Trichoderma reesei engineering strain for high yield and high performance cellulase and construction method and application thereof

Abstract

The present application relates to the field of biotechnology, in particular to a high-yield high-performance cellulase Trichoderma reesei engineering strain and a construction method and application thereof, the Trichoderma reesei engineering strain co-overexpresses beta-glucosidase gene Aabgl1 and expansin gene Trswo1, and knocks out transcriptional repressor gene Trace1, the Trichoderma reesei engineering strain has high cellulase yield, which is 100.15% higher than that of the starting strain Trichoderma reesei Rut C30.The high-performance cellulase produced by the Trichoderma reesei engineering strain has balanced enzyme system composition and excellent hydrolysis performance, when corn straw is hydrolyzed, the glucan conversion rate reaches 94.89%, the hydrolysis performance is equivalent to that of commercial enzyme preparation Cellic CTec2.0, and good industrial development prospect and practical application value are shown.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an engineered strain of Trichoderma reesei that produces high-performance cellulase, its construction method, and its application. Background Technology

[0002] Lignocellulose is the most abundant renewable biomass resource in nature. Its efficient conversion into fuel ethanol, high-value-added chemicals, and functional materials through biotechnology is a crucial pathway to promoting sustainable development and reducing dependence on fossil fuels. Cellulase plays a central role in this bioconversion process, degrading complex lignocellulose into fermentable sugars. Therefore, developing efficient and low-cost cellulase preparations is a key step in promoting the development of the biorefining industry. The filamentous fungus *Trichoderma reesei* has become a major strain for cellulase production due to its strong protein secretion capacity and relatively complete cellulase enzyme system. However, *Trichoderma reesei* still has significant shortcomings in industrial applications: low activity of key enzyme components (such as β-glucosidase), an imbalanced enzyme composition, and overall enzyme activity levels that are insufficient to meet the demands of large-scale industrial production. These limitations significantly restrict the economic feasibility of lignocellulose bioconversion. Therefore, researchers are using strategies such as enzyme system optimization, expression regulatory network modification, and secretion pathway enhancement to increase cellulase production from *Trichoderma reesei*, aiming to solve the problem of excessively high costs in lignocellulose bioconversion.

[0003] Chinese patent CN110951628B discloses a method for constructing and applying a high-β-glucosidase-activity Trichoderma reesei engineered strain for straw degradation. This patent optimizes the Trichoderma reesei enzyme system by expressing a β-glucosidase variant derived from Aspergillus niger in Trichoderma reesei, obtaining the engineered strain QMB1, whose β-glucosidase activity reaches 110 IU / mL, 55 times higher than the starting strain.

[0004] Chinese patent CN114686459B discloses the application of the transcriptional repressor 70351 of Trichoderma reesei cellulase and a method for increasing cellulase expression and activity. This patent modifies the expression regulatory network of Trichoderma reesei, knocking out the transcriptional repressor 70351. The resulting engineered strain Δ70351 exhibits an endonuclease activity of 2.42 IU / ml, which is 0.7 times higher than the original strain.

[0005] Although existing technologies have improved the enzyme production performance of Trichoderma reesei through single strategies such as enzyme system optimization, expression regulatory network modification, and secretion pathway enhancement, problems such as low cellulase yield, unbalanced enzyme system composition, and poor hydrolysis performance still exist, making it difficult to meet the needs of industrial applications.

[0006] In summary, there is an urgent need for a Trichoderma reesei strain with high cellulase yield, balanced enzyme composition, and excellent hydrolytic performance, as well as a method for its construction. Summary of the Invention

[0007] This invention addresses the problem of how to provide a Trichoderma reesei strain with high cellulase yield, balanced enzyme composition, and excellent hydrolysis performance, as well as its construction method.

[0008] To achieve the above objectives, the first aspect of the present invention provides a Trichoderma reesei engineered strain that produces high-performance cellulase, wherein the Trichoderma reesei engineered strain is named XZD4047.

[0009] The engineered Trichoderma reesei strains co-overexpressed the β-glucosidase gene Aabgl1 and the expansion hormone gene Trswo1, and knocked out the transcriptional repressor gene Trace1.

[0010] A second aspect of the present invention provides a method for constructing the above-mentioned high-yield, high-performance cellulase-producing Trichoderma reesei engineered strain, comprising the following steps:

[0011] S1. Using Trichoderma reesei PB3 genomic DNA as a template, the Aabgl1 gene fragment was amplified by PCR; using Trichoderma reesei Rut C30 genomic DNA as a template, the Trswo1 gene fragment was amplified by PCR. The Aabgl1 gene fragment and the Trswo1 gene fragment were inserted into the NcoI and AflII sites of the vector pCZF9, respectively, by seamless cloning to obtain the vector pCZFBS that overexpresses Aabgl1 and Trswo1.

[0012] S2. The vector pCZFBS overexpressing Aabgl1 and Trswo1 obtained in step S1 was transformed into Escherichia coli DH5α, and the vector pCZFBS was extracted. The extracted vector pCZFBS was then transformed into Agrobacterium rhizogenes competent cells AGL-1 to obtain Agrobacterium rhizogenes AGL-1 containing the vector pCZFBS.

[0013] S3. Through the mediation of Agrobacterium rhizogenes, Agrobacterium rhizogenes AGL-1 containing the vector pCZFBS was mixed with Trichoderma reesei RutC30 conidia, cultured, screened, and verified to obtain the Trichoderma reesei engineered strain XZD4010.

[0014] S4. Using Trichoderma reesei Rut C30 genomic DNA as a template, the upstream and downstream homologous arm fragments of the Trace1 gene were amplified by PCR. Using the pUC-Ble vector as a template, the bleomycin selection expression cassette was amplified by PCR. Using the pCZF8 vector as a template, the pCZF8 vector backbone was amplified by PCR. The upstream homologous arm fragment of the Trace1 gene, the bleomycin selection expression cassette, the downstream homologous arm fragment of the Trace1 gene, and the pCZF8 vector backbone were mixed and ligated using a seamless cloning method to obtain the knockout vector pCZF25.

[0015] S5. The knockout vector pCZF25 obtained in step S4 is transformed into Escherichia coli DH5α, and the vector pCZF25 is extracted. The extracted vector pCZF25 is transformed into Agrobacterium rhizogenes competent cells AGL-1 to obtain Agrobacterium rhizogenes AGL-1 containing the vector pCZF25.

[0016] S6. Through Agrobacterium rhizogenes-mediated mixing, Agrobacterium rhizogenes AGL-1 containing the vector pCZF25 was mixed evenly with conidia of engineered strain XZD4010, cultured, screened, and verified to obtain the engineered strain of Trichoderma reesei, XZD4047.

[0017] A third aspect of this invention provides the application of the above-mentioned high-yield, high-performance cellulase-producing Trichoderma reesei engineered strain in the fermentation production of high-performance cellulase, wherein the fermentation production of high-performance cellulase includes the following steps:

[0018] S101. The engineered strain of Trichoderma reesei, XZD4047, was inoculated into a sporulation medium and cultured at 25-30 ºC for 4-10 days. The spores were washed off with sterile water and filtered through gauze to obtain a spore suspension of the engineered strain XZD4047.

[0019] S201. Inoculate the spore suspension of the engineered strain XZD4047 obtained in step S101 into a seed culture medium and culture it at 25-30ºC and 100-300 rpm for 18-36 h to obtain the seed liquid of the engineered strain XZD4047; inoculate the seed liquid into a fermentation medium at an inoculation ratio of 5-10% and culture it at 25-30ºC and 100-300 rpm for 120-216 h.

[0020] S301. Centrifuge the fermentation broth obtained in step S201 at 5000 rpm-12000 rpm for 5 min-30 min, collect the supernatant, and obtain high-performance cellulase.

[0021] A fourth aspect of this invention provides the application of the aforementioned high-performance cellulase in improving the degradation efficiency of lignocellulosic biomass. Compared with the prior art, this invention has the following beneficial effects:

[0022] (1) This invention provides a high-yield, high-performance cellulase engineered strain of Trichoderma reesei, its construction method, and its application. This invention successfully constructed a high-yield, high-performance cellulase engineered strain XZD4047 by overexpressing the β-glucosidase gene Aabgl1 and the expansion hormone gene Trswo1 in Trichoderma reesei and knocking out the transcriptional repressor gene Trace1.

[0023] (2) The engineered strain XZD4047 was subjected to shake-flask fermentation and the cellulase yield and extracellular protein content of the engineered strain XZD4047 were determined. The results showed that the filter paper enzyme activity, β-glucosidase activity, exocellulase activity, endocellulase activity, xylanase activity and extracellular protein content of the engineered strain XZD4047 were significantly increased, and increased by 100.15%, 3128.07%, 1973.80%, 18.55%, 11.79% and 43.05% respectively compared with the starting strain Trichoderma reesei RutC30. Among them, the β-glucosidase activity and exocellulase activity were significantly increased, which effectively optimized the enzyme system composition ratio and solved the key problem of insufficient β-glucosidase secretion in the starting strain Trichoderma reesei RutC30.

[0024] (3) The high-performance cellulase produced by the engineered strain XZD4047 of the present invention was used to hydrolyze the pretreated lignocellulosic biomass. The dextran conversion rate reached 94.89%, which was 82.91% higher than that of the starting strain Trichoderma reesei Rut C30. The hydrolysis performance was comparable to that of the commercial enzyme preparation Cellic CTec2.0. The high-performance cellulase produced by the engineered strain provided by the present invention can achieve efficient hydrolysis of lignocellulosic biomass without the need for additional auxiliary enzymes and additives, which greatly simplifies the application process of enzyme preparations.

[0025] In summary, the engineered strain XZD4047 provided by this invention has the characteristics of high cellulase yield, balanced enzyme composition, and excellent hydrolysis performance. It can significantly reduce the production and application costs of cellulase preparations and has important industrial development prospects and practical application value in fields such as biomass refining, biofuel synthesis, and preparation of high-value-added chemicals based on lignocellulose. Attached Figure Description

[0026] Figure 1 This is the carrier map in Example 1.

[0027] Where: A is the spectrum of the pCZFBS vector; B is the spectrum of the pCZF25 vector.

[0028] Figure 2 The image shows the filter paper enzyme activity assay results of Trichoderma reesei Rut C30 and different engineered strains in Example 3.

[0029] Figure 3 The image shows the β-glucosidase activity assay results of Trichoderma reesei Rut C30 and different engineered strains in Example 3.

[0030] Figure 4 The graph shows the results of exocellulase activity assays for Trichoderma reesei Rut C30 and different engineered strains in Example 3.

[0031] Figure 5 The image shows the results of the endonuclease activity assay of Trichoderma reesei Rut C30 and different engineered strains in Example 3.

[0032] Figure 6 The graph shows the xylanase activity assay results of Trichoderma reesei Rut C30 and different engineered strains in Example 3.

[0033] Figure 7 The graph shows the results of extracellular protein content determination for Trichoderma reesei Rut C30 and different engineered strains in Example 3.

[0034] Figure 8 This is a graph showing the relative gene expression levels of the engineered Trichoderma reesei strains XZD4010 and XZD4047 in Example 4.

[0035] Wherein: A is the gene expression analysis diagram of engineered strain XZD4010 relative to the originating strain Trichoderma reesei Rut C30; B is the gene expression analysis diagram of engineered strain XZD4047 relative to engineered strain XZD4010.

[0036] Figure 9 The image shows the hydrolysis effect of Trichoderma reesei Rut C30 and different engineered strains of enzyme solution on alkali pretreated corn straw in Example 5.

[0037] Where A is the glucose concentration during the hydrolysis process; and B is the glucan conversion rate during the hydrolysis process. Detailed Implementation

[0038] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0039] The first aspect of this invention provides a Trichoderma reesei engineered strain that produces high-performance cellulase, wherein the Trichoderma reesei engineered strain is named XZD4047.

[0040] The engineered Trichoderma reesei strains co-overexpressed the β-glucosidase gene Aabgl1 and the expansion hormone gene Trswo1, and knocked out the transcriptional repressor gene Trace1.

[0041] According to the present invention, the β-glucosidase gene Aabgl1 is derived from Aspergillus aculeatus, and the nucleotide sequence of the β-glucosidase gene Aabgl1 is shown in SEQ ID NO.1.

[0042] According to the present invention, the swelling hormone gene Trswo1 is derived from Trichoderma reesei RutC30, and the nucleotide sequence of the swelling hormone gene Trswo1 is shown in SEQ ID NO.2.

[0043] According to the present invention, the transcription repressor gene Trace1 is derived from Trichoderma reesei Rut C30, and the nucleotide sequence of the transcription repressor gene Trace1 is shown in SEQ ID NO.3.

[0044] According to the present invention, the amino acid sequence of the β-glucosidase is shown in SEQ ID NO.4.

[0045] According to the present invention, the amino acid sequence of the swelling agent is shown in SEQ ID NO.5.

[0046] According to the present invention, the amino acid sequence of the transcriptional repressor ACE1 is shown in SEQ ID NO.6.

[0047] A second aspect of the present invention provides a method for constructing the above-mentioned high-yield, high-performance cellulase-producing Trichoderma reesei engineered strain, comprising the following steps:

[0048] S1. Using Trichoderma reesei PB3 genomic DNA as a template, the Aabgl1 gene fragment was amplified by PCR; using Trichoderma reesei Rut C30 genomic DNA as a template, the Trswo1 gene fragment was amplified by PCR. The Aabgl1 gene fragment and the Trswo1 gene fragment were inserted into the NcoI and AflII sites of the vector pCZF9, respectively, by seamless cloning to obtain the vector pCZFBS that overexpresses Aabgl1 and Trswo1.

[0049] S2. The vector pCZFBS overexpressing Aabgl1 and Trswo1 obtained in step S1 was transformed into Escherichia coli DH5α, and the vector pCZFBS was extracted. The extracted vector pCZFBS was then transformed into Agrobacterium rhizogenes competent cells AGL-1 to obtain Agrobacterium rhizogenes AGL-1 containing the vector pCZFBS.

[0050] S3. Through the mediation of Agrobacterium rhizogenes, Agrobacterium rhizogenes AGL-1 containing the vector pCZFBS was mixed with Trichoderma reesei RutC30 conidia, cultured, screened, and verified to obtain the Trichoderma reesei engineered strain XZD4010.

[0051] S4. Using Trichoderma reesei Rut C30 genomic DNA as a template, the upstream and downstream homologous arm fragments of the Trace1 gene were amplified by PCR. Using the pUC-Ble vector as a template, the bleomycin selection expression cassette was amplified by PCR. Using the pCZF8 vector as a template, the pCZF8 vector backbone was amplified by PCR. The upstream homologous arm fragment of the Trace1 gene, the bleomycin selection expression cassette, the downstream homologous arm fragment of the Trace1 gene, and the pCZF8 vector backbone were mixed and ligated using a seamless cloning method to obtain the knockout vector pCZF25.

[0052] S5. The knockout vector pCZF25 obtained in step S4 is transformed into Escherichia coli DH5α, and the vector pCZF25 is extracted. The extracted vector pCZF25 is transformed into Agrobacterium rhizogenes competent cells AGL-1 to obtain Agrobacterium rhizogenes AGL-1 containing the vector pCZF25.

[0053] S6. Through Agrobacterium rhizogenes-mediated mixing, Agrobacterium rhizogenes AGL-1 containing the vector pCZF25 was mixed evenly with conidia of engineered strain XZD4010, cultured, screened, and verified to obtain the engineered strain of Trichoderma reesei, XZD4047.

[0054] A third aspect of this invention provides the application of the above-mentioned high-yield, high-performance cellulase-producing Trichoderma reesei engineered strain in the fermentation production of high-performance cellulase, wherein the fermentation production of high-performance cellulase includes the following steps:

[0055] S101. The engineered strain of Trichoderma reesei, XZD4047, was inoculated into a sporulation medium and cultured at 25-30 ºC for 4-10 days. The spores were washed off with sterile water and filtered through gauze to obtain a spore suspension of the engineered strain XZD4047.

[0056] S201. Inoculate the spore suspension of the engineered strain XZD4047 obtained in step S101 into a seed culture medium and culture it at 25-30ºC and 100-300 rpm for 18-36 h to obtain the seed liquid of the engineered strain XZD4047; inoculate the seed liquid into a fermentation medium at an inoculation ratio of 5-10% and culture it at 25-30ºC and 100-300 rpm for 120-216 h.

[0057] S301. Centrifuge the fermentation broth obtained in step S201 at 5000 rpm-12000 rpm for 5 min-30 min, collect the supernatant, and obtain high-performance cellulase.

[0058] According to the present invention, in step S101, the sporulation culture medium contains 25-35 g / L malt extract powder, 15-20 g / L agar, water as solvent, and natural pH.

[0059] According to the present invention, the seed culture medium contains 10-30 g / L glucose, 4-6 g / L (NH4)2SO4, 10-20 g / L KH2PO4, 1-3 g / L peptone, 0.4-0.8 g / L MgSO4·7H2O, 0.4-0.6 g / L CaCl2, 0.0010-0.0018 g / L ZnSO4·7H2O, 0.0012-0.0020 g / L MnSO4·H2O, 0.003-0.006 g / L FeSO4·7H2O, and 0.001-0.003 g / L CoCl2, with water as the solvent and pH 5.5.

[0060] According to the present invention, the fermentation medium contains 10-30 g / L microcrystalline cellulose, 10-30 g / L corn steep liquor, 4-6 g / L (NH4)2SO4, 10-20 g / L KH2PO4, 0.4-0.8 g / L MgSO4·7H2O, 0.4-0.6 g / L CaCl2, 0.0010-0.0018 g / L ZnSO4·7H2O, 0.0012-0.0020 g / L MnSO4·H2O, 0.003-0.006 g / L FeSO4·7H2O, and 0.001-0.003 g / L CoCl2, with water as the solvent and pH 5.5.

[0061] The fourth aspect of this invention provides the application of the above-mentioned high-performance cellulase in improving the degradation efficiency of lignocellulosic biomass.

[0062] According to the present invention, the degradation of lignocellulosic biomass by the high-performance cellulase includes the following steps:

[0063] The lignocellulosic biomass is pretreated to obtain pretreated raw material, the high-performance cellulase is added, the pH is adjusted to 4.5-5.0, and hydrolysis is carried out at 45-55 ºC for 48-96 h.

[0064] In this invention, there is no particular limitation on the lignocellulosic biomass, which can be at least one of corn stalks, rice stalks, and corn cobs. In a preferred embodiment of this invention, the lignocellulosic biomass is corn stalks.

[0065] In this invention, there are no particular limitations on the pretreatment, which can be dilute sulfuric acid pretreatment, sodium hydroxide pretreatment, steam explosion pretreatment, or ionic liquid pretreatment. In a preferred embodiment of this invention, the pretreatment is sodium hydroxide pretreatment.

[0066] According to the present invention, the conditions for sodium hydroxide pretreatment are as follows: sodium hydroxide concentration is 0.5%-4%, the mass ratio of sodium hydroxide to corn stalk is 5:1-20:1, and the reaction conditions are 105 ºC-130 ºC for 30-120 min.

[0067] The present invention will be described in detail below through embodiments.

[0068] Example 1: Construction of vector pCZFBS and knockout vector pCZF25

[0069] The β-glucosidase gene Aabgl1 is derived from Aspergillus echinococcosis. The β-glucosidase gene Aabgl1 fragment is 2583 bp in size, and its nucleotide sequence is shown in SEQ ID NO.1, while its amino acid sequence is shown in SEQ ID NO.4.

[0070] The expansin gene Trswo1 is derived from Trichoderma reesei Rut C30. The Trswo1 fragment is 1482 bp in size, and its nucleotide sequence is shown in SEQ ID NO.2, while its amino acid sequence is shown in SEQ ID NO.5.

[0071] The transcription repressor gene Trace1 is derived from Trichoderma reesei Rut C30. The Trace1 fragment is 2247 bp in size, and its nucleotide sequence is shown in SEQ ID NO.3, while its amino acid sequence is shown in SEQ ID NO.6.

[0072] (1) Construction of vector pCZFBS

[0073] Using forward primer P1 and reverse primer P2 as primers, the β-glucosidase gene Aabgl1 fragment was amplified from the Trichoderma reesei PB3 genome by PCR. Using forward primer P3 and reverse primer P4 as primers, the expansion hormone gene Trswo1 fragment was amplified from the Trichoderma reesei RutC30 genome by PCR. The PCR reaction conditions and systems are shown in Tables 1 and 2.

[0074] Forward primer P1:

[0075] 5′-CAGCGCAGCTACAGCACAACTCAGTATGAAGCTCAGTTGGCTTGA-3′ (SEQ ID NO.11)

[0076] Reverse primer P2:

[0077] 5′-TATTCTCTAGACTAGCATCATGCTCATTGCACCTTCGGGAGCG-3′ (SEQ ID NO. 12)

[0078] Forward primer P3:

[0079] 5′-CAACAACTTCTCTCAACGGCAGCATGGCTGGTAAGCTTATCCT-3′ (SEQ ID NO.13)

[0080] Reverse primer P4:

[0081] 5′-GCGAGAATAATCGTCACGCTCACTCAATTCTGGCTAAACTGCA-3′ (SEQ ID NO.14)

[0082] Table 1 PCR reaction conditions

[0083]

[0084] Table 2 PCR reaction system

[0085]

[0086] The PCR products were electrophoresed on a 1% agarose gel, and the target fragment was recovered using a DNA purification kit after verification.

[0087] The pCZF9 vector was digested with the restriction endonuclease NcoI. The digestion products were electrophoresed on a 1% agarose gel. After verification, the target fragment was recovered using a DNA purification kit. The digestion system and reaction conditions are shown in Table 3. The β-glucosidase gene Aabgl1 fragment was ligated to the digested vector using a seamless cloning kit. The ligation reaction system and reaction conditions are shown in Table 4.

[0088] Table 3. Carrier Enzyme Digestion Reaction System

[0089]

[0090] Table 4. Carrier-linked reaction system

[0091]

[0092] Thaw *E. coli* DH5α competent cells on ice, add 10 µL of recombinant vector, gently shake to mix, and incubate on ice for 30 min. Heat shock at 42 ºC for 90 s, then quickly transfer to an ice bath to cool for 1 min. Add LB liquid medium preheated to 37 ºC and incubate at 37 ºC, 200 rpm for 45 min–1 h. Spread the incubated cells on LB plates containing 100 μg / mL Kan and incubate at 37 ºC for 13–15 h. Pick single colonies for PCR verification. Inoculate correctly verified transformants into LB liquid medium containing 100 μg / mL Kan and incubate at 37 ºC, 200 rpm for 10–14 h. Extract the vector and sequence it. If the sequencing is correct, obtain the pCZFB vector.

[0093] The pCZFB vector was digested with the restriction endonuclease AflII. The digestion products were electrophoresed on a 1% agarose gel. After verification, the target fragment was recovered using a DNA purification kit. The digestion system and reaction conditions are shown in Table 3. The Trswo1 fragment of the intumescent gene was ligated to the digested vector using a seamless cloning kit. The ligation reaction system and reaction conditions are shown in Table 4. The pCZFBS vector map is attached. Figure 1 .

[0094] Thaw *E. coli* DH5α competent cells on ice, add 10 µL of recombinant vector, gently shake to mix, and incubate on ice for 30 min. Heat shock at 42 ºC for 90 s, then quickly transfer to an ice bath to cool for 1 min. Add LB liquid medium preheated to 37 ºC and incubate at 37 ºC, 200 rpm for 45 min–1 h. Spread the incubated cells onto LB plates containing 100 μg / mL Kan and incubate at 37 ºC for 13–15 h. Pick single colonies for PCR verification. Inoculate correctly verified transformants into LB liquid medium containing 100 μg / mL Kan and incubate at 37 ºC, 200 rpm for 10–14 h. Extract the vector and sequence it. If the sequencing is correct, obtain the vector pCZFBS.

[0095] The vector pCZFBS was transformed into *Agrobacterium tumefaciens* competent cells AGL-1. The specific steps were as follows: 100 μL of *Agrobacterium tumefaciens* competent cells were taken, the corresponding vector was added, and the mixture was stirred by hand at the bottom of the tube. The cells were then incubated sequentially on ice for 5 min, in liquid nitrogen for 5 min, in a 37 ºC water bath for 5 min, and in an ice bath for 5 min. 700 μL of LB broth was added, and the cells were incubated at 28 ºC with shaking for 2-3 h. The cells were centrifuged at 6000 rpm for 1 min, and approximately 100 μL of the supernatant was collected and gently resuspended. The cells were then spread onto LB agar plates containing 100 μg / mL Kan and incubated at 28 ºC for 2-3 days. Single colonies were picked for PCR verification. After successful verification, *Agrobacterium tumefaciens* AGL-1 containing the vector pCZFBS was obtained.

[0096] (2) Construction of the knockout vector pCZF25

[0097] Using forward primer P5 and reverse primer P6 as primers, the upstream homologous arm fragment of the Trace1 gene was amplified from the Trichoderma reesei Rut C30 genome by PCR, and the nucleotide sequence is shown in SEQ ID NO.7. Using forward primer P7 and reverse primer P8 as primers, the downstream homologous arm fragment of the Trace1 gene was amplified from the Trichoderma reesei Rut C30 genome by PCR, and the nucleotide sequence is shown in SEQ ID NO.8. Using forward primer P9 and reverse primer P10 as primers, the bleomycin expression cassette was amplified from plasmid pUC-Ble by PCR, and the nucleotide sequence is shown in SEQ ID NO.9. Using forward primer P11 and reverse primer P12 as primers, the pCZF8 vector backbone was amplified from pCZF8 by PCR, and the nucleotide sequence is shown in SEQ ID NO.10. The PCR reaction conditions and systems are shown in Tables 1 and 2.

[0098] Forward primer P5:

[0099] 5′-TCGAGGTCGACGGTATCGATAGGGCATTGTTGGCCTCTTT-3′ (SEQ ID NO.15)

[0100] Reverse primer P6:

[0101] 5′-TACCGCTGTTGAGATCCAGGGGTAGTCTTAAGAGACAGGC-3′ (SEQ ID NO.16)

[0102] Forward primer P7:

[0103] 5′-ACACTAACGGATACTTTATCGAC-3′ (SEQ ID NO. 17)

[0104] Reverse primer P8:

[0105] 5′-ATAGAACTAGTGTATTCCCCGGGCTAACATCGTCTTCACCTTTTTG-3′ (SEQ ID NO. 18)

[0106] Forward primer P9:

[0107] 5′-CTGGATCTCAACAGCGGTA-3′ (SEQ ID NO.19)

[0108] Reverse primer P10:

[0109] 5′-GATAAAGTATCCGTTAGTGTTCTAGAAAGAAGGATTACCT-3′ (SEQ ID NO. 20)

[0110] Forward primer P11:

[0111] 5′-AGCCCGGGGAATACACTAGTTCTAT-3′ (SEQ ID NO. 21)

[0112] Reverse primer P12:

[0113] 5′-ATCGATACCGTCGACCTCGAGG-3′ (SEQ ID NO. 22)

[0114] The PCR products were electrophoresed on a 1% agarose gel, and the target fragment was recovered using a DNA purification kit after verification.

[0115] The upstream homologous arm fragment of the Trace1 gene, the bleomycin expression cassette, the downstream homologous arm fragment of the Trace1 gene, and the pCZF8 vector backbone were mixed at a molar ratio of 1:1:1:1. The fragments were then ligated using a seamless cloning kit to obtain the Trace1 gene knockout vector pCZF25. The ligation reaction system and conditions for the Trace1 gene knockout vector are shown in Table 5, and the pCZF25 knockout vector map is provided in the instruction manual. Figure 1 .

[0116] Table 5. Trace1 gene knockout vector ligation reaction system

[0117]

[0118] Thaw *E. coli* DH5α competent cells on ice, add 10 µL of recombinant vector, gently shake to mix, and incubate on ice for 30 min. Heat shock at 42 ºC for 90 s, then quickly transfer to an ice bath to cool for 1 min. Add LB liquid medium preheated to 37 ºC and incubate at 37 ºC, 200 rpm for 45 min–1 h. Spread the incubated cells on LB plates containing 100 μg / mL Kan and incubate at 37 ºC for 13–15 h. Pick single colonies for PCR verification. Inoculate correctly verified transformants into LB liquid medium containing 100 μg / mL Kan and incubate at 37 ºC, 200 rpm for 10–14 h. Extract the vector and sequence it. If the sequencing is correct, the knockout vector pCZF25 is obtained.

[0119] The knockout vector pCZF25 was transformed into *Agrobacterium tumefaciens* competent cells AGL-1. The specific steps were as follows: 100 μL of *Agrobacterium tumefaciens* competent cells were taken, the corresponding vector was added, and the mixture was stirred thoroughly by hand. The cells were then incubated sequentially on ice for 5 min, in liquid nitrogen for 5 min, in a 37 ºC water bath for 5 min, and in an ice bath for 5 min. 700 μL of LB broth was added, and the cells were incubated at 28 ºC with shaking for 2-3 h. The cells were centrifuged at 6000 rpm for 1 min, and approximately 100 μL of the supernatant was collected and gently resuspended. The cells were then spread onto LB agar plates containing 100 μg / mL Kan and incubated at 28 ºC for 2-3 days. Single colonies were picked for PCR verification. After successful verification, *Agrobacterium tumefaciens* AGL-1 containing the knockout vector pCZF25 was obtained.

[0120] Example 2 Construction of engineered Trichoderma reesei strains XZD4010 and XZD4047

[0121] (1) Construction of engineered Trichoderma reesei strain XZD4010

[0122] Agrobacterium rhizogenes containing the pCZFBS vector was picked and cultured in LB liquid medium containing 100 μg / mL Kan at 28 ºC with shaking at 200 rpm for 24 h. The cells were collected by centrifugation at 6000 rpm for 5 min and washed twice with sterile water. The OD was then transferred to IM medium containing 0.2 mM AS. 600 Dilute to 0.2 g / L, incubate at 28 °C and 200 rpm for OD. 600 Up to 0.6.

[0123] Trichoderma reesei Rut C30 was inoculated into seed culture medium at a 10% inoculum and cultured at 28 ºC with shaking at 200 rpm for 24 h. The activated bacterial solution was then spread onto a sporulation medium plate and incubated statically at 28 ºC for 7 days. The spores on the plate were washed off with distilled water, filtered through six layers of gauze to remove the mycelium, centrifuged at 8000 rpm for 5 min, and washed twice with sterile water. The culture was then resuspended in IM medium containing 0.2 mM AS and incubated at 24 ºC for 2-3 h for pre-germination.

[0124] Take 100 μL of the above-cultured Agrobacterium rhizogenes containing the pCZFBS vector and 100 μL of Trichoderma reesei Rut C30 conidia, mix them by blowing and aspiration, and incubate in the dark for 1 h. Spread the mixture onto an IM plate lined with an NC membrane and containing 0.2 mM AS, and incubate at 24 ºC for 3 days. Turn the NC membrane from the IM plate onto a PDA plate containing 300 μg / mL Hyg and 500 μg / mL Cef for preliminary screening and to kill Agrobacterium until transformants grow. Transfer the transformants to a PDA plate containing 200 μg / mL Hyg for secondary screening. Transfer the hyphae to a sporulation medium and incubate at 28 ºC for 3-5 days. Spores were transferred to a single-spore culture medium and cultured at 28 ºC for 3-5 days to obtain single colonies with individual spore growth. Single colonies were picked and cultured in a sporulation medium at 28 ºC for 3-5 days. Spores were collected and suspended in a 20% glycerol solution and stored at -80 ºC.

[0125] The transformed sporozoites obtained above were inoculated into seed culture medium and cultured at 28 ºC with shaking at 200 rpm for 36 h. The genome of the transformed sporozoites was extracted and verified by PCR. The correctly verified strain was named Trichoderma reesei engineered strain XZD4010.

[0126] (2) Construction of engineered Trichoderma reesei strain XZD4047

[0127] Agrobacterium rhizogenes containing the knockout vector pCZF25 was picked and cultured in LB liquid medium containing 100 μg / mL Kan at 28 ºC with shaking at 200 rpm for 24 h. The cells were collected by centrifugation at 6000 rpm for 5 min and washed twice with sterile water. The OD was then transferred to IM medium containing 0.2 mM AS. 600 Dilute to 0.2 g / L, incubate at 28 °C and 200 rpm for OD. 600 Up to 0.6.

[0128] The engineered Trichoderma reesei strain XZD4010 was inoculated into seed culture medium at a 10% inoculum and cultured at 28 ºC with shaking at 200 rpm for 24 h. The activated bacterial solution was then spread onto a sporulation medium plate and incubated statically at 28 ºC for 7 days. The spores on the plate were washed off with distilled water, filtered through six layers of gauze to remove the mycelium, centrifuged at 8000 rpm for 5 min, and washed twice with sterile water. The culture was then resuspended in IM medium containing 0.2 mM AS and incubated at 24 ºC for 2-3 h for pre-germination.

[0129] Take 100 μL of the above-cultured Agrobacterium rhizogenes containing the knockout vector pCZF25 and 100 μL of conidia from the engineered strain XZD4010, mix them thoroughly by blowing and aspiration, and incubate in the dark for 1 h. Spread the mixture onto an IM plate lined with an NC membrane and containing 0.2 mM AS, and incubate at 24ºC for 3 days. Then, flip the NC membrane from the IM plate onto a PDA plate containing 50 μg / mL Ble and 500 μg / mL Cef for preliminary screening and to kill Agrobacterium until transformants grow. Transfer the transformants to a PDA plate containing 50 μg / mL Ble for secondary screening. Transfer the hyphae to a sporulation medium and incubate at 28ºC for 3-5 days. Transfer the spores to a single-spore culture medium and incubate at 28ºC for 3-5 days to obtain single colonies with a single spore. Pick a single colony and place it in a sporulation medium. Incubate at 28 °C for 3-5 days. Collect the spores and suspend them in a 20% glycerol solution. Store at -80 °C.

[0130] The transformed sporozoites obtained above were inoculated into seed culture medium and cultured at 28 ºC with shaking at 200 rpm for 36 h. The genome of the transformed sporozoites was extracted and verified by PCR. The correctly verified strain was named Trichoderma reesei engineered strain XZD4047.

[0131] Example 3: Determination of cellulase, xylanase, and extracellular protein content and yield of engineered Trichoderma reesei strains XZD4010 and XZD4047

[0132] Fresh spores of the starting strain *Trichoderma reesei* Rut C30, and engineered strains *Trichoderma reesei* XZD4010 and XZD4047 were inoculated into 50 mL of seed culture medium and cultured at 28 ºC and 200 rpm for 24 h. The inoculum was then transferred at a 10% inoculation rate to 100 mL of fermentation medium and cultured at 28 ºC and 200 rpm. Every 24 h, 4 mL of fermentation broth was collected, centrifuged at 12000 rpm for 10 min, and the supernatant was used to determine cellulase activity, xylanase activity, and extracellular protein content.

[0133] The seed culture medium contains 20 g / L glucose, 5 g / L (NH4)2SO4, 15 g / L KH2PO4, 2 g / L peptone, 0.6 g / L MgSO4·7H2O, 0.6 g / L CaCl2, 0.0014 g / L ZnSO4·7H2O, 0.0016 g / L LmnSO4·H2O, 0.005 g / L FeSO4·7H2O, and 0.002 g / L CoCl2, with water as the solvent and a pH of 5.5.

[0134] The fermentation medium contains 20 g / L microcrystalline cellulose, 20 g / L corn steep liquor, 5 g / L (NH4)2SO4, 15 g / L KH2PO4, 0.6 g / L MgSO4·7H2O, 0.6 g / L CaCl2, 0.0014 g / L ZnSO4·7H2O, 0.0016 g / L LmnSO4·H2O, 0.005 g / L FeSO4·7H2O, and 0.002 g / L CoCl2, with water as the solvent and a pH of 5.5.

[0135] The methods for determining filter paper enzyme activity, β-glucosidase activity, exocellulase activity, endocellulase activity, xylanase activity, and extracellular protein content are as follows:

[0136] Filter paper enzyme activity assay: Add 0.5 mL of diluted enzyme solution, 1.0 mL of citrate buffer (50 mM, pH 4.8) and 50 mg of Whatman No.1 filter paper to a colorimetric tube, and react in a 50 ºC water bath for 60 min. Immediately after the reaction, add 3 mL of DNS reagent and boil in a water bath for 5 min to terminate the enzyme reaction. After cooling in cold water, measure the amount of reducing sugar generated, and calculate the filter paper enzyme activity based on the amount of reducing sugar generated.

[0137] β-glucosidase activity assay: Add 1.0 mL of diluted enzyme solution to 1.0 mL of 15 mM cellobiose solution (prepared with pH 4.8 and 50 mM citrate buffer), and react in a 50 ºC water bath for 30 min. After the reaction is completed, stop the reaction by boiling in a water bath for 5 min. Measure the glucose produced by the reaction using an SBA biosensor, and calculate the β-glucosidase activity based on the amount of glucose produced.

[0138] Method for determining exocellulase activity: Add 50 μL of 1 mg / mL pNPC solution to a 1.5 mL centrifuge tube, then add 100 μL of diluted enzyme solution. Incubate at 50 ºC for 30 min, and finally terminate the reaction by adding 150 μL of 10% Na2CO3 solution. Measure the absorbance at 420 nm using a microplate reader, and calculate the exocellulase activity based on the amount of p-nitrophenol generated.

[0139] Endocellulase activity assay: Add 0.5 mL of diluted enzyme solution and 1.0 mL of 1% sodium carboxymethyl cellulose (prepared with pH 4.8 50 mM citrate buffer) to a colorimetric tube, and react in a 50 ºC water bath for 30 min. Immediately after the reaction, add 3 mL of DNS reagent and boil in a water bath for 5 min to terminate the enzyme reaction. After cooling in cold water, measure the amount of reducing sugar generated, and calculate the endocellulase activity based on the amount of reducing sugar generated.

[0140] Xylanase activity assay: Add 0.5 mL of diluted enzyme solution to 1.0 mL of 1% xylan solution (prepared with pH 4.8 50 mM citrate buffer), react in a 50 ºC water bath for 30 min, immediately add 3 mL of DNS reagent and boil in a water bath for 5 min to terminate the enzyme reaction, cool in cold water, and measure the amount of reducing sugar generated. Calculate xylanase activity based on the amount of reducing sugar generated.

[0141] Extracellular protein content determination: BCA reagent and Cu reagent were mixed at a volume ratio of 50:1 to prepare BCA working solution. 10 μL of BSA standard was diluted to 100 μL with PBS. The standard was added to 96-well plates at concentrations of 0, 2, 4, 6, 8, 12, 16, and 20 μL, respectively, and the volume was brought to 20 μL with PBS. After diluting the sample, 20 μL was added to each well of the 96-well plate. 200 μL of BCA working solution was added to each well, and the plate was incubated at 37 ºC for 30 min. The A562 nm concentration was measured using a microplate reader, and the protein concentration was calculated based on the standard curve.

[0142] The results are as shown in the attached instruction manual. Figure 2-7 As shown, compared with the original strain *Trichoderma reesei* Rut C30, the engineered strains *Trichoderma reesei* XZD4010 and XZD4047 all showed varying degrees of increase in filter paper enzyme activity, β-glucosidase activity, exocellulase activity, endocellulase activity, xylanase activity, and extracellular protein content.

[0143] After 9 days of fermentation, the filter paper enzyme activity, β-glucosidase activity, exocellulase activity, endocellulase activity, xylanase activity, and extracellular protein content of the engineered *Trichoderma reesei* strain XZD4010 increased by 60.67%, 4813.68%, 548.53%, 27.06%, 18.84%, and 26.72%, respectively, compared to the starting strain *Trichoderma reesei* Rut C30. Furthermore, the filter paper enzyme activity, β-glucosidase activity, exocellulase activity, endocellulase activity, xylanase activity, and extracellular protein content of the engineered *Trichoderma reesei* strain XZD4047 increased by 100.15%, 3128.07%, 1973.80%, 18.55%, 11.79%, and 43.05%, respectively, compared to the starting strain *Trichoderma reesei* Rut C30.

[0144] This invention effectively optimizes the enzyme system composition of Trichoderma reesei by overexpressing the β-glucosidase gene Aabgl1 and the expansion hormone gene Trswo1, and knocking out the transcriptional repressor gene Trace1, thus significantly increasing cellulase production and effectively overcoming the inherent technical bottleneck of insufficient enzyme production capacity and unbalanced enzyme system composition in the starting strain Trichoderma reesei Rut C30.

[0145] Example 4: Analysis of relative gene expression levels in engineered Trichoderma reesei strains XZD4010 and XZD4047

[0146] Fresh spores of the starting strain *Trichoderma reesei* Rut C30, and engineered strains *Trichoderma reesei* XZD4010 and XZD4047 were inoculated into 50 mL of seed culture medium and cultured at 28 ºC and 200 rpm for 24 h. The inoculum was then transferred to 100 mL of fermentation medium at a 10% inoculation rate and cultured at 28 ºC and 200 rpm. Mycelia were collected at 36 h and 48 h of culture. 1 mL of Trizol reagent was added to the mycelia, and after cell disruption, total RNA was extracted and reverse transcribed into cDNA. RT-qPCR was then performed using the cDNA as a template.

[0147] Among them, the following genes were measured: exonuclease genes cbh1 and cbh2, endonuclease genes eg1 and eg2, xylanase genes xyn1 and xyn2, β-glucosidase genes Aabgl1 and bgl1, transcription factor genes xyr1, ace1, ace2, and ace3, and lignocellulose degradation accessory protein genes swo1, cel61a, cip1, and cip2.

[0148] The RT-qPCR primer sequences are as follows:

[0149] Forward primer P13:

[0150] 5′-TCCATCATGAAGTGCGAC-3′ (SEQ ID NO. 23)

[0151] Reverse primer P14:

[0152] 5′-GTAGAAGGAGCAAGAGCAGTG-3′ (SEQ ID NO. 24)

[0153] Forward primer P15:

[0154] 5′-CTTGGCAACGAGTTCTCTTT-3′ (SEQ ID NO.25)

[0155] Reverse primer P16:

[0156] 5′-TGTTGGTGGGATACTTGCTC-3′ (SEQ ID NO.26)

[0157] Forward primer P17:

[0158] 5′-TATATCGACACCATTCGTCAAATT-3′ (SEQ ID NO. 27)

[0159] Reverse primer P18:

[0160] 5′-AGTTGATGCACTCAAGGTAG-3′ (SEQ ID NO. 28)

[0161] Forward primer P19:

[0162] 5′-AACGAGATGGATATCCTGGA-3′ (SEQ ID NO. 29)

[0163] Reverse primer P20:

[0164] 5′-CGTAGTAGCTTTTGTAGCCG-3′ (SEQ ID NO.30)

[0165] Forward primer P21:

[0166] 5′-CTCAACAAGCTCATCAACTCCA-3′ (SEQ ID NO.31)

[0167] Reverse primer P22:

[0168] 5′-TTGAGCCGGTGAAGTTCTTC-3′ (SEQ ID NO.32)

[0169] Forward primer P23:

[0170] 5′-AAACTACCAAAACTGGCGG-3′ (SEQ ID NO.33)

[0171] Reverse primer P24:

[0172] 5′-TTGATGGGAGCAGAAGATCC-3′ (SEQ ID NO.34)

[0173] Forward primer P25:

[0174] 5′-CGGCTACTTCTACTCGTACTG-3′ (SEQ ID NO.35)

[0175] Reverse primer P26:

[0176] 5′-TTGATGACCTTGTTCTTGGTG-3′ (SEQ ID NO.36)

[0177] Forward primer P27:

[0178] 5′-ATGGACTGTCTTACACCAAG-3′ (SEQ ID NO.37)

[0179] Reverse primer P28:

[0180] 5′-ATTCTGGAACAGATCACTCG-3′ (SEQ ID NO.38)

[0181] Forward primer P29:

[0182] 5′-CCAAGCATTACATTCTCAAT-3′ (SEQ ID NO.39)

[0183] Reverse primer P30:

[0184] 5′-CATTTCATGAATGGTCTTGT-3′ (SEQ ID NO.40)

[0185] Forward primer P31:

[0186] 5′-TAATGATGACACATCCGAGT-3′ (SEQ ID NO.41)

[0187] Reverse primer P32:

[0188] 5′-GTTGATCTACTGTAGTGCCA-3′ (SEQ ID NO.42)

[0189] Forward primer P33:

[0190] 5′-ATGACATTGTCATCAACGGG-3′ (SEQ ID NO.43)

[0191] Reverse primer P34:

[0192] 5′-GTGAAACGAAGCCGTTGTCA-3′ (SEQ ID NO.44)

[0193] Forward primer P35:

[0194] 5′-GCTTGGATTCGGCTTCAGAC-3′ (SEQ ID NO.45)

[0195] Reverse primer P36:

[0196] 5′-GTTGTAGTCGAGAACTTGGCTT-3′ (SEQ ID NO.46)

[0197] Forward primer P37:

[0198] 5′-CGAGTCATTGATGCTCTTGAG-3′ (SEQ ID NO.47)

[0199] Reverse primer P38:

[0200] 5′-AACGATTCGTTTCTCGAAAG-3′ (SEQ ID NO.48)

[0201] Forward primer P39:

[0202] 5′-GGAAGAATTCAACGGGTACT-3′ (SEQ ID NO.49)

[0203] Reverse primer P40:

[0204] 5′-CAAGGCCGAATTCTATACAAT-3′ (SEQ ID NO.50)

[0205] Forward primer P41:

[0206] 5′-ACATTGACCCATCTGACGAG-3′ (SEQ ID NO.51)

[0207] Reverse primer P42:

[0208] 5′-CAATCTTTCCGGTGGCCATT-3′ (SEQ ID NO.52)

[0209] Forward primer P43:

[0210] 5′-ATGTCGCCTGCGTCTTTAGT-3′ (SEQ ID NO.53)

[0211] Reverse primer P44:

[0212] 5′-GCTCATGAGATCGAGCCAGT-3′ (SEQ ID NO.54)

[0213] Forward primer P45:

[0214] 5′-GAGATTGTGCGAGACATGCT-3′ (SEQ ID NO.55)

[0215] Reverse primer P46:

[0216] 5′-TGAATCCTGGTTGCGATGGA-3′ (SEQ ID NO.56)

[0217] The results are as shown in the attached instruction manual. Figure 8 As shown, compared with the original strain *Trichoderma reesei* Rut C30, at 36 h, the relative expression levels of β-glucosidase gene Aabgl1 and expansin gene Trswo1 in engineered strain XZD4010 were upregulated, reaching 11.55 times and 8.54 times that of the original strain *Trichoderma reesei* Rut C30, respectively. At 48 h, the relative expression levels of β-glucosidase gene Aabgl1 and expansin gene Trswo1 in engineered strain XZD4010 were further upregulated, reaching 353.30 times and 35.73 times that of the original strain *Trichoderma reesei* Rut C30, respectively.

[0218] Compared with engineered strain XZD4010, the relative expression levels of cellulase, xylanase, lignocellulose degradation accessory proteins, and transcription factor genes in engineered strain XZD4047 showed significant changes. At 36 h, the relative expression levels of exonuclease genes cbh1 and cbh2, endonuclease genes eg1 and eg2, xylanase genes xyn1 and xyn2, β-glucosidase gene bgl1, transcription factor genes xyr1 and ace3, and lignocellulose degradation accessory protein genes cel61a, cip1, and cip2 were much higher than those in engineered strain XZD4010.

[0219] The above results indicate that by overexpressing the β-glucosidase gene Aabgl1 and the expansion factor gene Trswo1 in *Trichoderma reesei* and knocking out the transcriptional repressor gene Trace1, this invention significantly upregulated the expression levels of cellulase, xylanase, lignocellulose degradation accessory proteins, and transcription factor genes in *Trichoderma reesei* at the transcriptional level, achieving precise regulation of genes related to the synthesis of lignocellulose degradation enzymes, thereby effectively enhancing the enzyme production capacity of *Trichoderma reesei*.

[0220] Example 5. Determination of the hydrolytic performance of enzyme solutions from engineered Trichoderma reesei strains XZD4010 and XZD4047 on alkali-pretreated corn straw.

[0221] Preparation of corn stalks pretreated with alkali: Mix 2% NaOH solution with corn stalks at a solid-liquid ratio of 10:1, place in a reaction vessel, and treat at 121 °C for 60 min. After pretreatment, perform solid-liquid separation, wash the solid with water until neutral, and dry for later use.

[0222] Alkali-pretreated corn stalk hydrolysis: The pretreated corn stalks were resuspended in 50 mM citrate buffer at pH 4.8 at a solid-liquid ratio of 10%. Enzyme solutions of Trichoderma reesei C30, XZD4010 and XZD4047 prepared in Example 3 with 10 FPU / g substrate were added. The mixture was placed in a water bath shaker and reacted at 50 ºC and 200 rpm for 72 h. After the reaction, the glucose content in the hydrolysate was analyzed by HPLC.

[0223] The results are as shown in the attached instruction manual. Figure 9 As shown, the high-performance cellulase produced by engineered strain XZD4047 achieved a dextran conversion rate of 94.89% when hydrolyzing corn stalks pretreated with alkali. This was 82.91% and 2.62% higher than that of the starting strain Trichoderma reesei Rut C30 and engineered strain XZD4010, respectively. The hydrolytic performance of the high-performance cellulase produced by engineered strain XZD4047 was comparable to that of the commercial enzyme preparation Cellic CTec2.

[0224] The enzyme solution produced by the engineered Trichoderma reesei strain XZD4047 constructed in this invention has excellent hydrolysis performance and can efficiently degrade lignocellulosic biomass such as corn stalks, showing good potential for industrial application.

[0225] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A high-yield, high-performance cellulase-producing Trichoderma reesei engineered strain, characterized in that, The engineered Trichoderma reesei strain was named XZD4047; The engineered Trichoderma reesei strains co-overexpressed the β-glucosidase gene Aabgl1 and the expansion hormone gene Trswo1, and knocked out the transcriptional repressor gene Trace1.

2. The *Trichoderma reesei* engineered strain with high-yield and high-performance cellulase according to claim 1, characterized in that, The β-glucosidase gene Aabgl1 is derived from Aspergillus aculeatus, and the nucleotide sequence of the β-glucosidase gene Aabgl1 is shown in SEQ ID NO.1; The swelling hormone gene Trswo1 is derived from Trichoderma reesei Rut C30, and the nucleotide sequence of the swelling hormone gene Trswo1 is shown in SEQ ID NO.2; The transcription repressor gene Trace1 is derived from Trichoderma reesei Rut C30, and the nucleotide sequence of the transcription repressor gene Trace1 is shown in SEQ ID NO.3; The amino acid sequence of the β-glucosidase is shown in SEQ ID NO.4; The amino acid sequence of the swelling agent is shown in SEQ ID NO.5; The amino acid sequence of the transcriptional repressor ACE1 is shown in SEQ ID NO.

6.

3. A method for constructing a high-yield, high-performance cellulase-producing Trichoderma reesei engineered strain as described in claim 1 or 2, characterized in that, Includes the following steps: S1. Using Trichoderma reesei PB3 genomic DNA as a template, the Aabgl1 gene fragment was amplified by PCR; using Trichoderma reesei Rut C30 genomic DNA as a template, the Trswo1 gene fragment was amplified by PCR. The Aabgl1 gene fragment and the Trswo1 gene fragment were inserted into the NcoI and AflII sites of the vector pCZF9, respectively, by seamless cloning to obtain the vector pCZFBS that overexpresses Aabgl1 and Trswo1. S2. The vector pCZFBS overexpressing Aabgl1 and Trswo1 obtained in step S1 was transformed into Escherichia coli DH5α, and the vector pCZFBS was extracted. The extracted vector pCZFBS was then transformed into Agrobacterium rhizogenes competent cells AGL-1 to obtain Agrobacterium rhizogenes AGL-1 containing the vector pCZFBS. S3. Through the mediation of Agrobacterium rhizogenes, Agrobacterium rhizogenes AGL-1 containing the vector pCZFBS was mixed with Trichoderma reesei conidia Rut C30, cultured, screened, and verified to obtain the Trichoderma reesei engineered strain XZD4010. S4. Using Trichoderma reesei Rut C30 genomic DNA as a template, the upstream homologous arm fragment and the downstream homologous arm fragment of the Trace1 gene were obtained by PCR amplification. Using pUC-Ble vector as a template, a bleomycin selection expression cassette was obtained by PCR amplification; Using pCZF8 as a template, the pCZF8 vector backbone was amplified by PCR. The upstream homologous arm fragment of the Trace1 gene, the bleomycin selection expression cassette, the downstream homologous arm fragment of the Trace1 gene and the pCZF8 vector backbone were mixed and ligated using a seamless cloning method to obtain the knockout vector pCZF25. S5. The knockout vector pCZF25 obtained in step S4 is transformed into Escherichia coli DH5α, and the vector pCZF25 is extracted. The extracted vector pCZF25 is transformed into Agrobacterium rhizogenes competent cells AGL-1 to obtain Agrobacterium rhizogenes AGL-1 containing the vector pCZF25. S6. Through Agrobacterium rhizogenes-mediated mixing, Agrobacterium rhizogenes AGL-1 containing the vector pCZF25 was mixed evenly with conidia of engineered strain XZD4010, cultured, screened, and verified to obtain the engineered strain of Trichoderma reesei, XZD4047.

4. The application of the *Trichoderma reesei* engineered strain with high yield and performance of cellulase as described in claim 1 or 2 in the fermentation production of high-performance cellulase, characterized in that, The fermentation production of high-performance cellulase includes the following steps: S101. The engineered strain of Trichoderma reesei, XZD4047, was inoculated into a sporulation medium and cultured at 25-30 ºC for 4-10 days. The spores were washed off with sterile water, filtered through gauze, and a spore suspension of engineered strain XZD4047 was obtained. S201. Inoculate the spore suspension of the engineered strain XZD4047 obtained in step S101 into a seed culture medium and culture it at 25-30 ºC and 100-300 rpm for 18-36 h to obtain the seed liquid of the engineered strain XZD4047; inoculate the seed liquid into a fermentation medium at an inoculation ratio of 5-10% and culture it at 25-30 ºC and 100-300 rpm for 120-216 h. S301. Centrifuge the fermentation broth obtained in step S201 at 5000 rpm-12000 rpm for 5 min-30 min, collect the supernatant, and obtain high-performance cellulase.

5. The application of the high-yield, high-performance cellulase engineered strain of *Trichoderma reesei* according to claim 4 in the fermentation production of high-performance cellulase, characterized in that, In step S101, the sporulation medium contains 25-35 g / L malt extract powder, 15-20 g / L agar, water as the solvent, and natural pH.

6. The application of the *Trichoderma reesei* engineered strain with high yield and performance of cellulase according to claim 4 in the fermentation production of high-performance cellulase, characterized in that, The seed culture medium contains 10-30 g / L glucose, 4-6 g / L (NH4)2SO4, 10-20 g / L KH2PO4, 1-3 g / L peptone, 0.4-0.8 g / L MgSO4·7H2O, 0.4-0.6 g / L CaCl2, 0.0010-0.0018 g / L ZnSO4·7H2O, 0.0012-0.0020 g / L MnSO4·H2O, 0.003-0.006 g / L FeSO4·7H2O, and 0.001-0.003 g / L CoCl2, with water as the solvent and a pH of 5.

5.

7. The application of the *Trichoderma reesei* engineered strain with high yield and performance of cellulase according to claim 4 in the fermentation production of high-performance cellulase, characterized in that, The fermentation medium contains 10-30 g / L microcrystalline cellulose, 10-30 g / L corn steep liquor, 4-6 g / L (NH4)2SO4, 10-20 g / L KH2PO4, 0.4-0.8 g / L MgSO4·7H2O, 0.4-0.6 g / L CaCl2, 0.0010-0.0018 g / L ZnSO4·7H2O, 0.0012-0.0020 g / L MnSO4·H2O, 0.003-0.006 g / L FeSO4·7H2O, and 0.001-0.003 g / L CoCl2, with water as the solvent and a pH of 5.

5.

8. The application of the high-performance cellulase according to any one of claims 4-7 in improving the degradation efficiency of lignocellulosic biomass.

9. The application of the high-performance cellulase according to claim 8 in improving the degradation efficiency of lignocellulosic biomass, characterized in that, The degradation of lignocellulosic biomass by the high-performance cellulase includes the following steps: The lignocellulosic biomass is pretreated to obtain pretreated raw material, and the high-performance cellulase is added to adjust the pH to 4.5-5.0, and hydrolyzed at 45-55 ºC for 48-96 h.

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