An engineered Trichoderma reesei strain secreting and expressing human lysozyme, and its construction method and application

Abstract

The present invention discloses a Trichoderma reesei engineering bacterium for secreting and expressing human lysozyme, and a construction method and application thereof, which relate to the fields of molecular biology and biotechnology. The construction method includes the step of transforming an expression module into a Trichoderma reesei original strain to construct the Trichoderma reesei engineering bacterium; the expression module includes a promoter, a fusion gene and a terminator which are sequentially connected; the fusion gene includes a secretion peptide gene, a peptide cleavage site gene and a human lysozyme gene which are sequentially connected. The present invention successfully constructs an engineering strain for secreting and expressing human lysozyme, and the engineering strain can be used for the production of human lysozyme preparations. Through experimental verification, the human lysozyme preparation can significantly kill Gram-positive and Gram-negative bacteria. The present invention provides a new human lysozyme preparation and a preparation method thereof for industries such as food and feed.

Description

Technical Field

[0001] The present invention relates to the fields of molecular biology and biotechnology, and particularly relates to a Trichoderma reesei engineering bacterium for secreting and expressing human lysozyme, a construction method thereof, and an application thereof. Background Art

[0002] Lysozyme was first discovered by British scientist Fleming in human saliva, tears and nasal mucus in 1922, and it got its name because it can dissolve the bacterial cell wall. Peptidoglycan is an important component of the cell wall of Gram-positive bacteria. Lysozyme can hydrolyze the β-1,4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycan, decomposing the insoluble mucopolysaccharide in the cell wall into soluble glycopeptides, resulting in the rupture of the cell wall and the overflow of the contents, thus lysing the bacteria. Therefore, lysozyme can kill most Gram-positive strains. Lysozyme can also directly bind to the negatively charged viral protein, form a complex salt with DNA, RNA, and apoprotein, inactivating the virus. Therefore, this enzyme has functions such as antibacterial, anti-inflammatory, antiviral, hemostatic, detumescence, and analgesic effects. These special properties of lysozyme make it have a wide range of application prospects in industries such as food, medicine, and livestock production. Currently, due to the reasons of being cheap and easily available, egg white lysozyme (abbreviation, egg white lysozyme) is the most widely used and commercialized lysozyme preparation.

[0003] Human lysozyme is a bactericidal protein in the human body itself. Compared with lysozymes from other sources, human lysozyme has many insurmountable advantages: such as the enzyme activity is 3 times that of egg white lysozyme; and it is derived from the human body, has natural compatibility with the human body, will not produce any side effects and immunogenicity, can be digested and absorbed as a nutrient in the gastrointestinal tract; it is non-toxic to the body and will not remain in the body, with higher safety. Therefore, it is more and more favored in the food, medicine, and feed industries.

[0004] Trichoderma reesei is an important industrial production strain, meeting the GRAS (Generally Regarded as Safe) standard, and the enzyme preparations produced by its fermentation have been widely used in industries such as food and feed. It is not reported in the known academic research field that Trichoderma reesei strains can ferment and produce human lysozyme.

[0005] Although there are successful cases of heterologous expression of egg white lysozyme using fungi such as yeast in the prior art, the technology of heterologous expression of human lysozyme using fungi (especially Trichoderma reesei) has not yet emerged. The reasons may be as follows: First, the activity of human lysozyme against glucans such as chitin is much higher than that of egg white lysozyme, so it has a certain killing effect on fungal hosts, resulting in the failure of heterologous expression. Second, for secretory expression, a secretory peptide is required. Commercialized strains (such as Escherichia coli, Pichia pastoris) have mature and efficient secretory peptides, while whether non-commercialized strains (such as Trichoderma reesei, etc.) have similar secretory peptides is still unknown.

[0006] The present invention intends to utilize Trichoderma reesei to secrete and express human lysozyme, thereby providing a new technical idea for the development of human lysozyme preparations. SUMMARY OF THE INVENTION

[0007] The object of the present invention is to provide an engineered Trichoderma reesei strain that secretes and expresses human lysozyme, and its construction method and application, so as to solve the problems existing in the above-mentioned prior art. This engineered Trichoderma reesei strain can secrete and express human lysozyme, and thus can be used for the production of human lysozyme preparations.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] The present invention provides a method for constructing an engineered Trichoderma reesei strain that secretes and expresses human lysozyme, including the step of transforming an expression module into the original Trichoderma reesei strain to construct the engineered Trichoderma reesei strain;

[0010] The expression module includes a promoter, a fusion gene, and a terminator connected in sequence; the fusion gene includes a secretion peptide gene, a peptide cleavage site gene, and a human lysozyme gene connected in sequence;

[0011] The secretion peptide gene is a gene encoding a secretion peptide, and the amino acid sequence of the secretion peptide is shown in any one of SEQ ID NO.6-13;

[0012] The peptide cleavage site gene is a gene encoding a peptide cleavage site, and the amino acid sequence of the peptide cleavage site is shown in any one of SEQ ID NO.14-18;

[0013] The human lysozyme gene is a gene encoding human lysozyme, and the amino acid sequence of the human lysozyme is shown in any one of SEQ ID NO.1-5. As is well known in the art, when substituting amino acids with similar or close properties, the function of the protein usually does not change. Also, for example, adding one or several amino acids at the C-terminus and / or N-terminus usually does not change the function of the protein. Therefore, the amino acid sequence of human lysozyme also includes variant forms of the SEQ ID NO.1-5 sequences that have the same function as human lysozyme. These variant forms include (but are not limited to): deletion, insertion, and / or substitution of several (usually 1-30, preferably 1-20, more preferably 1-10, most preferably 1-5) amino acids, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and / or N-terminus.

[0014] Further, the nucleotide sequence of the secretion peptide gene is shown as any one of SEQ ID NOs. 28 to 35; the nucleotide sequence of the peptide cleavage site gene is shown as any one of SEQ ID NOs. 36 to 40; the nucleotide sequence of the human lysozyme gene is shown as any one of SEQ ID NOs. 23 to 27.

[0015] Further, the original Trichoderma reesei strains include Trichoderma reesei QM6a, QM9414, RUT-C30, RL-P37, NG14 or PC-3-7.

[0016] Further, the promoter is the CBHI promoter or the CBHII promoter, and the terminator is the CBHI terminator or the CBHII terminator; when the promoter is the CBHI promoter, the terminator is the CBHI terminator; when the promoter is the CBHII promoter, the terminator is the CBHII terminator;

[0017] The nucleotide sequences of the CBHI promoter, the CBHI terminator, the CBHII promoter and the CBHII terminator are shown as SEQ ID NOs. 19 to 22 respectively.

[0018] Further, the nucleotide sequence of the secretion peptide gene is shown as SEQ ID NO. 34; the nucleotide sequence of the peptide cleavage site gene is shown as SEQ ID NO. 36; the nucleotide sequence of the human lysozyme gene is shown as SEQ ID NO. 23; the original Trichoderma reesei strain is PC-3-7.

[0019] The present invention also provides a Trichoderma reesei engineering bacterium constructed by the above construction method.

[0020] The present invention also provides the application of the above Trichoderma reesei engineering bacterium in the preparation of a human lysozyme preparation.

[0021] The present invention also provides a human lysozyme mutant, and the amino acid sequence is shown as any one of SEQ ID NOs. 2 to 5.

[0022] The present invention also provides the application of the above human lysozyme mutant in the preparation of an antibacterial enzyme preparation, and the pathogenic bacteria targeted by the antibacterial enzyme preparation include Salmonella enteritidis, Escherichia coli and / or Vibrio parahaemolyticus.

[0023] The present invention also provides an antibacterial enzyme preparation, and the active ingredient includes the above human lysozyme mutant.

[0024] Due to the existence of codon degeneracy (codon degeneracy refers to the sequence generated after one or more codons are replaced by degenerate codons encoding the same amino acid), degenerate sequences with as low as about 92% homology to the nucleotide sequences in SEQ ID NOs. 23 to 40 can also encode the said sequences. Therefore, the term "fusion gene" in the present invention also includes nucleic acid sequences with at least 92% homology to the nucleotide sequences in SEQ ID NOs. 23 to 40.

[0025] The present invention discloses the following technical effects:

[0026] The present invention constructs a human lysozyme fusion gene and an expression module, and transforms the expression module into a variety of Trichoderma reesei strains, successfully constructing an engineered strain that secretes and expresses human lysozyme. This engineered strain can be used for the production of human lysozyme preparations. Through experimental verification, this human lysozyme preparation can significantly kill Gram-positive and Gram-negative bacteria. The present invention provides a new human lysozyme preparation and its preparation method for industries such as food and feed. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0028] Figure 1 Schematic diagram of the construction process of the human lysozyme fusion gene (A), CBHI / II expression plasmid (B), and human lysozyme expression module (C);

[0029] Figure 2 Detection results of the activity of human lysozyme prepared in Example 3 using different secretion peptides;

[0030] Figure 3 Detection results of the activity of human lysozyme prepared in Example 3 using different peptide cleavage sites;

[0031] Figure 4 Detection results of the activity of human lysozyme prepared in Example 3 using different human lysozyme mutants;

[0032] Figure 5 Detection results of the activity of human lysozyme prepared in Example 3 using different promoters and Trichoderma reesei strains;

[0033] Figure 6 Inhibition zone experiment results of human lysozyme prepared from the CBHI-g12.14.1 (PC-3-7) engineered strain. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0034] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be construed as limiting the present invention, but rather as a more detailed description of certain aspects, features, and implementation manners of the present invention.

[0035] It should be understood that the terms described in the present invention are only for describing particular embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0036] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0037] Without departing from the scope or spirit of the present invention, various improvements and variations can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are merely exemplary.

[0038] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0039] The experimental methods without specific conditions noted in the following examples are generally carried out according to conventional conditions, such as those described in "Molecular Cloning: A Laboratory Manual" (New York: Cold Spring Harbor Laboratory Press, 1989).

[0040] The amino acid sequences of human lysozyme and its four mutants are shown in SEQ ID NO.1 - 5 respectively, and their corresponding nucleotide sequences are shown in SEQ ID NO.23 - 27 respectively.

[0041] The amino acid sequences of 8 secretion peptides are shown in SEQ ID NO.6 - 13 respectively, and their corresponding nucleotide sequences are shown in SEQ ID NO.28 - 35 respectively.

[0042] The amino acid sequences of 5 peptide cleavage sites are GGGGSKR (SEQ ID NO.14), GGGSDKR (SEQ ID NO.15), RSKR (SEQ ID NO.16), RYKR (SEQ ID NO.17), and NVISKR (SEQ ID NO.18); their corresponding nucleotide sequences are shown in SEQ ID NOs. 36 - 40 respectively.

[0043] The nucleotide sequence of the CBHI promoter is shown in SEQ ID NO.19; the nucleotide sequence of the CBHI terminator is shown in SEQ ID NO.20.

[0044] The nucleotide sequence of the CBHII promoter is shown in SEQ ID NO.21; the nucleotide sequence of the CBHII terminator is shown in SEQ ID NO.22.

[0045] Construction of the fusion gene in Example 1

[0046] In order to achieve efficient secretion and expression of human lysozyme in Trichoderma reesei, it is necessary to fuse the endogenous secretion peptide of the fungus at the N - terminus of the protein to direct the secretion and expression of heterologous human lysozyme. In addition to secretion, rapid cleavage and separation of lysozyme and the secretion peptide are also required, so a peptide cleavage site needs to be inserted between them. Therefore, the amino acid sequence structure encoded by the fusion gene is: secretion peptide - peptide cleavage site - human lysozyme, as shown in Figure 1 A in the figure. The present invention provides 8 secretion peptides, 5 peptide cleavage sites, and 5 human lysozymes, which can be arranged and combined to produce 200 different amino acid sequences and their encoding genes. For example, the fusion gene g12.KR.1 is the gene sequence encoding a protein with the amino acid sequence SEQ ID NO.12 (secretion peptide) + SEQ ID NO.14 (peptide cleavage site) + SEQ ID NO.1 (human lysozyme).

[0047] The method for obtaining the fusion gene is: providing the amino acid sequence and entrusting a conventional gene company to synthesize the corresponding fusion gene sequence. The synthesis rules of the present invention are based on the codon preference of the Trichoderma reesei host. Due to the existence of codon degeneracy (degeneracy refers to the sequence generated after one or more codons are replaced by degenerate codons encoding the same amino acid), degenerate sequences with as low as about 92% homology to the nucleotide sequences in SEQ ID NOs. 23 - 40 can also encode the said sequences. Therefore, the term "fusion gene sequence" also includes nucleic acid sequences with at least 92% homology to the nucleotide sequences in SEQ ID NOs. 23 - 40.

[0048] Construction of the expression module in Example 2

[0049] To transcriptionally express the fusion gene in Example 1 in Trichoderma reesei, a promoter and a terminator are required. The present invention provides two endogenous promoters with the highest expression levels in Trichoderma reesei and the sequences of their terminators (shown in SEQ ID NO.17-20 respectively). The construction scheme is as follows.

[0050] (1) Using the PCR method, the promoter and the terminator are ligated, and a restriction enzyme site SmiI is inserted at the ligation site for subsequent ligation of the fusion gene, that is, two DNA structures are constructed as follows - CBHI promoter - SmiI - CBHI terminator, CBHII promoter - SmiI - CBHII terminator, as Figure 1 shown in B. Since the specific DNA sequences have been provided in the present invention, the primer design and the PCR method can be carried out according to conventional research methods.

[0051] (2) The above two DNA structures are ligated into the Trichoderma reesei expression plasmid by seamless ligation, and two new expression plasmids - CBHI expression plasmid and CBHII expression plasmid can be constructed. The purpose of ligating into the plasmid is, firstly, to better preserve and replicate the above two DNA structures for subsequent large - scale acquisition of this structure; secondly, because there are resistance screening markers such as hygromycin on the plasmid, which is convenient for subsequent transformation of Trichoderma reesei.

[0052] The nucleotide sequences of the promoters and terminators of CBHI and CBHII are cloned and amplified by the PCR method, and the SmiI site (ATTTAAAT) is added to the primers to ensure seamless ligation into the structures of "CBHI promoter - SmiI - CBHI terminator" and "CBHII promoter - SmiI - CBHII terminator" finally.

[0053] A. Using primers CBHI - F1 (SEQ ID NO.41) and CBHI - F2 (SEQ ID NO.42), the nucleotide sequence of the CBHI promoter is amplified with the Trichoderma reesei genome as the template.

[0054] Amplification reaction system: 10×PCR Buffer for KOD - Plus - Neo 5 μL; 2 mM dNTPs 5 μL; 25 mM MgSO 4 3 μL; 10 μM primers CBHI - F1 / CBHI - F2 each 1.5 μL; genomic template (200 ng) 1 μL; KOD - Plus - Neo (1 U / μL) 1 μL.

[0055] Reaction program: 94°C for 2 min; 98°C for 10 sec, 58°C for 30 sec, 68°C for 60 sec, run 30 cycles; 68°C for 5 min.

[0056] B. Using primers CBHI-R1 (SEQ ID NO.43) and CBHI-R2 (SEQ ID NO.44), amplify the nucleotide sequence of the CBHI terminator with the Trichoderma reesei genome as the template.

[0057] Amplification reaction system: 5 μL of 10×PCR Buffer for KOD-Plus-Neo; 5 μL of 2 mM dNTPs; 25 mM MgSO 4 3 μL; 1.5 μL each of 10 μM primers CBHI-R1 / CBHI-R2; 1 μL of genomic template (200 ng); 1 μL of KOD-Plus-Neo (1 U / μL).

[0058] Reaction program: 2 min at 94°C; 10 sec at 98°C, 30 sec at 58°C, 40 sec at 68°C, run for 30 cycles; 5 min at 68°C.

[0059] C. Using primers CBHII-F1 (SEQ ID NO.45) and CBHII-F2 (SEQ ID NO.46), amplify the nucleotide sequence of the CBHII promoter with the Trichoderma reesei genome as the template.

[0060] Amplification reaction system: 5 μL of 10×PCR Buffer for KOD-Plus-Neo; 5 μL of 2 mM dNTPs; 25 mM MgSO 4 3 μL; 1.5 μL each of 10 μM primers CBHII-F1 / CBHII-F2; 1 μL of genomic template (200 ng); 1 μL of KOD-Plus-Neo (1 U / μL).

[0061] Reaction program: 2 min at 94°C; 10 sec at 98°C, 30 sec at 58°C, 40 sec at 68°C, run for 30 cycles; 5 min at 68°C.

[0062] D. Using primers CBHII-R1 (SEQ ID NO.47) and CBHII-R2 (SEQ ID NO.48), amplify the nucleotide sequence of the CBHII terminator with the Trichoderma reesei genome as the template.

[0063] Amplification reaction system: 5 μL of 10×PCR Buffer for KOD-Plus-Neo; 5 μL of 2 mM dNTPs; 25 mM MgSO 4 3 μL; 1.5 μL each of 10 μM primers CBHII-R1 / CBHII-R2; 1 μL of genomic template (200 ng); 1 μL of KOD-Plus-Neo (1 U / μL).

[0064] Reaction procedure: 94°C for 2 min; 98°C for 10 sec, 58°C for 30 sec, 68°C for 30 sec, run 30 cycles; 68°C for 5 min.

[0065] The expression plasmid uses LML2.0a (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) as the backbone to construct the expression vector. Single digestion is performed at the restriction enzyme SwaI site on the existing plasmid LML2.0a, and seamless ligation is carried out using the Vazyme OneStep Clone Kit, and the above two DNA structures - CBHI promoter - SmiI - CBHI terminator, CBHII promoter - SmiI - CBHII terminator are ligated respectively; the CBHI expression plasmid and the CBHII expression plasmid are constructed ( Figure 1 in B).

[0066] (3) The fusion gene is ligated into the expression plasmid to construct the expression module of the fusion gene. Single digestion is performed at the restriction enzyme SmiI site of the two CBHI expression plasmids and the CBHII expression plasmid, and seamless ligation is carried out using the Vazyme One Step Clone Kit, and various fusion genes are ligated to construct various expression modules of the fusion gene ( Figure 1 in C). For example, the CBHI - g12.14.1 expression module is: using the CBHI expression plasmid to express the g12.KR.1 fusion gene. Primers for cloning and amplifying the fusion gene are designed according to the specific sequence of the fusion gene synthesized by commission.

[0067] Example 3 The expression module is introduced into Trichoderma reesei to obtain an engineered strain expressing human lysozyme, and human lysozyme is produced

[0068] In the present invention, through conventional experimental operations such as electrotransformation, protoplast transformation, or Agrobacterium tumefaciens - mediated conjugation transfer, the expression module constructed in Example 2 is introduced into the Trichoderma reesei strain and integrated into the Trichoderma reesei genome. Through the selection marker on the expression module, the transformants are screened, and the correctness of the transformants is verified by detecting the lysozyme activity. The present invention takes the CBHI - g12.14.1 expression module and the Agrobacterium tumefaciens - mediated conjugation transfer scheme as an example.

[0069] (1) The CBHI-g12.14.1 expression module was electrotransformed into Agrobacterium tumefaciens, and then the successfully electrotransformed Agrobacterium tumefaciens was co-cultured with Trichoderma reesei host strains QM6a (ATCC 13631), QM9414 (ATCC 26921), RUT-C30 (ATCC 56765), RL-P37 (NRRL 15709), NG14 (ATCC 56767), and 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 Agrobacterium tumefaciens-mediated conjugation transfer. After two days of co-culture, the transformants were transferred to PDA plates containing cefotaxime (300 μg / mL) and hygromycin B (75 μg / mL) for screening until the transformants grew mycelia and spores, and then verification was carried out.

[0070] (2) The transformant CBHI-g12.14.1 (RUT-C30) of CBHI-g12.14.1 in Trichoderma reesei RUT-C30 was inoculated into 50 mL of lactose as the carbon source in a 250 mL Erlenmeyer flask for verification culture. The formula of the verification medium was: lactose 10 g / L, Tryptone 1 g / L, yeast extract 0.5 g / L, (NH 4 ) 2 SO 4 1.4 g / L, Urea 0.3 g / L, KH 2 PO 4 2 g / L, CaCl 2 ·2H 2 O 0.3 g / L, MgSO 4 ·7H 2 O 0.3 g / L, FeSO 4 ·7H 2 O 5 mg / L, MnSO 4 ·H 2 O 1.6 mg / L, ZnSO 4 ·7H 2 O 1.4 mg / L and CoCl 2 ·6H 2 O 2 mg / L. The inoculation amount was 10 8 spores / 50 mL of medium, and the culture was carried out at 28 °C and 200 rpm. Samples were taken on the 3rd day, and the activity of lysozyme was determined according to the national standard method GB / T 30990-2014 to verify its successful transformation. The results are shown in Figures 2 - 5 .

[0071] (3) Inoculate the engineered strain CBHI-g12.14.1 (PC-3-7) into the fermentation medium (lactose 37 g / L, glucose 5 g / L, corn steep liquor 27 g / L, Tryptone 1 g / L, (NH 4 ) 2 SO 4 5 g / L, CaCl 2 ·2H 2 O 0.5 g / L, MgSO 4 ·7H 2 O 1 g / L, FeSO 4 ·7H 2 O 5 mg / L, MnSO 4 ·H 2 O 1.6 mg / L, ZnSO 4 ·7H 2 O 1.4 mg / L and CoCl 2 ·6H 2 O 2 mg, and culture at 28 °C for 6 days. After fermentation, obtain the supernatant of the fermentation broth, which is the crude enzyme solution of human lysozyme. Spray dry (or freeze dry) the crude enzyme solution of human lysozyme to prepare human lysozyme enzyme powder, and the activity of the enzyme powder can reach 300 U / mg.

[0072] Example 4 Lysozyme Inhibitory Zone Experiment

[0073] Peptidoglycan is an important component of the cell wall of Gram-positive bacteria. Lysozyme can hydrolyze the β-1,4 glycosidic bond between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) in peptidoglycan, resulting in the rupture of the cell wall of Gram-positive bacteria under the action of osmotic pressure, and the contents overflow to dissolve the bacteria. Therefore, lysozyme can kill most Gram-positive strains. Human lysozyme has higher activity and can kill not only common Gram-positive strains but also a variety of Gram-negative bacteria. The purpose of this experiment: Through the inhibitory zone experiment method, evaluate the inhibitory effect of human lysozyme produced by Trichoderma reesei provided by the present invention on common pathogenic Gram-negative bacteria, and compare it with commercial egg white lysozyme.

[0074] Dissolve 1 g of the human lysozyme enzyme powder (300 U / mg) prepared using the CBHI-g12.14.1 (PC-3-7) engineering strain in Example 3 in 10 mL of ultrapure water to prepare a 10% (w / v) human lysozyme detection solution. Negative control: Dissolve 1 g of egg white lysozyme enzyme powder (commercial enzyme, 20,000 U / mg, purchased from Jierui Company) in 10 mL of ultrapure water to prepare a 10% egg white lysozyme control solution. According to the national standards "GB4789.28 Quality Requirements for Culture Media and Reagents" and "GB4789.4-2010 Salmonella Inspection", use: Salmonella enteritidis (CMCC(B) 50335), Escherichia coli (ATCC25922), Vibrio parahaemolyticus (ATCC17802) as indicator bacteria. Make 2 parallels for both the detection solution and the control solution.

[0075] Streak the indicator bacteria on an LB solid (5 g / L yeast powder, 10 g / L NaCl, 10 g / L peptone, and 20 g / L agar) plate and culture at 37 °C for 12 h. Pick a single colony from the plate into an LB liquid (5 g / L yeast powder, 10 g / L NaCl, and 10 g / L peptone) test tube and culture on a shaker at 37 °C for about 12 h to obtain the indicator bacteria seed solution. Then pipette 100 μL of the seed solution onto the surface of a new LB solid plate and spread the bacterial solution evenly with a spreader. Cut filter paper into 6 mm in diameter, sterilize and dry. Place the sterile filter paper on the surface of the LB solid plate coated with the indicator bacteria, and drop 10 μL of the detection solution or the control solution onto each filter paper. After the filter paper absorbs the liquid, flip the petri dish, make a mark, and place it in an incubator at 37 °C for about 12 h to observe and record the results. The results are as Figure 6 shown. The antibacterial effects of other mutants are summarized in Table 1.

[0076] Table 1 Antibacterial effects of the enzyme and bacteria

[0077]

[0078] Note: - represents no obvious antibacterial zone at the edge, + represents an obvious antibacterial zone.

[0079] Although the specific activity of the human lysozyme produced by Trichoderma reesei provided by the present invention is lower than that of commercial egg white lysozyme, but Figure 6 as can be seen from Table 1: The inhibitory effects of the human lysozyme provided by the present invention on common pathogenic bacteria such as Salmonella enteritidis, Escherichia coli, and Vibrio parahaemolyticus are significantly higher than those of commercial egg white lysozyme.

[0080] In summary, the present invention successfully secretes and expresses human lysozyme using Trichoderma reesei, and the bactericidal effect of the human lysozyme produced by the method of the present invention is significantly better than that of commercial egg white lysozyme.

[0081] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for constructing an engineered strain of Trichoderma reesei that secretes and expresses human lysozyme, characterized in that: The method comprises the steps of transforming the expression module into the original strain of Trichoderma reesei to construct the engineered strain of Trichoderma reesei; The expression module includes a promoter, a fusion gene and a terminator connected in sequence; the fusion gene includes a secretory peptide gene, a peptide cleavage site gene and a human lysozyme gene connected in sequence; The secretory peptide gene is a gene encoding a secretory peptide, and the amino acid sequence of the secretory peptide is shown in any one of SEQ ID NOs. 7 to 13; The peptide cleavage site gene is a gene encoding a peptide cleavage site, and the amino acid sequence of the peptide cleavage site is shown in any one of SEQ ID NOs. 14 to 18; The human lysozyme gene is a gene encoding human lysozyme, and the amino acid sequence of the human lysozyme is shown in any one of SEQ ID NOs. 2 to 4; The original strain of Trichoderma reesei includes Trichoderma reesei QM6a, QM9414, RUT-C30, RL-P37, NG14 or PC-3-7.

2. The construction method according to claim 1, characterized in that: The nucleotide sequence of the secretory peptide gene is shown in any one of SEQ ID NO.34; the nucleotide sequence of the peptide cleavage site gene is shown in any one of SEQ ID NO.36-40; the nucleotide sequence of the human lysozyme gene is shown in any one of SEQ ID NO.24-26.

3. The construction method according to claim 1, characterized in that: The promoter is a CBHI promoter or a CBHII promoter, and the terminator is a CBHI terminator or a CBHII terminator; when the promoter is a CBHI promoter, the terminator is a CBHI terminator; when the promoter is a CBHII promoter, the terminator is a CBHII terminator; The nucleotide sequences of the CBHI promoter, the CBHI terminator, the CBHII promoter and the CBHII terminator are shown in SEQ ID NOs. 19 to 22, respectively.

4. The construction method according to claim 2, characterized in that: The nucleotide sequence of the secretory peptide gene is shown in SEQ ID NO.34; the nucleotide sequence of the peptide cleavage site gene is shown in SEQ ID NO.36; the nucleotide sequence of the human lysozyme gene is shown in SEQ ID NO.24-26; and the original strain of Trichoderma reesei is PC-3-7.

5. An engineered strain of Trichoderma reesei constructed according to the construction method according to any one of claims 1 to 4.

6. Use of the engineered bacterium Trichoderma reesei as claimed in claim 5 in the preparation of a human lysozyme preparation.

7. A human lysozyme mutant, characterized in that: The amino acid sequence of the human lysozyme mutant is shown in any one of SEQ ID NOs. 2 to 4.

8. Use of the human lysozyme mutant according to claim 7 in the preparation of an antibacterial enzyme preparation, characterized in that: The pathogenic bacteria targeted by the antibacterial enzyme preparation include Salmonella enteritidis, Escherichia coli and / or Vibrio parahaemolyticus.

9. An antibacterial enzyme preparation, characterized in that: The active ingredient comprises the human lysozyme mutant according to claim 7.

Images