell concentration, evenly spread on an agar MRS plate containing ampicillin and erythromycin resistance, and statically culture at 37°C to obtain transformants; (6) Pick the transformants into a liquid MRS medium for culture, extract the genome and send it for sequencing to verify the editing results; (7) Inoculate the transformants with the correct editing results into a liquid MRS medium containing 10 ng / μL Nisin and culture overnight at 40°C.

3. The multi-gene editing method according to claim 2, characterized in that, In the step (2), the mass ratio of the plasmid pCRI02 to the DNA homologous repair template for the target gene is 1:2, and the volume ratio is 1:

1.

4. The multi-gene editing method according to claim 2, characterized in that, In step (2), the sgRNA expression module of the plasmid pCRI02 realizes the tandem insertion of multiple sgRNAs by using the XbaⅠ and SpeⅠ restriction enzyme sites through the BioBricks assembly method.

5. A method for editing a food-derived Lactobacillus strain using a gene editing method based on a CRISPR-Cas9 dual plasmid system, characterized in that, Using Lactobacillus parafarraginis as the starting strain, knockout the target gene by the multi-gene editing method described in any one of claims 2-4 to obtain an edited Lactobacillus parafarraginis strain.

6. The method for editing a food-like Lactobacillus strain by the gene editing method based on the CRISPR-Cas9 dual plasmid system according to claim 5, characterized in that, The target gene is at least one of LpaMA, UgpA, and UlpRTF1.

7. The method for editing a food-like Lactobacillus strain by the gene editing method based on the CRISPR-Cas9 dual plasmid system according to claim 5 or 6, characterized in that, The Lactobacillus parafarraginis is Lactobacillus parafarraginis LBM12001.

8. The method for editing a food-like Lactobacillus strain by the gene editing method based on the CRISPR-Cas9 dual plasmid system according to claim 7, characterized in that, In the gene LpaMA of the Lactobacillus parafarraginis LBM12001, alanine at the 375th position and serine at the 257th position are respectively mutated to proline, and at least one of the genes UgpA and UlpRTF1 is knocked out.

9. A Lactobacillus strain of food category, characterized in that, The Lactobacillus parafarraginis lacks at least one of the genes LpaMA, UgpA, and UlpRTF1.

10. The Lactobacillus strain of the food-like according to claim 9, characterized in that, The Lactobacillus parafarraginis is Lactobacillus parafarraginis LBM12001.

11. The food-like Lactobacillus strain according to claim 9, characterized in that, In the gene LpaMA of the Lactobacillus parafarraginis LBM12001, alanine at the 375th position and serine at the 257th position are respectively mutated to proline, and at least one of the genes UgpA and UlpRTF1 is knocked out.

12. Use of the edited Lactobacillus parafarraginis strain according to any one of claims 9-11 in food preparation.

13. Use of the edited Lactobacillus alimentarius strain according to any one of claims 9-11 as a food ingredient.

14. Use of the edited Lactobacillus alimentarius strain according to any one of claims 9-11 as a bread improver.

15. The application according to claim 14, characterized in that, By adding the edited Lactobacillus alimentarius strain defined in any one of claims 9 to 11 to the dough and maintaining it at 37 °C for at least 90 minutes.

16. A food composition comprising the edited Lactobacillus alimentarius strain according to any one of claims 9 to 11.

17. Use of the edited Lactobacillus alimentarius strain defined in any one of claims 9 to 11 as a sourdough starter.

18. A sourdough comprising the edited Lactobacillus alimentarius strain according to any one of claims 9 to 12.

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