CN117757643B

CN117757643B - A Trichoderma reesei engineered bacterium that secretes and expresses human lysozyme, and its construction method and application

Info

Publication number: CN117757643B
Application number: CN202410045920.9A
Authority: CN
Inventors: 王玮; 蔡万钏; 陈雨蒙
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2024-01-12
Filing date: 2024-01-12
Publication date: 2025-06-13
Anticipated expiration: 2044-01-12
Also published as: CN117757643A

Abstract

The invention discloses a trichoderma reesei engineering bacterium for secretory expression of human lysozyme and a construction method and application thereof, and relates to the fields of molecular biology and biotechnology. The construction method comprises the step of transforming an expression module into an original Trichoderma reesei strain to construct the Trichoderma reesei engineering strain, wherein the expression module comprises a promoter, a fusion gene and a terminator which are sequentially connected, and the fusion gene comprises a secretion peptide gene, a peptide cleavage site gene and a human lysozyme gene which are sequentially connected. The invention successfully constructs the engineering strain for secreting and expressing the human lysozyme, the engineering strain can be used for producing the human lysozyme preparation, and experiments prove that the human lysozyme preparation can obviously kill gram-positive and gram-negative bacteria. The invention provides a new human lysozyme preparation and a preparation method thereof for industries such as food, feed and the like.

Description

Trichoderma reesei engineering bacterium for secretory expression of human lysozyme and construction method and application thereof

Technical Field

The invention relates to the field of molecular biology and biotechnology, in particular to trichoderma reesei engineering bacteria for secretory expression of human lysozyme, a construction method and application thereof.

Background

Lysozyme was first found in 1922 by the uk scientist Fleming in human saliva, tears and nasal discharge, and is named because it can lyse the bacterial cell wall. Peptidoglycan is an important component of the cell wall of gram-positive bacteria. Lysozyme hydrolyzes the beta-1, 4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycan, causing the cell wall insoluble mucopolysaccharide to break down into soluble glycopeptides, resulting in the cell wall ruptured content overflowing to lyse the bacteria. Thus, lysozyme kills most gram-positive strains. The lysozyme can also be directly combined with virus proteins with negative charges to form double salts with DNA, RNA and apoprotein to inactivate viruses, so that the lysozyme has the functions of resisting bacteria, diminishing inflammation, resisting viruses, stopping bleeding, detumescence, easing pain and the like. The special properties of lysozyme lead the lysozyme to have wide application prospect in the industries of food, medicine, livestock production and the like. Currently, egg white lysozyme (abbreviated as egg white lysozyme) is the most widely used and commercialized lysozyme preparation for the reasons of low cost and easy availability.

Human lysozyme is a bactericidal protein of the human body itself. Compared with other sources of lysozyme, the human lysozyme has a plurality of insurmountable advantages, such as 3 times of enzyme activity than egg white lysozyme, natural compatibility with human body, no side effect and immunogenicity, capability of being digested and absorbed as nutrient substances in stomach and intestine, no toxicity to organisms, no residue in the body and higher safety. And are therefore increasingly favored in the food, pharmaceutical and feed industries.

Trichoderma reesei is an important industrial production strain, accords with GRAS (Generally Regarded as Safe) standards, and an enzyme preparation produced by fermentation of the Trichoderma reesei is widely applied to industries such as food, feed and the like. No report is made in the field of known academic research that Trichoderma reesei strains can be fermented to produce human lysozyme.

Although the prior art has the successful cases of using fungi such as yeast and the like to express egg white lysozyme in a heterologous way, the technology of using fungi (especially trichoderma reesei) to express human lysozyme in a heterologous way has not yet appeared. The reason for this may be that, first, human lysozyme has much higher activity on glucans such as chitin than egg white lysozyme, and thus produces a certain killing power on fungal hosts, resulting in heterologous expression failure. Second, secretory expression requires secretory peptides, and whether commercial strains (e.g., E.coli, pichia pastoris) have mature and efficient secretory peptides, but not commercial strains (e.g., trichoderma reesei, etc.) have similar secretory peptides is not known.

The invention aims to utilize the secretion of Trichoderma reesei to express human lysozyme, thereby providing a new technical idea for developing human lysozyme preparations.

Disclosure of Invention

The invention aims to provide a trichoderma reesei engineering bacterium capable of secreting and expressing human lysozyme, and a construction method and application thereof, so as to solve the problems in the prior art.

In order to achieve the above object, the present invention provides the following solutions:

The invention provides a construction method of Trichoderma reesei engineering bacteria for secretory expression of human lysozyme, which comprises the steps of transforming an expression module into an original Trichoderma reesei strain and constructing to obtain the Trichoderma reesei engineering bacteria;

The expression module comprises a promoter, a fusion gene and a terminator which are sequentially connected, wherein the fusion gene comprises a secretion peptide gene, a peptide cleavage site gene and a human lysozyme gene which are sequentially connected;

the secretory peptide gene is a gene for encoding secretory peptide, and the amino acid sequence of the secretory peptide is shown in any one of SEQ ID NO. 6-13;

The peptide cleavage site gene is a gene for 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;

The human lysozyme gene is a gene for encoding human lysozyme, and the amino acid sequence of the human lysozyme is shown in any one of SEQ ID NO. 1-5. It is well known in the art that substitution with similar or analogous amino acids does not generally alter the function of the protein. As another example, the addition of one or more amino acids at the C-terminus and/or N-terminus typically does not alter the function of the protein. Therefore, the amino acid sequence of human lysozyme also comprises a variant form of the sequence SEQ ID NO. 1-5, which has the same function as human lysozyme. Such variants include, but are not limited to, deletions, insertions and/or substitutions of several (typically l-30, preferably 1-20, more preferably l-10, most preferably l-5) amino acids, and the addition of one or several (typically less than 20, preferably less than l0, more preferably less than 5) amino acids at the C-terminus and/or N-terminus.

Further, the nucleotide sequence of the secretory peptide gene is shown in any one of SEQ ID NO. 28-35, the nucleotide sequence of the peptide cleavage site gene is shown in any one of SEQ ID NO. 36-40, and the nucleotide sequence of the human lysozyme gene is shown in any one of SEQ ID NO. 23-27.

Further, the Trichoderma reesei original strain comprises Trichoderma reesei QM6a, QM9414, RUT-C30, RL-P37, NG14 or PC-3-7.

Further, 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, and 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 respectively shown in SEQ ID NO. 19-22.

Further, the nucleotide sequence of the secretory 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, and the Trichoderma reesei original strain is PC-3-7.

The invention also provides the Trichoderma reesei engineering bacteria constructed by the construction method.

The invention also provides application of the trichoderma reesei engineering bacterium in preparation of a human lysozyme preparation.

The invention also provides a human lysozyme mutant, the amino acid sequence of which is shown in any one of SEQ ID NO. 2-5.

The invention also provides application of the human lysozyme mutant in preparing a bacteriostatic enzyme preparation, wherein the bacteriostatic enzyme preparation aims at pathogenic bacteria comprising salmonella enteritidis, escherichia coli and/or vibrio parahaemolyticus.

The invention also provides a bacteriostatic enzyme preparation, and the active ingredients comprise the human lysozyme mutant.

Because of the degeneracy of the codons (degeneracy refers to the sequence resulting from one or more codons replaced by degenerate codons encoding the same amino acid), degenerate sequences having as little as about 92% homology with the nucleotide sequences of SEQ ID NOS.23-40 are also capable of encoding said sequences. Thus, the term "fusion gene" in the present invention also includes nucleic acid sequences having at least 92% homology with the nucleotide sequences of SEQ ID NO. 23-40.

The invention discloses the following technical effects:

The invention constructs a human lysozyme fusion gene and an expression module, and converts the expression module into a plurality of Trichoderma reesei strains, and successfully constructs an engineering strain for secretion expression of human lysozyme. The engineering strain can be used for producing human lysozyme preparation, and experiments prove that the human lysozyme preparation can obviously kill gram-positive and gram-negative bacteria. The invention provides a new human lysozyme preparation and a preparation method thereof for industries such as food, feed and the like.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic diagram showing the construction process of a human lysozyme fusion gene (A), a CBHI/II expression plasmid (B) and a human lysozyme expression module (C);

FIG. 2 shows the results of the activity detection of human lysozyme prepared using different secretory peptides in example 3;

FIG. 3 shows the results of the activity assay of human lysozyme prepared using different peptide cleavage sites in example 3;

FIG. 4 shows the results of the activity detection of human lysozyme prepared by using different human lysozyme mutants in example 3;

FIG. 5 shows the results of the activity detection of human lysozyme obtained with different promoters and Trichoderma reesei strains in example 3;

FIG. 6 shows the experimental results of the inhibition zone of human lysozyme prepared by CBHI-g12.14.1 (PC-3-7) engineering strain.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, 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 invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

The experimental procedure, which is not specified in the following examples, is generally carried out according to conventional conditions, such as those described in the handbook of molecular cloning: laboratory (New York: cold Spring Harbor Laboratory Press, 1989).

The amino acid sequences of the human lysozyme and the four mutants are respectively shown as SEQ ID NO. 1-5, and the corresponding nucleotide sequences are respectively shown as SEQ ID NO. 23-27.

The amino acid sequences of the 8 secretory peptides are shown as SEQ ID NO. 6-13 respectively, and the corresponding nucleotide sequences are shown as SEQ ID NO. 28-35 respectively.

The amino acid sequences of the 5 peptide cleavage sites are GGGGSKR (SEQ ID NO. 14), GGGGSDKR (SEQ ID NO. 15), RSKR (SEQ ID NO. 16), RYKR (SEQ ID NO. 17) and NVISKR (SEQ ID NO. 18) respectively, and the corresponding nucleotide sequences are shown as SEQ ID NO. 36-40 respectively.

The nucleotide sequence of the CBHI promoter is shown as SEQ ID NO.19, and the nucleotide sequence of the CBHI terminator is shown as SEQ ID NO. 20.

The nucleotide sequence of the CBHII promoter is shown as SEQ ID NO.21, and the nucleotide sequence of the CBHII terminator is shown as SEQ ID NO. 22.

EXAMPLE 1 construction of fusion Gene

In order to realize efficient secretory expression of human lysozyme in Trichoderma reesei, it is necessary to fuse a secretory peptide endogenous to fungi at the N-terminus of the protein to guide secretory expression of heterologous human lysozyme. In addition to secretion, rapid cleavage and separation of lysozyme from the secreted peptide requires the insertion of a peptide cleavage site between the two. Thus, the amino acid sequence structure encoded by the fusion gene is secretory peptide-peptide cleavage site-human lysozyme, as shown in FIG. 1A. The invention provides 8 secretory peptides, 5 peptide cutting sites and 5 human lysozyme, and 200 different amino acid sequences and encoding genes thereof can be arranged and combined. For example, the fusion gene g12.KR.1 is the gene sequence of the protein with the amino acid sequence of SEQ ID NO.12 (secretory peptide) +SEQ ID NO.14 (peptide cleavage site) +SEQ ID NO.1 (human lysozyme).

The fusion gene is obtained by providing an amino acid sequence and entrusting a conventional gene company to synthesize a corresponding fusion gene sequence. The synthesis rules of the present invention are based on the codon preference of the Trichoderma reesei host. Because of the degeneracy of the codons (degeneracy refers to the sequence resulting from one or more codons replaced by degenerate codons encoding the same amino acid), degenerate sequences having as little as about 92% homology with the nucleotide sequences of SEQ ID NOS.23-40 are also capable of encoding said sequences. Thus, the term "fusion gene sequence" also includes nucleic acid sequences having at least 92% homology with the nucleotide sequences in SEQ ID NO. 23-40.

Example 2 construction of expression modules

In order to transcribe and express the fusion gene in example 1 in Trichoderma reesei, a promoter and a terminator are required. The invention provides two endogenous promoters with highest expression levels in Trichoderma reesei and sequences of terminator thereof (shown as SEQ ID No. 17-20 respectively). The construction scheme is as follows.

(1) By utilizing the PCR scheme, the promoter is connected with the terminator, and an enzyme cutting site SmiI is inserted at the connection part, so that the subsequent connection into a fusion gene is facilitated, namely, the following two DNA structures, namely a CBHI promoter-SmiI-CBHI terminator and a CBHII promoter-SmiI-CBHII terminator, are constructed, and the two DNA structures are shown in the diagram B in figure 1. The present invention has provided specific DNA sequences, and thus primer design and PCR protocols can be performed according to conventional research methods.

(2) The two DNA structures are connected into Trichoderma reesei expression plasmid in a seamless connection mode, so that two novel expression plasmids, namely CBHI expression plasmid and CBHII expression plasmid, can be constructed. The purpose of the plasmid is firstly to better preserve and replicate the two DNA structures, so that the structure is convenient to obtain in a large quantity subsequently, and secondly, the plasmid is provided with a hygromycin and other resistance screening mark, so that the trichoderma reesei can be transformed subsequently.

The nucleotide sequences of the CBHI and CBHII promoters and terminators are cloned and amplified by a PCR method, and a SmiI site (ATTTAAAT) is added on the primer to ensure that the structure of the CBHI promoter-SmiI-CBHI terminator and the CBHII promoter-SmiI-CBHII terminator is formed in a seamless connection mode.

A. The nucleotide sequence of the CBHI promoter was amplified using the primers CBHI-F1 (SEQ ID NO. 41) and CBHI-F2 (SEQ ID NO. 42) with the Trichoderma reesei genome as template.

The amplification reaction was 10X PCRBuffer for KOD-Plus-Neo 5. Mu.L, 2mM dNTPs 5. Mu.L, 25mM MgSO ₄. Mu.L, 10. Mu.M primer CBHI-F1/CBHI-F2 each 1.5. Mu.L, 1. Mu.L of genomic template (200 ng), and 1. Mu.L of KOD-Plus-Neo (1U/. Mu.L).

The reaction procedure was 94℃for 2min, 98℃for 10sec,58℃for 30sec,68℃for 60sec, 30 cycles, 68℃for 5min.

B. The nucleotide sequence of the CBHI terminator was amplified using the primers CBHI-R1 (SEQ ID NO. 43) and CBHI-R2 (SEQ ID NO. 44) with the Trichoderma reesei genome as template.

The amplification reaction was 10X PCRBuffer for KOD-Plus-Neo 5. Mu.L, 2mM dNTPs 5. Mu.L, 25mM MgSO ₄. Mu.L, 10. Mu.M primer CBHI-R1/CBHI-R2 each 1.5. Mu.L, 1. Mu.L of genomic template (200 ng), and 1. Mu.L of KOD-Plus-Neo (1U/. Mu.L).

The reaction procedure was 94℃for 2min, 98℃for 10sec,58℃for 30sec,68℃for 40sec, running 30 cycles, 68℃for 5min.

C. the nucleotide sequence of the CBHII promoter was amplified using the primers CBHII-F1 (SEQ ID NO. 45) and CBHII-F2 (SEQ ID NO. 46) with the Trichoderma reesei genome as template.

The amplification reaction system was 10X PCRBuffer for KOD-Plus-Neo 5. Mu.L, 2mM dNTPs 5. Mu.L, 25mM MgSO ₄. Mu.L, 10. Mu.M primers CBHII-F1/CBHII-F2 each 1.5. Mu.L, 1. Mu.L of genomic template (200 ng), and 1. Mu.L of KOD-Plus-Neo (1U/. Mu.L).

D. The nucleotide sequence of the CBHII terminator was amplified using the primers CBHII-R1 (SEQ ID NO. 47) and CBHII-R2 (SEQ ID NO. 48) with the Trichoderma reesei genome as template.

The amplification reaction system was 10X PCRBuffer for KOD-Plus-Neo 5. Mu.L, 2mM dNTPs 5. Mu.L, 25mM MgSO ₄. Mu.L, 10. Mu.M primers CBHII-R1/CBHII-R2 each 1.5. Mu.L, 1. Mu.L of genomic template (200 ng), and 1. Mu.L of KOD-Plus-Neo (1U/. Mu.L).

The reaction procedure was 94℃for 2min, 98℃for 10sec,58℃for 30sec,68℃for 30sec, running 30 cycles, 68℃for 5min.

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 3.0and OFN 1.0.Scientific Reports.2016,6:20761) as a framework to construct an expression vector, single enzyme digestion is carried out on a restriction enzyme SwaI site on the existing plasmid LML2.0a, and Vazyme One Step Clone Kit is utilized to carry out seamless connection, and the two DNA structures, namely a CBHI promoter-SmiI-CBHI terminator and a CBHII promoter-SmiI-CBHII terminator, are respectively connected to construct a CBHI expression plasmid and a CBHII expression plasmid (B in figure 1).

(3) And connecting the fusion gene into an expression plasmid to construct an expression module of the fusion gene. A single restriction enzyme cleavage is carried out at the restriction enzyme SmiI sites of the two CBHI expression plasmids and the CBHII expression plasmid, and Vazyme One Step Clone Kit is utilized for seamless connection, and various fusion genes are connected to construct expression modules of the various fusion genes (C in figure 1). For example, the CBHI-g12.14.1 expression module is to use CBHI expression plasmid to express g12.KR.1 fusion gene. Primers for cloning and amplifying the fusion gene are designed according to the specific sequence of the fusion gene which is synthesized in a entrusting way.

Example 3 production of human lysozyme by introducing an expression Module into Trichoderma reesei to obtain an engineering Strain expressing human lysozyme

The invention introduces the expression module constructed in the example 2 into Trichoderma reesei strain through routine experimental operations such as electrotransformation, protoplast transformation or Agrobacterium tumefaciens combined transfer, integrates the expression module into Trichoderma reesei genome, screens to obtain a transformant strain through screening by a screening mark on the expression module, and verifies the correctness of the transformant strain through lysozyme activity detection. The invention is illustrated by CBHI-g12.14.1 expression modules and Agrobacterium tumefaciens binding transfer protocols.

(1) CBHI-g12.14.1 expression modules were electrotransferred to Agrobacterium tumefaciens, and the electrotransferred Agrobacterium was then screened 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), respectively, in IM plate (Covert et al.Agrobacterium tumefaciens-mediated transformation of Fusarium circinatum.Mycol.Res.105(3):259-264) for Agrobacterium tumefaciens-mediated binding transfer, after two days of co-culture the transformants were transferred to PDA plates containing cefotaxime (300. Mu.g/mL) and hygromycin B (75. Mu.g/mL) until the transformants developed hyphae and spores, and then verified.

(2) Transformant CBHI-g12.14.1 in Trichoderma reesei RUT-C30 CBHI-g12.14.1 (RUT-C30) was inoculated into 50mL flasks with lactose as carbon source for validation culture. The culture medium formulation was verified to be lactose 10g/L、Tryptone 1g/L、yeast extract 0.5g/L、(NH₄)₂SO₄1.4 g/L、Urea 0.3g/L、KH₂PO₄2 g/L、CaCl₂·2H₂O0.3g/L、MgSO₄·7H₂O 0.3g/L、FeSO₄·7H₂O 5mg/L、MnSO₄·H₂O 1.6mg/L、ZnSO₄·7H₂O 1.4mg/L and CoCl ₂·6H₂ O2 mg/L. The inoculum size was 10 ⁸ spores/50 mL medium, 28℃and 200 rpm. Sampling on the 3 rd day, and measuring the activity of lysozyme according to the national standard method GB/T30990-2014 to verify the success of the transformation, wherein the result is shown in figures 2-5.

(3) The CBHI-g12.14.1 (PC-3-7) engineering strain is inoculated in a fermentation culture medium (lactose 37g/L, glucose 5g/L, corn steep liquor 27g/L、Tryptone 1g/L、(NH₄)₂SO₄5 g/L、CaCl₂·2H₂O 0.5g/L、MgSO₄·7H₂O 1g/L、FeSO₄·7H₂O 5mg/L、MnSO₄·H₂O 1.6mg/L、ZnSO₄·7H₂O 1.4mg/L and CoCl ₂·6H₂ O2 mg, and cultured for 6 days at 28 ℃), after fermentation is finished, a fermentation broth supernatant is obtained, namely the human lysozyme crude enzyme solution is subjected to spray drying (or freeze drying), and the human lysozyme crude enzyme solution is prepared into human lysozyme enzyme powder, wherein the activity of the enzyme powder can reach 300U/mg.

Example 4 lysozyme bacteriostasis zone experiment

Peptidoglycan is an important component of the cell wall of gram-positive bacteria. Lysozyme hydrolyzes the beta-1, 4 glycosidic bond in N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) in peptidoglycan, resulting in rupture of the cell wall of gram positive bacteria under osmotic pressure, and the content overflows to solubilize the bacteria. Thus, lysozyme kills most gram-positive strains. The human lysozyme has higher activity, and can kill various gram-negative bacteria besides common gram-positive strains. The method is used for evaluating the inhibition effect of the human lysozyme produced by trichoderma reesei on gram-negative bacteria of common pathogens, and comparing the inhibition effect with commercial egg white lysozyme.

1G of human lysozyme powder (300U/mg) prepared by using CBHI-g12.14.1 (PC-3-7) engineering strain of example 3 was dissolved in 10mL of ultrapure water to prepare a 10% (w/v) human lysozyme test solution. Negative control 1g egg white lysozyme powder (commercial enzyme, 20000U/mg, purchased by Jieli Co.) was dissolved in 10mL of ultrapure water to prepare a 10% egg white lysozyme control solution. Salmonella enteritidis (CMCC (B) 50335), escherichia coli (ATCC 25922) and Vibrio parahaemolyticus (ATCC 17802) are adopted as indicator bacteria according to national standard GB4789.28 quality requirements of culture media and reagents and GB4789.4-2010 salmonella test. The detection liquid and the control liquid are respectively 2 paralleled.

The indicator bacteria were streaked on LB solid (yeast powder 5g/L, naCl g/L, peptone 10g/L and agar 20 g/L) plates and cultured at 37℃for 12 hours. Single colonies are picked from the flat plate and put into LB liquid (yeast powder 5g/L, naCl g/L and peptone 10 g/L) test tubes for shake cultivation at 37 ℃ for about 12 hours, and then the indicator bacteria seed liquid is obtained. Then 100. Mu.L of seed solution was sucked onto the surface of a new LB solid plate, and the bacterial solution was uniformly coated with a coating rod. Cutting filter paper sheets with the diameter of 6mm, sterilizing and drying. Sterile filter paper sheets were placed on the surface of the above LB solid plate coated with the indicator bacteria, and 10. Mu.L of the test solution or the control solution was added dropwise to each filter paper sheet. After the filter paper sheet absorbs the liquid, the plate is turned over, marked and placed in a 37 ℃ incubator for culturing for about 12 hours, and the recorded result can be observed, and the result is shown in fig. 6. The bacteriostatic effects of the other mutants are collated in table 1.

TABLE 1 bacteriostatic Effect of the enzymes

Note that-represents a clear zone of inhibition without edges, + represents a clear zone of inhibition.

Although the specific activity of the human lysozyme produced by using trichoderma reesei provided by the invention is lower than that of commercial egg white lysozyme, as shown in fig. 6 and table 1, the inhibition effect of the human lysozyme on common pathogenic bacteria such as salmonella enteritidis, escherichia coli, vibrio parahaemolyticus and the like provided by the invention is obviously higher than that of commercial egg white lysozyme.

In conclusion, the invention successfully utilizes trichoderma reesei to secrete and express human lysozyme, and the sterilization effect of the human lysozyme produced by the method is obviously superior to that of commercial egg white lysozyme.

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit 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 it comprises the steps of transforming an expression module into an 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 is 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 to 40; the nucleotide sequence of the human lysozyme gene is shown in any one of SEQ ID NO.24 to 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 is characterized in that the nucleotide sequence of the secretory 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.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 pathogens 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.