Trichoderma reesei engineering bacteria for secreting egg white lysozyme and construction method thereof

Abstract

The application discloses a Trichoderma reesei engineering bacterium for secreting and expressing egg white lysozyme and a construction method thereof, and belongs to the field of molecular biology and biotechnology. The application firstly provides an egg white lysozyme mutant, and the amino acid sequence is shown in any one of SEQ ID NO. 2-4. The construction method of the Trichoderma reesei engineering bacterium for secreting and expressing egg white lysozyme comprises the following steps: taking Trichoderma reesei as a starting strain, and transforming an expression module into the Trichoderma reesei to construct the Trichoderma reesei engineering bacterium; the expression module comprises a promoter, a secretion peptide gene, an egg white lysozyme gene and a terminator which are sequentially connected. By transforming the coding gene of the egg white lysozyme into the Trichoderma reesei genome, the Trichoderma reesei engineering strain for secreting and expressing egg white lysozyme is successfully constructed. It is found that different egg white lysozyme variants can bring different effects, and when the Trichoderma reesei RUT-C30 is used as the starting strain, the effect is better than that of QM6a.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology and biotechnology, in particular to a Trichoderma reesei engineering bacterium for secreting expression of egg white lysozyme and a construction method thereof. BACKGROUND

[0002] Egg white lysozyme has various biological functions: 1. Antibacterial effect. Egg white lysozyme can hydrolyze the β-1, 4 glycosidic bond of mucopolysaccharide in bacterial cell wall, decompose the insoluble mucopolysaccharide into soluble glycopeptide, cause the bacterial cell wall to rupture, and the contents to escape, thereby playing a bactericidal effect. The antibacterial effect on gram-positive bacteria is particularly significant, such as Staphylococcus aureus, etc. It can help the human body to resist bacterial infection and has a certain effect on the prevention and adjuvant therapy of infectious diseases in the oral cavity, respiratory tract, etc., and can be used for chronic rhinitis, chronic pharyngolaryngitis, oral ulcer, etc. 2. Anti-viral effect. Lysozyme can directly combine with negatively charged viral proteins, form a complex with DNA, RNA and apo-protein, and inactivate the virus, thereby playing a certain anti-viral effect. 3. Improve food quality. In the food industry, it can be used as a natural food preservative to prolong the shelf life of food. For example, adding lysozyme to dairy products, meat products, fruit and vegetable products and other foods can inhibit the growth and reproduction of microorganisms and maintain the quality and safety of food. 4. Help egg white foamability. In egg white, lysozyme can improve the strength and stability of the foam film and has a positive effect on the foamability of egg white.

[0003] Egg white lysozyme can be purified from chicken egg white by affinity chromatography, ion exchange resin method, direct crystallization of salt, polyacrylic acid precipitation method, ultrafiltration method, etc. The content of egg white lysozyme in chicken egg white is only about 0.3%-0.35%, and the raw material cost and extraction cost of egg white lysozyme are relatively high. Even if the content of egg white lysozyme is not high, the extraction cost is high, but since egg white lysozyme is a natural protein, it is non-toxic to the human body and will not remain in the body, and it is a highly safe bactericide and food preservative, which has a broad application prospect.

[0004] Trichoderma reesei is a thermophilic saprophytic filamentous fungus, which is derived from the original strain QM6a and further mutagenized and screened for high-yield cellulase strains, such as RUT-C30. Trichoderma reesei is widely used because of its ability to naturally secrete a large amount of endogenous proteins into the extracellular space, and the yield of endogenous cellulase after fermentation can be as high as 100 g / L.

[0005] In order to reduce the production cost of egg white lysozyme, there are research reports on obtaining egg white lysozyme by fermentation of Pichia pastoris. However, Pichia pastoris is not a food safety strain, and methanol is needed to induce gene expression, which does not meet the food safety standard. Therefore, using a food safety strain to express egg white lysozyme is a technical problem to be solved at present. SUMMARY

[0006] The purpose of the present application is to provide a Trichoderma reesei engineering bacterium for secreting and expressing egg white lysozyme and a construction method thereof, so as to solve the problems existing in the prior art. The present application successfully constructs a Trichoderma reesei engineering strain for secreting and expressing egg white lysozyme, which can be used for the production of egg white lysozyme.

[0007] In order to achieve the above-mentioned purpose, the present application provides the following scheme:

[0008] The present application provides an egg white lysozyme mutant, and the amino acid sequence of the egg white lysozyme mutant is shown in any one of SEQ ID NO. 2-4.

[0009] The present application also provides a gene encoding the egg white lysozyme mutant, and the nucleotide sequence is shown in any one of SEQ ID NO. 6-8.

[0010] The present application also provides a construction method of a Trichoderma reesei engineering bacterium for secreting and expressing egg white lysozyme, comprising the step of transforming an expression module into Trichoderma reesei to construct the Trichoderma reesei engineering bacterium, using Trichoderma reesei as a starting strain.

[0011] The expression module comprises a promoter, a secretion peptide gene, an egg white lysozyme gene and a terminator connected in sequence.

[0012] Optionally, the nucleotide sequence of the promoter is shown in SEQ ID NO. 10.

[0013] The nucleotide sequence of the secretion peptide gene is shown in SEQ ID NO. 9.

[0014] The nucleotide sequence of the egg white lysozyme gene is shown in any one of SEQ ID NO. 5-8.

[0015] The nucleotide sequence of the terminator is shown in SEQ ID NO. 11.

[0016] Optionally, the Trichoderma reesei comprises Trichoderma reesei RUT-C30, Trichoderma reesei QM6a or a derivative strain of Trichoderma reesei QM6a.

[0017] Preferably, the Trichoderma reesei comprises Trichoderma reesei RUT-C30.

[0018] The present invention also provides engineered Trichoderma reesei strains that secrete and express egg white lysozyme according to the construction method described above.

[0019] The present invention also provides the application of the engineered Trichoderma reesei strain that secretes and expresses egg white lysozyme in the production of egg white lysozyme.

[0020] The present invention also provides a method for producing egg white lysozyme, comprising the step of fermentation using the engineered Trichoderma reesei strain that secretes and expresses egg white lysozyme.

[0021] The present invention discloses the following technical effects:

[0022] This invention discovers three variants of egg white lysozyme, with amino acid sequences as shown in any one of SEQ ID NO. 2-4 and nucleotide sequences as shown in any one of SEQ ID NO. 6-8.

[0023] This invention marks the first time that egg white lysozyme has been expressed in the secretion of *Trichoderma reesei*. By transforming the encoding gene for egg white lysozyme (either the original sequence or the nucleotide sequence of a mutant) into the *Trichoderma reesei* genome to replace the cellulase CBHI in *Trichoderma reesei*, an engineered *Trichoderma reesei* strain expressing egg white lysozyme was successfully constructed. Furthermore, it was found that different egg white lysozyme variants exhibited different effects, with *Trichoderma reesei* RUT-C30 showing superior performance compared to QM6a when used as the starting strain.

[0024] The Trichoderma reesei engineered strain that secretes and expresses egg white lysozyme constructed in this invention can be used for the production of egg white lysozyme. The produced egg white lysozyme meets food safety standards and is more suitable for the actual needs of the food, feed and textile industries. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is a diagram illustrating the construction of the egg white lysozyme expression module in this invention;

[0027] Figure 2 This is a flowchart illustrating the construction of the engineered strain of egg white lysozyme in this invention;

[0028] Figure 3 This refers to the lysozyme activity secreted and expressed by the engineered strain of egg white lysozyme in this invention. Detailed Implementation

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0032] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0033] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0034] In embodiments of the method of the present invention, the formulations of the culture medium and reagents are as follows:

[0035] (1) Luria Bertani (LB) medium formula: 5g yeast powder, 10g peptone, 10g sodium chloride, tap water to 1L, natural pH.

[0036] (2) Formula for glucose or xylose PDA medium: 6g potato extract powder, 20g glucose (or xylose), 16g agar, tap water to 1L, natural pH.

[0037] (3) Formula of Trichoderma reesei fermentation medium (1L): 5g glucose, 37g lactose, 6g ammonium sulfate, 10g cellulose powder, 5g wheat bran, 20g corn steep liquor, 2g peptone, 1g yeast powder, 10g KH2PO4, 0.6g MgSO4·7H2O, 0.5g CaCl2, 1mL Mandels trace element solution and 1mL Tween 80.

[0038] (4) Formula of Mandels trace element solution (1000×): 5g FeSO4·7H2O, 2g CoCl·6H2O, 1.4g ZnSO4·7H2O, 1.6g MnSO4·H2O, and diluted with purified water to 1L.

[0039] (5) Agrobacterium conjugation transfer of Trichoderma reesei: The expression module was electroporated into Agrobacterium rhizogenes, and then the successfully electroporated Agrobacterium was co-cultured with the Trichoderma reesei host strain on IM plate medium (Covert et al.Agrobacterium tumefaciens-mediated transformation of Fusarium circinatum.Mycol.Res.105(3):259-264) to carry out Agrobacterium rhizogenes-mediated conjugation transfer. After co-culturing for 2 days, the strains were screened on glucose PDA plates with cefotaxime (300 μg / mL) and hygromycin B (75 μg / mL) until hyphae and spores grew. Then, PCR verification was performed to prove that the grown cells were the correct transformants.

[0040] (6) Trichoderma reesei shuttle plasmid and resistance marker deletion scheme: After eliminating 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 OFN1.0. Scientific Reports. 2016, 6:20761), the next round of gene modification can be carried out. The principle is as follows: Hygromycin resistance will be deleted when cultured and passaged in xylose PDA plates, with a deletion efficiency of close to 70-100%; the deleted strains cannot grow in PDA plates containing hygromycin, and this characteristic can be used to verify the deleted strains.

[0041] The Trichoderma reesei starting strains used in the embodiments of the present invention are Trichoderma reesei QM6a (ATCC 13631) and RUT-C30 (ATCC 56765). This scheme is also applicable to other Trichoderma reesei strains, such as QM9414 (ATCC 26921), RL-P37 (NRRL15709) and NG14 (ATCC 56767) and their derivative strains.

[0042] (7) Method for measuring the activity of egg white lysozyme: The activity of lysozyme was determined according to the national standard GB / T 30990-2014.

[0043] The plasmid extraction kit was purchased from AXYGEN, the gel extraction kit from MAGEN, the seamless cloning kit from TransGen, and the DNA restriction endonuclease and ligase from NEB, or similar products from other companies.

[0044] Experimental methods in the following examples, unless otherwise specified, were performed under standard conditions, such as those described in Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).

[0045] The original egg white lysozyme sequence (PDB:193L_A) is shown in SEQ ID NO.1.

[0046] SEQ ID NO.1:

[0047] KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFNTQATNRNTDGSTDYGILQINSRWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSDGNGMNAWVAWRNRCKGTDVQAWIRGCRL.

[0048] The amino acid sequences of the egg white lysozyme variant proteins in the following examples are shown in SEQ ID NO. 2-4. This invention is the first discovery of a novel mutant of egg white lysozyme.

[0049] SEQ ID NO.2:

[0050] KVFGRCELAAAMKKHGLDNYRGYSLGNWVCAAKFESNFNTQATNRNTDGSTDYGIL QINSRWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSDGNGMNAWVAWRNRCK GTDVQAWIRGCRL.

[0051] SEQ ID NO.3:

[0052] KVFGRCELAAAMKSHGLDNYRGYSLGNWVCAAKFESNFNTQATNRNTDGSTDYGIL QINSRWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSDGNGMNAWVAWRNRCK GTDVQAWIRGCRL.

[0053] SEQ ID NO.4:

[0054] KVFGRCELAAAMKSHGLDNYRGYSLGNWVCAAKFESNFNTQATNRNTDGSTDYGIL QINSRWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKSIVSDGNGMNAWVAWRNRCK GTDVQAWIRGCRL.

[0055] The nucleotide sequence encoding egg white lysozyme protein, as shown in SEQ ID NO.5-8, is an optimized nucleotide sequence based on the codon preference of Trichoderma reesei.

[0056] SEQ ID NO.5:

[0057] AAGGTCTTCGGCCGATGCGAGCTGGCTGCTGCTATGAAGCGCCACGGCCTCGATAACTACCGCGGCTACTCCCTCGGCAACTGGGTCTGCGCTGCTAAGTTCGAGAGCAACTTCAACACCCAGGCCACCAACCGCAACACCGACGGCAGCACCGATTACGGCATCCTGCAGATCAACTCCCGCTGGTGGTGCAA CGACGGCCGAACCCCTGGCAGCCGAAACCTCTGCAACATCCCCTGCTCCGCCCTGCTCTCTAGCGATATCACCGCCAGCGTCAACTGCGCCAAGAAGATCGTCTCCGACGGCAACGGCATGAACGCCTGGGTCGCTTGGCGAAACCGCTGCAAGGGCACTGACGTCCAGGCTTGGATCCGCGGCTGCCGACTC.

[0058] SEQ ID NO.6:

[0059] AAGGTCTTCGGCCGATGCGAGCTCGCTGCTGCTATGAAGAAGCACGGCCTCGACAACTACCGCGGCTACTCTCTCGGCAACTGGGTCTGCGCTGCCAAGTTCGAGTCCAACTTCAACACCCAGGCCACCAACCGCAACACCGACGGCAGCACCGATTACGGCATCCTCCAGATCAACTCCCGCTGGTGGTGCAACGACGGCCGAACCCCTGGCAGCCGAAACCTCTGCAACATCCCCTGCTCCGCCCTGCTCTCTTCCGATATCACCGCCTCCGTCAACTGCGCCAAGAAGATCGTCTCCGACGGCAACGGCATGAACGCCTGGGTCGCTTGGCGAAACCGCTGCAAGGGCACTGACGTCCAGGCTTGGATCCGCGGCTGCCGACTC。

[0060] SEQ ID NO.7:

[0061] AAGGTCTTCGGCCGATGCGAGCTCGCTGCTGCTATGAAGAGCCACGGCCTCGACAACTACCGCGGCTACTCTCTCGGCAACTGGGTCTGCGCTGCCAAGTTCGAGTCCAACTTCAACACCCAGGCCACCAACCGCAACACCGACGGCAGCACCGATTACGGCATCCTCCAGATCAACTCCCGCTGGTGGTGCAACGACGGCCGAACCCCTGGCAGCCGAAACCTCTGCAACATCCCCTGCTCCGCCCTGCTCTCTTCCGATATCACCGCCTCCGTCAACTGCGCCAAGAAGATCGTCTCCGACGGCAACGGCATGAACGCCTGGGTCGCTTGGCGAAACCGCTGCAAGGGCACTGACGTCCAGGCTTGGATCCGCGGCTGCCGACTC。

[0062] SEQ ID NO.8:

[0063] AAGGTCTTCGGCCGATGCGAGCTCGCTGCTGCTATGAAGAGCCACGGCCTCGACAACTACCGCGGCTACTCTCTCGGCAACTGGGTCTGCGCTGCCAAGTTCGAGTCCAACTTCAACACCCAGGCCACCAACCGCAACACCGACGGCAGCACCGATTACGGCATCCTCCAGATCAACTCCCGCTGGTGGTGCAA CGACGGCCGAACCCCTGGCAGCCGAAACCTCTGCAACATCCCCTGCTCCGCCCTGCTCTCTTCCGATATCACCGCCTCCGTCAACTGCGCCAAGAGCATCGTCTCCGACGGCAACGGCATGAACGCCTGGGTCGCTTGGCGAAACCGCTGCAAGGGCACTGACGTCCAGGCTTGGATCCGCGGCTGCCGACTC.

[0064] The secretory peptide sequence enables the secretion of egg white lysozyme into Trichoderma reesei cells. The nucleotide sequence of the gene encoding the secretory peptide is shown in SEQ ID NO.9. This sequence is an optimized nucleotide sequence according to the codon preference of Trichoderma reesei.

[0065] SEQ ID NO.9:

[0066] ATGATCGTCGGCATCCTCACCACCCTGGCCACTCTCGCTACCCTCGCTGCTTCTGTCCCTCGAGGAGCGACAGGCTTGCAGCAGCGTCTGGGGCCAGTGCGGCGGCCAGAACTGGAGCGGCCCTACCTGCTGC GCTTCTGGCAGCACCTGCGTCTACTCCAACGACTACTACAGCCAGTGCCTGCCCGGCGCTGCTAGCAGCAGCAGCTCCACCCGAGCTGCTTCTACCACCTCCCGAGTCTCCCCTACCACCTCTCGAGACAAGCGC.

[0067] The promoter and terminator sequences of the cellulase gene CBHI are shown in SEQ ID NO.10 and 11, respectively.

[0068] SEQ ID NO.10 (promoter):

[0069]

[0070] SEQ ID NO.11 (Terminator):

[0071]

[0072] Example 1: Obtaining the egg white lysozyme gene and secretory peptide gene

[0073] (1) The method for obtaining the egg white lysozyme encoding gene is as follows: The amino acid sequence (SEQ ID NO. 1-4) is provided, and a conventional gene company is commissioned to synthesize the corresponding nucleotide encoding sequence. The synthesis rules can be based on the host's codon preference. Due to host differences, codon preferences also vary. Based on the codon preference of *Trichoderma reesei*, the final synthesized DNA sequence is shown in SEQ ID NO. 5-8. Because codon preference is a range, other DNA sequences that can encode the same amino acid sequence, similar to the sequence shown in SEQ ID NO. 5-8, can also be used.

[0074] (2) The method for obtaining the gene encoding the secreted peptide is as follows: Provide the amino acid sequence (SEQ ID NO.9), and entrust a conventional gene company to synthesize the same nucleotide coding sequence. This is because codon bias is a range. Similar to the sequence shown in SEQ ID NO.9, other DNA sequences that can encode the same amino acid sequence can also be used.

[0075] Example 2: Construction of an egg white lysozyme expression module

[0076] To transcribe the gene from Example 1 into Trichoderma reesei, a promoter and a terminator are required. This invention provides the endogenous promoter with the highest expression level in Trichoderma reesei, and the sequences of its terminator (as shown in SEQ ID NO. 10 and 11, respectively).

[0077] The construction scheme is as follows: Using PCR, the promoter, secretory peptide, and egg white lysozyme gene fragments were cloned separately. Using the Vazyme One Step Clone Kit, these three fragments were ligated into the PacI / XbaI site of the *Trichoderma reesei* shuttle plasmid LML2.0a (Zhang et al. Light-inducible genetic engineering and control of non-homologous end-joining in industrial eukaryotic microorganisms: LML3.0 and OFN 1.0. Scientific Reports. 2016, 6:20761) to construct an intermediate plasmid. Using PCR, the terminator was cloned. Using the seamless ligation kit, the terminator fragment was ligated into the SmiI single restriction site of the intermediate plasmid, thus forming the egg white lysozyme expression module. The entire process is as follows: Figure 1 As shown.

[0078] The specific experimental procedures are as follows.

[0079] A. The nucleotide sequence of the CBHI promoter was amplified using the Trichoderma reesei genome as a template, using primers CBHI-F1 and CBHI-F2. An 18-base sequence from the Trichoderma reesei shuttle plasmid LML2.0a (underlined) was added to the 5' end of primer CBHI-F1 for seamless ligation.

[0080] CBHI-F1: 5'- TTACGAATTCTTAATTAA TTCTGGAGACGGCTTGTTGAATCAT-3'(SEQ IDNO.12);

[0081] CBHI-F2: 5'-CATGATGCGCAGTCCGCGGTTGA-3' (SEQ ID NO. 13).

[0082] Amplification reaction system: 5 μL of 10×PCR Buffer for KOD-Plus-Neo, 5 μL of 2mM dNTPs, 3 μL of 25mM MgSO4, 1.5 μL each of 10 μM primers CBHI-F1 / CBHI-F2, 1 μL of genomic template (200 ng), and 1 μL of KOD-Plus-Neo (1 U / μL).

[0083] Reaction program: 94℃ for 2 min; 98℃ for 10 sec, 58℃ for 30 sec, 68℃ for 30 sec, run for 30 cycles; 68℃ for 5 min.

[0084] B. Using primers CBHI-R1 and CBHI-R2, the nucleotide sequence of the CBHI terminator was amplified using the *Trichoderma reesei* genome as a template. A 16-base sequence from the *Trichoderma reesei* shuttle plasmid LML2.0a (underlined) was added to the 5' end of both primers CBHI-R1 and CBHI-R2 for seamless ligation.

[0085] CBHI-R1: 5'- ACTAGTGAGCTCATTT AGCTCCGTGGCGAAAGCCT-3' (SEQ ID NO. 14);

[0086] CBHI-R2: 5'- AGTGCCAAGCTTATTT CATCGTAACCGAGAATCCAGAGCTG-3' (SEQ ID NO. 15).

[0087] Amplification reaction system: 5 μL of 10×PCR Buffer for KOD-Plus-Neo, 5 μL of 2mM dNTPs, 3 μL of 25mM MgSO4, 1.5 μL each of 10 μM primers CBHI-R1 / CBHI-R2, 1 μL of genomic template (200 ng), and 1 μL of KOD-Plus-Neo (1 U / μL).

[0088] Reaction program: 94℃ for 2 min; 98℃ for 10 sec, 58℃ for 30 sec, 68℃ for 30 sec, run for 30 cycles; 68℃ for 5 min.

[0089] C. The nucleotide sequence of the secretory peptide was amplified using primers C2C-F1 and C2C-F2 with SEQ ID NO.9 as a template. A 15-base sequence from the CBHI promoter (underlined) was added to the 5' end of primer C2C-F1 for seamless ligation.

[0090] C2C-F1: 5'- GGACTGCGCATCATG ATCGTCGGCATCCTCACCA-3' (SEQ ID NO. 16);

[0091] C2C-F2: 5'-GCGCTTGTCTCGAGAGGTGGTA-3' (SEQ ID NO. 17).

[0092] Amplification reaction system: 5 μL of 10×PCR Buffer for KOD-Plus-Neo, 5 μL of 2mM dNTPs, 3 μL of 25mM MgSO4, 1.5 μL each of 10 μM primers C2C-F1 / C2C-F2, 1 μL of genomic template (200 ng), and 1 μL of KOD-Plus-Neo (1 U / μL).

[0093] Reaction program: 94℃ for 2 min; 98℃ for 10 sec, 58℃ for 30 sec, 68℃ for 30 sec, run for 30 cycles; 68℃ for 5 min.

[0094] D. Using primers HEWL-F1 and HEWL-F2, the nucleotide sequences of four egg white lysozymes were amplified using SEQ ID NO. 5–8 as templates. A 15-base sequence from a secreted peptide (underlined) was added to the 5' end of primer HEWL-F1 for seamless ligation. A 22-base sequence from the *Trichoderma reesei* shuttle plasmid LML2.0a (underlined) was added to the 5' end of primer HEWL-F2 for seamless ligation.

[0095] HEWL-F1: 5'- TCTCGAGACAAGCGCAAGGTCTTCGGCCGATGCGA-3' (SEQ ID NO. 18);

[0096] HEWL-F2: 5'- CATACATTATACGAAGTTATTA GAGTCGGCAGCCGCGGAT-3' (SEQ ID NO. 19).

[0097] Amplification reaction system: 5 μL of 10×PCR Buffer for KOD-Plus-Neo, 5 μL of 2mM dNTPs, 3 μL of 25mM MgSO4, 1.5 μL each of 10μM primers HEWL-F1 / HEWL-F2, 1 μL of genomic template (200 ng), and 1 μL of KOD-Plus-Neo (1 U / μL).

[0098] Reaction program: 94℃ for 2 min; 98℃ for 10 sec, 58℃ for 30 sec, 68℃ for 30 sec, run for 30 cycles; 68℃ for 5 min.

[0099] Expression modules were constructed using the Trichoderma reesei shuttle plasmid LML2.0a as the backbone. The plasmid was double-digested with the restriction endonucleases PacI / XbaI, and then seamlessly ligated using the Vazyme One Step Clone Kit to insert the promoter, secretory peptide, and egg white lysozyme DNA fragments. Next, the plasmid was digested with the restriction endonuclease SwaiI, and then seamlessly ligated using the Vazyme One Step Clone Kit to insert the unique terminator DNA fragment, thus forming the egg white lysozyme expression module. Four egg white lysozyme expression modules were constructed.

[0100] Example 3: Construction of an engineered Trichoderma reesei strain expressing egg white lysozyme

[0101] The four egg white lysozyme expression modules from Example 2 were transformed into *Trichoderma reesei* QM6a (ATCC 13631) and RUT-C30 (ATCC 56765) respectively to obtain *Trichoderma reesei* engineered strains expressing egg white lysozyme. In this invention, *Trichoderma reesei* strain X (QM6a or RUT-C30) was transformed using the *Agrobacterium tumefaciens-mediated transformation of *Fusarium circinatum*. Mycol. Res. 105(3):259-264). Egg white lysozyme replaced cellulase CBHI, resulting in first-generation *Trichoderma reesei* engineered strains X-HEWL(1-4)-1. After culturing in xylose-PDA medium and removing the hygromycin resistance marker, the final resistance-free *Trichoderma reesei* engineered strain X-HEWL(1-4)-2 expressing egg white lysozyme was obtained. In the engineered strain, the most important cellulase cbh1 was replaced by the egg white lysozyme gene, and the entire construction process was as follows: Figure 2 As shown.

[0102] The final engineered Trichoderma reesei strain X-HEWL(1-4)-2 was fermented in Trichoderma reesei fermentation medium to induce the secretion and expression of egg white lysozyme. The fermentation method followed 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 fermentation, the fermentation broth was centrifuged at 12500 rpm for 10 min, and the supernatant was collected as the crude egg white lysozyme solution. The activity of the egg white lysozyme secreted by each strain was determined according to the national standard GB / T 30990-2014. The results are shown in Table 1 and [Table data missing]. Figure 3 As shown, the Trichoderma reesei starting strains QM6a and RUT-C30 do not possess lysozyme activity. Lysozyme activity can only be detected in the fermentation broth after the cellulase CBHI gene is replaced with the egg white lysozyme gene, indicating that the promoter and secretory peptide can effectively express and secrete egg white lysozyme extracellularly.

[0103] In terms of host cell expression of egg white lysozyme, Trichoderma reesei RUT-C30 (ATCC 56765) is superior to QM6a (ATCC 13631).

[0104] Different egg white lysozyme variants showed varying levels of activity in the egg white lysozyme produced during shake-flask fermentation. The results indicated that mutations at different sites could produce different effects. Among them, the egg white lysozyme variant with the amino acid sequence shown in SEQ ID NO.2, when using Trichoderma reesei RUT-C30 as the starting strain, had the best effect and produced the highest activity of secreted egg white lysozyme.

[0105] Table 1 Lysozyme activity of each strain

[0106] Strain Lysozyme activity (U / mL) QM6a 21 QM6a-HEWL(1)-2 502 QM6a-HEWL(2)-2 801 QM6a-HEWL(3)-2 895 QM6a-HEWL(4)-2 825 RUT-C30 45 RUT-C30-HEWL(1)-2 1532 RUT-C30-HEWL(2)-2 2350 RUT-C30-HEWL(3)-2 2130 RUT-C30-HEWL(4)-2 1940

[0107] As can be seen from the results of the above embodiments, the present invention provides an engineered strain that secretes and expresses egg white lysozyme, and a method for producing egg white lysozyme.

[0108] In summary, this invention discloses the sequences of three egg white lysozyme variants and the genetic modification process of *Trichoderma reesei*, successfully constructing for the first time an engineered strain that secretes and expresses egg white lysozyme. This engineered strain has the potential to replace traditional egg extraction processes for producing food-grade egg white lysozyme.

[0109] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An egg white lysozyme mutant, characterized in that, The amino acid sequence of the egg white lysozyme mutant is shown in SEQ ID NO.

2.

2. The gene encoding the egg white lysozyme mutant of claim 1, characterized in that, The nucleotide sequence is shown in SEQ ID NO.

6.

3. A method for constructing an engineered Trichoderma reesei strain that secretes and expresses egg white lysozyme, characterized in that, The process includes the step of using Trichoderma reesei as the starting strain and converting the expression module into Trichoderma reesei to construct the Trichoderma reesei engineered strain. The expression module includes a promoter, a secretory peptide gene, an egg white lysozyme mutant gene, and a terminator connected in sequence. The nucleotide sequence of the promoter is shown in SEQ ID NO.10; The nucleotide sequence of the secreted peptide gene is shown in SEQ ID NO.9; The nucleotide sequence of the egg white lysozyme mutant gene is shown in SEQ ID NO. 6; The nucleotide sequence of the terminator is shown in SEQ ID NO.

11.

4. The construction method as described in claim 3, characterized in that, The Trichoderma reesei includes Trichoderma reesei RUT-C30, Trichoderma reesei QM6a, or a derivative strain of Trichoderma reesei QM6a.

5. The engineered Trichoderma reesei strain that secretes and expresses egg white lysozyme, obtained by the construction method as described in claim 3 or 4.

6. The application of the engineered Trichoderma reesei strain that secretes and expresses egg white lysozyme as described in claim 5 in the production of egg white lysozyme.

7. A method for producing egg white lysozyme, characterized in that, The process includes the step of fermentation using the engineered Trichoderma reesei strain that secretes and expresses egg white lysozyme as described in claim 5.

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