Pediococcus pentosaceus capable of producing novel IIa bacteriocin, application of pediococcus pentosaceus and construction of secretory engineering bacteria

Abstract

The invention provides a Pediococcus pentosaceus ZPP083 strain, which is preserved in the General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms, and the preservation number is CGMCC No.31944. The Pediococcus pentosaceus ZPP083 strain has the advantages that the Pediococcus pentosaceus ZPP083 strain can be used for preparing the Pediococcus pentosaceus ZPP083 strain; the pediococcus pentosaceus provided by the invention has a relatively strong antibacterial effect, can produce novel IIa bacteriocin, and has the characteristics of acid resistance, high temperature resistance, protease resistance and the like. Genetically engineered bacteria are constructed by using the bacteriocin, and the expression quantity is improved.

Description

Technical Field

[0001] The 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 phenomenon of drug-resistant bacteria caused by the abuse of antibiotics and drug residues have made feed safety and human health issues increasingly prominent. The research and development of effective drugs for treating drug-resistant bacteria are included in the global research priorities announced by the World Health Organization. Therefore, in order to control drug residues, improve food safety and the sustainable development of animal husbandry, it is urgent to develop new antibiotic alternatives.

[0003] Bacteriocin is a type of low-molecular protein or peptide produced by bacteria during metabolism that can inhibit the growth of other bacteria except the producing bacteria. It is considered to be a potential substitute for antibiotics due to its high antibacterial activity, non-toxicity, and non-resistance. Antibiotics are not easily degraded, which can easily lead to bacterial resistance, and can kill sensitive bacteria regardless of whether they are beneficial or harmful. In contrast, bacteriocin is an antibacterial substance produced by bacterial ribosomes. As a protein, bacteriocin not only has strong antibacterial activity, but is also relatively stable, selectively inhibits or kills sensitive bacteria, is biodegradable, digestible, and highly safe. In recent years, research on bacteriocin has gradually increased, and bacteriocin has become a research hotspot in the fields of animal antibiotic substitutes, food biopreservation, and biopharmaceuticals. In 1988, the US FDA approved lactic acid bacteria (Nisin) and other products as biological preservatives, and the research on bacteriocin has attracted more attention. Bacteriocin is currently widely used in food production, but in the field of animal husbandry, although there have been studies, it is rarely used in production. In addition, the yield of bacteriocin produced by wild fungi is low, which seriously restricts its application in actual production. 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 effects 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; The second object of the present invention is to provide the use of the Pediococcus pentosaceus; The third object of the present invention is to provide a genetically engineered bacterium based on the Pediococcus pentosaceus bacteriocin and its application.

[0006] The present invention screened a strain of Pediococcus pentosaceus ( Pediococcus pentosus ) ZPP083, and deposited it in the General Microbiology Center of China Microbiological Culture Collection Administration with the deposit number CGMCC No. 31944.

[0007] The present invention Pediococcus pentosaceus ( Pediococcus pentosus ) ZPP083 can produce class IIa bacteriocin, which still has antibacterial activity after treatment at pH 4.1 and catalase, and after treatment at high temperature (40-100°C), and after treatment with proteases (trypsin, papain, proteinase K, pepsin).

[0008] Furthermore, the present invention provides a bacterial agent comprising the above strain. The bacterial agent can be a single agent consisting of the above strain and a carrier, or a composite bacterial agent consisting of the above strain and other probiotics, wherein the probiotics preferably have the property of being resistant to the above bacteriocin.

[0009] Furthermore, the present invention also provides a bacteriocin produced by the Pediococcus pentosaceus. Its amino acid sequence is shown in SEQ ID No. 2. (MKKIEKLTEKEMANIIGGKYYGTGLSCGIHSFSVDWGNATTCIRNNGAMAWATGGHQGTHKC) The bacteriocin of the present invention also includes a bacteriocin with equivalent activity formed by replacing, deleting or adding one or more amino acids in the sequence.

[0010] Furthermore, the present invention also provides a gene encoding the above bacteriocin, and 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, considering factors such as the degeneracy of codons, 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 of the sequence shown in SEQ ID No. 1.

[0011] Furthermore, the present invention also includes complementary sequences of the above-mentioned coding genes, transcribed RNA, and the like.

[0012] Furthermore, the present invention also provides a recombinant vector comprising the encoding gene. The vector may be a cloning vector or an expression vector.

[0013] Furthermore, the present invention also provides a genetically engineered bacterium for transforming the vector. The starting bacterium of the genetically engineered bacterium may be a cerevisiae yeast. In one embodiment of the present invention, the starting bacterium of the genetically engineered bacterium is a yeast.

[0014] Furthermore, the present invention also provides products comprising the above Pediococcus pentosaceus or the above bacterial agent or the above bacteriocin or engineered bacteria. The products include but are not limited to: feed additives, feeds, food additives, foods, preservatives, medicines or health products.

[0015] 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.

[0016] Furthermore, the present invention also provides a method for preparing bacteriocin, which is obtained by culturing the above-mentioned Pediococcus pentosaceus or engineered bacteria.

[0017] The present invention Pediococcus pentosaceus ( Pediococcus pentosus ) ZPP083 has good culture characteristics and good antibacterial ability. The bacteriocin has the characteristics of acid and high temperature resistance, and still has antibacterial activity after being treated with protease. The genetically engineered bacteria constructed by the present invention can further improve the expression of bacteriocin and improve the antibacterial ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the culture growth curve of the strain of the present invention; FIG2 is a stability test result of bacteriocin of the strain of the present invention, wherein Figure 2A Shown is the effect of catalase treatment, Figure 2B Shown is the effect of different pH treatments. Figure 2C Shown is the effect 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; Figure 3 It is the electrophoresis diagram of the engineering bacteria induced to express bacteriocin of the present invention. DETAILED DESCRIPTION

[0019] 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.

[0020] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0021] Example 1. Strain screening and identification Using MRS medium as the basic medium, collect pig feces samples, weigh 25g, place in a glass beaded flask containing 225mL of sterile saline, and shake thoroughly to obtain bacterial solution. Pipette 1.0mL of the bacterial solution into a test tube containing 9.0mL of sterile saline and mix well. 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 for 24 to 48 hours under anaerobic conditions. Typical colonies were picked to observe the growth performance and acid production performance. A total of 15 strains with better performance were isolated. Antibacterial tests were carried out on the 15 strains (after activating the Pediococcus pentosaceus ZPP083 strain, it was inoculated in MRS liquid medium (Beijing Aoboxing) at a rate of 1% (v / v). 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 live bacteria in the bacterial solution was adjusted to 10 7 CFU / mL, the fermentation broth and supernatant were diluted and diluted 2 times, 180 μL of each dilution was taken and added to the Oxford cup. The indicator bacteria were cultured at 37°C and the inhibition diameter was determined. The results are shown in Table 1. Among them, ZPP083 grew for 24hOD 600nm The highest value was 2.122, and the number of viable bacteria was 3.55×10 9 CFU / g, the lowest pH value was 4.01, and the highest antibacterial diameter for Salmonella was 25.25mm. The 16Sr DNA of the strain was amplified by PCR and sequenced. After the above tests, a strain with excellent growth performance and acid and bile resistance was screened, which was ZPP083. Colony morphology observation, physiological and biochemical characteristics analysis and 16Sr DNA molecular identification were performed. The 16Sr DNA of the strain was amplified by PCR (primers 27F SEQID NO.3: 5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (SEQ ID NO.4: 5'-TACGACTTAACCCCAATCGC-3') and sequenced, and the sequencing results of the amplified products were sequenced. Pediococcus pentosaceus was grown in MRS medium and cultured anaerobically at 37℃ for 24 h. The colonies were white, round, moist and opaque. 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 ).

[0022] The strain ZPP083 was sent to the General Microbiology Center of China Microbiological Culture Collection Administration (address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101) for patent deposit on September 11, 2024, with the deposit number: CGMCC No. 31944, the deposit name: ZPP083, and the classification name: Pediococcus pentosaceus Pediococcus pentosus .

[0023] Table 1 Growth characteristics and antibacterial screening test results

[0024] 2. Growth curve of Pediococcus pentosaceus After activation, Pediococcus pentosaceus ZPP083 was inoculated into MRS liquid medium (available from Beijing Aoboxing) at inoculation amounts of 0.5% (v / v), 1% (v / v), 2% (v / v), 3% (v / v), and 5% (v / v), respectively, and cultured at 37°C for 48 h. The OD of the bacterial solution from 0 to 48 h was measured using the fully automatic growth curve analyzer Bioscreen C (Shanghai Weizai Technology Co., Ltd.). 600nm Value, draw a growth curve. Figure 1 As shown, the growth of Pediococcus pentosaceus was optimal when the inoculation amount was 0.5%.

[0025] Example 2 Determination of the stability of Pediococcus pentosaceus bacteriocin After the bacteria were activated, they were inoculated into MRS broth at a 1% inoculum and cultured at 37°C for 24 hours. After the culture was completed, the culture was centrifuged at 6000 r / min for 15 minutes. The supernatant was filtered with a 0.22 μm filter membrane and stored at 4°C for later use.

[0026] (1) Organic acid exclusion The pH of the supernatant was adjusted to pH 4.1 using 1 mol / L NaOH and HCl solutions. The blank control group was the untreated supernatant. The antibacterial test was carried out using Salmonella as the indicator bacteria, and the diameter of the inhibition zone was measured to evaluate the effect of organic acids on the antibacterial effect of bacteriocins.

[0027] (2) Hydrogen peroxide removal The pH of the bacterial supernatant was adjusted to 7.0 using 1 mol / L NaOH and HCl solutions, and 10 mg / mL of 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 an antibacterial test. The supernatant not treated with catalase was used as a blank control.

[0028] (3) Protease stability assay Take 4 portions of 1 mL of supernatant and add 10 mg / mL of trypsin, papain, pepsin and 50 μL of proteinase K respectively. Adjust the pH of the solution to their respective optimal values ​​(trypsin: 7.4; papain: 6.5; pepsin: 4.5; proteinase K: 7.5). After incubating the solution in a 37°C water bath for 2 hours, adjust the pH back to the original value and perform the antibacterial test. The untreated supernatant was used as a blank control.

[0029] (4) Thermal stability determination The supernatant was treated in a water bath at 40℃, 60℃, 80℃ and 100℃ respectively. The water bath was 30 minutes at 100℃ and 4 hours at other temperature gradients. The supernatant at room temperature was used as a blank control for the antibacterial test.

[0030] (5) pH stability determination Take 4 1 mL portions of the concentrated supernatant of isolated bacteria and adjust the pH of the supernatant to 3.0, 4.0, 5.0 and 6.0 with 1 mol / L NaOH and HCl solutions, respectively. After standing at room temperature for 1 hour, restore the pH to the initial pH with NaOH and HCl solutions. Determine the antibacterial activity of bacteriocins under different pH treatments, and use the untreated supernatant as the blank control.

[0031] The stability test of bacteriocin produced by the strain is shown in Figure 2. The results show that after treatment with pH 3.0-6.0 and temperature (40℃, 60℃, 80℃ and 100℃), its antibacterial activity did not decrease significantly ( 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.

[0032] Example 3 Construction of bacteriocin heterologous expression engineering bacteria The complete genome sequence of Pediococcus pentosaceus was obtained and its gene sequence was compared using NCBI blast to determine the CDS sequence of the bacteriocin gene as shown in SEQ ID No. 1 and its amino acid sequence as shown in SEQ ID No. 2. Primers were designed based on this sequence, and a heterologous expression strain producing bacteriocin was constructed by PCR cloning, purification, and insertion into an expression vector.

[0033] (1) Inoculate Pediococcus pentosaceus into MRS liquid medium, culture at 37℃ for 18h, harvest the bacteria by centrifugation, extract total DNA according to the DNA extraction kit instructions, and store at -20℃ for later use. Use snapgene 6.0.2 software to design relevant primers, which are synthesized by a biological company: Purify the PCR amplification product (F (Seq ID No.6) according to the PCR product purification kit: 5-CACCGTTAATTAACCCGGGGATCCATGAAAAAAATTGAAAAATTAACT-3'; R (Seq ID No. 7): 5'- CGTCAAGGAGAAAAAACCCCGGATCCCTAGCATTTATGAGTACCTTGATGT-3').

[0034] The linear plasmid fragment pAM1 and the gene fragment were connected by one-step method and transferred into E. coli DH5α for cloning. A single clone was selected and placed in 500 μL LB liquid medium (containing ampicillin antibiotics) and cultured at 37°C and 220 rpm for 1 hour. Colony PCR was performed, and the single colony that was positive for colony PCR was sent to a biological company for sequencing.

[0035] Table 2 Connection system Connecting components Volume (4 μL) Linearized plasmid 0.5μL Purpose fragment 1.5μL 2× recombinase 2μL The reaction temperature of the recombinase (2×MultiF Seamless Assembly Mix) was 50°C and the reaction time was 45 min. After the reaction, centrifugation was required for E. coli transformation.

[0036] (2) Transfer into yeast and verify its function Yeast cell transformation: (1) Inoculate YPG30 yeast cells in 10 mL YPD medium and culture overnight at 30°C, 220 rpm; (2) Dilute the bacterial solution 10 times and culture at 30°C, 220 rpm for about 5 h until the logarithmic growth phase is reached; (3) Centrifuge at 800 × g for 2 min, discard the supernatant, and add 3 mL ddHO. 2 O and resuspend by aspiration, centrifuge at 800 × g for 2 min, and discard the supernatant: Place 2 mg / mL ssDNA in a 95°C metal bath in advance, heat for 5 min, and then precool on ice; (4) Stand at room temperature for 30 min, heat shock in a 42°C water bath for 15 min, and ice bath for 5 min. Centrifuge at 800 × g for 2 min, discard the supernatant, and add 200 μL ddHO 2 Resuspend the cells in 5% paraformaldehyde and transfer them to SD medium (without Ura3) plates and culture at 30°C for 3 days.

[0037] Yeast function verification: The constructed engineered bacteria were transferred to 5 mL YPR medium and cultured at 30°C 220 rpm for 16 h. After 16 h, 1 mL YPR medium was transferred to 3 mL YPG medium and cultured at 30°C 220 rpm for 4 h.

[0038] (3) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) The YPG bacterial solution was centrifuged at 12000 rpm for 1 min and the supernatant was discarded. The protein in the sterilized supernatant was extracted 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 molecular weight of the bacteriocin was 27 kDa ( Figure 3 ), protein bands appeared after galactose induction.

[0039] (4) Antibacterial test of engineered bacteria The engineered bacterial solution was centrifuged at 4°C and 6000×g for 30 min, and the supernatant was aspirated for Oxford cup test to identify its antibacterial effect. The results showed that the engineered bacteria had good antibacterial effect on the four harmful bacteria, among which the best effect was on Salmonella, with an antibacterial diameter of 28.0 mm.

[0040] Table 3 Diameter of inhibition zone of engineered bacteria against harmful bacteria (mm)

[0041] Example 4 Comparison with existing recombinant bacteriocins Among the currently disclosed bacteriocins, bacteriocin pediocin PA-1 (GenBank: KY038164.1, hereinafter referred to as bacteriocin pediocin PA-1, DNA sequence as shown in SEQID No.8) is relatively close to the sequence of the present invention. The present invention synthesizes its sequence by a sequence synthesis method based on its DNA sequence, and performs sequencing verification. Then, according to the method of the above-mentioned Example 3, a bacteriocin heterologous expression engineering bacterium is constructed, and induced expression is performed. The YPG bacterial solution is centrifuged at 12000 rpm for 1min, and the supernatant is discarded. The protein in the sterilized supernatant is extracted using a protein extraction kit, and the extracted protein is then identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results show that the molecular weight of the bacteriocin is 32kDa, and a protein band appears after galactose induction.

[0042] Comparison of antibacterial activity and stability of bacteriocins 1. Test methods Construction of a simulated porcine gastric juice system: Take 0.2 mol / L sodium dihydrogen phosphate solution, adjust the pH value 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.

[0043] Experimental grouping and test: simulated gastric fluid was used as negative control group 1 (simulated gastric fluid direct test), pediocinPA-1 engineered bacteria were induced to express and centrifuged at 4℃ 8000×g for 30min to take the supernatant as the control group, and PP083 engineered bacteria were induced to express and centrifuged at 4℃ 8000×g for 30min to take the supernatant as the experimental group. 1ml of supernatant was taken from the treatment group, 1ml of simulated gastric fluid was added to mix, and constant temperature oscillation (100 rpm) was maintained at 37℃ for 1 hour to simulate the mechanical peristalsis environment of the stomach. 1ml of distilled water was added to each group of the negative control group and the group before engineering bacteria treatment, and the mixture was stirred at 37℃ for 1 hour to simulate the mechanical peristalsis environment of the stomach. The Oxford cup method was used to test, with 6 replicates in each group, to determine the antibacterial activity of different bacteriocins against Salmonella, and the average antibacterial diameter was calculated.

[0044] 2. Test results The test results are shown in Table 4.

[0045] Table 4 Comparison of the stability of antibacterial effects of different bacteriocins (mm)

[0046] The above results show that the bacteriocin of the present invention has good antibacterial ability compared with the control, and after being treated with simulated gastric juice, 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 juice 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 the bacteriocin engineering bacteria of the present invention by 15 percentage points. In general, the bacteriocin of the present invention and the control bacteriocin have excellent antibacterial properties, and 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 juice environment.

Claims

1. Pediococcus pentosaceus Pediococcus pentosus ) ZPP083, whose accession number is CGMCC No. 31944.

2. A bacterial agent containing the Pediococcus pentosaceus according to claim 1.

3. A bacteriocin, whose amino acid sequence is: the amino acid sequence shown in SEQ ID No. 2; or a sequence with equivalent function formed by replacing, deleting or adding one or more amino acids in the sequence shown in SEQ ID No.

2.

4. A gene encoding the bacteriocin according to claim 3.

5. A vector containing the coding gene according to claim 4.

6. An engineered bacterium transformed with the vector according to claim 5.

7. A product containing the Pediococcus pentosaceus according to claim 1, the bacterial agent according to claim 2, the bacteriocin according to claim 3 or the engineered bacteria according to claim 6.

8. The product according to claim 7, characterized in that The product is a feed additive, feed, food additive, food, preservative, medicine or health product.

9. Use of the Pediococcus pentosaceus according to claim 1, the bacterial agent according to claim 2, the bacteriocin according to claim 3 or the engineered bacteria according to claim 6 in the preparation of antibacterial products.

10. A method for preparing bacteriocin, which is obtained by culturing the Pediococcus pentosaceus according to claim 1 or the engineered bacteria according to claim 6.

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