Method for improving extracellular protein production level of filamentous fungi

Abstract

The invention discloses a method for improving extracellular protein production of filamentous fungi such as thermophilic fungi and a recombinant strain obtained by the method. The method comprises the following steps: carrying out genetic manipulation on thermophilic fungi to delete or reduce the activity of a specific gene, so as to improve the production capacity of cellulase, hemicellulase, amylolytic enzyme or other proteins of the thermophilic fungi and remarkably improve the production level of the cellulase, hemicellulase and glucoamylase, compared with the original strain, the protein secretion levels of the modified strain under cellulose induction are respectively and obviously improved by about 39.6% and 48.6%, the activity of cellulase is improved by about 45.2% and 52.4%, the activity of hemicellulase is obviously improved by about 35.1% and 44.8%, and the application value is higher.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering and biotechnology. Specifically, the present invention relates to a method for promoting the production level of extracellular proteins of filamentous fungi such as Myceliophthora, and specifically to the genetic modification of Myceliophthora. Background Art

[0002] The protein expression and secretion system of filamentous fungi has great development potential and broad patent prospects. As a cell factory, it can efficiently express and secrete extracellular proteins to produce valuable products (such as industrial proteins and organic acids, etc.). Myceliophthora thermophila is a thermophilic filamentous fungus that can secrete a variety of lignocellulose hydrolases, has the characteristics of high temperature fermentation, fast growth and metabolic rate, and has great application potential in industrial production.

[0003] However, the protein production level of natural strains of filamentous fungi is low, and multiple mutagenesis breeding or multiple rounds of molecular genetic modification are required to improve the expression and secretion level of proteins in order to obtain industrial strains that can be used for protein production. With the rapid development and maturity of genomics technology and genome editing technology of filamentous fungi, industrial production strains with improved traits have been obtained through molecular genetics and metabolic engineering technology. However, the number of target genes that can be used for genetic engineering modification is very limited. Patent CN110205334B provides a method for improving the expression level of cellulase by modifying regulatory factors in thermophilic myceliophthora. However, the molecular mechanism of protein expression and secretion of filamentous fungi is very complex, and there are many genes involved in the expression and secretion of lignocellulose hydrolase genes, which have not yet been clearly resolved.

[0004] However, high-yield strains must not only increase the RNA abundance of genes at the expression level, but also require us to further understand the effects of proteins in the secretion process, reduce or even eliminate the bottleneck of the secretion pathway, and further improve the production level of filamentous fungal proteins. Therefore, it is necessary to identify the key regulatory factors in this secretion pathway and use gene editing technology to obtain recombinant strains with significantly improved cellulase production levels, thereby developing technologies and methods that can improve the production of proteins such as cellulase, which is innovative. Summary of the invention

[0005] The present inventors have conducted extensive and in-depth research, aiming to provide a strain modification method capable of improving the cellulase production capacity and protein secretion level of filamentous fungi, which is to improve the cellulase production and protein secretion level of filamentous fungi by genetically modifying genes related to the protein secretion pathway of filamentous fungi. The present invention uses CRISPR-Cas9 genome editing technology to knock out and edit the genes Mycth_2300902 and Mycth_2306768 of the thermophilic Myceliophthora strain, and obtains a recombinant strain with missing genes Mycth_2300902 and Mycth_2306768. The recombinant strain can significantly improve the secretion level of protein, and significantly improve the production capacity and enzyme activity level of cellulase, hemicellulase and saccharifying enzyme.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides a method for obtaining a deletion mutant strain of genes Mycth_2300902 and Mycth_2306768 by genetic modification technology, which is a gene-edited mutant strain obtained by knocking out the genes of a thermophilic Myceliophthora strain by using CRISPR-Cas9 genome editing technology. The construction method comprises the following steps: constructing two guide RNA (sgRNA) expression elements for the targeting genes Mycth_2300902 and Mycth_2306768, whose nucleotide sequences are shown in SEQ ID NO.5 (Mycth_2300902-sgRNA) and SEQ ID NO.6 (Mycth_2306768-sgRNA), respectively; constructing homologous donor DNA sequences of the genes Mycth_2300902 and Mycth_2306768 containing the G418 resistance marker, whose nucleotide sequences are shown in SEQ ID NO.7 (donor-Mycth_2300902) and SEQ ID NO.8 (donor-Mycth_2306768). After the expression frame of Cas9 protein, Mycth_2300902-sgRNA and donor DNA donor-Mycth_2300902; the expression frame of Cas9 protein, Mycth_2306768-sgRNA and donor DNA donor-Mycth_2306768 are co-transformed into the protoplast cells of the host strain, gene-edited mutant strains can be obtained through homologous recombination.

[0008] On the other hand, the present invention provides a recombinant strain for producing cellulase, saccharifying enzyme, or other proteins, which has a stronger secretion capacity of cellulase, saccharifying enzyme or other proteins and the ability to degrade cellulose and starch than the starting strain; the recombinant strain is a gene mutant strain named ΔMycth_2300902 and ΔMycth_2306768, which is a Myceliophthora strain with knocked out genes Mycth_2300902 and Mycth_2306768. More specifically, the present invention provides a recombinant Myceliophthora thermophila with enhanced secretion of cellulase and saccharifying enzyme, which is a recombinant Myceliophthora thermophila with deletion mutations of genes Mycth_2300902 and Mycth_2306768 obtained by gene editing Myceliophthora thermophila genes Mycth_2300902 and Mycth_2306768 through the gene editing method of the present invention.

[0009] Wherein, the host cell is Myceliophthora. In a more specific embodiment, the host cell is selected from but not limited to Myceliophthora, and most preferably Myceliophthora thermophila.

[0010] In the present invention, the meanings of the terms used are explained.

[0011] When "recombinant" is used to refer to a strain, cell, nucleic acid, protein or vector, it means that the strain, cell, nucleic acid, protein or vector has been modified by introducing a heterologous nucleic acid or protein or by altering a native nucleic acid or protein. For example, a recombinant strain expresses genes that are not found in the native (non-recombinant) form of the strain, or expresses native genes.

[0012] "Host strain" or "host cell" refers to a suitable host for expression vectors or DNA constructs, and specifically, the host strain is preferably Myceliophthora, most preferably Myceliophthora thermophila. The host cell can be a Myceliophthora host cell or a genetically modified host cell.

[0013] "Genome editing" refers to the deletion, insertion or replacement of the genomic DNA of an organism in order to achieve the purpose of modifying the target sequence.

[0014] "CRISPR-Cas9 editing technology" refers to the ability of Cas9 protein to cut specific DNA sequences in the genome of an organism under the guidance of the guide sgRNA, creating double-stranded DNA breaks, thereby triggering the cell's own repair mechanism and performing non-homologous end joining (NHEJ) or homologous recombination (HR) repair at the DSB site, thereby performing gene knockout, gene inactivation, gene knock-in and gene replacement, achieving the purpose of efficient genome editing.

[0015] "Target site" or "protospacer" refers to a nucleic acid sequence that defines a portion of a nucleic acid, and refers to a 20-base sequence at the 5' end of the guide RNA (sgRNA). This sequence is identical to the target DNA sequence. Under conditions sufficient for binding, the sgRNA requires this sequence to bind to the target DNA, and the complex of Cas9 and sgRNA shears the target DNA.

[0016] "Sequence" refers to a nucleotide sequence of any suitable length, which may be DNA or RNA; may be linear, circular or branched, and may be single-stranded or double-stranded. The term "donor DNA sequence" refers to a nucleotide sequence that is inserted into a genome. The donor sequence may be of any length, for example, preferably between about 500 and 3,000 nucleotides in length (or any integer value therebetween).

[0017] The present invention obtains the following beneficial effects: the present invention studies multiple genes of the sterol regulatory element binding protein (SREBP) pathway, and finally finds that genetic manipulation of Myceliophthora cells to delete or reduce the activity of Mycth_2300902 and Mycth_2306768 genes can significantly improve the production level of cellulase and starch hydrolyzing enzyme. In a specific experiment, the present invention uses CRISPR-Cas9 editing technology to knock out the thermophilic Myceliophthora genes Mycth_2300902 and Mycth_2306768 to obtain gene editing deletion mutant strains. The obtained mutant strains ΔMycth_2300902 and ΔMycth_2306768 can significantly improve the production level of cellulase and starch hydrolyzing enzyme, wherein the protein secretion level is increased by about 33% compared with the starting strain, the activity of cellulase is increased by about 36% compared with the starting bacteria, and the activity of saccharifying enzyme is increased by about 24% compared with the starting bacteria, which has great application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of knocking out the target genes Mycth_2300902 and Mycth_2306768 using CRISPR-Cas9 editing technology.

[0019] Figure 2 Nucleic acid electrophoresis diagram for PCR identification of target genes Mycth_2300902 and Mycth_2306768 knocked out by CRISPR-Cas9 editing technology.

[0020] Figure 3The protein secretion amount of the Mycth_2300902 and Mycth_2306768 deletion mutant strains and the wild strain WT under cellulose induction conditions.

[0021] Figure 4 The cellulase activities of the Mycth_2300902 and Mycth_2306768 deletion mutant strains and the wild strain WT of Myceliophthora thermophila.

[0022] Figure 5 The cellulase activities of the Mycth_2300902 and Mycth_2306768 deletion mutant strains and the wild strain WT of Myceliophthora thermophila.

[0023] Figure 6 The protein secretion level and saccharifying enzyme activity of the Mycth_2300902 and Mycth_2306768 deletion mutant strains and the wild strain WT under starch induction conditions. DETAILED DESCRIPTION

[0024] To further illustrate the technical means and effects of the present invention, the technical solution of the present invention is further described below in conjunction with the preferred embodiments of the present invention. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0025] The methods used in the following examples are all conventional methods unless otherwise specified. For specific steps, please refer to: "Molecular Cloning: A Laboratory Manual" (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).

[0026] The methods for obtaining various biological materials described in the examples are only provided as an experimental method to achieve the specific disclosed purpose, and should not be construed as limiting the sources of the biological materials of the present invention. In fact, the sources of the biological materials used are extensive, and any biological material that can be obtained without violating laws and moral ethics can be replaced and used according to the instructions in the examples.

[0027] The original starting strain Myceliophthora thermophila ATCC 42464 used in the examples was purchased from the American type culture collection and obtained through commercial channels.

[0028] If the specific technology or conditions are not specified in the embodiments, the technology or conditions described in the literature in the field or the product instructions shall be followed. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased through regular channels. Among them, the percentage concentrations that appear are all mass percentage concentrations unless otherwise specified. The primers used and nucleic acid sequencing were completed by Suzhou GENEWIZ Biotechnology Co., Ltd. GENEWIZ. Among them, "MYCTH_..." is the gene site number of Thermophilic Myceliophthora.

[0029] Example 1. Construction of CRISPR-Cas9-mediated editing vectors for Myceliophthora thermophila genes Mycth_2300902 and Mycth_2306768

[0030] (1) Construction of sgRNA expression vectors for genes Mycth_2300902 and Mycth_2306768

[0031] The target sites of target genes Mycth_2300902 and Mycth_2306768 were designed by software sgRNACas9 tool. The U6p promoter, target site and sgRNA backbone of Myceliophthora thermophila were connected together by fusion PCR, and the sgRNA expression cassette vector was constructed by gene overlap extension (SOE) method. The primer sequences required for the sgRNA expression cassette vector are shown in Table 1.

[0032] The PCR reaction system was: 5× phusion HF buffer 10 μL, 10 mM dNTPs 1 μL, upstream / downstream primers 1.0 μL, template DNA 1 μL, Phusion DNA polymerase 0.5 μL, ddH 2 O 35.0 μL.

[0033] The PCR reaction conditions were as follows: first 98°C for 30 s; then 98°C for 10 s, 65°C for 30 s, 72°C for 30 s, for 34 cycles; and finally 72°C for 6 min, 4°C for 10 min.

[0034] The sgRNA expression plasmids Mycth_2300902-sgRNA and Mycth_2306768-sgRNA were formed by SOE-PCR amplification, and their sequences are shown in SEQ ID No.5 and SEQ ID No.6, respectively.

[0035] (2) Construction of homologous donor DNA vector

[0036] In this example, the homologous donor DNA fragments were respectively composed of the upstream and downstream homologous fragments (966 and 876 bp) of the target gene Mycth_2300902 and the glufosinate resistance gene (bar) expression frame PtrpC-bar fragment, which were connected to the plasmid pUC118 linearized by restriction endonucleases EcoRI and Hind III by the Gibson Assembly method, and finally the donor DNA fragment donor-Mycth_2300902 was constructed, and its nucleic acid sequence is shown in SEQ ID No.7. In this embodiment, the homologous donor DNA fragments are respectively composed of the upstream and downstream (806 and 878 bp) homologous fragments of the target gene Mycth_2306768 and the geneticin (G418) resistance gene expression frame PtrpC-neo fragment, which are connected to the plasmid pUC118 linearized by restriction endonucleases EcoR I and HindIII by the Gibson Assembly method, and finally the donor DNA fragment donor-Mycth_2306768 is constructed, and its nucleic acid sequence is shown in SEQ ID No. 8. The PCR primer sequences required for constructing the donor DNA fragment are shown in Table 1.

[0037] Table 1: Primers used in Example 1

[0038]

[0039]

[0040] Example 2: Gene knockout of Myceliophthora thermophila genes Mycth_2300902 and Mycth_2306768 using the CRISPR-Cas9 system

[0041] (1) Protoplast transformation of Myceliophthora thermophila

[0042] A. Mycelium Preparation

[0043] The mature spores of Myceliophthora thermophila were collected with 0.05% Tween-80 sterilized water, filtered through lens paper to remove the mycelium, and then inoculated into MM solid culture medium and cultured at 35°C for 20 hours.

[0044] B. Protoplast Preparation

[0045] After collecting the mycelium, place it in 20mL lysis solution (formula: 0.15g lysis enzyme, 30mL solution A, filtered and sterilized; solution A: 1.0361g potassium dihydrogen phosphate, 21.864g sorbitol, dissolved in 90mL deionized water, pH adjusted to 5.6, quantitative to 100mL, high temperature sterilization), lyse at 30℃ for 2h. Then filter with cellophane, centrifuge at 4℃ 2000rpm for 10min, discard the supernatant, add 4mL solution B (formula: 0.735g calcium chloride, 18.22g sorbitol, 1mL Tris-HCl (1M, pH 7.5), dissolved in 90mL deionized water, pH adjusted to 7.6, quantitative to 100mL, high temperature sterilization), centrifuge at 2000rpm 4℃ for 10min; discard the supernatant, and add a certain volume of solution B at 200μL / plasmid.

[0046] C. Protoplast transformation

[0047] In a precooled 15mL centrifuge tube, add 50μL precooled PEG (12.5g PEG6000, 0.368g calcium chloride, 500μL Tris HCl (1M pH 7.5) in sequence, mix the DNA fragments to be transformed and add them to 200μL protoplasts. After placing on ice for 20min, add 2mL precooled PEG liquid, place at room temperature for 5min, and then add 4mL solution B and mix gently. Take 3mL of the above solution and add it to 12mL melted MM medium containing glufosinate or G418, spread it on a plate, and culture it at 35℃. After 3d, pick a single mycelium and grow it on the corresponding resistance plate.

[0048] (2) CRISPR-Cas9 system for gene knockout at Mycth_2300902 and Mycth_2306768 loci

[0049] After the Cas9 protein expression element p0380-Ptef1-Cas9, Mycth_2300902-sgRNA and homologous donor DNA fragment donor-Mycth_2300902 were mixed in equal proportions and co-transformed into the thermophilic Myceliophthora ATCC 42464 protoplast cells; after the Cas9 protein expression element p0380-Ptef1-Cas9, donor-Mycth_2306768 and homologous donor DNA fragment donor-Mycth_2306768 were mixed in equal proportions and co-transformed into the thermophilic Myceliophthora ATCC 42464 protoplast cells. Figure 1As shown, Cas9, under the mediation of sgRNA, recognizes the target site for cutting by pairing the target sequence with the DNA chain of the target gene on the host cell genome. Subsequently, the donor DNA fragment undergoes homologous recombination with the sequences on both sides of the target site, thereby achieving the purpose of editing the genome. Transformants are selected by adding phosphinothricin or G418 to the plate.

[0050] (3) Verification of Thermophilic Myceliophthora transformants

[0051] A. Extraction of the Myceliophthora thermophila genome

[0052] The genomic DNA was extracted from the transformants selected in the above transformation process using the phenol-chloroform method, which specifically included the following operations:

[0053] 1) Add 200 mg of zirconium beads and 1 mL of lysis solution (formula: 0.2 M Tris·HCl (pH 7.5), 0.5 M NaCl, 10 mM EDTA, 1% SDS (w / v)) to a 2.0 mL sterile DNA extraction tube, and pick the hyphae of Myceliophthora thermophila growing on the plate and put them into the DNA extraction tube;

[0054] 2) Place all DNA extraction tubes on the grinding machine and oscillate at the maximum speed for 30 seconds, repeat twice;

[0055] 3) Incubate in a 65°C water bath for 30 min, vortexing every few minutes during the water bath;

[0056] 4) After the water bath, take out the tubes and add 80 μL of 1M Tris·HCl (pH 7.5) to each tube for neutralization;

[0057] 5) Add 400 μL of phenol:chloroform (1:1) and centrifuge at 13000 rpm for 5 minutes;

[0058] 6) Take 300 μL of supernatant and put it into a new 1.5 mL EP tube, add 600 μL of 95% ethanol (DNA grade);

[0059] 7) Incubate on ice for one hour, then centrifuge at 4°C and 13,000 rpm. White DNA precipitate can be seen at the bottom of the EP tube.

[0060] 8) Wash with 400 μL of 75% alcohol (DNA grade), centrifuge at 13,000 rpm at 4°C, and gently remove the supernatant;

[0061] 9) Place the EP tube in a vacuum concentrator to remove residual alcohol;

[0062] 10) Add 50 μL ddH2O to dissolve the DNA, measure the DNA concentration with NanoDrop, and store the extracted DNA in a -20°C refrigerator for PCR verification in the next step.

[0063] B. PCR verification of transformants

[0064] The genomic DNA extracted above was used as a template, and the transformants were verified by gene PCR using primers Mycth_2300902-ko-F / R and Mycth_2306768-ko-F / R. PCR reaction reagents were purchased from Nanjing Novozyme Biotechnology Co., Ltd.

[0065] The PCR reaction system was: 2 μL of 10× Taq Buffer, 0.2 μL of 10 mM dNTP Mix, 0.4 μL of upstream / downstream primers, 1 μL of DNA template, 0.2 μL of Taq DNA Polymerase, and 15.8 μL of water.

[0066] The PCR reaction conditions were: 94°C for 5 min, then 94°C for 30 s, 55°C for 30 s, 72°C for 1.2 min, 30 cycles, and finally 72°C for 7 min, 4°C for 10 min.

[0067] Experimental results: The PCR amplification products were subjected to 1% agarose gel electrophoresis (120V voltage, 30 minutes), and obvious gene amplification bands were displayed under the gel imaging system. The PCR amplification band of the Mycth_2300902 knockout mutant was 2000bp, and the target band of the wild-type strain was 1200bp; the PCR amplification band of the Mycth_2306768 knockout mutant was 2000bp, and the target band of the wild-type strain was 1100bp. The results are as follows Figure 2 As shown, it shows that the donor DNA fragment undergoes homologous recombination with the sequences on both sides of the target site, thereby obtaining a gene-edited mutant strain. Among the 20 Mycth_2300902-edited transformants obtained, the gene Mycth_2306768 of 18 transformants was successfully knocked out, and the knockout efficiency was as high as 90%; among the 18 Mycth_2306768-edited transformants obtained, the gene Mycth_2306768 of 16 transformants was successfully knocked out, and the knockout efficiency was as high as 88%.

[0068] Example 3. Phenotypic analysis of cellulase production by gene-edited deletion strains under cellulose induction conditions

[0069] (1) Cultivation of strains in microcrystalline cellulose induction medium: The Mycth_2300902 deletion mutant strains (ΔMycth_2300902-1, ΔMycth_2300902-2 and ΔMycth_2300902-3), Mycth_2306768 deletion mutant strains (ΔMycth_2306768-1, ΔMycth_2306768-2 and ΔMycth_2306768-3) and wild-type strain WT obtained above were inoculated into 100 mL of culture medium in a 250 mL Erlenmeyer flask. Medium formula: 2 mL of 50×Vogel's salt, 2 g of microcrystalline cellulose, fixed volume to 100 mL, autoclaved, inoculation size of 2.5*10 5 / mL, cultured at 45℃ and 150rpm for 5 days, the samples were centrifuged to obtain the supernatant, and the protein concentration and cellulase activity were determined.

[0070] (2) Determination of secreted protein concentration: The protein concentration in the supernatant was measured using the Bio-Rad Bradford Protein Rapid Test Kit. The results were as follows: Figure 3 As shown, compared with the starting strain wild type WT, the protein production of all mutant strains was significantly improved. Under cellulose culture conditions, compared with the wild strain WT, the Mycth_2300902 deletion mutant strain and the Mycth_2306768 deletion mutant strain were significantly increased by ~39.6% and ~48.6%, respectively.

[0071] (3) Method for determining the activity of CMCase (endo-1,4-β-glucanase): dilute the crude enzyme solution with 0.1M sodium acetate buffer to an appropriate multiple to a final volume of 0.5mL, preheat in a 50℃ water bath, take out, add 0.5mL MegazymeAZO-CM-CELLULOSE substrate solution, mix well, incubate at 50℃ for 10min, terminate the reaction with 2.5mL precipitation solution, let stand at room temperature for 10min, mix well, centrifuge at 1000g for 10min, and measure OD at 590nm. The blank group uses inactivated enzyme solution as a control. Activity definition: The amount of enzyme that produces 1μmol of product by hydrolyzing the substrate with 1mL of enzyme solution per min is defined as 1 unit of enzyme activity. The results are as follows Figure 4 As shown, compared with the starting wild strain WT, the cellulase activity of the Mycth_2300902 and Mycth_2306768 deletion mutant strains increased by 45.2% and 52.4%, respectively.

[0072] (4) Determination of XYLANase (xylanase) enzyme activity: Dilute the crude enzyme solution with 0.1M sodium acetate buffer to an appropriate multiple to a final volume of 0.5mL, preheat in a 50℃ water bath, take out and add 0.5mL Megazyme AZO-XYLAN substrate solution, mix well, incubate at 50℃ for 10min, terminate the reaction with 2.5mL precipitation solution, let stand at room temperature for 10min, mix well, centrifuge at 1000g for 10min, and measure OD at 590nm. The blank group uses inactivated enzyme solution as a control. Activity definition: The amount of enzyme that produces 1μmol of product by hydrolyzing the substrate with 1mL of enzyme solution per min is defined as 1 unit of enzyme activity. The results are as follows: Figure 5 As shown, compared with the starting strain, the hemicellulase activities of the Mycth_2300902 and Mycth_2306768 deletion mutant strains were increased by ∼35.1 and ∼44.8%, respectively.

[0073] Example 4. Analysis of protein production and saccharifying enzyme activity of gene-edited deletion strains under starch induction conditions

[0074] The Mycth_2300902 deletion mutant strain, Mycth_2306768 deletion mutant strain and wild-type strain WT obtained above were cultured in 2% (2 g / 100 mL) water-soluble starch medium (formula: 50×Vogel's salt 2 mL, water-soluble starch 2 g, fixed volume to 100 mL, high pressure sterilization) at 45°C for 5 days, and the samples were centrifuged to obtain the supernatant, and the protein concentration and saccharification enzyme activity were determined.

[0075] (1) Determination of secreted protein concentration:

[0076] The protein concentration in the supernatant was tested using the Bio-Rad Bradford Protein Rapid Test Kit. Figure 6 As shown, under water-soluble starch culture conditions, compared with the starting strain wild-type WT, the protein production of the knockout mutant strains of the target genes Mycth_2300902 and Mycth_2306768 was significantly improved, and their protein production was as high as ~2.6 and ~3.5 times that of the wild-type, respectively.

[0077] (2) Determination of saccharifying enzyme activity: Dilute the sample enzyme solution with 0.05M pH 4.6 sodium acetate buffer to a suitable multiple to a final volume of 0.25 mL, preheat in a 40°C water bath, take out, add 0.25 mL of 1% soluble starch (Difco) substrate solution preheated in a 40°C water bath, mix well, react at 50°C for 10 min, terminate the reaction with 0.5 mL of DNS solution, boil for 10 min, cool on ice, add distilled water to 2.5 mL, shake up and down, and determine the amount of glucose released by the DNS method. Determine the OD value at a wavelength of 540 nm. The blank group uses inactivated enzyme solution as a control. Definition of saccharifying enzyme activity: The amount of enzyme that hydrolyzes soluble starch to produce 1 μmol of glucose per min in 1 mL of enzyme solution at 40°C and pH 4.6 is one enzyme activity unit (U).

[0078] The results are as follows Figure 6 As shown, compared with the starting strain, the saccharifying enzyme activity of the deletion mutant strains of genes Mycth_2300902 and Mycth_2306768 was significantly increased by 2.8 and 3.4 times, respectively.

Claims

1. A method for increasing the yield of filamentous fungi, such as Myceliophthora ( Myceliophthora ) A method for producing extracellular protein at the level of The method is to inactivate or reduce one or both of the genes Mycth_2300902 and Mycth_2306768 of Myceliophthora to enhance its ability to secrete proteins.

2. The method according to claim 1, characterized in that The amino acid sequence of the gene Mycth_2300902 is shown in SEQ ID NO.1, and the amino acid sequence of the gene Mycth_2306768 is shown in SEQ ID NO.

2.

3. The method according to claim 1, characterized in that The nucleotide sequence of the gene Mycth_2300902 is shown in SEQ ID No.3, and the nucleotide sequence of the gene Mycth_2306768 is shown in SEQ ID No.

4.

4. The method according to claim 1, characterized in that The Mycth_2300902 and Mycth_2306768 genes are inactivated or reduced by completely or partially deleting their gene expression cassettes, by insertional inactivation or by rendering the gene non-functional for its intended purpose to prevent the gene from expressing functional proteins, or by reducing the expression of functional proteins.

5. The method according to claim 4, wherein the genes Mycth_2300902 and Mycth_2306768 in Myceliophthora cells are knocked out by genetic manipulation, more specifically by gene editing.

6. The method according to any one of claims 1 to 5, characterized in that: The Myceliophthora is a thermophilic Myceliophthora ( Myceliophthora thermophila ).

7. The method according to any one of claims 1 to 5, characterized in that: The increased secretory proteins refer to proteins involved in lignocellulose degradation and starch hydrolysis.

8. The method according to claim 7, characterized in that The protein is a cellulase or a saccharifying enzyme.

9. A recombinant strain obtained by the method according to any one of claims 1 to 8.

10. A method for degrading lignocellulosic materials using the recombinant strain according to claim 9, characterized in that: The recombinant strain according to claim 9 is cultured in the presence of a lignocellulose raw material or a lignocellulose inducer, thereby degrading lignocellulose.

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