Antimicrobial protein, and coding gene and use thereof

Abstract

The present invention belongs to the technical field of genetic engineering. Provided are an antimicrobial protein, and a coding gene and the use thereof. An amino acid sequence of the antimicrobial protein is a sequence as set forth in any one of SEQ ID NOs. 1-12, or a sequence with the same function as the sequence as set forth in any one of SEQ ID NOs. 1-12 after the substitution and / or deletion and / or addition of 1-30 amino acids, or a sequence with the same function as the sequence as set forth in any one of SEQ ID NOs. 1-12 after the addition of 1-20 amino acids to the C-terminus and / or N-terminus thereof. The produced antimicrobial protein provided can hydrolyze bacterial cell walls, enabling lysis of Micrococcus luteus. Upon verification, the antimicrobial protein can significantly inhibit or kill Gram-positive and Gram-negative bacteria and yeast, thereby providing a new antimicrobial protein preparation and a preparation method therefor for industries such as daily chemicals, food, and feeds.

Description

Bactericidal protein and its encoding gene and application TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, in particular to a bactericidal protein and its encoding gene and application. BACKGROUND

[0002] The cell wall of microorganisms is composed of polysaccharides, mainly including peptidoglycan, glucan, mannan, chitin and cellulose, etc. The genome of fungi contains a large number of unknown proteins with glycoside hydrolase activity. If the polysaccharides mentioned above can be hydrolyzed, the cell wall of microorganisms can be hydrolyzed, and the effect of killing bacteria can be achieved. The known academic research field has not reported the related research of fungal bactericidal protein.

[0003] Trichoderma reesei is an important industrial production strain, which meets the GRAS (Generally Regarded as Safe) standard. The enzyme preparation produced by fermentation has been widely used in food, feed and other industries. The known academic research field has not reported that the Trichoderma reesei strain can ferment and produce fungal bactericidal protein.

[0004] The present application intends to use Trichoderma reesei to express and secrete bactericidal protein, thereby developing a new technical idea and providing a new technical scheme for the field of pathogenic bacteria killing. SUMMARY

[0005] The present application provides a bactericidal protein and its encoding gene and application to solve the problems existing in the prior art. The bactericidal protein produced by the present application can hydrolyze the cell wall of bacteria and cause the lysis of Micrococcus luteus. It has been verified that the bactericidal protein can significantly inhibit or kill gram-positive bacteria, gram-negative bacteria and yeast, thereby providing a new bactericidal protein preparation and its preparation method for the daily chemical, food, feed and other industries.

[0006] To achieve the above-mentioned purpose, the present application provides the following scheme:

[0007] The present application provides a bactericidal protein, wherein the amino acid sequence of the bactericidal protein is any one of the sequences shown in SEQ ID NO. 1-12, or a sequence with the same function after 1-30 amino acid substitutions and / or deletions and / or additions of any one of the sequences shown in SEQ ID NO. 1-12, or a sequence with the same function after adding 1-20 amino acids to the C-terminal and / or N-terminal of any one of the sequences shown in SEQ ID NO. 1-12.

[0008] In the art, when a substitution is made with a similar or similar amino acid, the function of the protein is usually not changed; the addition of one or several amino acids at the C-terminus and / or N-terminus also usually does not change the function of the protein. Therefore, in the present application, the amino acid sequence of the bactericidal protein also includes variants of any one of SEQ ID NO. 1-12 having the same function as the bactericidal protein. These variants include substitution and / or deletion and / or addition of several (l-30, preferably 1-20, further preferably 1-10, and further preferably 1-5) amino acids, and addition of one or several (1-20, preferably 1-10, and further preferably 1-5) amino acids at the C-terminus and / or N-terminus of any one of SEQ ID NO. 1-12.

[0009] The present application also provides a gene encoding the above-mentioned bactericidal protein, and the nucleotide sequence of the gene is any one of the sequences shown in SEQ ID NO. 13-24, or a sequence having 85% or more homology with any one of the sequences of SEQ ID NO. 13-24.

[0010] Due to the degeneracy of codons (degeneracy refers to a sequence produced after one or more codons are replaced by degenerate codons that encode the same amino acid), in the present application, the nucleotide sequence of the gene encoding the bactericidal protein also includes sequences having 85% or more homology with any one of the sequences of SEQ ID NO. 13-24.

[0011] The present application also provides a recombinant expression vector comprising the nucleotide sequence encoding the above-mentioned bactericidal protein, or comprising the above-mentioned gene.

[0012] The present application also provides a recombinant microorganism comprising the above-mentioned recombinant expression vector.

[0013] Preferably, the chassis microorganism of the recombinant microorganism is a Trichoderma.

[0014] Preferably, the Trichoderma is Trichoderma reesei QM6a, QM9414, RUT-C30, RL-P37, NG14 or PC-3-7.

[0015] The present application also provides the use of the above-mentioned gene, the above-mentioned recombinant expression vector, or the above-mentioned recombinant microorganism in the production of a bactericidal protein.

[0016] The present application also provides a method for producing a bactericidal protein, which comprises fermenting the above-mentioned recombinant microorganism, collecting the fermentation broth, and obtaining the bactericidal protein.

[0017] The application also provides application of the bactericidal protein produced by the production method in preparation of a bactericide.

[0018] The application has the following beneficial effects:

[0019] The application discloses an amino acid sequence of a bactericidal protein and a gene sequence for coding the bactericidal protein. The coding gene of the bactericidal protein is transformed into a Trichoderma genome by using a Trichoderma expression vector CBH1T1.0, and a recombinant Trichoderma strain for secreting and expressing the bactericidal protein is successfully constructed. The recombinant Trichoderma strain can be used for production of the bactericidal protein. Through experiments, it is verified that the bactericidal protein produced by the application can significantly kill gram-positive bacteria, gram-negative bacteria and yeast, and the effect is better than that of common bactericidal proteins in the prior art. The application provides a new bactericidal protein preparation and a preparation method thereof, and application of the bactericidal protein preparation in pathogenic bacteria killing. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description only constitute some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative effort.

[0021] Fig. 1 is a schematic diagram of the structure of the recombinant expression vector in Example 1;

[0022] Fig. 2 is a schematic diagram of the construction process of the recombinant Trichoderma strain in Example 2;

[0023] Fig. 3 is the experimental result of the bacteriostatic circle of the bactericidal protein F5 and commercial lysozyme in Example 4. DETAILED DESCRIPTION

[0024] The various exemplary embodiments of the present application will be described in detail below, which should not be considered as limiting the present application, but should be understood as a more detailed description of some aspects, characteristics and embodiments of the present application.

[0025] It should be understood that the terms described in the present application are only for describing the specific embodiments, and are not used to limit the present application. In addition, for the numerical range in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or intermediate value in the stated range, and any other stated value or intermediate value in the stated range, is also included in the present application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range.

[0026] All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains unless otherwise specifically defined herein. Although preferred methods and materials are described herein, any method and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in full the methods and / or materials which are described herein. In case of conflict between the content of the specification and that of any document incorporated herein by reference, the content of the specification prevails.

[0027] Many modifications and variations of the present application described in the specific embodiments of the application can be made by those skilled in the art without departing from the spirit or scope of the application. Other implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.

[0028] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0029] In the embodiments of the method of the present application, the formulations of the culture medium and reagents are as follows:

[0030] (1) Luria Bertani (LB) medium formulation: yeast powder 5 g, peptone 10 g, sodium chloride 10 g, tap water to 1 L, natural pH.

[0031] (2) PDA medium formulation: potato 200 g, glucose (or xylose) 20 g, agar 20 g, tap water to 1 L, natural pH.

[0032] (3) Trichoderma fermentation medium formulation (1 L): glucose 30 g, lactose 10 g, cellulose powder 10 g, bran 5 g, corn syrup 5 g, peptone 3 g, yeast powder 2 g, KH2PO4 2.0 g, CaCl2 0.34 g, MgSO4·7H2O 0.3 g, Mandels trace element solution 1 mL, Tween 80 1 mL.

[0033] (4) Mandels trace element solution (1000x) formulation: FeSO4·7H2O 5 g, CoCl·6H2O 2 g, ZnSO4·7H2O 1.4 g, MnSO4·H2O 1.6 g, purified water to 1 L.

[0034] (5) Agrobacterium-mediated transformation of Trichoderma: The expression vector was electroporated into Agrobacterium tumefaciens, and then the electroporated Agrobacterium was co-cultured with Trichoderma strains QM6a (ATCC 13631), QM9414 (ATCC 26921), RUT-C30 (ATCC 56765), RL-P37 (NRRL 15709), NG14 (ATCC 56767), PC-3-7 (ATCC 66589) on IM plates (Covert et al. Agrobacterium tumefaciens-mediated transformation of Fusarium circinatum. Mycol. Res. 105(3): 259-264) for two days, and then the co-cultured plates were added with cefotaxime (300 μg / mL) and hygromycin B (75 μg / mL) for screening, until the mycelium and spores were grown, and then PCR verification was performed to prove that the grown mycelium was the correct transformant.

[0035] The plasmid extraction kit was purchased from AXYGEN Company, the gel recovery kit was purchased from MAGEN Company, the seamless cloning kit was selected from Quansheng Company, and the DNA restriction endonuclease and ligase were selected from NEB Company. Other similar products of other companies can also be used instead.

[0036] The experimental methods in the following examples not specified in the specific conditions are generally carried out according to the conventional conditions, such as the conditions described in Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).

[0037] The amino acid sequences of the bactericidal proteins in the following examples are shown in SEQ ID NO. 1-12.

[0038] The synthesis method of the bactericidal protein coding gene provided in the embodiments of the present application is as follows:

[0039] The amino acid sequences (SEQ ID NO. 1-12) are provided, and the corresponding nucleotide coding sequences (SEQ ID NO. 13-24) are synthesized by Gene Company. The synthesis rule is based on the codon bias of the host. Because of the difference of the host, the codon bias is also different (SEQ ID NO. 13-24 is the coding gene of the bacteriocidal protein expressed by Trichoderma). The empty expression plasmid without connecting the gene is provided to the Gene Company, and the enzyme cutting site required to connect the expression gene and the DNA sequence upstream and downstream of the enzyme cutting site are informed to avoid frame shift mutation. The gene can be directly synthesized on the provided plasmid to construct the final expression vector for expressing the reducing sugar oxidase, which saves the steps of PCR, enzyme cutting, and ligation.

[0040] It should be noted that the N-terminus of the bacteriocidal protein amino acid sequence (SEQ ID NO. 1-12) provided by the present application contains its own secretion peptide sequence; it can be secreted and expressed in Trichoderma.

[0041] The bacteriocidal protein coding gene sequences in the following examples are shown in SEQ ID NO. 13-24:

[0042] The bacteriocidal protein and the lysozyme extracted from commercial egg white can hydrolyze the cell wall of the indicator bacteria, causing the death of the indicator bacteria. The process is manifested as the gradual decrease of the absorbance of the bacterial suspension of the indicator bacteria, and the bacterial suspension becomes clear. Therefore, in the following examples, the bacteriocidal activity of the bacteriocidal protein is determined by using the determination method of GB1886.257-2016, and the bacteriocidal activity is simply defined as follows: one bacteriocidal activity unit is defined as the solution volume or mass of the bacteriocidal protein required to cause a decrease of 0.001 in absorbance per minute at 450 nm using Micrococcus luteus suspension under the condition of 25℃ and pH 6.2. The bacteriocidal activity can directly reflect the expression level of the bacteriocidal protein.

[0043] Example 1 Construction of bacteriocidal protein expression vector

[0044] The present application utilizes the previously constructed Trichoderma reesei expression plasmid CBH1T1.0 (recorded in the patent document “202211596367.5 Construction and application of xylanase and its secreting expression strain”) to construct a recombinant Trichoderma strain, so that the bacteriocidal protein is secreted and expressed in the recombinant Trichoderma strain.

[0045] The application entrusts Shanghai Jetstar Gene Synthesis Company to directly synthesize 12 bacteriocin protein coding gene sequences (named F1 (SEQ ID NO. 13), F2 (SEQ ID NO. 14), F3 (SEQ ID NO. 15), F4 (SEQ ID NO. 16), F5 (SEQ ID NO. 17), F6 (SEQ ID NO. 18), F7 (SEQ ID NO. 19), F8 (SEQ ID NO. 20), F9 (SEQ ID NO. 21), F10 (SEQ ID NO. 22), F11 (SEQ ID NO. 23), F12 (SEQ ID NO. 24) in turn) at the XbaI enzyme cutting site (TCTAGA) of CBH1T1.0 plasmid, that is, to construct 12 corresponding recombinant expression vectors, named CBH1T1.0-F1~12 (the structure of the recombinant expression vector is shown in FIG. 1) in turn.

[0046] The upstream and downstream sequences near the XbaI enzyme cutting site (the insertion position of the target gene) of the CBH1T1.0 plasmid are shown in SEQ ID NO. 25, the TCTAGA gene insertion site is underlined, and the insertion position is represented by “ / ”. The ATG at the front end of the bacteriocin protein coding gene sequence is the start codon of the coding gene, and the TAG in the second half of the XbaI enzyme cutting site of the CBH1T1.0 plasmid is the stop codon.

[0047] Example 2 Construction of recombinant Trichoderma strains expressing bacteriocin proteins

[0048] The 12 recombinant expression vectors constructed in Example 1 are respectively transformed into Trichoderma strains QM6a (ATCC 13631), QM9414 (ATCC 26921), RUT-C30 (ATCC 56765), RL-P37 (NRRL 15709), NG14 (ATCC 56767), PC-3-7 (ATCC 66589) by using Agrobacterium tumefaciens-mediated transformation (Covert et al. Agrobacterium tumefaciens-mediated transformation of Fusarium circinatum. Mycol. Res. 105 (3): 259-264), to obtain recombinant Trichoderma strains.

[0049] In this example, Trichoderma RUT-C30 (ATCC 56765) is used as the host strain to illustrate the detailed process of constructing the recombinant Trichoderma strain RUT-C30-CBH1T1.0-F1~12; when other Trichoderma strains are used as the host strain, the construction process is completely consistent.

[0050] The process is as follows:

[0051] The recombinant expression vector CBH1T1.0-F1-12 is transformed into Trichoderma strain RUT-C30 by Agrobacterium tumefaciens-mediated method, and the endogenous cellulase gene cbh1 is replaced by homologous recombination to construct 12 kinds of recombinant Trichoderma strains expressing bacteriocin protein (the construction process is shown in Figure 2); the recombinant Trichoderma strains eliminate the hygromycin resistance marker according to the literature scheme (Zhang et al. Light-inducible genetic engineering and control of non-homologous end-joining in industrial eukaryotic microorganisms: LML 3.0 and OFN 1.0. Scientific Reports. 2016, 6: 20761).

[0052] Example 3 Production of Bacteriocin Protein

[0053] The recombinant Trichoderma strains are fermented in Trichoderma fermentation medium to induce the secretion and expression of bacteriocin protein. The fermentation tank culture method refers to the literature “Chen et al. Engineering of Trichoderma reesei for enhanced degradation of lignocellulosic biomass by truncation of the cellulase activator ACE3. Biotechnol Biofuels. 2020, 13: 62”. After 8 days of fermentation, the fermentation broth is taken and centrifuged at 12500 rpm for 10 min, and the supernatant is taken as the crude extract of bacteriocin protein. The crude extract is spray dried, and then a general protective agent (starch or cyclodextrin) is added at a weight ratio of 1:1 to prepare a solid fungal bacteriocin protein powder. The activity of the crude extract and the fungal bacteriocin protein powder is determined according to the national standard GB1886.257-2016, and the results are shown in Table 1. The activity of the fungal bacteriocin protein powder is 100-400 U / mg.

[0054] Table 1 shows that the activity of the bacteriocin protein produced by Trichoderma RUT-C30 (ATCC 56765) as the host strain is higher. a Staphylococcus aureus is abbreviated as S. aureus.

[0055] Table 1 shows that the activity of the bacteriocin protein produced by Trichoderma RUT-C30 (ATCC 56765) as the host strain is higher.

[0056] Example 4 Application of bacteriocin protein

[0057] The bacteriocin protein powder prepared in Example 3 was used for the inhibition zone experiment, and a commercial egg white lysozyme powder (from Ningxia Xiasen Industrial Group Co., Ltd., with a vitality of about 3000 U / mg) was used as a control. The inhibition effect of the bacteriocin protein on common pathogenic bacteria was evaluated by the inhibition zone experiment method.

[0058] The experimental method is as follows:

[0059] Taking the bacteriocin protein powder F5 produced by RUT-C30-CBH1T1.0-F5 as an example, a proper amount of F5 and commercial lysozyme powder was dissolved in ultrapure water to prepare a test liquid and a control liquid with a concentration of 40 U / μL, and filtration was performed to remove bacteria. Three indicator bacteria were selected: Candida albicans, Staphylococcus aureus, and Escherichia coli. The indicator bacteria were streaked on LB plates and cultured at 37°C for about 12 h. The single colonies on the plates were picked into LB test tubes and cultured at 37°C on a shaker for about 12 h to obtain the indicator seed liquid, which was stored in a refrigerator for use.

[0060] After sterilization of the LB medium, the medium was cooled to slightly above body temperature, 100 μL of the above indicator seed liquid was taken and shaken in the un-solidified LB medium, and then quickly poured into a plate, with 15 mL of medium in each plate, and waited for solidification.

[0061] Filter paper pieces with a diameter of 6 mm were cut, high-temperature sterilized, and sterile-dried. Each filter paper piece was allowed to absorb 10 μL of the bacteriocin protein test liquid or the commercial lysozyme control liquid. The filter paper pieces were picked up with sterile tweezers and attached to the surface of the plate. The plate was turned over, labeled, and placed in a 37°C incubator for culture, and the inhibition zone could be observed after about 12-24 h.

[0062] The results of the inhibition zone experiment are shown in FIG. 3. It can be seen that the bacteriocin protein can significantly kill or inhibit the growth of gram-positive bacteria (Staphylococcus aureus resistant to egg white lysozyme) and yeast (Candida albicans), and produce a significant inhibition zone, while egg white lysozyme cannot inhibit these two strains; the bacteriocin protein can significantly kill or inhibit gram-negative bacteria (Escherichia coli), and its effect is better than that of egg white lysozyme, and a larger inhibition zone can be produced. The sizes of the inhibition zones produced by other bacteriocin protein powders are shown in Table 1.

[0063] In summary, after genetic recombination and modification of the Trichoderma sp., a recombinant Trichoderma sp. strain secreting and expressing bacteriocin protein was successfully constructed. The bacteriocin protein produced by the recombinant Trichoderma sp. strain can significantly kill or inhibit gram-positive bacteria, gram-negative bacteria, and yeast, and provides a new bacteriocin protein preparation and a preparation method thereof for the daily chemical, food, and feed industries.

[0064] The above described embodiments are only to illustrate the preferred modes of the present application, and are not intended to limit the scope of the present application. Any modification and improvement made by those skilled in the art to the technical solutions of the present application without departing from the design spirit of the present application shall fall within the protection scope of the present application as defined by the claims.

Claims

1. A bacteriocin protein, characterized in that, The amino acid sequence of the bactericidal protein is any one of SEQ ID NO. 1-12, or a sequence with 1-30 amino acid substitutions and / or deletions and / or additions to any one of SEQ ID NO. 1-12, or a sequence with 1-20 amino acid additions to the C-terminal and / or N-terminal of any one of SEQ ID NO. 1-12.

2. A gene encoding the bacteriocin of claim 1, wherein, The nucleotide sequence of the gene is any one of SEQ ID NO. 13-24, or a sequence with more than 85% homology to any one of SEQ ID NO. 13-24.

3. A recombinant expression vector, characterized in that, The recombinant expression vector comprises a nucleotide sequence encoding the bactericidal protein of claim 1, or the gene of claim 2.

4. A recombinant microorganism, characterized in that, The recombinant microorganism comprises the recombinant expression vector of claim 3.

5. The recombinant microorganism of claim 4, wherein, The chassis microorganism of the recombinant microorganism is Trichoderma.

6. The recombinant microorganism of claim 5, wherein, The Trichoderma is Trichoderma reesei QM6a, QM9414, RUT-C30, RL-P37, NG14 or PC-3-7.

7. Use of the gene of claim 2, the recombinant expression vector of claim 3, or the recombinant microorganism of any one of claims 4-6 in the production of a bactericidal protein.

8. A method for producing a bactericidal protein, characterized by, The recombinant microorganism of any one of claims 4-6 is fermented, and the fermentation broth is collected to obtain the bactericidal protein.

9. Use of the bactericidal protein produced by the production method of claim 8 in the preparation of a bactericide.

Images