Method for preparing cellulase and application thereof

Abstract

The invention provides a method for preparing cellulase and application of the cellulase. The method for preparing the cellulase comprises the following steps: S1) improving the expression level of an SNC1 gene in trichoderma reesei to obtain trichoderma reesei engineering bacteria; s2, the trichoderma reesei engineering bacteria are cultured and collected to obtain the cellulase, the problem that in the prior art, a large amount of cellulase is difficult to produce through trichoderma reesei under the culture condition containing glucose can be solved, and the method is suitable for the technical field of microorganisms.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically, to a method for preparing cellulase and its application. Background Technology

[0002] Cellulose is a homopolysaccharide composed of glucose molecules linked by β-1,4 glycosidic bonds. It is a major component of plant cell walls and one of the most abundant renewable resources on Earth. Cellulase is a complex enzyme system capable of degrading cellulose into glucose. Based on their sites of action on cellulose, they can be classified into endoglucanases, exoglucanases, and β-glucosidases. When cellulose is used as a substrate, endoglucanases can recognize and cleave the β-1,4 glucosidic bonds in the amorphous regions of cellulose, releasing short-chain cellulosic and cellopolysaccharides of varying lengths, thus providing substrates for exoglucanases and β-glucosidases. Therefore, endoglucanases play a crucial role in the hydrolysis of cellulose, and their activity directly affects the efficiency of cellulose hydrolysis by cellulase.

[0003] Currently, *Trichoderma reesei* is an important enzyme production strain in industries such as biofuels, food and feed, and paper and textile manufacturing due to its ability to produce cellulase. It possesses rich genomic data and potential for synthetic biology modification, and also demonstrates significant application value in biocontrol and plant symbiosis. However, in actual production, utilizing *Trichoderma reesei* for cellulase production still faces several challenges, such as the need to further improve enzyme yield and the inhibition of cellulase synthesis by carbon metabolism repression. Carbon metabolism repression refers to the phenomenon where microorganisms preferentially utilize easily degradable carbon sources such as glucose in a mixed carbon source containing glucose and other sugars, while inhibiting the expression of genes related to the metabolism of other carbon sources. This repression effect limits the efficient expression of cellulase in industrial production, especially when using inexpensive and readily available carbon sources. Therefore, how to improve the enzyme activity of *Trichoderma reesei* cellulase and alleviate the inhibition of cellulase synthesis by carbon metabolism repression are urgent problems to be solved in the field of cellulase production. Summary of the Invention

[0004] The main objective of this invention is to provide a method for preparing cellulase and its application. By increasing the expression of the SNC1 gene in Trichoderma reesei, an engineered Trichoderma reesei strain for cellulase production is constructed to solve the problem in the prior art that Trichoderma reesei is difficult to produce cellulase in large quantities under culture conditions containing glucose.

[0005] To achieve the above objective, according to a first aspect of the present invention, a method for preparing cellulase is provided, the method comprising: S1) increasing the expression level of the SNC1 gene in Trichoderma reesei to obtain an engineered Trichoderma reesei strain; S2) culturing the engineered Trichoderma reesei strain and collecting the cellulase.

[0006] Furthermore, the above-mentioned SNC1 gene sequence includes: the SNC1 gene sequence derived from Trichoderma reesei QM6a, or the SNC1 gene of Trichoderma reesei itself.

[0007] Furthermore, the protein encoded by the SNC1 gene is the amino acid sequence shown in SEQ ID NO: 1, or a protein that has more than 70% homology with the above amino acid sequence.

[0008] Furthermore, the SNC1 gene described above is the nucleic acid sequence shown in SEQ ID NO: 2, or a sequence that has more than 70% homology with the above nucleic acid sequence.

[0009] Furthermore, the above-mentioned culture includes: culturing the above-mentioned Trichoderma reesei engineered strain in a fermentation medium, wherein the carbon source in the fermentation medium includes glucose, and the concentration of the glucose is 10-30 g / L.

[0010] Furthermore, the aforementioned fermentation medium comprises 1-3% glucose, 0.8-3% corn steep liquor, 0.1-1% ammonium sulfate, 0.05-0.6% dipotassium hydrogen phosphate, 0.02-0.18% magnesium sulfate, and 0.02-0.1% calcium chloride. All components in the culture medium of this application are expressed as mass-volume ratios; for example, a glucose content of 1-3% is equivalent to 1-3% (w / v).

[0011] Furthermore, the glucose in the above-mentioned fermentation medium is added to the above-mentioned fermentation medium in the form of fermentation syrup; the preparation method of the above-mentioned fermentation syrup includes: mixing glucose and 0.05-1.5% β-glucosidase to obtain a reaction system, reacting the above-mentioned reaction system under stirring conditions to obtain the above-mentioned fermentation syrup.

[0012] Furthermore, the temperature of the above reaction system is 50-60℃, the pH of the above reaction system is 4.0-5.0, the reaction time under the above stirring conditions is 70-80h, and the stirring speed is 80-150rpm.

[0013] Furthermore, the concentration of glucose in the fermented syrup is 400-600 g / L, and the concentration of β-glucosidase is 10000-15000 IU / g.

[0014] Further, S1 above includes: introducing an exogenous SNC1 gene into the above-mentioned Trichoderma reesei, and / or increasing the expression level of the endogenous SNC1 gene in the above-mentioned Trichoderma reesei.

[0015] Furthermore, the method for transferring the exogenous SNC1 gene into Trichoderma reesei includes: transferring an SNC1 expression cassette into Trichoderma reesei, wherein the SNC1 expression cassette includes a promoter and the SNC1 gene connected sequentially from 5' to 3', and the promoter includes Pcdna1.

[0016] Furthermore, the method for transferring the exogenous SNC1 gene into the above-mentioned Trichoderma reesei includes: transferring a plasmid into the above-mentioned Trichoderma reesei, the plasmid containing the above-mentioned SNC1 expression cassette, the plasmid including pPTRII.

[0017] To achieve the above objective, according to a second aspect of the present invention, a modification method for relieving the carbon metabolism repression effect of Trichoderma reesei is provided, the modification method comprising: increasing the expression level of the SNC1 gene in Trichoderma reesei.

[0018] Furthermore, the steps of the above modification method include any one or more of the following: transforming the SNC1 gene into the above-mentioned Trichoderma reesei, or increasing the copy number of the SNC1 gene in the above-mentioned Trichoderma reesei, or replacing the promoter of the SNC1 gene in the above-mentioned Trichoderma reesei with a strong promoter.

[0019] Furthermore, the SNC1 gene mentioned above includes the SNC1 gene used in the above-described method for preparing cellulase.

[0020] To achieve the above objectives, according to a third aspect of the present invention, a method for preparing cellulase as described above, or a modification method for relieving the carbon metabolism repression effect of Trichoderma reesei as described above, or the application of the SNC1 gene as described above in Trichoderma reesei culture or cellulase production is provided.

[0021] By applying the technical solution of this invention and utilizing the above-mentioned method for preparing cellulase, and by constructing an engineered Trichoderma reesei strain, it is possible to efficiently utilize glucose and achieve rapid strain proliferation while relieving the carbon metabolism repression effect of glucose, and still obtain a large quantity of cellulase in the presence of glucose in the culture medium. Attached Figure Description

[0022] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0023] Figure 1 A schematic diagram of the pPTR-SNC1 carrier structure according to Embodiment 1 of the present invention is shown.

[0024] Figure 2 The figure shows the results of enzyme activity determination in the supernatant of shake-flask fermentation broth according to Example 2 of the present invention.

[0025] Figure 3 The graph shows the results of protein content determination in shake-flask fermentation broth according to Example 2 of the present invention.

[0026] Figure 4 The figure shows the results of endoglucanase activity assay in the fermentation broth when glucose was used as the sole carbon source according to Example 3 of the present invention.

[0027] Figure 5 The graph shows the residual sugar content in the fermentation broth when glucose is the sole carbon source according to Example 3 of the present invention.

[0028] Figure 6 The SDS-PAGE results of cellulase content in the fermentation broth supernatant are shown in Example 3 of the present invention, where glucose is the sole carbon source.

[0029] Figure 7 The diagram shows the transcriptome results of endoplasmic reticulum stress-related genes according to Example 4 of the present invention.

[0030] Figure 8 A graph showing the transcriptome results of the cellulase gene according to Example 4 of the present invention is presented.

[0031] Figure 9 The diagram shows the transcriptome results of cellulase-regulated genes according to Example 4 of the present invention. Detailed Implementation

[0032] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0033] As mentioned in the background section, it is difficult to prepare cellulase from *Trichoderma reesei* under glucose conditions in the prior art. Therefore, in this application, the inventors attempted to develop a method for preparing cellulase. By constructing engineered *Trichoderma reesei* strains, it is possible to efficiently utilize glucose and achieve rapid strain proliferation, while eliminating the carbon metabolism repression effect that occurs when glucose is present in the culture medium, thus improving the flexibility and economy of cellulase production. Therefore, a series of protection schemes are proposed in this application.

[0034] In a first typical embodiment of this application, a method for preparing cellulase is provided, the method comprising: S1) increasing the expression level of the SNC1 gene in Trichoderma reesei to obtain an engineered Trichoderma reesei strain; S2) culturing the engineered Trichoderma reesei strain and collecting the cellulase.

[0035] Carbon metabolism repression refers to the phenomenon where microorganisms preferentially utilize readily biodegradable carbon sources such as glucose in a mixed carbon source containing glucose and other sugars, while simultaneously inhibiting the expression of genes related to the metabolism of other carbon sources. This repression effect greatly limits the efficient expression of cellulase in industrial production.

[0036] In this application, the inventors obtained an engineered Trichoderma reesei strain with significantly enhanced cellulase yield and activity by increasing the expression level of the SNC1 gene in Trichoderma reesei. The increased expression level of the SNC1 gene effectively improves the cellulase activity and exoprotein content of Trichoderma reesei. Especially when glucose is used as the sole carbon source, it effectively overcomes the inhibition of cellulase synthesis by carbon metabolism repression, significantly increasing the cellulase production rate in the early stages of fermentation, shortening the production cycle, and enhancing the application value of Trichoderma reesei in industrial production.

[0037] In a preferred embodiment, the sequence of the SNC1 gene includes: the sequence of the SNC1 gene derived from Trichoderma reesei QM6a, or the SNC1 gene of Trichoderma reesei itself.

[0038] In this application, the inventors discovered that when the SNC1 gene originates from, but is not limited to, *Trichoderma reesei* QM6a, or the *Trichoderma reesei*'s own SNC1 gene, high gene adaptability and expression efficiency are ensured. The SNC1 gene within its genome has undergone long-term evolution and adaptation, achieving high compatibility with the *Trichoderma reesei* secretion system. By integrating this gene source in a preferred scheme, a significant increase in cellulase activity is promoted, optimizing cellulose degradation efficiency. Based on the genetic background of *Trichoderma reesei* E619, a significant increase in cellulase yield is achieved, especially under the condition of using glucose as the sole carbon source. This effectively overcomes the inhibitory effect of carbon metabolism repression on cellulase synthesis, improves enzyme yield in the early stages of fermentation, shortens the enzyme production cycle, and enhances the application potential of *Trichoderma reesei* in the field of biomass conversion. Precise insertion of the SNC1 gene nucleotide sequence makes cellulase production more efficient, especially under the condition of using glucose as the sole carbon source, significantly alleviating the inhibition of carbon metabolism repression on cellulase synthesis, thereby improving cellulase production efficiency under a wider range of carbon source conditions. Simultaneously, production costs are reduced, improving economic benefits. This improvement is of great significance for broadening the industrial application range of cellulase.

[0039] In a preferred embodiment, the protein encoded by the SNC1 gene is the amino acid sequence shown in SEQ ID NO: 1, or a protein having more than 70% homology with the above amino acid sequence.

[0040] The above-mentioned amino acid sequences were experimentally investigated in the embodiments of this application. Compared with the parent protein having the amino acid sequence shown in SEQ ID NO: 1, all of them have the function of improving the preparation of cellulase by *Trichoderma reesei* under the condition of glucose presence in the culture medium. All of the above-mentioned mutation sites are mutations performed around the active amino acid sites. Such mutations can improve the binding ability of the mutant to the substrate and / or its catalytic activity. Mutations far from the active site have less impact on the enzyme's catalytic activity; therefore, proteins with 80% or more homology to the above-mentioned amino acid sequences and the same catalytic activity can be obtained.

[0041] In this specification, homology refers to the "homology" between amino acid sequences, that is, the total ratio of the same type of amino acid residues in the amino acid sequence. The homology of amino acid sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.

[0042] SEQ ID NO: 1:

[0043] MADAPYDPYVPKAGADQSGGQSRTQALQGEIDATVQVMRKNIENVAQRGDRLDVLQDKTDNLAESAQGFRRGANRVRKQMWWKDMKMRVCIVVGIILLLVVIIVPSGELPLASEDLPSKEQKKLTRQTTVVATR.

[0044] In a preferred embodiment, the SNC1 gene is the nucleic acid sequence shown in SEQ ID NO: 2, or a sequence that has more than 70% homology with the above nucleic acid sequence.

[0045] The above-mentioned nucleic acid sequences were all experimentally investigated in the embodiments of this application. Compared with the parent material with the nucleic acid sequence shown in SEQ ID NO: 2, the sequences with more than 70% homology with the above-mentioned nucleic acid sequences all have the function of improving the preparation of cellulase by Trichoderma reesei under the condition of glucose presence in the culture medium. They effectively overcome the inhibitory effect of carbon metabolism repression on cellulase synthesis, improve the enzyme yield in the early stage of fermentation, shorten the enzyme production cycle, and enhance the application potential of Trichoderma reesei in the field of biomass conversion.

[0046] SEQ ID NO: 2:

[0047] atggccgacgctccgtacgatccctacgttcccaaggccggcgccgaccagtccggcggccagtcgcgcacgcaggcgcttcaaggtgaaatcgacgcaac ggtccaagtgatgcgaaagaacattgaaaacgtggctcagcgtggtgaccgcctggacgtcctgcaagacaagaccgataacctggcggaatccgcacaggg cttccgccggggcgcaaaccgagtgcgaaagcagatgtggtggaaggacatgaagatgcgcgtctgcatcgttgttggaatcatcctcctcctggttgtca ttatcgttccatcaggtgagctaccacttgcgagcgaggatttgcccagcaaagaacagaaaaaactgacaaggcaaaccacagtcgttgccacccgttaa.

[0048] In a preferred embodiment, the above-mentioned culture includes: culturing the above-mentioned Trichoderma reesei engineered strain in a fermentation medium, wherein the carbon source in the fermentation medium includes glucose, and the concentration of the glucose is 10-30 g / L.

[0049] In this application, the inventors further discovered that when the glucose concentration in the fermentation medium is, for example, 10-30 g / L (including but not limited to 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L, 26 g / L, 27 g / L, 28 g / L, 29 g / L, or 30 g / L), the engineered *Trichoderma reesei* strain can still maintain highly efficient cellulase expression and secretion under high glucose concentration conditions. Increasing glucose concentration usually leads to carbon metabolism repression, inhibiting cellulase synthesis. However, overexpression of the SNC1 gene, in a preferred manner, overcomes this challenge, enabling the engineered strain to maintain high levels of cellulase expression even under excessively high glucose concentrations. This strategy significantly increases enzyme yield by enhancing the transcriptional efficiency of the cellulase gene and thus increasing the activity of the protein secretion pathway. Overexpression of the SNC1 gene effectively alleviates the inhibitory effect of carbon metabolism repression on cellulase synthesis, enhancing cellulase synthesis capacity, improving production efficiency, and increasing the economic value of the enzyme. The optimized scheme improves the overall cellulase production process in the basic scheme, increasing enzyme yield and activity, and providing a more efficient and stable cellulase production solution for industrial applications.

[0050] In a preferred embodiment, the fermentation medium comprises 1-3% glucose, 0.8-3% corn steep liquor, 0.1-1% ammonium sulfate, 0.05-0.6% dipotassium hydrogen phosphate, 0.02-0.18% magnesium sulfate, and 0.02-0.1% calcium chloride.

[0051] In this application, the inventors discovered that the components of the fermentation medium include, but are not limited to, glucose, corn steep liquor, ammonium sulfate, dipotassium hydrogen phosphate, magnesium sulfate, and calcium chloride. The glucose content is, for example, 1-3% (including but not limited to 1%, 1.5%, 2%, 2.5%, or 3%), and the corn steep liquor content is, for example, 0.8-3% (including but not limited to 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 2.2%, 2.4%, 2.6%, 2.8%, or 3%). The content of ammonium sulfate is, for example, 0.1-1% (including but not limited to 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1%), the content of dipotassium hydrogen phosphate is, for example, 0.05-0.6% (including but not limited to 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55% or 0.6%), and the content of magnesium sulfate is, for example, 0.1-1% (including but not limited to 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55% or 0.6%). For example, a calcium chloride content of 0.02-0.18% (including but not limited to 0.02%, 0.04%, 0.06%, 0.08%, 0.1%, 0.12%, 0.14%, 0.16%, or 0.18%) can provide the optimal nutrient environment for the growth of *Trichoderma reesei* engineered bacteria and the synthesis of cellulase. All components in the culture medium of this application are expressed as mass-volume ratios; for example, a glucose content of 1-3% is equivalent to 1-3% (w / v). Glucose, as the main carbon source, supports the rapid growth of microorganisms and the large-scale synthesis of enzymes; corn steep liquor is rich in various vitamins and trace elements, which helps to improve microbial metabolism and enzyme activity; ammonium sulfate, dipotassium hydrogen phosphate, magnesium sulfate, and calcium chloride are essential inorganic salts in the process of microbial growth and enzyme synthesis. They maintain the osmotic pressure and pH stability of the culture medium, promote the nutrient absorption and metabolic balance of microorganisms, and thus improve the yield of cellulase.

[0052] Overall, by precisely controlling the aforementioned nutrient components and combining this with the SNC1 gene overexpression technology in the basic scheme, the inventors achieved a significant increase in the yield and activity of cellulase in engineered Trichoderma reesei during fermentation. The synergistic effect of these two methods greatly shortened the enzyme production cycle, increased the total enzyme yield, and effectively overcame carbon metabolism repression under conditions where glucose was the sole carbon source, ensuring that enzyme synthesis and secretion were not inhibited. This is of great significance for improving the economic benefits and application value of cellulase in industrial production.

[0053] In a preferred embodiment, the glucose in the fermentation medium is added to the fermentation medium in the form of fermentation syrup. The preparation method of the fermentation syrup includes: mixing glucose and 0.05-1.5% β-glucosidase to obtain a reaction system, reacting the reaction system under stirring conditions to obtain the fermentation syrup.

[0054] In this application, the inventors mixed glucose with β-glucosidase (including but not limited to 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, or 1.5%) at a concentration of, for example, 0.05-1.5% to obtain a reaction system. The reaction was carried out under stirring conditions to produce a fermentation syrup. This fermentation syrup effectively promotes the growth of *Trichoderma reesei* engineered strains and the synthesis of cellulase during fermentation. Overexpression of the SNC1 gene increases the secretion and activity of cellulase, while the specific preparation method of the fermentation syrup ensures that the strain can rapidly initiate cellulase production in the early stages of fermentation, unaffected by the inhibitory effect of glucose, thereby improving the overall yield and production efficiency of cellulase. The implementation of this technical solution ensures that the engineered *Trichoderma reesei* strain maintains continuous production of highly active cellulase under glucose-based carbon source conditions, significantly increasing cellulase yield and activity, while shortening the enzyme production cycle and significantly improving the production rate. This provides a more efficient and economical approach for the industrial production of cellulase. The fermentation syrup preparation step in the optimized scheme, combined with SNC1 gene overexpression, works synergistically with the cultivation process of the engineered *Trichoderma reesei* strain in the basic scheme. By optimizing the carbon source supply method, the inhibition of carbon metabolism repression on cellulase synthesis is effectively overcome, enhancing the adaptability of the strain and the enzyme production efficiency. Under glucose as the sole carbon source, both cellulase activity and protein content are significantly improved, achieving a dual improvement in the flexibility and economy of cellulase production.

[0055] In a preferred embodiment, the temperature of the reaction system is 50-60°C, the pH of the reaction system is 4.0-5.0, the reaction time under the stirring conditions is 70-80 hours, and the stirring speed is 80-150 rpm.

[0056] In this application, the inventors discovered that when the reaction temperature of the fermentation syrup preparation system is within a temperature range of, for example, 50-60℃ (including but not limited to 51℃, 52℃, 53℃, 54℃, 55℃, 56℃, 57℃, 58℃, 59℃, or 60℃), the transglycosylation reaction of glucose by β-glucosidase can be thermally promoted while maintaining the stability of enzyme activity. The technical principle is that β-glucosidase exhibits high catalytic activity within this temperature range, effectively converting glucose into oligosaccharides, and subsequently into fermentation syrup. The effects are manifested in that the optimized temperature selection accelerates the glucose production rate, improves the yield and quality of the fermentation syrup, and provides a high-quality carbon source for subsequent microbial fermentation. More importantly, this temperature range avoids irreversible enzyme inactivation caused by excessively high temperatures, ensuring the enzyme's continuous and efficient operation throughout the fermentation process, thereby improving the overall production process efficiency.

[0057] In this application, the inventors discovered that when the pH of the reaction system is within a range of, for example, 4.0-5.0 (including but not limited to, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0), the β-glucosidase can maintain optimal activity during glucose conversion, thereby promoting the efficient preparation of fermented syrup. Within this pH range, the enzyme's stability is enhanced, enabling it to more effectively cleave glucosinolate bonds and generate higher concentrations of glucose, providing a higher-quality carbon source for subsequent microbial fermentation. Furthermore, a lower pH value can inhibit the growth of harmful microorganisms, reduce potential contamination during fermentation, and ensure the quality of the fermented syrup. The implementation of this technical feature is reflected in improved yield and purity of fermented syrup, thereby enhancing the yield and quality of cellulase in the subsequent fermentation process of *Trichoderma reesei*. In summary, by precisely controlling the pH value of the reaction system, not only can the activity of β-glucosidase be guaranteed and the efficient preparation of syrup be promoted, but also contamination by other microorganisms can be effectively prevented, ensuring the quality of fermentation raw materials, thereby indirectly improving the production and economic benefits of cellulase. The introduction of this technical feature provides a more stable and controllable foundation for the large-scale industrial production of cellulase.

[0058] In this application, the inventors further discovered that in the system for preparing fermented syrup, a reaction time under stirring conditions within, for example, 70-80 hours (including but not limited to 71 hours, 72 hours, 73 hours, 74 hours, 75 hours, 76 hours, 77 hours, 78 hours, 79 hours, or 80 hours) ensures sufficient contact between glucose and β-glucosidase, guaranteeing the thoroughness of the enzymatic hydrolysis reaction. This increases the glucose concentration in the fermented syrup, providing a sufficient carbon source for subsequent cellulase production. Within this time range, the substances in the reaction system reach an optimal equilibrium state, ensuring sufficient enzymatic hydrolysis efficiency while avoiding the increased costs caused by excessively long reaction times, which could negatively impact the growth of *Trichoderma reesei*. By precisely controlling the reaction time, the technical solution of this embodiment not only improves the quality of the fermented syrup but also provides strong support for high-yield cellulase production.

[0059] In this application, the inventors discovered that limiting the stirring speed to the range of 80-150 rpm (including but not limited to 80 rpm, 85 rpm, 90 rpm, 95 rpm, 100 rpm, 105 rpm, 110 rpm, 115 rpm, 120 rpm, 125 rpm, 130 rpm, 135 rpm, 140 rpm, 145 rpm, or 150 rpm) can promote effective contact and reaction between glucose and β-glucosidase, thereby improving enzymatic hydrolysis efficiency and glucose conversion rate. Within this speed range, it can ensure that the materials in the reaction system are fully mixed without being too vigorous, avoiding the generation of a large amount of foam in the reaction system caused by over-stirring, while also preventing uneven substrate distribution and limited reaction rate caused by insufficient stirring.

[0060] In a preferred embodiment, the concentration of glucose is 400-600 g / L, and the concentration of β-glucosidase is 10000-15000 IU / g.

[0061] In this application, the inventors discovered that a glucose concentration of, for example, 400-600 g / L (including but not limited to 400 g / L, 450 g / L, 500 g / L, 550 g / L, or 600 g / L) and a β-glucosidase concentration of, for example, 10000-15000 IU / g (including but not limited to 10000 IU / g, 11000 IU / g, 12000 IU / g, 13000 IU / g, 14000 IU / g, or 15000 IU / g) can effectively optimize the preparation conditions of fermented syrup. When the glucose concentration in the system is high, β-glucosidase catalyzes the transglycosylation reaction: using glucose as a glycosyl donor and acceptor, oligosaccharides are formed through β-glycosidic bonds, and the product contains a certain amount of sophorose. Sophora japonica syrup, as an important carbon source, can effectively induce the synthesis and secretion of cellulase in *Trichoderma reesei*. The amount of β-glucosidase added within the range specified in this application ensures sufficient sophora japonica syrup production while avoiding the increased cost associated with excessive enzyme dosage. When the glucose and β-glucosidase concentrations are within the aforementioned preferred ranges, the efficiency and quality of fermentation syrup preparation are improved, providing an ideal carbon source and environment for subsequent efficient fermentation of *Trichoderma reesei*. Under these conditions, the SNC1 gene expression of the engineered *Trichoderma reesei* strain is more stable and efficient, promoting cellulase expression and significantly increasing cellulase activity and yield. Overexpression of the SNC1 gene enhances the transcription and translation efficiency of the cellulase gene and mitigates the repression effect of glucose metabolism on cellulase synthesis. This optimization not only accelerates the enzyme production cycle of the engineered strain but also increases cellulase yield under glucose as the sole carbon source. This optimized scheme further improves cellulase production performance within the framework of the basic scheme, especially achieving efficient cellulase expression under carbon metabolism repression conditions, thus improving the flexibility and economy of cellulase production.

[0062] In a preferred embodiment, S1) above includes: introducing an exogenous SNC1 gene into the above-mentioned Trichoderma reesei, and / or increasing the expression level of the endogenous SNC1 gene in the above-mentioned Trichoderma reesei.

[0063] The "exogenous SNC1 gene" in this application includes genes derived from other fungal species rather than the host cell itself, including but not limited to genes derived from different fungal species or subspecies. In this application, the inventors discovered that by introducing an exogenous SNC1 gene into *Trichoderma reesei* or by other means to increase the expression level of its endogenous SNC1 gene, the yield and activity of cellulase were significantly increased. Overexpression of the SNC1 protein effectively enhances the cellulase activity and extracellular protein secretion of *Trichoderma reesei*, especially under conditions where glucose is the sole carbon source, significantly alleviating the inhibition of cellulase synthesis by carbon metabolism repression, thereby improving cellulase production efficiency under a wider range of carbon source conditions. This application not only increases cellulase yield but also optimizes enzyme yield and production rate, particularly in the environment where glucose is the sole carbon source, enhancing the flexibility and economy of industrial production and providing a new approach for the efficient production of cellulase.

[0064] In a preferred embodiment, the method of transferring an endogenous SNC1 gene into the Trichoderma reesei includes: transferring an SNC1 expression cassette into the Trichoderma reesei, the SNC1 expression cassette including a promoter and the SNC1 gene connected sequentially from 5' to 3', the promoter including Pcdna1.

[0065] In this application, the inventors discovered that engineered Trichoderma reesei strains, when containing promoters including but not limited to Pcdna1, can effectively promote the expression of the SNC1 protein. In the recombinant plasmid, the SNC1 gene is placed under the control of the constitutive strong promoter Pcdna1. Overexpression of the SNC1 gene not only significantly increases the yield of cellulase but also exhibits the characteristic of alleviating carbon metabolism repression. This allows the engineered strains to maintain high cellulase activity even under conditions where glucose is the sole carbon source, breaking through traditional production limitations and improving production flexibility. Furthermore, the engineered strains in the preferred embodiment show a significantly accelerated enzyme production rate in the early stages of fermentation. Especially under conditions where glucose is the sole carbon source, they can significantly alleviate the inhibition of cellulase synthesis by carbon metabolism repression, thereby improving cellulase production efficiency under a wider range of carbon source conditions, helping to shorten the overall production cycle and improve cellulase production efficiency.

[0066] In a preferred embodiment, the method of transferring the exogenous SNC1 gene into the above-mentioned Trichoderma reesei includes: transferring a plasmid into the above-mentioned Trichoderma reesei, the plasmid containing the above-mentioned SNC1 expression cassette, the plasmid including pPTRII.

[0067] In this application, the inventors discovered that a recombinant plasmid was obtained by inserting a specific nucleotide sequence fragment of the SNC1 gene into the pPTRII plasmid. Overexpression of the SNC1 gene was achieved by precisely positioning its expression cassette within the plasmid to ensure efficient expression in *Trichoderma reesei*. This design not only promotes cellulase synthesis, but also significantly alleviates the inhibition of cellulase synthesis by carbon metabolism repression, particularly when glucose is used as the sole carbon source, thereby improving cellulase production efficiency under a wider range of carbon source conditions. This results in a significant increase in cellulase yield and activity in the basic scheme.

[0068] In a second typical embodiment of this application, a modification method is provided to relieve the carbon metabolism repression effect of Trichoderma reesei, the modification method comprising: increasing the expression level of the SNC1 gene in Trichoderma reesei.

[0069] This application provides a modification method to overcome the carbon metabolism repression effect in *Trichoderma reesei*, the core of which lies in increasing the expression level of the SNC1 gene in *Trichoderma reesei*. The SNC1 protein plays a crucial role in fungal secretion pathways, especially in the secretion of cellulase. Increased SNC1 gene expression effectively promotes enhanced cellulase activity. In industrial production, this modification strategy effectively solves the problem of low cellulase yield in *Trichoderma reesei* strains, particularly under conditions where glucose is the sole carbon source, significantly alleviating the inhibition of cellulase synthesis by carbon metabolism repression, thus increasing production flexibility and economic efficiency. Therefore, this method, through the regulation of SNC1 gene expression, achieves a significant increase in cellulase yield and has important industrial application value.

[0070] In a preferred embodiment, the steps of the above modification method include any one or more of the following: converting the SNC1 gene into the above-mentioned Trichoderma reesei, or increasing the copy number of the SNC1 gene in the above-mentioned Trichoderma reesei, or replacing the promoter of the SNC1 gene in the above-mentioned Trichoderma reesei with a strong promoter.

[0071] In a preferred embodiment, the SNC1 gene described above includes the SNC1 gene used in the method for preparing cellulase described above.

[0072] The aforementioned strong promoters refer to one or more promoters that can further enhance the expression level of the SNC1 gene compared to the original promoter of the SNC1 gene in Trichoderma reesei.

[0073] In this application, the inventors achieved overexpression of the SNC1 gene by either increasing the copy number of the SNC1 gene in *Trichoderma reesei* or replacing the promoter of the SNC1 gene in *Trichoderma reesei* with a strong promoter, thereby increasing the yield and activity of cellulase. Overexpression of the SNC1 protein increased cellulase production. After preliminary resistance screening and sequencing verification, a *Trichoderma reesei* mutant strain was obtained. This strain exhibited higher cellulase activity, especially in a culture medium with glucose as the sole carbon source, effectively alleviating carbon metabolism repression and significantly increasing enzyme yield in the early stages of fermentation. This application, through precise control of gene expression and optimized transformation methods, not only accelerates the cellulase production cycle but also increases the overall enzyme yield and reduces production costs, providing a superior microbial platform for the large-scale production and application of cellulase and enhancing the competitiveness of *Trichoderma reesei* in the industrial production of cellulase.

[0074] In a third typical embodiment of this application, a method for preparing cellulase as described above, a modification method for relieving the carbon metabolism repression effect of Trichoderma reesei as described above, or the application of the SNC1 gene in Trichoderma reesei culture or cellulase production is provided.

[0075] In this application, the inventors provide a method for applying the aforementioned SNC1 gene, the aforementioned method for preparing cellulase, or the aforementioned modification method for relieving the carbon metabolism repression effect of *Trichoderma reesei* in the cultivation of *Trichoderma reesei* or the production of cellulase, aiming to increase cellulase yield and alleviate the carbon metabolism repression effect. Overexpression of the SNC1 gene in *Trichoderma reesei* can increase the yield of extracellular cellulase. Furthermore, overexpression of the SNC1 gene can alleviate the glucose-induced carbon metabolism repression effect, enabling *Trichoderma reesei* to maintain high cellulase activity even in media where glucose is the sole carbon source, thereby enhancing cellulase synthesis and secretion under a wider range of carbon source conditions. This application not only improves the production efficiency of cellulase but also enhances the flexibility of *Trichoderma reesei* in industrial applications, particularly in the fields of cellulase biofuels, food and feed, papermaking, and textiles, providing a new strategy for the industrial production of cellulase.

[0076] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0077] Example 1

[0078] Construction of recombinant vectors and protoplast transformation and validation:

[0079] The vector is as follows: The selected vector backbone is PptrII, containing the pyridine thiamine resistance gene ptrA, and the Pcdna1-SNC1-TrpC expression cassette. The pPTR-SNC1 vector is derived from the pPTRII vector (Takara; Code No. 3622) and carries the pyridine thiamine resistance gene ptrA as a selection marker. The pPTR-SNC1 vector map is shown below. Figure 1 As shown in Table 1, the primers used to construct the vector are also shown.

[0080] Table 1

[0081]

[0082] Enzymes and reagents: Phanta Max Super-Fidelity DNA Polymerase (Nanjing Novizan Biotechnology Co., Ltd.) was used for PCR amplification; NEBuilder HiFi DNA Assembly Cloning Kit (NEB) was used for in vitro assembly of the constitutive strong promoter cdna1 of Trichoderma reesei with the TrpC terminator sequence of Aspergillus nidulans, the SNC1 gene, and the pPTRII backbone; Escherichia coli TOP 10 competent cells (Takara) were used for transformation of the assembly products; LB + ampicillin sodium (final concentration 100 μg / mL) solid medium and LB + ampicillin sodium (final concentration 100 μg / mL) liquid medium were used for culturing the transformation products and expanding the positive transformants, respectively; SP Fungi DNA kit (Omega) was used to extract genomic DNA from the strains for subsequent verification.

[0083] Preparation and transformation of protoplasts: Trichoderma reesei mycelium (the starting strain Trichoderma reesei E619 was deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M20242076); Escherichia coli strain TOP10 was used for the construction of recombinant plasmids and was purchased from Takara.

[0084] (1) Take a fresh Trichoderma reesei spore plate (no more than two weeks old), add sterile physiological saline (0.9% NaCl, 0.05% Tween) to the plate, scrape off the spores with a glass rod, and set aside.

[0085] (2) Inoculate 1-2 mL of spore suspension into 50 mL of PDB medium and incubate at 28℃ and 200 rpm for about 12-15 h. Add 10 mg / mL lyase (Sigma #L-1412), 10 mg / mL lysozyme, and 5 mg / mL to 20 mL of Solution I, shake gently, and filter sterilize through a 0.22 μm filter into a 50 mL centrifuge tube (sterile).

[0086] (3) Filter the Trichoderma reesei mycelia cultured for 12-15h using a nylon cloth funnel to remove the culture medium. Place the filtered Trichoderma reesei mycelia in a 9cm diameter (sterile) petri dish, add 15-20 mL of the prepared lysis buffer, and gently blow with a sterile pipette tip until completely dispersed. Place the petri dish in a shaker at 30℃ and enzymatically digest at 70 rpm for 1.5-2h. Take samples regularly for microscopic observation to prevent over-digestion.

[0087] Note: During enzymatic hydrolysis, the centrifuge and rotor can be pre-cooled to 4°C.

[0088] (4) After the enzymatic hydrolysis, filter the protoplast suspension into a 50 mL centrifuge tube placed on ice, and then rinse with a few milliliters of solution I. The total volume of the filtrate should not exceed 40 mL.

[0089] (5) Centrifuge at 3000 rpm and 4℃ for 10 min (make sure to keep the centrifuge rotor at 4℃ and all operations are performed on ice), carefully remove the supernatant and resuspend the protoplasts in 4 mL of solution II (must be at 4℃).

[0090] (6) Centrifuge at 3000 rpm and 4℃ for 10 min, carefully remove the supernatant, and resuspend the protoplasts in 0.2-0.5 mL of solution II (4℃). Place the protoplasts on ice. Transform the above recombinant plasmid into Trichoderma reesei strain E619 by PEG / CaCl2-mediated protoplast transformation. Take 200 μL of the above protoplasts, add 5 μg of recombinant plasmid and pre-cooled PEG solution, mix gently, and let stand on ice for 20 min. First add 1 mL of PEG solution, incubate at 25℃ for 5 min, then slowly add 2 mL of solution II and mix gently. Finally, mix the transformation products and transfer them to MMS + pyridine thiamine (final concentration 500 ng / mL) medium. After cooling and solidification, incubate upside down at 28℃ for 5 days.

[0091] Solution I consists of: 1.2 M sorbitol, 0.1 M KH2PO4, and pH 5.6.

[0092] Solution II consists of: 1M sorbitol, 50mM CaCl2·2H2O, 10mM Tris-HCl, pH 7.5;

[0093] The composition of the PEG solution is: 25% (w / v) PEG 6000, 50mM CaCl2·2H2O, 10mM Tris-HCl, pH 7.5;

[0094] The MM medium is prepared as follows: 2% glucose, 0.5% ammonium sulfate, 1.5% potassium dihydrogen phosphate, 0.06% calcium chloride, 0.06% magnesium sulfate, 0.2% peptone, 0.0037 g / L CoCl2·6H2O, 0.005 g / L FeSO4·7H2O, 0.0014 g / L ZnSO4·7H2O, 0.0016 g / L MnSO4·H2O, 1.8% agar powder, pH 4.5~5.5;

[0095] The components of MMS medium are: MM medium (liquid), 1M sorbitol, 0.7% agarose, pH 5.6.

[0096] The components in the culture medium or in this application are all in mass-volume ratio, such as 2% glucose content is 2% (w / v).

[0097] Single colonies were picked from MMS+pyrithiamine (final concentration 500 ng / mL) medium transformation plates and inoculated onto PDA (200 g / L potato juice, 20 g / L glucose, 1.8% agar powder) plates and cultured at 29°C for 5-7 days. Spores were washed away with sterile physiological saline and inoculated at a ratio of 1% (v / v) onto PDB liquid medium (200 g / L potato juice, 20 g / L glucose) and cultured at 29°C, 200 rpm for 48 hours. Mycelia were collected. The mycelia were collected through nylon cloth and transferred to 2 ml centrifuge tubes. After drying, the mycelia were homogenized using a tissue homogenizer, and the genome was extracted using the Omega® SP Fungi DNA kit.

[0098] Using the genome of the above-mentioned transformant as a template, genomic PCR was performed using primers Pcdna1-F (SEQ ID NO: 4) and SNC1-R (SEQ ID NO: 8) for verification. At the same time, the constructed expression vector plasmid was used as a template as a positive control, and the genomic DNA of the originating strain E619 was used as a template as a negative control. The PCR products with the correct band size were sent to Qingke Biotechnology Co., Ltd. for sequencing.

[0099] Example 2

[0100] Fermentation validation of recombinant strains:

[0101] (1) Spore collection: The recombinant strain with correct sequencing was inoculated into PDA medium for sporulation culture and cultured at 30℃ for 4 days. The spore suspension was collected and the number of spores per milliliter of suspension was calculated using a hemocytometer.

[0102] (2) Seed culture: Inoculate the spore resuspension into 50 mL of seed culture medium (2-5% glucose, 0.2-2% yeast extract, 0.05-0.6% dipotassium hydrogen phosphate, 0.05-0.6% potassium dihydrogen phosphate, 0.02-0.18% magnesium sulfate, 0.02-0.1% calcium chloride), with a final spore concentration of 10. 6 Cells / mL, cultured at 30℃ and 200rpm until the mycelial morphology matures.

[0103] (3) Shake-flask fermentation: The above seed culture was inoculated into the fermentation medium (1-3% fermentation syrup, 0.8-3% corn steep liquor, 0.1-1% ammonium sulfate, 0.3-0.8% dipotassium hydrogen phosphate, 0.3-0.6% potassium dihydrogen phosphate, 0.02-0.18% magnesium sulfate, and 0.02-0.1% calcium chloride) at a 10% inoculation rate. Shake-flask fermentation was carried out at 30℃ and 220 rpm. The fermentation supernatant was collected during the fermentation process. The fermentation syrup was prepared by adding 0.5-1.5% β-glucosidase (12000 IU / g) to 400-600 g / L glucose and stirring the mixture at 50-60℃ and pH 4.0-5.0 for 70-80 h, followed by high-temperature inactivation.

[0104] Referring to "NY / T912-2020 Determination of Cellulase Activity in Feed Additives - Spectrophotometric Method", the standard specifies the use of the reducing sugar colorimetric method to determine the activity of cellulase.

[0105] DNS reagent: Weigh 3.15g of 3,5-dinitrosalicylic acid, add 500mL of water and stir. Heat in a water bath to 45℃, slowly add 100mL of sodium hydroxide solution (200g / L) while stirring continuously until completely dissolved. Then gradually add 91.0g of potassium sodium tartrate tetrahydrate, 2.5g of phenol and 2.5g of sodium sulfite, stirring until dissolved. Stop heating, cool to room temperature, add water to make up to 1000mL, shake well, filter, and store in the dark.

[0106] 0.1M sodium acetate buffer (pH=5.5): Weigh 23.14g of sodium acetate trihydrate, dissolve in water, add 1.70mL of glacial acetic acid, dilute with water to 2000mL, and adjust the pH to 5.5 with acetic acid solution or sodium acetate solution.

[0107] Glucose solution: Weigh 0.5g of anhydrous glucose (accurate to 0.001g), place it in a 50mL volumetric flask, dissolve it in acetate-sodium buffer solution and dilute to the mark, then shake well.

[0108] Plotting the standard curve:

[0109] (1) Take 4.0 ml of 0.1 M sodium acetate buffer (pH=5.5), add 5.0 mL of DNS reagent, heat in a boiling water bath for 5 min; cool to room temperature with tap water. Make up to 25.0 mL with water to prepare a standard blank sample;

[0110] (2) Take 1.00 mL, 2.00 mL, 3.00 mL, 4.00 mL, 5.00 mL, 6.00 mL and 7.00 mL of 10 g / L glucose solution respectively, and make up to 100 mL with sodium acetate buffer to prepare glucose standard solutions with a concentration of 0.10 mg / mL to 0.70 mg / mL;

[0111] (3) Take 2.00 ml of each of the above concentration series of glucose standard solutions (make 2 parallels), add them to the graduated test tubes respectively, then add 2 ml of water and 5 ml of DNS reagent, vibrate electromagnetically for 3 s, and heat in a boiling water bath for 5 min. Then, cool to room temperature with tap water, and then dilute to 25 ml with water. Use the standard blank sample as a control to zero the volume, and measure the absorbance OD value at 540 nm.

[0112] (4) Plot a standard curve with glucose concentration as the Y-axis and absorbance OD value as the X-axis. A new standard curve needs to be plotted each time DNS reagent is prepared. Specific method: Take 2 mL of sodium carboxymethyl cellulose solution (15 mg / mL), add 2 mL of appropriately diluted enzyme solution, and mix well. After reacting at 37℃ for 30 min, add 5 mL of DNS reagent, vortex for 3 s to mix, and terminate the enzymatic reaction. Add 1 mL of 0.3 mg / mL glucose solution, heat in a boiling water bath for 5 min, add distilled water to a final volume of 25 mL, vortex to mix, and measure the absorbance at 540 nm. For the blank control, add DNS first, then add the enzyme solution.

[0113] Endoglucanase plays a crucial role in the hydrolysis of cellulose, and its activity directly affects the hydrolysis efficiency of cellulose by cellulase. Endoglucanase activity (CMCase): One unit (U) of enzyme activity is defined as the amount of enzyme required to produce 1 μmol of reducing sugar per minute from a 7.5 mg / mL sodium carboxymethyl cellulose solution at 37°C and pH 5.5.

[0114] The engineered strain ES27 reached its peak endoglucanase activity at 138 h of fermentation, while the control strain E619 reached its peak at 164 h, shortening the enzyme production cycle by approximately 24 h. The peak endoglucanase activity in the ES27 fermentation supernatant reached 2024 U / mL, 2.31 times that of the control strain E619, and the maximum secretion of extracellular protein was also increased by 72.7% compared to the control strain E619. Furthermore, the rate of endoglucanase production was significantly increased in the early fermentation stage (24-60 h), reaching 5.6 times that of the control strain E619. The endoglucanase activity in the shake-flask fermentation supernatant was as follows... Figure 2 As shown, the changes in protein content in the supernatant of shake-flask fermentation are as follows: Figure 3 As shown.

[0115] Example 3

[0116] Fermentation validation using glucose as the sole carbon source in recombinant strains:

[0117] The seed culture medium using glucose as the sole carbon source was the same as that in Example 2. The fermentation medium was modified by replacing the fermentation syrup with 4.2% glucose as the sole carbon source, while other components remained unchanged. Sterilization was performed at 121°C for 20 minutes. Samples were taken every 24 hours to determine CMCase enzyme activity and conduct relevant analyses. The fermentation process was the same as in Example 2. The CMCase enzyme activity during glucose-only fermentation was as follows... Figure 4 As shown in the results, the enzyme activity of the control strain E619 increased slowly at a low level throughout the early stage (0-48h), almost zero, and only slowly increased to about 50 U / mL in the middle and late stages (72-120h). In contrast, strain ES27 showed a rapid increase-peak-decline trend, reaching a peak (approximately 200 U / mL) around 70-80h, and then gradually decreasing. The overall enzyme activity level was significantly higher than that of E619, indicating a stronger ability to synthesize and secrete cellulase. The changes in residual sugar during fermentation with glucose as the sole carbon source are shown in the figure. Figure 5 As shown in the figure, the experimental results indicate that even with a high glucose concentration within 24 hours, cellulase production by strain ES27 was almost unaffected by glucose concentration, significantly relieving the inhibition of cellulase genes by glucose and increasing endoglucanase activity (CMCase) by 4-fold. SDS-PAGE results of the 120-hour fermentation supernatant showed that cellulase production by strain ES27 was almost unaffected by high glucose concentrations, while cellulase activity in the control strain E619 only appeared after 72 hours, indicating a significant inhibitory effect. The experimental results are as follows... Figure 6 Sn2 and Sn3 were parallel experiments of strain ES27. ES27, Sn2 and Sn3 all showed excellent results in that the expression level of cellulase was not affected by glucose.

[0118] Example 4

[0119] Relative transcriptional level analysis of recombinant strain ES27 compared to control strain E619:

[0120] Preparation and sequencing of transcriptome sequencing samples: Samples of ES27 and E619 strains were fermented with glucose as the sole carbon source for 40 h, centrifuged at 12000 r / min for 3 min at 4℃, washed three times with PBS, and then flash-frozen in liquid nitrogen for 30 min and stored at -80℃. Each sample was performed in triplicate. The E619 strain fermented for 40 h served as the control group, and the ES27 strain fermented for 40 h served as the experimental group. Total RNA was extracted using the Megan Universal RNA Extraction Kit. After passing quality control, the samples were sent to Suzhou Genewiz Biotechnology Co., Ltd. for library construction and sequencing.

[0121] In a comparison of the transcriptome levels of endoplasmic reticulum stress-related genes (pdi1, bip1, ire1) in strains E619 and ES27, the relative transcription levels of pdi1 and bip1 were slightly increased in ES27 compared to the control strain E619, while the relative transcription level of ire1 remained significantly unchanged. The results are as follows: Figure 7 As shown in the figure. In the comparison of the transcriptome levels of cellulase genes (egl1, egl2, cbh1, cbh2, bgl1) between strains E619 and ES27, the relative transcription levels of egl1, egl2, cbh1, cbh2, and bgl1 were significantly increased in ES27 compared to the control strain E619, as shown in the figure. Figure 8 As shown in the figure. In the comparison of transcriptome levels of cellulase regulatory genes (snc1, xyr1, ace1, ace2, cre1) between strains E619 and ES27, the relative transcription levels of xyr1 and ace2 were significantly higher in ES27 than in the control strain E619, while the transcription levels of ace1 and cre1 were lower. The transcription level of the SNC1 gene in strain ES27 was 1.58 times that of E619, which significantly increased the transcription level of cellulase genes under glucose as a carbon source (relative transcription level refers to the fold change of a specific gene expression in the experimental group relative to the control group). The results are shown in the figure. Figure 9 As shown.

[0122] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects:

[0123] (1) Cellulase production was significantly increased. By overexpressing the SNC1 gene, the extracellular protein secretion and cellulase production of Trichoderma reesei were effectively increased. The experimental results showed that the total extracellular protein secretion of the engineered strain ES27 was increased by 72.7% compared with the control strain, and the highest cellulase activity reached 2024 U / mL, which was 2.31 times that of the control strain.

[0124] (2) The engineered Trichoderma reesei strain overexpressing the SNC1 gene showed good cellulase expression in a medium containing glucose as the sole carbon source, with an endoglucanase expression level of 150 U / mL after 48 h. Under the same conditions, the wild-type strain was repressed by carbon metabolism, and the endoglucanase expression level was 0, indicating that cellulase synthesis was significantly inhibited. This suggests that overexpression of the SNC1 gene can alleviate the inhibitory effect of carbon repression on cellulase synthesis, enabling Trichoderma reesei to synthesize cellulase under a wider range of carbon source conditions, thus improving the flexibility and economy of cellulase production.

[0125] (3) The constructed Trichoderma reesei engineered strain showed a significant increase in enzyme production rate in the early stage of fermentation (24-60h), which was 5.6 times that of the control strain, further enhancing the application value of cellulase in industrial production.

[0126] (4) Compared with the control strain E619, the relative transcription levels of pdi1 and bip1 of endoplasmic reticulum stress-related genes in ES27 were slightly increased. Compared with the control strain E619, the relative transcription levels of egl1, egl2, cbh1, cbh2, and bgl1 of cellulase genes in ES27 were significantly increased. Compared with the control strain E619, the relative transcription levels of xyr1 and ace2 of cellulase regulatory genes in ES27 were significantly higher than those in E619, while the transcription levels of ace1 and cre1 of cellulase regulatory genes were lower. The transcription level of SNC1 of cellulase regulatory gene in ES27 was 1.58 times that of E619, which can significantly improve the transcription level of cellulase genes under glucose as a carbon source.

[0127] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing cellulase, characterized in that, The method includes: S1) Increase the expression level of the SNC1 gene in Trichoderma reesei to obtain engineered Trichoderma reesei strains; S2) Cultivate the engineered Trichoderma reesei strain and collect the cellulase; The cultivation includes: culturing the engineered Trichoderma reesei in a fermentation medium, wherein the carbon source in the fermentation medium includes glucose, and the concentration of glucose is 10-30 g / L.

2. The method according to claim 1, characterized in that, The SNC1 gene sequence includes: the SNC1 gene sequence derived from Trichoderma reesei QM6a, or the SNC1 gene of Trichoderma reesei itself.

3. The method according to claim 1, characterized in that, The protein encoded by the SNC1 gene has the amino acid sequence shown in SEQ ID NO: 1, or a protein that has more than 70% homology with the amino acid sequence.

4. The method according to claim 1 or 3, characterized in that, The SNC1 gene is the nucleic acid sequence shown in SEQ ID NO: 2, or a sequence that has more than 70% homology with the nucleic acid sequence.

5. The method according to claim 1, characterized in that, The fermentation medium includes 1-3% glucose, 0.8-3% corn steep liquor, 0.1-1% ammonium sulfate, 0.05-0.6% dipotassium hydrogen phosphate, 0.02-0.18% magnesium sulfate, and 0.02-0.1% calcium chloride.

6. The method according to claim 5, characterized in that, The glucose in the fermentation medium is added to the fermentation medium in the form of fermentation syrup; The method for preparing the fermented syrup includes: mixing glucose and 0.05-1.5% β-glucosidase to obtain a reaction system, reacting the reaction system under stirring conditions to obtain the fermented syrup.

7. The method according to claim 6, characterized in that, The temperature of the reaction system is 50-60℃, the pH of the reaction system is 4.0-5.0, the reaction time under the stirring conditions is 70-80h, and the stirring speed is 80-150rpm.

8. The method according to claim 6, characterized in that, The concentration of glucose in the fermented syrup is 400-600 g / L, and the concentration of β-glucosidase is 10000-15000 IU / g.

9. The method according to claim 1, characterized in that, S1) includes: introducing an exogenous SNC1 gene into the Trichoderma reesei, and / or increasing the expression level of the endogenous SNC1 gene in the Trichoderma reesei.

10. The method according to claim 9, characterized in that, A method for transferring an exogenous SNC1 gene into the Trichoderma reesei includes: transferring an SNC1 expression cassette into the Trichoderma reesei, the SNC1 expression cassette comprising a promoter and the SNC1 gene connected sequentially from 5' to 3', the promoter comprising Pcdna1.

11. The method according to claim 10, characterized in that, A method for transferring an exogenous SNC1 gene into the Trichoderma reesei includes: transferring a plasmid into the Trichoderma reesei containing the SNC1 expression cassette, the plasmid comprising pPTRII.

12. A method for modifying Trichoderma repens to relieve carbon metabolism repression, characterized in that, The modification method includes: increasing the expression level of the SNC1 gene in Trichoderma reesei.

13. The modification method according to claim 12, characterized in that, The modification method includes any one or more of the following steps: The SNC1 gene is transformed into the Trichoderma reesei, or the copy number of the SNC1 gene in the Trichoderma reesei is increased, or the promoter of the SNC1 gene in the Trichoderma reesei is replaced with a strong promoter.

14. The modification method according to claim 12 or 13, characterized in that, The SNC1 gene includes the SNC1 gene used in the method for preparing cellulase according to any one of claims 2-4.

15. The method for preparing cellulase according to any one of claims 1-11, or the modification method for relieving the carbon metabolism repression effect of Trichoderma reesei according to any one of claims 12-14, or the application of the SNC1 gene in Trichoderma reesei culture or cellulase production.

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