Pediococcus pentosaceus producing novel type IIa bacteriocin, its application and construction of secretory engineered bacteria

Abstract

The present invention provides a Pediococcus pentosaceus ( Pediococcus pentosus ) strain ZPP083, which is deposited with the General Microbiology Center of the China National Center for Microbiological Culture Collection under the CGMCC No. 31944. The Pediococcus pentosaceus provided by the present invention has a strong antibacterial effect and produces a novel Class IIa bacteriocin with acid, heat, and protease resistance. The present invention also constructs a genetically engineered bacterium using this bacteriocin, increasing its expression.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to Pediococcus pentosaceus producing type IIa bacteriocin, application thereof and engineering bacteria construction. Background Art

[0002] The overuse of antibiotics, which has led to drug-resistant bacteria and drug residues, has become an increasingly prominent concern for feed safety and human health. The World Health Organization has listed the research and development of effective drugs to treat drug-resistant bacteria as one of its global research priorities. Therefore, to control drug residues, improve food safety, and maintain sustainable development of the livestock industry, there is an urgent need to develop new alternatives to antibiotics.

[0003] Bacteriocins are a class of low-molecular-weight proteins or peptides produced by bacteria during metabolism that inhibit and kill the growth of bacteria other than the producing bacteria. Due to their highly effective antibacterial activity, non-toxicity, and resistance-resistant properties, they are considered potential alternatives to antibiotics. Antibiotics are difficult to degrade, which can easily lead to bacterial resistance and kill sensitive bacteria, regardless of whether they are beneficial or harmful. In contrast, bacteriocins are antimicrobial substances produced by bacterial ribosomes. As proteins, bacteriocins not only have strong antibacterial activity but are also relatively stable, selectively inhibiting or killing sensitive bacteria. They are biodegradable, digestible, and highly safe. Research on bacteriocins has increased in recent years, and bacteriocins have become a research hotspot in areas such as animal antibiotic alternatives, food biopreservation, and biopharmaceuticals. The US FDA approved nisin, a lactic acid bacterium, as a biopreservative in 1988, further garnering significant attention. Bacteriocins are currently widely used in food production, but while research has been conducted in the livestock sector, their practical application is minimal. Furthermore, the low yield of bacteriocins produced by wild mushrooms severely restricts their practical application. Using molecular biological methods to increase the production of bacteriocin through exogenous expression is an effective and feasible method.

[0004] Patent document CN110982745A discloses a Pediococcus pentosaceus Z-1, whose bacteriocin has an antibacterial effect, but it is sensitive to proteinase K, pepsin, trypsin and papain, and is easily degraded by proteases and loses its activity, which makes it unable to produce an effect in animals. Summary of the Invention

[0005] The first object of the present invention is to provide a Pediococcus pentosaceus that produces a novel type IIa bacteriocin;

[0006] The second object of the present invention is to provide the use of the Pediococcus pentosaceus;

[0007] The third object of the present invention is to provide a genetically engineered bacterium based on the Pediococcus pentosaceus bacteriocin and its application.

[0008] The present invention screened a strain of Pediococcus pentosaceus ( Pediococcus pentosus ) ZPP083, and deposited it in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the accession number CGMCC No. 31944.

[0009] The present invention Pediococcus pentosaceus ( Pediococcus pentosus ) ZPP083 can produce class IIa bacteriocin, which retains antibacterial activity after treatment at pH 4.1 and catalase, and after treatment at high temperature (40-100°C), as well as after treatment with proteases (trypsin, papain, proteinase K, pepsin).

[0010] Furthermore, the present invention provides a bacterial agent comprising the above-mentioned strain. The bacterial agent can be a single agent consisting of the above-mentioned strain and a carrier, or a composite agent consisting of the above-mentioned strain and other probiotics, wherein the probiotics are preferably resistant to the above-mentioned bacteriocin.

[0011] Furthermore, the present invention provides a bacteriocin produced by the aforementioned Pediococcus pentosaceus. Its amino acid sequence is shown in SEQ ID No. 2: (MKKIEKLTEKEMANIIGGKYYGTGLSCGIHSFSVDWGNATTCIRNNGAMAWATGGHQGTHKC). The present invention also includes bacteriocins with equivalent activity formed by replacing, deleting, or adding one or more amino acids in the aforementioned sequence.

[0012] Furthermore, the present invention also provides a gene encoding the above-mentioned bacteriocin. In one embodiment of the present invention, the nucleotide sequence of the gene is shown in SEQ ID No. 1. It should be understood in the art that, taking into account factors such as codon degeneracy, the nucleotide sequence of the encoding gene also includes a sequence capable of expressing an equivalent active protein formed by replacing, deleting, or adding one or more nucleotides to the sequence shown in SEQ ID No. 1.

[0013] Furthermore, the present invention also includes complementary sequences of the above-mentioned coding genes, transcribed RNA, and the like.

[0014] Furthermore, the present invention also provides a recombinant vector comprising the encoding gene. The vector can be a cloning vector or an expression vector.

[0015] Furthermore, the present invention also provides a genetically engineered bacterium transformed with the vector. The starting bacterium of the genetically engineered bacterium may be Saccharomyces cerevisiae. In one embodiment of the present invention, the starting bacterium of the genetically engineered bacterium is a yeast.

[0016] Furthermore, the present invention also provides products comprising the above-mentioned Pediococcus pentosaceus or the above-mentioned bacterial agent or the above-mentioned bacteriocin or engineered bacteria. The products include but are not limited to: feed additives, feeds, food additives, foods, preservatives, medicines or health products.

[0017] Furthermore, the present invention also provides uses of the above-mentioned Pediococcus pentosaceus or the above-mentioned bacterial agent or the above-mentioned bacteriocin or engineered bacteria, including but not limited to use in the preparation of antibacterial products.

[0018] Furthermore, the present invention also provides a method for preparing bacteriocin, which is obtained by culturing the above-mentioned Pediococcus pentosaceus or engineered bacteria.

[0019] The present invention Pediococcus pentosaceus ( Pediococcus pentosus ZPP083 has excellent culture characteristics and good antibacterial activity. This bacteriocin is acid- and temperature-resistant and retains antibacterial activity even after protease treatment. The genetically engineered bacteria constructed by this invention can further enhance bacteriocin expression and improve antibacterial activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the culture growth curve of the strain of the present invention;

[0021] FIG2 is a stability test result of the bacteriocin of the strain of the present invention, wherein Figure 2A Shown is the effect of catalase treatment, Figure 2B Shown are the effects of different pH treatments. Figure 2C Shown are the effects of different temperature treatments. Figure 2D Shown are the effects of different protease treatments, Figure 2E Shown is the antibacterial effect at pH 4.1;

[0022] Figure 3 It is the electrophoresis diagram of the engineering bacteria induced to express bacteriocin. DETAILED DESCRIPTION

[0023] The following specific embodiments are used to further illustrate the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention.

[0024] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0025] Example 1. Strain screening and identification

[0026] Using MRS medium as the base medium, collect pig feces sample, weigh 25g, place it in a glass bead flask filled with 225mL sterile saline, shake it thoroughly to obtain bacterial solution. Pipette 1.0mL of the bacterial solution into a test tube filled with 9.0mL sterile saline and mix it evenly. This dilution is 10 -1 Repeat the above steps to make 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 Select 10 -4 , 10 -5 , 10 -6 Three dilutions were made, and 0.1 mL of bacterial liquid was taken from each dilution and dropped onto MRS medium plates (Beijing Aoboxing). The culture was carried out at 37°C under anaerobic conditions for 24 to 48 hours. Typical colonies were picked and their growth and acid production performances were observed. A total of 15 strains with better performance were isolated. An antibacterial test was carried out on the 15 strains (after activating the Pediococcus pentosaceus ZPP083 strain, it was inoculated into MRS liquid medium (Beijing Aoboxing) at a 1% (v / v) inoculation volume. After static culture at 37°C for 24 hours, an appropriate amount of fermentation liquid was taken, and the supernatant was taken after centrifugation at 6000 rpm for 10 minutes. The indicator bacteria Escherichia coli ( Escherichia coli C83905), Salmonella ( Salmonella A-72), Staphylococcus aureus ( Staphylococcus aureus 8028) The number of viable bacteria in the bacterial solution was adjusted to 10 7 CFU / mL, the fermentation broth and supernatant were diluted 2-fold, and 180 μL of each dilution was pipetted and added to an Oxford cup. The indicator bacteria were cultured at 37°C and the inhibition diameter was measured. The results are shown in Table 1. ZPP083 grew for 24 hOD 600nm The highest value was 2.122, and the number of viable bacteria was 3.55×10 9The lowest pH value was 4.01, and the highest inhibitory diameter against Salmonella was 25.25 mm. 16S rDNA of the strains was amplified by PCR and sequenced. After these tests, a strain with excellent growth performance and acid and bile salt tolerance was identified, identified as ZPP083. Colony morphology, physiological and biochemical characteristics, and 16S rDNA molecular identification were performed. 16S rDNA of strains 27F (SEQ ID NO. 3: 5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (SEQ ID NO. 4: 5'-TACGACTTAACCCCAATCGC-3') was amplified by PCR and sequenced. Sequencing of the amplified products was performed. Pediococcus pentosaceus was grown in MRS medium and incubated anaerobically at 37°C for 24 hours. White, round colonies with a moist, opaque surface were observed. The bacteria were spherical, 0.8-1.0 μm, arranged singly or in pairs, and Gram-positive. The strain ZPP083 was identified as Pediococcus pentosaceus ( Pediococcus pentosus ).

[0027] The strain ZPP083 was sent to the General Microbiology Center of China Culture Collection Administration for Microorganisms (Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, Postal Code: 100101) for patent deposit on September 11, 2024. The deposit number is CGMCC No. 31944, the deposit name is ZPP083, and the taxonomic name is Pediococcus pentosaceus. Pediococcus pentosus .

[0028] Table 1 Growth characteristics and antibacterial screening test results

[0029]

[0030]

[0031] 2. Growth curve of Pediococcus pentosaceus

[0032] After activation, Pediococcus pentosaceus ZPP083 was inoculated into MRS liquid medium (available from Beijing Aoboxing) at inoculum levels of 0.5% (v / v), 1% (v / v), 2% (v / v), 3% (v / v), and 5% (v / v), respectively, and incubated at 37°C for 48 h. The OD values of the bacterial cultures from 0 to 48 h were measured using the fully automated growth curve analyzer Bioscreen C (Shanghai Weizai Technology Co., Ltd.). 600nm Value, draw the growth curve. Figure 1 As shown in the figure, the best growth was achieved when the inoculation amount of Pediococcus pentosaceus was 0.5%.

[0033] Example 2 Determination of the stability of Pediococcus pentosaceus bacteriocin

[0034] After the bacteria were activated, they were inoculated into MRS broth at a 1% inoculum volume and cultured at 37°C for 24 h. After the culture was completed, the supernatant was centrifuged at 6000 rpm for 15 min. The supernatant was filtered with a 0.22 μm filter membrane and stored at 4°C for later use.

[0035] (1) Organic acid exclusion

[0036] The pH of the supernatant was adjusted to pH 4.1 using 1 mol / L NaOH and HCl solutions. The blank control group consisted of untreated supernatant. An antibacterial test was performed using Salmonella as an indicator bacteria. The diameter of the inhibition zone was measured to evaluate the effect of organic acids on the antibacterial effect of bacteriocins.

[0037] (2) Hydrogen peroxide elimination

[0038] The pH of the bacterial supernatant was adjusted to 7.0 using 1 mol / L NaOH and HCl solutions, and 10 mg / mL catalase was added. After incubation in a 37°C water bath for 2 h, the pH was adjusted back to the original pH 4.1 for antibacterial testing. The supernatant not treated with catalase served as a blank control.

[0039] (3) Protease stability assay

[0040] Take four 1 mL aliquots of the supernatant and add 10 mg / mL of trypsin, papain, or pepsin, respectively, and 50 μL of proteinase K. Adjust the pH of the solutions to their respective optimal values (trypsin: 7.4; papain: 6.5; pepsin: 4.5; proteinase K: 7.5). Incubate the solutions in a 37°C water bath for 2 hours, then return the pH to its original value for antibacterial testing. Untreated supernatant served as a blank control.

[0041] (4) Thermal stability determination

[0042] The supernatant was treated in a water bath at 40°C, 60°C, 80°C, and 100°C. The 100°C water bath was used for 30 minutes, and the remaining temperature gradients were used for 4 hours. The room temperature supernatant served as a blank control for the antibacterial test.

[0043] (5) pH stability determination

[0044] Take four 1 mL aliquots of the concentrated supernatant of the isolated bacteria and adjust the pH to 3.0, 4.0, 5.0, and 6.0 using 1 mol / L NaOH and HCl solutions, respectively. After standing at room temperature for 1 hour, return the pH to the initial pH using NaOH and HCl solutions. Determine the antibacterial activity of the bacteriocin under different pH treatments, using the untreated supernatant as a blank control.

[0045] The stability test of bacteriocin produced by the strain is shown in Figure 2. The results show that its antibacterial activity did not decrease significantly after treatment with pH 3.0-6.0 and temperature (40℃, 60℃, 80℃ and 100℃). Figure 2B , C); 4 kinds of proteases were added. Compared with the control group, trypsin was more sensitive, but the antibacterial diameter was still above 20mm (Figure D). In addition, after the supernatant was treated with pH 4.1 and catalase, the antibacterial diameter was still above 21mm ( Figure 2E , A), indicating that the antibacterial substance in the supernatant is bacteriocin, and bacteriocin has strong acid-resistant and high-temperature-resistant activity.

[0046] Example 3 Construction of bacteriocin heterologous expression engineering bacteria

[0047] The complete genome sequence of Pediococcus pentosaceus was obtained and its gene sequences were aligned using NCBI blast analysis. The CDS sequence encoding the bacteriocin gene was determined as SEQ ID No. 1, and its amino acid sequence was determined as SEQ ID No. 2. Based on this sequence, primers were designed, and a heterologous expression strain producing the bacteriocin was constructed by PCR cloning, purification, and insertion into an expression vector.

[0048] (1) Pediococcus pentosaceus was inoculated into MRS liquid medium and cultured at 37°C for 18 h. The bacteria were harvested by centrifugation and total DNA was extracted using a DNA extraction kit according to the operating instructions. The DNA was stored at -20°C for later use. The relevant primers were designed using the software snapgene 6.0.2 and synthesized by a biological company: The PCR amplification product (F (Seq ID No. 6)) was purified using the PCR product purification kit:

[0049] 5-CACCGTTAATTAACCCGGGGATCCATGAAAAAAATTGAAAAATTAACT-3'; R (Seq ID No. 7): 5'- CGTCAAGGAGAAAAAACCCCGGATCCCTAGCATTTATGAGTACCTTGATGT-3').

[0050] The linearized plasmid fragment pAM1 and the gene fragment were ligated using a one-step method and transformed into E. coli DH5α for cloning. A single colony was selected and incubated in 500 μL of LB liquid medium (containing ampicillin) at 37°C with a shaker at 220 rpm for 1 hour. Colony PCR was performed, and positive colonies were sent to a biotechnology company for sequencing.

[0051] Table 2 Connection system

[0052] Connecting components Volume (4 μL) Linearized plasmid 0.5μL Destination fragment 1.5 μL 2× recombinase 2μL

[0053] The recombinase (2×MultiF Seamless Assembly Mix) reaction temperature is 50°C, the reaction time is 45 min, and centrifugation is required after completion for E. coli transformation.

[0054] (2) Transfer into yeast and verify its function

[0055] Yeast cell transformation: (1) Inoculate YPG30 yeast cells in 10 mL of YPD medium and culture at 30°C, 220 rpm overnight; (2) Dilute the bacterial solution 10-fold and culture at 30°C, 220 rpm for about 5 h to reach the logarithmic growth phase; (3) Centrifuge at 800 × g for 2 min, discard the supernatant, add 3 mL of ddH2O, pipette and resuspend, centrifuge at 800 g for 2 min, discard the supernatant: Place 2 mg / mL ssDNA in a 95°C metal bath in advance, heat for 5 min, and then pre-cool on ice; (4) Let it stand at room temperature for 30 min, heat shock in a 42°C water bath for 15 min, and then place it on ice for 5 min. Centrifuge at 800 × g for 2 min, discard the supernatant, add 200 μL of ddH2O to resuspend the bacteria, and transfer them to SD medium (without Ura3) plates and culture at 30°C for 3 days.

[0056] Yeast function verification: The engineered bacteria were transferred to 5 mL of YPR medium and cultured at 30°C, 220 rpm for 16 h. After 16 h, 1 mL of YPR medium was transferred to 3 mL of YPG medium and cultured at 30°C, 220 rpm for 4 h.

[0057] (3) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

[0058] The YPG bacterial solution was centrifuged at 12,000 rpm for 1 minute and the supernatant was discarded. Proteins in the sterilized supernatant were extracted using a protein extraction kit and then identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that the molecular weight of the bacteriocin was 27 kDa ( Figure 3 ), protein bands appeared after galactose induction.

[0059] (4) Antibacterial test of engineered bacteria

[0060] The engineered bacteria solution was centrifuged at 6000×g for 30 minutes at 4°C, and the supernatant was aspirated for Oxford cup test to identify its antibacterial effect. The results showed that the engineered bacteria had good antibacterial effects on all four harmful bacteria, among which the best effect was on Salmonella, with an antibacterial diameter of 28.0 mm.

[0061] Table 3 Diameter of inhibition zone of engineered bacteria against harmful bacteria (mm)

[0062]

[0063] Comparison of Example 4 with existing recombinant bacteriocins

[0064] Among the currently available bacteriocins, pediocin PA-1 (GenBank: KY038164.1, hereinafter referred to as pediocin PA-1, with a DNA sequence as shown in SEQ ID No. 8) is relatively close to the sequence of the present invention. Based on its DNA sequence, the present invention synthesized its sequence by sequence synthesis and verified it by sequencing. Then, according to the method of Example 3 above, an engineered bacterium expressing the bacteriocin was constructed and induced for expression. The YPG bacterial solution was centrifuged at 12,000 rpm for 1 minute, and the supernatant was discarded. Protein was extracted from the sterilized supernatant using a protein extraction kit, and the extracted protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that the bacteriocin had a molecular weight of 32 kDa, and a protein band appeared upon induction with galactose.

[0065] Comparison of antibacterial activity and stability of bacteriocins

[0066] 1. Test methods

[0067] Construction of a simulated porcine gastric juice system: Take 0.2 mol / L sodium dihydrogen phosphate solution, adjust the pH to 4.0 with sodium hydroxide test solution, and add pepsin K to 0.3% (w / v) to simulate the digestive function of gastric juice.

[0068] Experimental Grouping and Testing: Simulated gastric fluid served as the negative control group (direct simulated gastric fluid test). After induction of expression in the engineered pediocin PA-1 bacteria, the supernatant was centrifuged at 8000×g for 30 minutes at 4°C and collected as the control group. After induction of expression in the engineered PP083 bacteria, the supernatant was centrifuged at 8000×g for 30 minutes at 4°C and collected as the experimental group. In the treatment group, 1 ml of supernatant was added to 1 ml of simulated gastric fluid, mixed, and incubated at 37°C with constant shaking (100 rpm) for 1 hour to simulate gastric peristalsis. In the negative control group and the engineered bacteria pre-treatment group, 1 ml of distilled water was added to each group, mixed, and incubated at 37°C with constant shaking (100 rpm) for 1 hour to simulate gastric peristalsis. The antibacterial activity of different bacteriocins against Salmonella was determined using the Oxford cup assay with six replicates per group, and the mean inhibitory diameter was calculated.

[0069] 2. Test results

[0070] The test results are shown in Table 4.

[0071] Table 4 Comparison of the stability of antibacterial effects of different bacteriocins (mm)

[0072]

[0073] The above results show that the bacteriocin of the present invention has good antibacterial ability compared with the control, and after treatment with simulated gastric fluid, its antibacterial activity is still above 28mm, without significant decrease, and the antibacterial activity of the control bacteriocin is lower than that of the present invention; In addition, the stability of the supernatant of the present invention before and after treatment with simulated gastric fluid is as high as 88.46%, while the stability of the control group before and after treatment is only 73.63%, which is significantly lower than that of the bacteriocin engineered bacteria of the present invention by 15 percentage points. Overall, the bacteriocin of the present invention and the control bacteriocin both have excellent antibacterial properties. At the same time, the bacteriocin of the present invention is significantly better than the control bacteriocin in terms of protease stability and pH stability in the simulated gastric fluid environment.

Claims

1. A bacteriocin, the amino acid sequence of which is the amino acid sequence shown in SEQ ID No.

2.

2. A gene encoding the bacteriocin according to claim 1.

3. A vector containing the coding gene according to claim 2.

4. An engineered bacterium containing the vector according to claim 3.

5. A product containing the bacteriocin according to claim 1 or the engineered bacteria according to claim 4, wherein the product is a feed additive, feed or medicine.

6. Use of the bacteriocin according to claim 1 or the engineered bacteria according to claim 4 in the preparation of an antibacterial product, wherein the product is a feed additive, feed or medicine, and the bacteria is Escherichia coli, Salmonella or Staphylococcus aureus.

7. A method for preparing bacteriocin, which is obtained by culturing the engineered bacteria according to claim 4.

Images