Food-like lactobacillus double-plasmid multi-gene editing system based on crisper / cas9 technology and application thereof

Abstract

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

Description

Technical Field

[0001] This invention relates to gene editing plasmids based on CRISPR / Cas9 technology and gene editing methods for food-like lactobacilli, belonging to the fields of genetic engineering and microbial genetics. Background Technology

[0002] Food-like Lactobacillus ( L. paralimentarius As an important type of lactic acid bacteria, it is deeply involved in the production process of traditional fermented foods such as sourdough, kimchi, and cheese. Its metabolites have a significant impact on the flavor, shelf life, and functional properties of these foods. 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 biomedicine fields due to their safety, probiotic properties, and tolerance to acidic environments. Their metabolic regulation capabilities directly affect food quality. Therefore, developing a suitable... L. paralimentarius Gene editing tools not only help to reveal metabolic regulation mechanisms, but also provide 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 technologies such as homologous recombination, transposon mutation, and zinc finger nucleases (ZFNs) are gradually being replaced by emerging CRISPR / Cas9-based gene editing technologies due to their low efficiency, cumbersome procedures, and difficulty in targeting multiple sites. Compared with traditional gene editing technologies, CRISPR / Cas9 gene editing technology has higher efficiency, stronger specificity, and wider applicability in lactic acid bacteria. Traditional gene editing relies on inefficient homologous recombination, while CRISPR / Cas9 guides the Cas9 enzyme to precisely cut target genes through sgRNA, greatly improving editing efficiency and supporting simultaneous editing of multiple genes, making it suitable for complex metabolic engineering of lactic acid bacteria. In addition, CRISPR / Cas9 can achieve scarless mutation through label-free homologous recombination repair (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, improves editing accuracy, and significantly shortens the experimental cycle compared to traditional methods. In our previous study, we used CRISPR / Cas9 in *Lactobacillus plantarum* (… L. plantarum The established gene-editing plasmid pHSP02 has been successfully used in... L. paralimentarius The gene was successfully knocked out in China. LpaMAThe efficiency reached 75%; however, when performing multi-gene editing tasks, the number of transformants and gene editing efficiency decreased sharply with the increase of target genes. It is speculated that this is because the mounting of the repair template resulted in an excessively large plasmid size, affecting transformation efficiency. The decrease in editing efficiency is attributed to the host's inability to adapt to the repair pressure caused by multi-site DNA double-strand breaks induced by the multi-sgRNA-Cas9 protein complex. The effectiveness of existing gene editing tools is limited to single sites, which severely restricts their potential application as cell factories.

[0004] Key strategies for achieving multi-gene editing based on CRISPR / Cas9 technology include reducing plasmid size, optimizing transformation efficiency, and introducing auxiliary mechanisms to improve homology repair capabilities. Summary of the Invention

[0005] One of the objectives of this invention is to provide a food-like lactobacillus dual-plasmid multi-gene editing system based on CRISPR / Cas9 technology.

[0006] The technical solution to achieve the above-mentioned invention objective is as follows: a food-like lactobacillus dual-plasmid multi-gene editing system based on CRISPR / Cas9 technology, comprising:

[0007] (1) Plasmid pCRI01 contains the following elements: Cas9 protein-coding gene; temperature-sensitive replicon pSC101; sgRNA expression module targeting plasmid pCRI02 replicon, regulated by the inducible promoter Pnis; ampicillin resistance;

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

[0009] The dual plasmid binding λ-Red recombination system described in this invention, the λ-Red recombination system Exo , Beta and Gam Genes have been codon-optimized to adapt to the host expression environment of food-like lactobacilli, which is beneficial for improving the expression of food-like lactobacilli (… L. paralimentarius (Multi-gene editing efficiency)

[0010] The second objective of this invention is to provide a multi-gene editing method for the aforementioned food-like Lactobacillus dual-plasmid multi-gene editing system based on CRISPR / Cas9 technology.

[0011] The technical solution to achieve the above-mentioned invention objective is as follows: A multi-gene editing method based on a CRISPR / Cas9-based food-like Lactobacillus dual-plasmid multi-gene editing system, comprising the following steps:

[0012] (1) pCRI01 was electroconverted into competent food-like Lactobacillus strains by electroconversion to obtain competent food-like Lactobacillus strains containing plasmid pCRI01;

[0013] (2) Construct plasmid pCRI02 containing at least one sgRNA corresponding to a target gene;

[0014] (3) Construct DNA homology repair templates for targeted genes;

[0015] (4) The plasmid pCRI02 and the target gene DNA homology repair template were co-electrotransformed into competent cells of food-like Lactobacillus containing plasmid pCRI01, incubated, the cell concentration was concentrated, and the cells were evenly spread on MRS agar plates containing ampicillin and erythromycin resistance. The cells were then incubated at 37 °C to obtain transformants.

[0016] (5) Select the transformants and culture them in liquid MRS medium, extract the genome and send it for sequencing to verify the editing results;

[0017] (6) Take the transformants with the correct editing results and inoculate them into liquid MRS medium containing 10 ng / μL Nisin and incubate overnight at 40°C.

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

[0019] In step (2), the sgRNA expression module of plasmid pCRI02 is assembled using the BioBricks method, and multiple sgRNAs are tandemly inserted using the XbaⅠ and SpeⅠ restriction sites.

[0020] Specifically, plasmid pCRI02 was used as a template for PCR, with the P23 promoter and sgRNA module as upstream and downstream components. The 3' primer of P23 contained an XbaI restriction site, and the 5' primer of the sgRNA module contained a SpeI restriction site and the target gene gRNA. The PCR product was a linearized pCRI02 plasmid. Using pCRI02-1 as a template, PCR was performed with the sgRNA module and P23 as upstream and downstream components. The 5' primer of the sgRNA module contained a SpeI restriction site and the target gene gRNA, and the 3' primer of P23 contained an XbaI restriction site. Multiple PCRs were performed for multi-sgRNA editing. All PCR products were purified and recovered using a DNA column and then assembled into BioBricks using the XbaI-SpeI restriction site to ensure consistent orientation of the target fragment.

[0021] The third objective of this invention is to provide a method for editing food-grade Lactobacillus strains using a gene editing method based on the CRISPR-Cas9 dual plasmid system.

[0022] Specifically, the method for editing food-like Lactobacillus strains using gene editing based on the CRISPR-Cas9 dual plasmid system involves using food-like Lactobacillus as the starting strain and knocking out the target gene using the aforementioned multi-gene editing method to obtain edited food-like Lactobacillus strains.

[0023] Target genes are at least LpaMA , UgpA and UlpRTF1 one of them.

[0024] The preferred food-grade lactobacillus is LBM12001. The food-grade lactobacillus (… Lactobacillus paralimentarius LBM12001 was deposited with the Guangdong Provincial Center for Microbial Culture Collection on July 21, 2022, and the collection registration number GDMCC 62232 was received. The deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0025] Furthermore, the gene of the aforementioned food-like Lactobacillus LBM12001 LpaMA The alanine at position 375 and the serine at position 257 were mutated to proline, and at least the gene was knocked out. UgpA and UlpRTF1 one of them.

[0026] A fourth objective of this invention is to provide edited food-like Lactobacillus strains.

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

[0028] The preferred food-grade Lactobacillus is Lactobacillus LBM12001. Further, the gene of said food-grade Lactobacillus LBM12001... LpaMA The alanine at position 375 and the serine at position 257 of the amino acid sequence were mutated to proline, and at least the gene was knocked out. UgpA and UlpRTF1 one of them.

[0029] A fifth objective of this invention is to provide the application of the edited food-like Lactobacillus strain.

[0030] Specifically, this relates to the application of the edited food-like Lactobacillus strains in food preparation; the application of the food-like Lactobacillus strains as food ingredients; and the application of the edited food-like Lactobacillus strains as bread improvers.

[0031] In one embodiment, when the edited food-like lactobacillus strain is used as a bread improver, it is added to dough and kept at 37°C for at least 90 minutes.

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

[0033] The present invention also provides the use of the above-described edited food-like lactobacillus strain as a sourdough starter; and sourdough comprising the above-described edited food-like lactobacillus strain.

[0034] In this invention, the food is dough or dough product, or paste or paste product, baked product, or bakery or pastry shop product.

[0035] Preferably, the baked products of this invention are bakery or pastry shop products. Bread or pastry products known in the art include, but are not limited to, bread, mochi, soft rolls, bagels, donuts, Danish pastries, hamburger rolls, pizza, pita bread, ciabatta, sponge cake, cream cake, pound cake, muffins, cupcakes, steamed cakes, waffles, brownies, cake donuts, yeast-leavened donuts, baguettes, bread rolls, savory biscuits, sweet biscuits, pie crusts, rusks, and / or other baked products. More preferably, the baked products are bread, baguettes, and / or bread rolls.

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

[0037] 1. This invention, by splitting the large-volume plasmid pIBE01 into the smaller-volume pCRI01 and pCRI02 dual-plasmid systems, significantly increases the number of transformants after resistance screening. Thanks to the assistance of the λ-Red recombinase system, the gene editing efficiency mediated by the pCRI plasmid is greatly improved. Furthermore, when performing single-gene editing, the single-plasmid system requires 6 days; when editing multiple genes, the time required is (4+2N) days. The dual-plasmid editing systems pCRI01 and pCRI02 require 5 days for single-gene editing. For multi-gene editing, to ensure efficiency, the strategy should be adjusted according to the number of genes being edited, with multiple rounds of editing performed in groups of three genes. Therefore, gene editing mediated by the dual-plasmid system greatly reduces the time required for large-scale genetic engineering. Attached Figure Description

[0038] Figure 1 This is a structural diagram of the dual plasmid system of the present invention.

[0039] Figure 2 This is a schematic diagram of the principle of the dual plasmid system of the present invention.

[0040] Figure 3 The conversion rate of the dual plasmid system of this invention is given.

[0041] Figure 4 Modification of the A375P / S257P site to superimpose UlpRTF1 transcriptional repressor silencing expression L. paralimentarius Graph showing changes in bread storage of wild-type Lactobacillus and blank control.

[0042] Note: Control represents regular wheat bread, WT-Sourdough represents bread with added wild wheat varieties. L. paralimentarius The sourdough wheat bread and WHU-Sourdough bread produced were modified with LpaMA A375P / S257P sites and silenced by UlpRTF1 transcriptional repressor. L. paralimentarius Sourdough wheat bread made from this strain.

[0043] Figure 5 The optimal pH for the activity of wild-type maltose amylase was determined.

[0044] Figure 6 The optimal operating temperature and hydrolytic activity of wild-type and mutant A375P, S257P, and A375P / S257P were determined.

[0045] Figure 7 Temperature stability of wild-type and mutant A375P, S257P, and A375P / S257P at 60℃ was determined.

[0046] Figure 8 The half-life of wild-type and mutant A375P, S257P, and A375P / S257P at 60°C ( t 1 / 2 Fitting. Detailed Implementation

[0047] Various exemplary embodiments 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, features, and embodiments of the present invention.

[0048] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0049] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0050] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0051] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0052] The dual plasmid gene editing system based on CRISPR / Cas9 technology provided by this invention, such as Figure 1As shown, this invention can significantly reduce the experimental procedures and time required for multi-gene editing while maintaining high editing efficiency. Two plasmids were constructed in this invention: pCRI01 (8880 bp) and pCRI02 (6740 bp). pCRI01 is primarily used to express the Cas9 protein. To facilitate plasmid elimination, a temperature-sensitive replicon, pSC101, was introduced. This replicon cannot replicate at 37 ℃, ensuring convenient elimination of pCRI01. Furthermore, to facilitate the elimination of plasmid pCRI02, pCRI01 was equipped with an sgRNA targeting the pCRI02 replicon. This sgRNA is induced by the Pnis promoter. After pCRI02 completes sgRNA expression, sgRNA expression in pCRI01 is induced with a final concentration of 100 ng / µL Nicin, cleaving the pCRI02 replicon to eliminate the plasmid. pCRI02 is loaded with sgRNA to guide the Cas9 protein to the target gene. In addition, a species-codon-optimized λ-Red recombinase system derived from λ phage was introduced into pCRI02. λ-Red recombination is mediated by three proteins: Exo, Beta, and Gam. Exo is an exonuclease that degrades exogenous double-stranded DNA along the 5'-3' direction, producing single-stranded DNA. Beta is a single-stranded DNA-binding protein that protects single-stranded DNA and promotes homologous pairing with chromosomal DNA. Gam is an inhibitor that prevents the bacteria's own RecBCD enzyme from degrading exogenous DNA, thus eliminating the need for homology repair templates to be encoded on plasmids, further improving gene editing efficiency. To facilitate the loading of multiple sgRNAs, another version of pCRI02-1 was also constructed. In this version, the P23-sgRNA module expression element is oriented in the opposite direction to the original version, and the loading of multiple sgRNAs adopts the classic BioBricks method, the principle of which is illustrated in the diagram below. Figure 2 As shown.

[0053] Specifically, plasmid pCRI02 was used as a template for PCR, with the P23 promoter and sgRNA module as upstream and downstream components. The 3' primer of P23 contained an XbaI restriction site, and the 5' primer of the sgRNA module contained a SpeI restriction site and the target gene gRNA. The PCR product was a linearized pCRI02 plasmid. Using pCRI02-1 as a template, PCR was performed with the sgRNA module and P23 as upstream and downstream components. The 5' primer of the sgRNA module contained a SpeI restriction site and the target gene gRNA, and the 3' primer of P23 contained an XbaI restriction site. Multiple PCRs were performed for multi-sgRNA editing. All PCR products were purified and recovered using a DNA column and then assembled into BioBricks using the XbaI-SpeI restriction site to ensure consistent orientation of the target fragment.

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

[0055] (1) Preservation of glycerol tubes L. paralimentarius The strain was activated by streaking on MRS agar plates, and after static incubation at 37°C, a single colony was picked and transferred to 20 mL of liquid MRS medium, and then incubated at 37°C for 24 h.

[0056] (2) Take 2 mL of overnight culture and inoculate it into 50 mL of MRS liquid medium (containing 0.4 mol·L⁻¹ sucrose). -1 5 g·L glycine -1 ), 37 ℃, rotation speed 200 r·min -1 Cultured to OD 600 It is 0.3-0.4.

[0057] (3) Pour the bacterial culture into a pre-cooled centrifuge tube and let it stand on ice for 30 min.

[0058] (4) 4 ℃, 5000 r·min -1 Centrifuge for 10 min to collect the bacterial cells and discard the supernatant.

[0059] (5) Electroconversion buffer (containing 0.4 mol·L⁻¹ sucrose) -1 5 g·L glycine -1 4 ℃, 5000 r·min -1 Centrifuge for 5 min, wash the cells twice, discard the supernatant, collect the cells into a pre-cooled centrifuge tube, and let stand on ice for 10 min.

[0060] (6) Resuspend the bacterial cells in electroporation buffer. 4 ℃, 5000 r·min -1 Centrifuge for 10 min and discard the supernatant.

[0061] (7) Resuspend the bacterial cells by adding 1 mL of electroporation buffer, aliquot 100 μL into each pre-cooled 1.5 mL EP tube, and store at -80 ℃.

[0062] Example 2: Knockout of the LpaMA gene

[0063] 1. Contains pCRI01 plasmid L. paralimentarius Preparation of competent strains

[0064] (1) Take 10 μL of plasmid pCRI01 and add it to 100 μL of pre-thawed liquid. L. paralimentarius Electroporation was performed in competent cells at 2.5 kV and 5 ms. After electroporation, the bacterial culture was added to 900 μL of MRS medium and incubated at 37 °C for 2 h for recovery.

[0065] (2) After the culture is completed, the bacterial culture is placed in a 1.5 mL centrifuge tube and centrifuged at 8000 r / min for 1 min. 900 μL of supernatant is discarded, and the remaining bacterial culture is evenly spread on an agar MRS plate containing ampicillin resistance and incubated at 37 ℃ for 48 h.

[0066] (3) Select transformants from the agar plate and prepare competent cells according to the method described in Example 1.

[0067] 2. pCRI02-Δ LpaMA plasmid construction

[0068] (1) The sgRNA of the corresponding gene was designed using GeniousPrime 8.0.1. The sgRNA was inserted into the corresponding position by whole plasmid PCR.

[0069] (2) The whole plasmid PCR conditions were 98 ℃ for 1 min, (98 ℃ for 30 s, 58 ℃ for 15 s, 72 ℃ for 3 min) × 30 cycles, 72 ℃ for 5 min, and 16 ℃ for 5 min. 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. PCR products containing the correct gel electrophoresis bands were purified using a purification kit (ThermoScientific).

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

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

[0072] (3) The purified gene sample was digested with Dpn1 to remove the empty plasmid template. The digested mixture was incubated at 37°C for 1 hour and then purified again using a purification kit (Thermo Scientific). The purified product was then transformed into E. coli using chemical transformation. E. coli In DH5α, the bacterial culture was spread onto LB agar plates containing kanamycin resistance and incubated overnight at 37 °C.

[0073] (4) Select transformants and inoculate them into antibiotic-free LB liquid medium. After culturing for 8 h, extract plasmid pCRI02-Δ according to the instructions of the plasmid extraction kit (Vazyme). LpaMA .

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

[0075] 3. Δ LpaMA Construction of homology repair template

[0076] (1) Select LpaMA The upstream and downstream 500 bp bases form homologous arms, with L. paralimentarius Using the genome as a template, the PCR reaction was performed at 98 °C for 1 minute, followed by 30 cycles of (98 °C for 30 seconds, 58 °C for 15 seconds, 72 °C for 15 seconds), then at 72 °C for 5 minutes, and finally at 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, 25 μL of RimeSTAR MAX DNA polymerase, and 22 μL of ultrapure water. Homologous arms were amplified separately, and the PCR products were purified using a Thermo Scientific purification kit.

[0077] Upstream homologous arm forward primer: 5'-TCCCAATGGCTTTGGATGAA-3';

[0078] Upstream homologous arm reverse primer: 5'-TGAAATAACCTCTCATTTTTAAAAATTTG-3';

[0079] Downstream homologous arm forward primer: 5'-AAAAATGAGAGGTTATTTCAAGAGGTGCTTAATTTGCAAAAACA-3';

[0080] Downstream homologous arm reverse primer: 5'-AGAGGTGAAGATAGTATTGACCAGTCTT-3'.

[0081] (2) The purified products of the upstream and downstream homologous arms were ligated by overlap extension PCR. The ligation products were verified by gel electrophoresis, and the purified PCR products were used as homology repair templates. The overlap method was as follows: the upstream and downstream homologous arms were diluted to 10 ng / uL. The first round of PCR used a 10 μL system, which included 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 were 98 ℃ for 1 min, (98 ℃ for 30 s, 58 ℃ for 15 s, 72 ℃ for 30 s) × 10 cycles, 72 ℃ for 5 min, and 16 ℃ for 5 min. The second round of PCR used a 50 μL system, which included 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 PCR product, 25 μL of PrimeSTAR MAX DNA polymerase, and 22 μL of ultrapure water. The reaction conditions were 98 °C for 1 min, followed by 30 cycles of (98 °C for 30 s, 58 °C for 15 s, 72 °C for 30 s), then 72 °C for 5 min, and 16 °C for 5 min. The product was validated by gel electrophoresis and then purified using a purification kit (ThermoScientific).

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

[0083] 4. L. paralimentarius LBM12001 Δ lpaMA Construction of strains

[0084] (1) Take 10 μL of plasmid pCRI02-Δ lpaMA 10 μL Δ LpaMA Homologous repair template was modified by adding 100 μL of pre-thawed pCRI01 plasmid. L. paralimentarius The competent cells of the strain were electroporated at 2.5 kV for 5 ms. After electroporation, the bacterial culture was added to 900 μL of MRS medium and incubated at 37 °C for 2 h for recovery.

[0085] (2) After the culture is completed, the bacterial culture is placed in a 1.5 mL centrifuge tube and centrifuged at 8000 r / min for 1 min. 900 μL of supernatant is discarded, and the remaining bacterial culture is evenly spread on an MRS agar plate containing erythromycin resistance and incubated at 37 ℃ for 48 h.

[0086] (3) Transformants were picked from agar plates and inoculated into antibiotic-free liquid MRS medium and cultured for 24 h. The genomes of the transformants were extracted according to the method described in the genomic extraction kit (Vazyme). The extracted genomes were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing verification. Figure 3 The comparison results with single plasmid editing tools are as follows: Figure 3 As shown.

[0087] 5. Plasmid elimination

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

[0089] The results showed that the dual plasmid editing system produced 63±11 transformants in the single gene knockout experiment, which was a significant increase compared to the 24±2 transformants produced by the single plasmid, and the gene editing positive rate remained at 75%.

[0090] Example 3: LpaMA, UgpA Double gene knockout

[0091] The specific steps are as follows:

[0092] 1. Contains pCRI01 plasmid L. paralimentarius Preparation of competent strains

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

[0094] 2. pCRI02-Δ LpaMA Δ UgpA plasmid construction

[0095] (1) According to Figure 2 The method is shown. Using pCRI02 as a template, and p23 and the sgRNA module as the boundary, the plasmid pCRI02 was linearized. When designing primers, an XbaI restriction site was introduced at the 3' end of p23, and a SpeI restriction site was introduced at the 5' end of the sgRNA module. UgpA The corresponding gRNA; using pCRI02-1 as a template, the sgRNA module-P23 fragment was amplified by PCR. When designing primers, a SpeⅠ restriction site was introduced at the 5' end of the sgRNA module. LpaMA The corresponding gRNA has an XbaI restriction site introduced 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 gRNA sequence to form the vector pCRI02-Δ LpaMA Δ UgpA The subsequent work shall refer to the operation in Example 2.

[0097] 3. Δ LpaMA Δ UgpA Construction of homology repair template

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

[0099] 4. L. paralimentarius LBM12001 Δ lpaMA Δ UgpA Construction of strains

[0100] (1) Take 10 μL of plasmid pCRI02-Δ lpaMA 5 μL Δ LpaMA With, 5 μL Δ UgpA Homologous repair template was modified by adding 100 μL of pre-thawed pCRI01 plasmid. L. paralimentarius The competent cells of the strain were electroporated at 2.5 kV for 5 ms. After electroporation, the bacterial culture was added to 900 μL of MRS medium and incubated at 37 °C for 2 h for recovery.

[0101] (2) After the culture is completed, the bacterial culture is placed in a 1.5 mL centrifuge tube and centrifuged at 8000 r / min for 1 min. 900 μL of supernatant is discarded, and the remaining bacterial culture is evenly spread on an MRS agar plate containing erythromycin resistance and incubated at 37 ℃ for 48 h.

[0102] (3) Transformants were picked from agar plates and inoculated into antibiotic-free liquid MRS medium and cultured for 24 h. The genomes of the transformants were extracted according to the method described in the genomic extraction kit (Vazyme). The extracted genomes were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing verification. Figure 3 The comparison results with single plasmid editing tools are as follows: Figure 3 As shown.

[0103] 5. Plasmid elimination

[0104] Follow the method described in Example 2.

[0105] The results showed that: during knockout LpaMA , UgpA In the dual-gene experiment, the single-plasmid editing system produced 7±1 transformants, while the dual-plasmid editing system produced 60±7 transformants, nearly 10 times the number of single-plasmid transformants. In terms of gene editing positivity, the dual-plasmid editing system maintained 75%, while the single-plasmid editing system only reached 13%±12%.

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

[0107] The specific steps are as follows:

[0108] 1. Contains pCRI01 plasmid L. paralimentarius Preparation of competent strains

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

[0110] 2. pCRI02-Δ LpaMA Δ UgpA Δ UlpRTF 1. Construction of plasmids

[0111] (1) According to Figure 2 The method is shown. Using pCRI02 as a template, and p23 and the sgRNA module as the boundary, the plasmid pCRI02 was linearized. When designing primers, an XbaI restriction site was introduced at the 3' end of p23, and a SpeI restriction site was introduced at the 5' end of the sgRNA module. UlpRTF The corresponding gRNA; using pCRI02-1 as a template, the sgRNA module-P23 fragment was amplified by PCR. When designing primers, a SpeⅠ restriction site was introduced at the 5' end of the sgRNA module. LpaMA ( UgpA The corresponding gRNA has an XbaI restriction site introduced at the 3' end of P23.

[0112] (2) The linearized vector pCRI02 and the sgRNA module-P23 fragment were ligated by T4 DNA ligase in gRNA sequence to form the vector pCRI02-Δ LpaMA Δ UgpA Δ UlpRTF 1. Follow the steps in Example 2 for subsequent work.

[0113] 3. Δ LpaMA Δ UgpA Δ UlpRTF 1. Construction of the Homologous Repair Template

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

[0115] 4. L. paralimentarius LBM12001 Δ lpaMA Δ UgpA Δ UlpRTF Construction of strain 1

[0116] (1) Take 10 μL of plasmid pCRI02-Δ lpaMA 3.3 μL Δ LpaMA With, 3.3 μL Δ UgpA3.3 μLΔ UlpRTF 1. Add 100 μL of pre-thawed pCRI01 plasmid to the homology repair template. L. paralimentarius The competent cells of the strain were electroporated at 2.5 kV for 5 ms. After electroporation, the bacterial culture was added to 900 μL of MRS medium and incubated at 37 °C for 2 h for recovery.

[0117] (2) After the culture is completed, the bacterial culture is placed in a 1.5 mL centrifuge tube and centrifuged at 8000 r / min for 1 min. 900 μL of supernatant is discarded, and the remaining bacterial culture is evenly spread on an MRS agar plate containing erythromycin resistance and incubated at 37 ℃ for 48 h.

[0118] (3) Transformants from agar plates were inoculated into antibiotic-free liquid MRS medium and cultured for 24 h. The genomes of the transformants were extracted according to the method described in the Vazyme genome extraction kit. The extracted genomes were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing verification. Comparison results with single plasmid editing tools are shown below. Figure 3 As shown.

[0119] 5. Plasmid elimination

[0120] Follow the method described in Example 2.

[0121] The results showed that: during knockout lpaMA , UgpA , UlpRTF In the three-gene experiment, the dual-plasmid system still showed significant advantages. In terms of transformation efficiency, the dual-plasmid system produced 57±8 transformants, while the single-plasmid system had a near-zero transformation efficiency due to its large plasmid volume. In terms of editing efficiency, the dual-plasmid system achieved a positive mutation rate of 46%±7%, which remained at a relatively high level.

[0122] Example 5: L. paralimentarius Construction of strains with LpaMA A375P / S257P gene modification and UlpRTF1 transcriptional repressor silencing expression and their application in bread anti-aging.

[0123] 1. Construction of engineered strains

[0124] The engineered strain is described in Example 4.

[0125] L. paralimentarius LpaMA A375P / S257P gene modification:

[0126] Wild-type maltose amylase is derived from food-like lactobacilli. Lactobacillus paralimentarius .

[0127] The gene encoding maltose amylase was chemically synthesized and transferred into Escherichia coli in the vector pET28a. E. coli BL21(DE3), forming the wild-type pET28a-LpaMA / maltose amylase E. coli BL21, whose NCBI accession number is WP_025085606.1, was used to perform site-directed mutagenesis on the recombinant plasmid pET28a-LpaMA as a template, using whole plasmid PCR technology to obtain mutants A375P, S257P, and A375P / S257P.

[0128] amino acid sequence:

[0129] MGNTPAIYHRPDSEFAYLYKDNLMHIRLRTARGDIKEVGLLHGDPYTLDTDHWQDQPTPMKRYLTTDLYDFWTIEVTEPFKRISYAFKVTGNDGITIFYGDHGVFSFEKSVYDSPDNYFRLPYFHEVDRFKAPEWVKKTVWYQIFPERFANGDKSNDPENTLPWGSKTPSATDFFGGDLQ GVIDHLDHLTDLGVNGIYFCPIFKAKSNHKYDTIDYMEIDPAFGDKATFKKLVQECHKRGIKVMLDAVFNHMGDSSPQWLDVVKNGKNSKYADWFHINKFPVSYEDDGFDQAKNISYDTFAFTPHMPKLNTANPEVKEYLLKIAKYWIEEFDIDAWRLDVANEVDHEFWRDFRRACDSVKK DFYILGEVWHSSQPWLQGDQFSAVMNYAYTDSISKYLIKKQIPIEKMVSDINDQLMLYRDQTDQIQFNVLDSH DTARLLTETNDDKDLMKQVLAFTYLQPGVPCIYYGDEIGMDGGNDPECRKCMVWDKSQQDLNLYDFFKHLI AFRKDNQKVLSEGEMVWEKVCDDGLLILSRKLNGTEVKVILNAGDTPKQVTTNQSVVLSNLVTENNHELTVD PKGFALVK.

[0130] Nucleotide sequence:

[0131] ATGGGCAATACACCAGCAATTTATCATCGCCCAGACAGCGAATTTGCATATCTATACAAAGATAATTTAATGCATATTAGATTAAGAACCGCTCGCGGAGACATTAAAGAGGTAGGTTTACTTCACGGTGATCCGTATACGCTAGATACAGACCACTGGCAAGATCAACCTACGCCGATGAAACGTTATTTAACGACTGATTTGTATGATTTTTGGACTATTGAAGTTACAGAGCCGTTCAAACGTATTTCCTATGCTTTCAAGGTTACTGGTAACGATGGCATTACCATATTCTATGGTGATCATGGTGTGTTCTCATTTGAAAAGAGCGTCTACGATAGTCCTGATAATT ACTTTAGATTGCCATATTTCCACGAAGTAGATCGTTTCAAGGCACCAGAGTGGGTCAAAAAGACTGTCTG GTATCAAATTTTCCCCGAAAGATTTGCTAATGGGGATAAATCTAATGATCCCGAAAATACTTTGCCATGG GGTTCTAAAACTCCAAGTGCTACGGATTTCTTTGGTGGTGATCTACAAGGTGTTATTGACCATCTAGATCA TTTAACTGATTTGGGTGTCAATGGAATCTATTTCTGTCCTATCTTCAAAGCTAAGTCTAATCACAAATACG ACACAATTGATTATATGGAAATAGATCCAGCCTTTGGCGACAAAGCTACTTTTAAAAAATTAGTTCAAGA ATGTCACAAACGTGGCATTAAAGTCATGCTAGATGCGGTTTTCAATCATATGGGTGACAGTTCACCGCAA TGGTTGGATGTCGTTAAAAATGGTAAGAATTCTAAATACGCCGATTGGTTCCATATCAATAAATTCCCAGTTTCATATGAAGATGATGGCTTTGATCAAGCTAAGAATATTTCCTATGATACTTTTGCCTTCACACCACATA TGCCAAAATTGAATACTGCTAATCCAGAAGTTAAGGAGTATTTGTTGAAGATTGCTAAGTATTGGATTGAAGAATTCGATATTGATGCTTGGAGATTAGATGTGGCAAATGAAGTTGATCATGAATTCTGGCGTGACTTT AGAAGAGCATGTGACAGTGTTAAAAAGGATTTCTATATTCTAGGTGAAGTTTGGCACTCCTCACAACCGT GGCTCCAAGGAGATCAGTTTAGTGCTGTAATGAATTATGCGTATACTGATTCAATTTCCAAATACTTAATTAAAAAACAAATTCCAATTGAAAAAATGGTTTCAGATATCAATGATCAATTGATGCTCTATCGTGATCAAA CAGATCAAATTCAATTCAATGTTTTGGATTCACATGATACAGCTCGATTGTTGACTGAAACAAATGATGAT AAAGATTTAATGAAACAAGTACTAGCTTTCACATATCTACAACCAGGTGTTCCATGTATATATTATGGTGA TGAAATCGGTATGGATGGCGGTAATGATCCTGAATGTCGTAAGTGTATGGTTTGGGATAAATCTCAACAA GATTTAAATCTATATGATTTCTTCAAACACTTGATTGCTTTTAGAAAAGATAATCAAAAAGTGCTTTCTGA AGGAGAGATGGTTTGGGAGAAGGTCTGTGATGATGGATTATTGATCTTGAGTCGTAAGTTGAATGGTACT GAAGTAAAGGTTATTTTGAACGCAGGCGATACTCCTAAACAAGTAACAACTAATCAAAGCGTAGTTTTAA GTAACTTAGTTACAGAAAATAATCATGAGTTAACGGTTGATCCTAAAGGATTTGCATTAGTTAAA.

[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 S257P mutant (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 under the following conditions: pre-denaturation at 94°C for 4 min; followed by 30 cycles: denaturation at 98°C for 10 s, annealing at 55°C for 5 s, extension at 72°C for 5 min; and final extension at 72°C for 10 min, followed by incubation at 4°C.

[0139] PCR amplification products were detected by 1% agarose gel electrophoresis. After detection, 0.5 μL of methylation template digestion enzyme was added to 10 μL of amplification products. Dpn I) Mix thoroughly by blowing and sucking with a pipette tip, and react at 37°C for 1.5 hours. Dpn The amplification products after I treatment were transformed into E. coli E. coli BL21(DE3) transformation products were plated on LB solid medium and cultured at 37°C for 8-10 h. Twenty transformants were picked from the LB solid medium and inoculated into LB liquid medium. After culturing at 37°C for 10 h, plasmids were extracted and sequenced. If the sequencing was correct, recombinant E. coli containing genes encoding mutants A375P, S257P, and A375P / S257P were obtained.

[0140] (2) Induction culture and protein purification of maltose amylase mutant

[0141] Using maltose amylase with the amino acid sequence shown in SEQ ID NO.1 as wild-type, the obtained recombinant Escherichia coli pET28a-LpaMA / E. coliBL21 and recombinant *E. coli* containing genes encoding mutants A375P, S257P, and A375P / S257P were plated on LB agar 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 and 200 rpm for 6–8 h to obtain seed culture. The seed culture was inoculated into LB liquid medium at an inoculum rate of 2% (v / v) and cultured at 37°C and 200 rpm for 2–3 h. IPTG was then added to the fermentation broth to a final concentration of 0.1 mmol / L, and the culture was further induced at 17°C and 200 rpm for 12–17 h to obtain the fermentation broth. Centrifuge the fermentation broth at 4°C and 8000 rpm for 5 min, discard the supernatant, and wash the precipitate twice with 9% physiological saline to obtain wild-type, mutant A375P, S257P, and A375P / S257P wet cells.

[0142] The wet bacterial cell mass was resuspended in buffer A and sonicated. The mixture was then centrifuged at 10,000 rpm and 4°C for 30 min to obtain the crude enzyme solution. After filtration through a 0.22 μm aqueous filter, the solution was slowly loaded onto a Ni-NAT affinity chromatography column. After loading, the column was washed with buffer A, followed by gradient elution with buffer B. The elution peak corresponding to 300 mmol / L imidazole was collected. The imidazole was then removed from the purified enzyme using a desalting column to obtain pure enzymes of wild-type, mutant A375P, S257P, and A375P / S257P.

[0143] (3) Hydrolytic activity of different maltose 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) to obtain reaction systems. 500 μL of the reaction system was incubated at 40-70°C for 3 min, and then 100 μL of purified enzymes of wild-type, mutant A375P, S257P, and A375P / S257P obtained in Example 2 was added to initiate the reaction. The control group contained no enzyme solution, and all other components were the same. The reaction was carried out at 40-70°C for 10 min. 10 μL of the product was diluted 20 times and an equal volume of 200 μL of DNS (3,5-dinitrosalicylic acid) was added to stop the reaction. After boiling in a water bath at 100°C for 5 min, the mixture was placed on ice to cool to room temperature. The absorbance change at 540 nm was recorded to obtain the hydrolytic activity of 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] Where Ew is the change in absorbance at 540 nm within 10 min; k is the standard curve coefficient of reducing sugar, in μmol / mL; V is the volume of the enzyme activity assay reaction system, in mL, which is 0.6 here; t is the reaction time, in min, which is 10 here; N is the enzyme dilution factor; L is the volume of enzyme solution added, in mL, which is 0.1 here; the protein concentration of the enzyme solution was determined using the Bradford Protein Kit (for determining the protein concentration using the Bradford Protein Kit, please refer to: Zhou-Pan XR, E Sérée, Zhou XJ, et al. Involvement of Human LiverCytochrome P450 3A in Vinblastine Metabolism: Drug Interactions1[J]. CancerResearch, 1993, 53(21):5121-5126.), in mg / mL.

[0147] The results showed that the optimal operating temperature and pH for the wild type were 50°C and 5.0, respectively, with a maximum hydrolytic activity of 274.79 U·mg⁻¹; the optimal operating temperatures for the mutants A375P, S257P, and A375P / S257P were 60, 60, and 65°C, respectively, with maximum hydrolytic activities of 425.89, 325.82, and 430.15 U·mg⁻¹, respectively.

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

[0149] The wild-type, mutant A375P, S257P, and A375P / S257P enzyme solutions obtained in Example 2 were incubated in a 60°C water bath for 60 min. Samples were taken at intervals, and the catalytic activity of the wild-type, mutant A375P, S257P, and A375P / S257P maltose amylase on starch was measured at 50°C after incubation. The activity before incubation was taken as 100%, and the residual activity after incubation was compared to this to calculate the relative activity, in order to examine the temperature stability and half-life of the wild-type, mutant A375P, S257P, and A375P / S257P maltose amylase. The calculated residual activity was linearly fitted to the corresponding incubation time using Origin software to obtain the enzyme half-life. t1 / 2 ).

[0150] The results showed that the residual relative activities of mutants A375P, S257P, and A375P / S257P were 70.63%, 47.59%, and 95.27%, respectively, with half-lives of 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 is evident that the double-point mutant A375P / S257P, building upon the improved thermal stability of the single-point mutants A375P and S257P, further exhibits excellent temperature stability.

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

[0152] 100 μL of pure enzymes of wild-type and double-point mutant A375P / S257P obtained in Example 2 were added to a phosphate buffer (50 mmol / L, pH 5.0) containing 0–30 mg / mL β-cyclodextrin to begin the reaction. The control group contained no enzyme solution, and other components were the same. The reaction was carried out at the optimal temperature for 10 min to obtain the hydrolytic activity of wild-type and double-point mutant A375P / S257P against β-cyclodextrin. The calculated hydrolytic activity was nonlinearly fitted to the corresponding substrate concentration using Origin software to obtain the enzyme's catalytic efficiency. k cat / K m).

[0153] The results showed that the catalytic efficiency of wild-type β-cyclodextrin was ( k cat / K The catalytic efficiency (m) of the two-point mutant A375P / S257P for β-cyclodextrin was 13.36 mM⁻¹·s⁻¹; k cat / K The value of m is 30.42 mM-1·s-1.

[0154] 2. Preparation of sourdough

[0155] Engineering renovation L. paralimentarius The bacterial strain was activated for two generations and cultured to a stabilizer. 100 µL of the bacterial suspension was inoculated into 5 mL of MRS medium and cultured at 37 ℃ for 16 h. After centrifugation at 8000 r / min for 5 min, the culture was washed twice with sterile physiological saline to obtain bacterial sludge. The centrifuged bacterial sludge was resuspended in sterile water and inoculated into wheat substrate for culture. The dough yield (DY value) was 250. Each 10 mL of MRS medium containing the centrifuged bacterial sludge was inoculated into 100 g of sourdough and cultured in a 30 ℃, 85% humidity incubator for 24 h to obtain sourdough. The DY value represents the mass relationship between the substrate powder and water, calculated using the following formula:

[0156]

[0157] In the formula: The mass (g) of the matrix powder; The mass of water is expressed in grams (g).

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

[0159] A 300g bread recipe uses the following ingredients: 300g wheat flour, 4.5g yeast, 18g sugar, 3g salt, and 180g water. The specific bread-making process is as follows:

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

[0161] (2) Take out the dough and knead it manually to a suitable extent, then cover it with plastic wrap for 5 minutes;

[0162] (3) After the dough has been relaxed, it is divided into pieces of about 90 g each. After the initial shaping and degassing, the dough is covered with plastic wrap and allowed to rise for the first time at room temperature (5 min).

[0163] (4) Roll out each small dough ball in turn, making sure all air bubbles are removed, then roll it up and place it in the baking mold. Let it rise a second time (90 min) at 37°C and 85% relative humidity.

[0164] (5) Bake the proofed bread at 170 °C top heat and 210 °C bottom heat for 21 min;

[0165] The recipe for sourdough bread is: 255 g flour, 112.5 g sourdough, 4.5 g yeast, 18 g sugar, 3 g salt, and 112.5 g water. The specific processing steps for the bread are similar to those described above.

[0166] 4. Engineering renovation L. paralimentarius Baking anti-aging performance test of the prepared sourdough

[0167] After baking, the bread was left to cool at room temperature for 2 hours, and then its hardness was determined by Texture Profile Analysis (TPA). The remaining bread was stored at 4 °C. The hardness changes of the bread after 1, 3, 5, and 7 days of storage were measured to evaluate its anti-aging properties. The bread was allowed to warm to room temperature for 2 hours before hardness measurement. (Regular wheat bread and bread with added wild-type wheat were included.) L. paralimentarius The bread made from sourdough contains engineered ingredients. L. paralimentarius The change in hardness of the sourdough bread is as follows: Figure 4 As shown.

[0168] The results show:

[0169] After 7 days of storage at 4℃, the hardness of ordinary wheat bread was 2070.7 g ± 92.5 g, the WT-Sourdough group had a hardness of 1770.3 g ± 31.4 g, while the WHU-Sourdough group's hardness decreased to 1208.7 g ± 38.5 g. This indicates that the hardness of wild-type wheat bread... L. paralimentariusThe sourdough produced has a significant inhibitory effect on the aging of wheat bread, and the genetically engineered sourdough... L. paralimentarius The resulting sourdough has stronger anti-aging properties.

[0170] Targeting the maltose amylase gene LpaMA Thermal stability modifications were performed, and pure enzyme baking application experiments confirmed that the heat-resistant mutant A375P / S257P of LpaMA exhibits higher enzyme activity and heat resistance, demonstrating superior anti-aging properties in bread baking applications; a new enzyme was discovered and identified. LpaMA transcriptional repressors of genes UlpRTF1 Its efficacy was verified by RT-qPCR experiments. LpaMA The transcriptional repression effect was observed. Using the aforementioned dual-plasmid gene editing system, a dual-trait superimposed mutant was rapidly constructed. Sourdough made from this mutant strain 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 variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

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

2. The method of multi-gene editing of the food-like Lactobacillus bifidus dual-plasmid multi-gene editing system based on the CRISPR / Cas9 technology according to claim 1, characterized in that, Comprise the following steps: (1) The pCRI01 is electroporated into the competent food-grade lactobacillus strain by the method of electroporation, and the food-grade lactobacillus strain containing plasmid pCRI01 is obtained; (2) Constructing plasmid pCRI02 containing at least one sgRNA targeting the corresponding target gene; (3) Constructing a DNA homologous repair template targeting the target gene; (4) Co-electroporating the plasmid pCRI02 and the DNA homologous repair template targeting the target gene into the food-grade lactobacillus containing plasmid pCRI01 competent, incubating, concentrating the bacterial concentration, uniformly coating on the agar MRS plate containing ampicillin and erythromycin resistance, and incubating at 37℃ to obtain the transformant; (5) Picking the transformant in liquid MRS medium for culture, extracting the genome for sequencing verification of editing results; (6) Inoculating the transformant with correct editing results into liquid MRS medium containing 10 ng / μL Nisin, and incubating at 40℃ overnight.

3. The method of multi-gene editing of claim 2, wherein, In the step (2), the mass ratio of the plasmid pCRI02 to the DNA homologous repair template targeting 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 the step (2), the sgRNA expression module of the plasmid pCRI02 is inserted into the multiple sgRNA by the BioBricks assembly method using XbaⅠ and SpeⅠ enzyme digestion sites.

5. A method for editing a food-like Lactobacillus strain based on a CRISPR-Cas9 dual plasmid system, characterized by, Using the food-grade lactobacillus as the starting strain, the multi-gene editing method of any one of claims 2-4 is used to knockout the target gene, and the edited food-grade lactobacillus strain is obtained.

6. The method of claim 5, wherein the method of editing a food-grade Lactobacillus strain based on a CRISPR-Cas9 dual plasmid system is characterized by, The targeting gene is LpaMA .

7. The method of claim 5 or 6, wherein the method of editing a Lactobacillus strain of the genus of food-grade bacteria using a CRISPR-Cas9 dual plasmid system is characterized by, The food-grade lactobacillus is food-grade lactobacillus LBM12001, and the preservation number is GDMCC 62232.

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