CN119876203B

CN119876203B - Over-expression bacteriocin synthesis regulation gene, and construction method and application of recombinant lactobacillus plantarum thereof

Info

Publication number: CN119876203B
Application number: CN202510352460.9A
Authority: CN
Inventors: 曾小群; 何维宁; 周钰雯; 潘道东; 吴振; 杜启伟; 涂茂林
Original assignee: Ningbo University
Current assignee: Ningbo University
Priority date: 2025-03-25
Filing date: 2025-03-25
Publication date: 2025-08-12
Anticipated expiration: 2045-03-25
Also published as: CN119876203A

Abstract

The invention discloses an over-expression bacteriocin synthesis regulation gene, a construction method and application of recombinant lactobacillus plantarum thereof, which is characterized in that the gene is derived from a gene of lactobacillus plantarum ZY-1 for encoding L (+) -tartaric acid dehydratase, and the nucleotide sequence is shown as SEQ ID NO.1, and the specific steps are that 1) the genome DNA of lactobacillus plantarum is taken as a template, ttdB gene is amplified, the double enzyme cutting glue of an expression vector pMG36e is recovered, and a PCR amplification product is inserted into the expression vector pMG36e to construct a recombinant expression vector; 2) the over-expression plasmid is extracted, then is electrically transformed and guided into the lactobacillus plantarum, the erythromycin resistance plate is coated after resuscitated, and then the transformant is screened and verified by PCR, so that the recombinant lactobacillus plantarum ZY-1-ttdB which over-expresses the L (+) -tartaric acid dehydratase gene is obtained, and the advantage is that the bacteriocin yield of the lactobacillus plantarum can be obviously improved.

Description

Over-expression bacteriocin synthesis regulation gene, and construction method and application of recombinant lactobacillus plantarum thereof

Technical Field

The invention belongs to the technical fields of bioengineering and microbial fermentation, and particularly relates to a construction method and application of an over-expressed bacteriocin synthesis regulatory gene (ttdB) and recombinant lactobacillus plantarum thereof.

Background

Bacteriocins are polypeptides or proteins synthesized by ribosomes during the growth metabolism of certain bacteria, which have an antibacterial effect. The bacteriocin produced by the lactobacillus has the advantages of wide sources, high safety, good stability, wide bacteriostasis spectrum, no pollution and the like, and has great potential as a novel biological preservative in the food industry. However, the yield of the bacteriocin is low, the molecular weight is small, the pure egg is difficult to separate and purify, and the quorum sensing genes of the bacteriocin-producing lactobacillus are diversified, so that no universal bacteriocin regulating system exists at present, and the application and development of the bacteriocin are influenced.

The current method for improving the synthesis amount of the lactic acid bacteria bacteriocin mainly comprises the steps of high-yield strain screening, fermentation condition optimization, mutation breeding, protoplast fusion, genetic engineering, regulation and co-culture by using a quorum sensing system and the like. The co-culture is common in multi-bacteria fermentation, and the method for improving the synthesis amount of bacteriocin has the advantages of short time, high equipment utilization rate, easiness in operation, relatively low cost and the like. The synthesis of bacteriocins requires the environment to provide additional metabolic energy, which adds to the "cost" of bacterial growth, and many bacteria "selectively" synthesize bacteriocins according to environmental changes to promote self-growth and conserve energy. The interaction of the microorganisms during co-culture, such as synergistic metabolism, nutrition supply, substrate competition, pH change and the like, has certain influence on the yield of target products and the production of new substances in a co-culture system.

At present, the stimulation of the biosynthesis of the induction lactobacillus bacteriocins by adding exogenous specific microorganism co-culture as an environmental condition has been more reported, but the screening of key differential expression genes by researching the co-culture condition is rarely reported. Therefore, the differential genes influencing bacteriocin synthesis under the co-culture condition are screened out through transcriptome, and the bacterial strain is modified through molecular biology to improve the bacteriocin yield of the lactobacillus plantarum, so that the method has important economic value and social significance.

Disclosure of Invention

The invention aims to solve the technical problem of providing an over-expression bacteriocin synthesis regulation gene capable of remarkably improving the yield of bacteriocin, and a construction method and application of recombinant lactobacillus plantarum thereof.

The invention solves the technical problems by adopting the technical scheme that the over-expression bacteriocin synthesis regulation gene is derived from a gene (ttdB gene for short) of lactobacillus plantarum ZY-1 coding L (+) -tartaric acid dehydratase, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1.

The invention also provides a construction method of the recombinant lactobacillus plantarum which overexpresses the bacteriocin synthesis regulatory gene, which comprises the following specific steps:

(1) Construction of recombinant expression vector pMG-ttdB

Designing ttdB upstream and downstream amplification primers by taking Lactobacillus plantarum ZY-1 genome DNA as a template, amplifying ttdB gene fragments, carrying out double enzyme digestion on an expression vector pMG36e by using restriction enzymes Sac I and Hind III, then carrying out agarose gel electrophoresis, and connecting ttdB gene fragments with a linear vector pMG36e by using a recombinase connection method after gel digestion recovery to construct a recombinant plasmid pMG36e-ttdB;

(2) Construction of recombinant Lactobacillus plantarum

The recombinant plasmid pMG36e-ttdB is electrically transformed and introduced into the lactobacillus plantarum ZY-1, after resuscitating for 1-5 hours at 35-40 ℃, erythromycin resistance plates are coated, then the transformant is cultured for 36-48 hours at 35-40 ℃, and the transformant is screened and verified by PCR, so that the recombinant lactobacillus plantarum ZY-1-ttdB which over-expresses bacteriocin synthesis regulatory genes is obtained.

Further, the nucleotide sequence of the ttdB gene upstream amplification primer in the step (1) is shown as SEQ ID NO.2 to be AAAAATTCGTAATTCGAGCTCATGAAAACTTACCACTTAACCACCC, and the nucleotide sequence of the ttdB gene downstream amplification primer is shown as SEQ ID NO.3 to be GTTTTCAGACTTTGCAAGCTTTTATTTAATGAATTTAACTTGTTCGTTGA.

Further, the PCR amplification procedure in the step (1) is as follows (1) 95℃for 3min, (2) 95℃for 15 s, 60℃for 15 s, 72℃for 60 s; 35 cycles are repeated, (3) 72℃for 5 min, and the PCR reaction system is as follows, 50-400 ng of template DNA, 2X Phanta Max Master Mix. Mu.L of ttdB gene upstream amplification primer 2. Mu.L and ttdB gene downstream amplification primer 2. Mu.L, ddH ₂ O being made up to 50. Mu.L.

Further, the specific steps of the electric conversion are as follows:

(1) Preparation of Phytosterium plantarum competence

Inoculating overnight activated lactobacillus plantarum ZY-1 in a 50 mL competent medium at a volume ratio of 1%, placing the strain in a 37 ℃ incubator for expansion culture to a logarithmic phase, placing the cultured strain in ice water to cool cells, centrifuging at a low speed to collect the strain, adding a precooled washing buffer to resuspend the strain, centrifuging at a low speed, repeating washing twice, and finally adding a precooled washing buffer with the same volume as the strain to resuspend the strain to obtain a lactobacillus plantarum ZY-1 competent cell solution;

(2) Electrotransformation of lactobacillus plantarum

1 Mug of recombinant plasmid pMG-ttdB and 100 mug of lactobacillus plantarum ZY-1 competent cell solution are gently mixed, the mixture of the recombinant plasmid and competent cells is transferred to a precooled electric shock cup by standing 10min on ice, electric shock is carried out after the electric shock is finished, 900 mug of precooled reviving culture medium is immediately added to be mixed evenly after the electric shock is finished, the mixture is transferred to a centrifuge tube by a1 mL sterile injector, after the shaking table 200 rpm is revived for 3 hours at 37 ℃, supernatant is discarded after low-speed centrifugation, sterile MRS broth culture medium of 1 mL is added again, a pipetting gun is used for blowing and resuspension, each 100 mug of culture solution is coated on an MRS flat plate containing 5 mug/mL erythromycin, 37 ℃ is cultured for 36-48 h, milky white single colony is picked up for culturing 12-16 h in MRS broth containing 5 mug/mL erythromycin, and turbid bacterial solution is taken for 1 mug of bacterial solution verification to obtain recombinant lactobacillus plantarum ZY-35 which overexpresses bacteriocin synthesis genes.

Further, the competent medium was formulated as MRS broth 52.24g/L, sorbitol 136.64g/L and glycine 10g/L, the wash buffer was formulated as sucrose 326g/L and hexahydrate and magnesium chloride 0.72g/L, and the revitalization medium was formulated as MRS broth 52.24g/L, sorbitol 136.64g/L and calcium chloride 1.12g/L.

The invention also provides application of the recombinant lactobacillus plantarum over-expressing the bacteriocin synthesis regulation gene in bacteriocin preparation.

Compared with the prior art, the invention has the advantages that the invention discloses an over-expression L (+) -tartaric acid dehydratase (L (+) -TARTRATE DEHYDRATASE) gene capable of obviously improving bacteriocin yield for the first time, and a construction method and application of recombinant lactobacillus plantarum ZY-1-ttdB thereof. The L (+) -tartaric acid dehydratase gene (ttdB) fragment of the cloned lactobacillus plantarum (Lactiplantibacillus plantarum) ZY-1 is connected to a pMG36e expression vector, and is introduced into lactobacillus plantarum by an electrotransformation method, and recombinant bacteria with target genes, namely recombinant lactobacillus plantarum ZY-1-ttdB over-expressing the L (+) -tartaric acid dehydratase gene (ttdB) are obtained through erythromycin resistance screening and identification. Compared with the empty control ZY-1-0 strain, the yield of the bacteriocin of the recombinant lactobacillus plantarum is improved by 18%, and the recombinant lactobacillus plantarum ZY-1 which can obviously improve the yield of the bacteriocin and over-express the L (+) -tartaric acid dehydratase gene is constructed for the first time, so that an important basis is provided for further research and application of the bacteriocin.

The lactobacillus plantarum (Lactiplantibacillus plantarum) ZY-1 strain has a preservation number of CGMCC No. 29542 and is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) on the day 08 of 2024, and has a preservation address of North Star Xiya No. 1 and 3 in the Korean region of Beijing city.

Drawings

FIG. 1 shows the result of agarose gel electrophoresis detection of PCR products over-expressing bacteriocin synthesis regulatory genes (ttdB genes), wherein 1 represents Lane 1, lane 1 is the amplified product of the genes ttdB, lane is a Lane, M is Marker, and M is a molecular weight standard reference substance;

FIG. 2 is a diagram of construction flow of recombinant plasmid pMG36 e-ttdB;

FIG. 3 shows the result of colony PCR products of pMG36e-ttdB positive clones, wherein 1 represents Lane 1,2 represents Lane 2,3 represents Lane 3, lane (1-3) is colony PCR product of erythromycin-screened pMG36e-ttdB positive clones, lane is Lane, M represents Marker, and is a molecular weight standard reference;

FIG. 4 shows the results of double cleavage verification of recombinant plasmids pMG36e-ttdB and pMG36e, wherein 1 represents Lane1, 2 represents Lane2, 3 represents Lane3, 4 represents Lane4, lane1 is pMG36e-ttdB double cleavage product, lane2 is pMG36e-ttdB uncleaved product, lane3 is pMG36e double cleavage product, lane4 is pMG36e uncleaved product, lane is Lane, M is Marker, and is a molecular weight standard reference;

FIG. 5 shows the result of the electrotransformation PCR verification, wherein (a) shows the electrophoretogram of the bacterial liquid PCR product of pMG36e electrotransformed into Lactobacillus plantarum ZY-1, M is Marker, which is a molecular weight standard reference, and 1-4 is bacterial liquid PCR product, (b) shows the electrophoretogram of the bacterial liquid PCR product of pMG36e-ttdB electrotransformed into Lactobacillus plantarum ZY-1, M is Marker, which is a molecular weight standard reference, and 1-4 is bacterial liquid PCR product;

FIG. 6 shows the relative quantitative results of qPCR of Lactobacillus plantarum genetically engineered bacteria ZY-1-ttdB and ZY-1-0, wherein P <0.0001 is shown in dashed lines, and the relative expression level of control ZY-1-0 is 1;

FIG. 7 shows the cell morphology under a scanning electron microscope, wherein (a) is wild type Lactobacillus plantarum ZY-1, (b) is Lactobacillus plantarum ZY-1-0, and (c) is Lactobacillus plantarum ZY-1-ttdB;

Fig. 8 is a diagram showing bacteriocin inhibitory changes produced by lactobacillus plantarum genetically engineered bacteria ZY-1-ttdB and ZY-1-0, wherein P <0.01, P <0.001, P <0.0001, escherichia coli, human staphylococci, staphylococcus aureus.

Detailed Description

The invention is described in further detail below with reference to the embodiments of the drawings.

The construction method of the recombinant expression vector pMG-ttdB in example 1 comprises the following steps:

Step 1, designing a PCR primer for amplifying the over-expressed L (+) -tartaric acid dehydratase (ttdB) gene fragment, wherein the sequence of the PCR primer is shown as follows, the nucleotide sequence of an amplification primer at the upstream of ttdB gene is shown as SEQ ID NO.2, AAAAATTCGTAATTCGAGCTCATGAAAACTTACCACTTAACCACCC, and the nucleotide sequence of an amplification primer at the downstream of ttdB gene is shown as SEQ ID NO.3, GTTTTCAGACTTTGCAAGCTTTTATTTAATGAATTTAACTTGTTCGTTGA.

Step 2, using the genome DNA of Lactobacillus plantarum (Lactiplantibacillus plantarum) ZY-1 (the Lactobacillus plantarum has been preserved in China general microbiological culture Collection center with accession number CGMCC No. 29542) as a template, performing the following PCR procedures (1) 95 ℃ 3 min, (2) 95 ℃ 15 s,60 ℃ 15 s,72 ℃ 60 s, and repeating 35 cycles (3) 72 ℃ 5 min. The PCR reaction was carried out by adding 2X Phanta Max Master Mix. Mu.L of the amplification primer upstream of the 10. Mu. M ttdB gene, 2. Mu.L of the amplification primer downstream of the 10. Mu. M ttdB gene, 50-400 ng of the template DNA and adding ddH ₂ O to a total volume of 50. Mu.L. FIG. 1 shows the result of agarose gel electrophoresis detection of the PCR amplification product of the target gene, wherein Lane1 is the amplified product of the gene ttdB PCR, and the molecular weight of the product is approximately correct as can be seen from the band position of the product. Then the gel cutting recovery and sequencing are carried out, and the nucleotide sequence of the ttdB gene is shown as SEQ ID NO.1 ：ATGAAAACTTACCACTTAACCACCCCAATTTCTGATGACGATATTAAGGACATTCGTATCGGCGACATCGTCTACTTGAATGGCTCACTAACAACCTGTCGGGACGTTGCCCATCGGCGGCTCGTCGAAGAACATCGTCCCCTACCCGTTAACGTGACAGACCGAGCAATTCTCCACGCTGGCCCGATCATTAAAGATCTCGGCCATGACAAGTACGAAATGGTCGCAGTCGGGCCAACGACTAGTATGCGGATGGAAAAATTTGAAGAAGAATTCGTAAAACAAACCCGTGTCAAACTAATCGTTGGTAAAGGCGGCATGGGACCGGGAACCGAACGGGCCTGCAAGAAATATCACGCCTTACACCTCGTCTACCCCGCTGGCAATGCTGTTTATGCCGCCCTGCACGTCAATAAGATTGTTGATGCCCAGTGGAAAGACCTCGGAATGCCCGAAACACTCTGGGCGTGTGAGGTCAAAGACTTCGGTCCGTTAATTGTCTCCATCGATACGAATGGCGACAACTTGTTTGAAAAGAACAAGGTCATCTTCAACGAACGTAAAGAAGCTGAGATTAAAAAAATCAACGAACAAGTTAAATTCATTAAATAA.

And 3, double enzyme cutting of the expression vector pMG36e by using restriction enzymes Sac I and Hind III, agarose gel electrophoresis and gel cutting recovery. And (3) connecting the target fragment (ttdB) PCR amplified product obtained in the step (2) with a linear vector (pMG 36 e) by utilizing a recombinase connection method, and constructing a recombinant plasmid pMG36e-ttdB at 37 ℃ for 30 min at 4 ℃ and 10 min. FIG. 2 is a diagram of the construction scheme of recombinant plasmid pMG36e-ttdB.

FIG. 3 shows the result of colony PCR product electrophoresis of pMG36e-ttdB positive clones, lane (1-3) was colony PCR product of erythromycin-screened pMG36e-ttdB positive clones. The colony PCR product is single, and the measurement result with the size of about 1000 bp is matched with the recombinant plasmid sequence.

FIG. 4 shows the results of double cleavage verification of recombinant plasmids pMG36e-ttdB and pMG36e, lane1 was a double cleavage product of pMG36e-ttdB, lane2 was a double cleavage product of pMG36e-ttdB, lane3 was a double cleavage product of pMG36e, lane4 was a double cleavage product of pMG36e, and the results showed a linear plasmid band and a target gene band. The above shows that the recombinant plasmid pMG36e-ttdB was constructed successfully.

EXAMPLE 2 construction of Lactobacillus plantarum genetically engineered bacterium ZY-1-ttdB, ZY-1-0

The pMG-ttdB over-expression plasmid prepared in example 1 is extracted by a plasmid mass extraction kit, is subjected to electric shock transformation, is introduced into lactobacillus plantarum ZY-1, is subjected to recovery at 37 ℃ for 3 hours, is coated with an erythromycin resistance plate, is cultured at 37 ℃ for 36-48 hours, is screened, and is subjected to PCR verification, so that recombinant lactobacillus plantarum ZY-1-ttdB over-expressing L (+) -tartaric acid dehydratase gene ttdB is obtained.

Meanwhile, after the untreated expression vector pMG36e is extracted by a large number of plasmid extraction kits, electric shock transformation is conducted to lactobacillus plantarum ZY-1, erythromycin resistance plates are coated after recovery for 3 hours at 37 ℃, then the transformants are cultured for 36-48 hours at 37 ℃, and the transformants are screened and verified by PCR, so that the empty lactobacillus plantarum ZY-1-0 is obtained.

The specific steps of the electric conversion are as follows:

(1) Preparation of Phytosterium plantarum competence

The overnight activated lactobacillus plantarum ZY-1 was inoculated in a 1% volume ratio into 50 mL competent medium and placed in a 37℃incubator for expansion to log phase (OD ₆₀₀ reached 0.3-0.6). And (3) placing the cultured bacterial liquid in ice water to cool the cells for 30min, centrifuging at a low speed of 6000 g for 5min at a temperature of 4 ℃ and collecting the bacterial cells. The cells were resuspended in pre-chilled wash buffer, centrifuged at 4℃and 3000 rpm min at low speed and washed twice repeatedly. Finally, adding a pre-cooled equal volume of washing buffer solution to resuspend the thalli, sub-packaging 100 mu L of the thalli into a 1.5 mL centrifuge tube, quick-freezing 15 to min by liquid nitrogen, and storing in a refrigerator at-80 ℃. Wherein the competent medium comprises MRS broth 52.24g/L, sorbitol 136.64g/L and glycine 10g/L, the washing buffer comprises sucrose 326g/L, hexahydrate and magnesium chloride 0.72g/L, and the revitalization medium comprises MRS broth 52.24g/L, sorbitol 136.64g/L and calcium chloride 1.12g/L;

(2) Electrotransformation of lactobacillus plantarum

The frozen competent cells and plasmids of the lactobacillus plantarum are taken out and put on an ice box for thawing. 1 mug of recombinant plasmid pMG-ttdB is taken and mixed with 100 mug of competent cell solution of lactobacillus plantarum ZY-1 gently, the mixture is stood on ice for 10 min, the mixture of the recombinant plasmid and the competent cell is transferred to a precooled electric shock cup, and electric shock is carried out after the ice bath stands for 10 min, wherein the electric shock parameters are 1.2 KV and 4 ms. Immediately after the electric shock was completed, 900 μl of pre-chilled resumption medium was added and mixed well, the mixture was transferred to a 1.5 mL centrifuge tube with a1 mL sterile syringe, after 3h resuscitation with a 37 ℃ shaker 200 rpm, the supernatant was centrifuged at 3000 rpm low speed and 2 min was discarded, and 1 mL sterile MRS broth was added again and resuspended by pipetting with a pipette. Each 100. Mu.L of the culture broth was plated on MRS plates containing 5. Mu.g/mL erythromycin and incubated at 37℃for 36-48 h. A milky white single colony is selected and cultured in MRS broth containing 5 mu g/mL erythromycin at 1 mL for 12-16 h, 1 mu L of turbid bacterial liquid is taken for bacterial liquid PCR verification, sequencing and comparison are carried out on PCR products, the nucleotide sequence of a forward identification primer pMG36e-F is shown as SEQ ID NO.4 and CAATCTGCCTCCTCATCCT, and the nucleotide sequence of a reverse identification primer pMG36e-ttdB-R is shown as SEQ ID NO.5 and TCCACTGGGCATCAACAAT. mu.L of colony PCR products were taken for agarose gel electrophoresis detection (agarose concentration 2%), and PCR products showing the target bands from the results of nucleic acid electrophoresis were sent for detection.

In FIG. 5, (a) shows an electrophoresis pattern of a bacterial liquid PCR product of the electro-transferred lactobacillus plantarum ZY-1 of pMG36e, a verification primer pMG36e-F/R of pMG36e is positioned at two ends of a cloning site, the product length is 350 bp, the product electrophoresis band position accords with the theoretical size of the product, wherein the nucleotide sequence of the reverse verification primer pMG36e-R is shown as SEQ ID NO. 6: GCCACCTTCGTTTTCAGACT, in FIG. 5, (b) shows an electrophoresis pattern of a bacterial liquid PCR product of the electro-transferred lactobacillus plantarum ZY-1 of pMG36e-ttdB, the primer pMG36e-F, pMG e-ttdB-R designed by pMG36e-ttdB is positioned on a plasmid, the downstream primer is positioned on an inserted target gene, the product length is 716 bp, and the product electrophoresis band position accords with the theoretical size of the product.

Example 3 verification of Gene level of genetically engineered bacterium ZY-1-ttdB.

The over-expression strain ZY-1-ttdB obtained in example 2 is subjected to RNA extraction and reverse transcription to obtain cDNA product, a ttdB gene RT-qPCR primer is designed, the ttdB gene real-time quantitative PCR forward primer has a nucleotide sequence shown as SEQ ID NO.7 of 5'-CGCCTTACACCTCGTCTACC-3', a ttdB gene real-time quantitative PCR reverse primer has a nucleotide sequence shown as SEQ ID NO.8 of 5'-CAAGTTGTCGCCATTCGTATCG-3', 16S rRNA is used as an internal reference gene, the internal reference gene real-time quantitative PCR forward primer has a nucleotide sequence shown as SEQ ID NO.9 of GGCGTGCTATTCATACCAGTC, and the internal reference gene real-time quantitative PCR reverse primer has a nucleotide sequence shown as SEQ ID NO.10 of CAGGTGTTATCCCGTGCTTC. The real-time quantitative PCR system was 1.5. Mu.L of cDNA template, 0.4. Mu.L of upstream primer, 0.4. Mu.L of downstream primer, 2X Taq Pro Universal SYBR QPCR MASTER Mix 10. Mu.L, and was made up to 20. Mu.L with dd H ₂ O. The amplification procedure was set to (1) pre-denaturation (95 ℃,30 s), (2) denaturation (95 ℃,10 s), annealing extension (60 ℃,30 s), 40 cycles, and (3) cooling (37 ℃,10 min). The relative expression level of the target gene was calculated using the 2 ^-△△CT method.

FIG. 6 shows the relative quantitative result of RT-qPCR of ttdB, ZY-1-0 is used as a control bacterium, the relative expression amount is always 1, and the result shows that compared with ZY-1-0, the expression amount of ttdB of the over-expression strain ZY-1-ttdB is obviously increased by more than 6.54 times, and ttdB is proved to be over-expressed on the gene level.

EXAMPLE 4 scanning electron microscopy of morphological changes of ZY-1, ZY-1-0 and ZY-1-ttdB

After overnight incubation of the strain ZY-1-0 obtained in example 2 with ZY-1-ttdB and wild-type ZY-1 in MRS broth, ZY-1-0 and ZY-1-ttdB bacterial pellet was collected by centrifugation, washed and resuspended in 2.5% glutaraldehyde at 4℃for 3 hours. The cells were then washed 3 times with 0.1M phosphate buffered saline (PBS, pH 7.2) and dehydrated in gradient ethanol (30, 50, 70, 80, 90, 100%) for 15 minutes each. Finally, the cells were lyophilized (-49 ℃, 24 h, and 9 Pa) in vacuo after resuspension with 1 mL dry t-butanol. And (3) bonding the dried sample on a Scanning Electron Microscope (SEM) sample stage by using conductive adhesive, and observing the cell morphology in a scanning electron microscope after metal spraying.

FIG. 7 (a) shows the cell morphology of wild-type Lactobacillus plantarum ZY-1 under a scanning electron microscope, FIG. 7 (b) shows the cell morphology of Lactobacillus plantarum ZY-1-0 under a scanning electron microscope, and FIG. 7 (c) shows the cell morphology of Lactobacillus plantarum ZY-1-ttdB under a scanning electron microscope. ZY-1-0 and ZY-1-ttdB have a broader cell diameter compared to wild-type Lactobacillus plantarum ZY-1. This change in cell diameter may be related to the transformation process of the exogenous plasmid, and the ttdB gene may not be directly involved in cell proliferation or synthesis of the major components of the membrane, so its effect on cell morphology is not significant.

Example 5 determination of bacteriocin Activity of genetically engineered bacterium ZY-1-ttdB.

Strains ZY-1-0 and ZY-1-ttdB obtained in example 2 were subjected to single colony activation 16-h to obtain seed solutions with the same OD ₆₀₀ value, inoculated into MRS broth in the same proportion, cultured at 37 ℃ for 24 h, the fermentation supernatant was sterilized by a sterile 0.22 μm filter membrane, concentrated by freeze-drying in sterile water for 15 times, pH was adjusted to 6.5-7.0 by a solution of 5M NaOH, sterilized by a sterile 0.22 μm filter membrane, and the sterilized catalase solution was added to the fermentation supernatant to a concentration of 1 mg/mL, and water bath 2h at 37 ℃. A bacteria inhibition plate is taken, a sterile oxford cup is placed in the bacteria inhibition plate, 200 mu L of supernatant liquid with acid and hydrogen peroxide removed is added into the oxford cup, and the diameter of a bacteria inhibition zone is measured after the bacteria inhibition plate is cultured at 37 ℃ for 10 h. MRS broth adjusted to pH 6.5-7.0 was added as a blank.

FIG. 8 is a diagram showing bacteriostasis changes of the genetically engineered bacteria ZY-1-ttdB and ZY-1-0 bacteriocins. Under three different indicator bacteria, ZY-1-ttdB has a good antibacterial effect. Wherein the inhibitory activity against Staphylococcus aureus (abbreviated as Staphylococcus aureus) and Staphylococcus hominis (abbreviated as Staphylococcus hominis) is improved by 18% and 10%, respectively.

The above description is not intended to limit the invention, nor is the invention limited to the examples described above. Variations, modifications, additions, or substitutions will occur to those skilled in the art and are therefore within the spirit and scope of the invention.

Claims

1. A bacteriocin synthesis regulation gene is characterized in that the gene is derived from lactobacillus plantarum (Lactiplantibacillus plantarum) ZY-1 with the preservation number of CGMCC No.29542, and a gene ttdB for encoding L (+) -tartaric acid dehydratase, and the nucleotide sequence of the gene is shown as SEQ ID No. 1.

2. A method for constructing recombinant lactobacillus plantarum which overexpresses the bacteriocin synthesis regulatory gene of claim 1, which is characterized by comprising the following specific steps:

(1) Construction of recombinant expression vector pMG36e-ttdB

Designing upper and downstream PCR amplification primers of the L (+) -tartaric acid dehydratase gene ttdB in claim 1 by taking Lactobacillus plantarum (Lactiplantibacillus plantarum) ZY-1 genome DNA with the preservation number of CGMCC NO. 29542 as a template, amplifying ttdB gene fragments, carrying out double enzyme digestion on an expression vector pMG36e by using restriction enzymes Sac I and Hind III, carrying out agarose gel electrophoresis, carrying out gel digestion recovery, and connecting ttdB gene fragments with a linear vector pMG36e by using a recombinase connection method to construct a recombinant plasmid pMG36e-ttdB;

(2) Construction of recombinant Lactobacillus plantarum

3. The construction method according to claim 2, wherein the nucleotide sequence of the ttdB gene upstream amplification primer in the step (1) is shown as SEQ ID NO.2 to AAAAATTCGTAATTCGAGCTCATGAAAACTTACCACTTAACCACCC, and the nucleotide sequence of the ttdB gene downstream amplification primer is shown as SEQ ID NO.3 to GTTTTCAGACTTTGCAAGCTTTTATTTAATGAATTTAACTTGTTCGTTGA.

4. The construction method according to claim 3, wherein the PCR amplification in the step (1) is carried out by (1) 95℃for 3min, (2) 95℃for 15 s, 60℃for 15 s, 72℃for 60 s, and (3) 35 cycles, and (3) 72℃for 5 min, and the PCR reaction system is as follows, 50-400 ng of template DNA, 2X Phanta Max Master Mix. Mu.L of ttdB gene upstream amplification primer 2. Mu.L and ttdB gene downstream amplification primer 2. Mu.L and ddH ₂ O up to 50. Mu.L.

5. The construction method according to claim 2, characterized in that the specific steps of the electrical transformation are as follows:

(1) Inoculating overnight activated lactobacillus plantarum ZY-1 in a 50mL competent medium according to a volume ratio of 1%, placing the strain in a 37 ℃ incubator for expansion culture to a logarithmic phase, placing the cultured strain liquid in ice water to cool cells, collecting the strain body through low-speed centrifugation, adding a precooled washing buffer solution to resuspend the strain body, repeating washing twice after low-speed centrifugation, and finally adding the precooled washing buffer solution which is equal to the strain suspension in volume to resuspend the strain body to obtain lactobacillus plantarum ZY-1 competent cell solution;

(2) The electrotransfer of the plant lactobacillus comprises the steps of taking 1 mug of recombinant plasmid pMG36e-ttdB and 100 mug of plant lactobacillus ZY-1 competent cell solution, gently mixing the mixture, standing the mixture on ice for 10min, transferring the mixture to a precooled electric shock cup, carrying out electric shock after the ice bath stands for 10min, immediately adding 900 mug of precooled reviving culture medium after the electric shock is finished, uniformly mixing, transferring the mixture into a centrifuge tube by using a1 mL sterile injector, carrying out resuscitation for 3 hours by using a 37 ℃ shaker 200 rpm, carrying out low-speed centrifugation, discarding supernatant, re-adding a1 mL sterile MRS broth culture medium, carrying out blowing and resuspension by using a pipetting gun, coating each 100 mug of culture solution on an MRS plate containing 5 mug/mL erythromycin, culturing the culture solution at 37 ℃ for 36-48 h, picking up 1 mug of opalescent single colony in MRS containing 1 mL mug/mL erythromycin, culturing the 12-16 to h, and carrying out bacterial solution verification and PCR (polymerase chain reaction) to obtain the recombinant plant lactobacillus ZY-35 strain expressing the bacteriocin synthesized gene.

6. The method of claim 5, wherein the competent medium comprises MRS broth 52.24g/L, sorbitol 136.64g/L and glycine 10g/L, the wash buffer comprises sucrose 326g/L and hexahydrate and magnesium chloride 0.72g/L, and the revitalization medium comprises MRS broth 52.24g/L, sorbitol 136.64g/L and calcium chloride 1.12g/L.

7. Use of recombinant lactobacillus plantarum, constructed by the method of any of claims 2-6, to overexpress a bacteriocin synthesis regulatory gene in the preparation of bacteriocin.