Trichoderma reesei recombinant strain, construction method and application thereof

Abstract

The application discloses a recombinant Trichoderma reesei strain TriY120601 and a construction method and application thereof. Rut‑C30 The application discloses a recombinant Trichoderma reesei strain TriY120601 and a construction method and application thereof. Rut‑C30 The application discloses a recombinant Trichoderma reesei strain TriY120601 and a construction method and application thereof. Rut‑C30 The application discloses a recombinant Trichoderma reesei strain TriY120601 and a construction method and application thereof. Rut‑C30 The application discloses a recombinant Trichoderma reesei strain TriY120601 and a construction method and application thereof. Rut‑C30 The application discloses a recombinant Trichoderma reesei strain TriY120601 and a construction method and application thereof. Rut‑C30 The application discloses a recombinant Trichoderma reesei strain TriY120601 and a construction method and application thereof. Rut‑C30 The application discloses a recombinant Trichoderma reesei strain TriY120601 and a construction method and application thereof. Rut‑C30 The application discloses a recombinant Trichoderma reesei strain TriY120601 and a construction method and application thereof. Rut‑C30 The application discloses a recombinant Trichoderma reesei strain TriY120601 and a construction method and application thereof. Rut‑C30 The application discloses a recombinant Trichoderma reesei strain TriY120601 and a construction method and application thereof.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, specifically to a recombinant Trichoderma reesei strain, its construction method, and its application. Background Technology

[0002] Lignocellulose materials exhibit resistance to biodegradation due to their robust properties, such as the protective effect of lignin and the stable cross-linking of hemicellulose. Therefore, efficient saccharification technologies are needed to break down their dense structure, thereby improving their digestibility and biodegradability. While lignocellulose can be degraded into glucose through high-temperature heating and acid / alkali treatment, these physicochemical methods are energy-intensive and environmentally polluting. To effectively utilize this important biomass, green and safe bioenzymatic degradation technologies need to be developed. Because of the complex and dense structure of lignocellulose biomass, complex enzyme systems composed of multiple enzymes and proteins are often required to saccharify it into reducing sugars that can be utilized by microorganisms.

[0003] Cellulases are a group of enzymes that degrade cellulose to produce oligosaccharides or monosaccharides. With societal development, the applications of cellulases are continuously expanding across various fields, including food, ethanol fuel, feed processing, textiles, agriculture, and medicine. This necessitates higher demands for industrial strains capable of producing high levels of cellulase. Reducing the production cost of cellulase requires the selection of strains with strong enzyme-producing capabilities; therefore, genetic modification of filamentous fungi has received widespread attention. The *Trichoderma reesei* Rut-C30 mutant strain is a high-cellulase-producing strain. In this strain, the gene cre1, which regulates carbon catabolic repression (CCR), has been modified, weakening the CCR effect to some extent and thus increasing cellulase production. *Trichoderma reesei* Δku70 Rut-C30 It is a genetically engineered bacterium that started with Rut-C30 and knocked out the KU70 gene that mediates non-homologous end joining, thereby improving the homologous recombination efficiency of the strain (Cai W et al. A three-gene cluster in Trichoderma reesei reveals a potential role of dmm2 in DNA repair and cellulase production. Biotechnology for Biofuels and Bioproducts. 2022, 15(1): 34).

[0004] Currently, the construction of high-cellulase-producing *Trichoderma reesei* strains is mostly limited to the discovery and modification of transcription factors involved in regulating cellulase synthesis at the transcriptional regulation level, including transcription activators and repressors. However, cellulase biosynthesis and secretion are subject to multi-level molecular regulation. Although there has been extensive research on the genome, transcriptome, proteome, and secretome of *Trichoderma* enzyme production, and some key genes regulating enzyme production have been identified (Yang J, et al. Fungal strain improvement for efficient cellulase production and lignocellulosic biorefinery: current status and future prospects. Bioresource Technology. 2023, 385: 129449), due to the complexity of genetic manipulation in *Trichoderma*, its functional genomics research is not yet in-depth, and more enzyme production regulatory elements need to be developed.

[0005] Small proteins are short peptides or small proteins transcribed and translated from small open reading frames (sORFs), generally not exceeding 100 amino acids in eukaryotes. The discovery of small proteins benefited from the development of deep transcriptome sequencing methods, such as RNA-seq and Ribo-seq. Due to their generally short sequences, incomplete homologous sequences, domain mismatches with known databases, and the fact that some functional small proteins are only induced under specific conditions, the functions of many small proteins are difficult to define. Currently identified small proteins are widely involved in DNA replication and repair, transcription, protein synthesis and folding, substance transport, mitochondrial composition, and metabolic regulation. However, research on using small protein overexpression to enhance enzyme production in *Trichoderma* is still in its early stages.

[0006] The production of cellulase from filamentous fungi requires induction. Microcrystalline cellulose is a commonly used solid inducer, but it has disadvantages such as high cost and difficulty in addition during fermentation. Utilizing soluble inducers produced by the glucosidase-transglycosylation reaction (Mixture of glucose and β-disaccharides, abbreviated as MGD, Li YH, et al. Overproduction of cellulase by Trichoderma reesei RUT C30 through batch-feeding of synthesized low-cost sugar mixture. Bioresource Technology 2016, 216: 503-510) or (Acid-catalytic synthesized mixture, abbreviated as ACM, Liu Chenguang, Deng Lin, Bai Fengwu, A highly efficient cellulase inducer and its preparation and application method, application number 202210201654.5), containing oligosaccharide inducers such as sophorose to induce enzyme production, can reduce the cost of inducing enzyme production. Intracellular comparative proteomics and transcriptomics analysis reveals that the protein quantity or transcription level of some small proteins changes significantly under enzyme-inducing conditions. Further gene overexpression or knockout can reveal small proteins that affect enzyme production, thus leading to this invention. Summary of the Invention

[0007] The purpose of this invention is to address the shortcomings of existing technologies by providing a recombinant Trichoderma reesei strain, its construction method, and its applications, achieved through the following technical solution:

[0008] A method for constructing a recombinant Trichoderma reesei strain, using Trichoderma reesei strain Δku70 Rut-C30Using this as the starting strain, the coding gene for the dynamin-related protein M419DRAFT_36429 was replaced with the coding gene for the small protein M419DRAFT_120601. The small protein M419DRAFT_120601 gene was overexpressed using the inducible promoter Pcbh1 to construct a recombinant strain producing high levels of cellulase, which was named TriY120601 in the laboratory. The nucleotide sequence of the inducible promoter Pcbh1 is shown in SEQ ID No. 1, the nucleotide sequence of the coding gene for the small protein M419DRAFT_120601 is shown in SEQ ID No. 2, and the amino acid sequence of the small protein M419DRAFT_120601 is shown in SEQ ID No. 3. The nucleotide sequence of the coding gene for M419DRAFT_36429 is shown in SEQ ID No. 4, the nucleotide sequence of the upstream homologous arm of 36429 is shown in SEQ ID No. 5, and the nucleotide sequence of the downstream homologous arm of 36429 is shown in SEQ ID No. 6. The small protein M419DRAFT_120601 is a very unique protein in ascomycetes, found only in Trichoderma reesei Rut-C30.

[0009] The recombinant strain TriY120601 has the accession number CGMCC No.41526, is classified as *Trichoderma reesei*, and is deposited at the China General Microbiological Culture Collection Center on September 24, 2024. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] The recombinant strain TriY120601 can efficiently produce cellulase during fermentation using enzyme inducers microcrystalline cellulose, lactose, and soluble inducers ACM or MGD, with an optimal dosage of 20 g / L for each. The crude cellulase solution produced during fermentation can be used to degrade lignocellulosic biomass.

[0011] The enzyme-producing inducer ACM (Acid-catalytic synthesized mixture) is an inducer for the synthesis of a mixed sugar containing disaccharides and oligosaccharides through acid-catalyzed glucose-to-glycoside reaction. The preparation of ACM is based on the patent "A Highly Efficient Cellulase Inducer and Its Preparation and Application Method," application number 202210201654.5. The enzyme-producing inducer MGD (Mixture of glucose and β-disaccharides) is a glucose-sophorose mixture synthesized through β-glucosidase-catalyzed glucose-to-glycoside reaction. The preparation of MGD is based on the patent "A Method for Highly Efficiently Producing Cellulase Based on Glucose-to-Glycoside Reaction," application number 201610309126.6. The addition of enzyme-producing inducers enables the strain to produce cellulase efficiently, increasing enzyme activity and its enzymatic hydrolysis performance on cellulose.

[0012] The recombinant strain TriY120601 provided by this invention, and the inoculum containing this strain, can be used for the fermentation production of cellulase and can be applied in the field of degrading lignocellulosic biomass. The lignocellulosic biomass includes, but is not limited to, various crop straws, cellulose-containing food (processing) waste, and domestic and industrial forestry waste.

[0013] The advantages of this invention are:

[0014] 1. This invention is the first to discover the regulatory function of an unknown functional small protein in cellulase biosynthesis, and proves that overexpression of the M419DRAFT_120601 gene using the inducible promoter Pcbh1 can significantly improve the cellulase production capacity of Trichoderma reesei. It provides a new method to improve the cellulase production capacity of Trichoderma reesei and provides a recombinant strain TriY120601, which has good potential for industrial application.

[0015] 2. This invention discovered the knockout strain Δku70 Rut-C30 The gene encoding the dynamin-related protein M419DRAFT_36429 does not affect the growth of the strain or its cellulase production capacity, providing a suitable integration site for the successful ectopic overexpression of the small protein M419DRAFT_120601.

[0016] 3. The addition of an enzyme inducer to the recombinant strain TriY120601 during fermentation significantly improves cellulase production. Compared with the control strain, the cellulase production capacity of the recombinant strain TriY120601 is increased by at least 46%. Attached Figure Description

[0017] Figure 1 It is strain Δ36429-Δku70 Rut-C30 and Δku70 Rut-C30Comparison of filter paper enzyme activity during shake-flask fermentation under 20 g / L ACM induction conditions. (Strain Δku70) Rut-C30 It is the original strain, Δ36429-Δku70 Rut-C30 It is Δku70 Rut-C30 Blank control strain with the M419DRAFT_36429 coding gene knocked out.

[0018] Figure 2 It is the recombinant strains TriY120601 and Δ36429-Δku70 Rut-C30 Comparison of filter paper enzyme activity during shake-flask fermentation under 20 g / L ACM induction conditions.

[0019] Figure 3 It is the recombinant strains TriY120601 and Δ36429-Δku70 Rut-C30 Comparison of exonuclease activity during shake-flask fermentation under 20 g / L ACM induction conditions.

[0020] Figure 4 It is the recombinant strains TriY120601 and Δ36429-Δku70 Rut-C30 Comparison of xylanase activity during shake-flask fermentation under 20 g / L ACM induction conditions.

[0021] Figure 5 It is the recombinant strains TriY120601 and Δ36429-Δku70 Rut-C30 Comparison of extracellular protein content in shake-flask fermentation under 20 g / L ACM induction conditions.

[0022] Figure 6 It is the recombinant strains TriY120601 and Δ36429-Δku70 Rut-C30 Comparison of β-glucosidase activity during shake-flask fermentation under 20 g / L ACM induction conditions.

[0023] Figure 7 It is the recombinant strains TriY120601 and Δ36429-Δku70 Rut-C30 Comparison of endonuclease activity during shake-flask fermentation under 20 g / L ACM induction conditions.

[0024] Figure 8 It is the recombinant strains TriY120601 and Δ36429-Δku70 Rut-C30 Comparison of filter paper enzyme activity during shake-flask fermentation under 20 g / L lactose induction conditions.

[0025] Figure 9 It is the recombinant strains TriY120601 and Δ36429-Δku70 Rut-C30 Comparison of enzyme activity in filter paper during shake-flask fermentation under 20 g / L microcrystalline cellulose induction conditions.

[0026] Figure 10 It is the recombinant strains TriY120601 and Δ36429-Δku70 Rut-C30 Comparison of filter paper enzyme activity during shake-flask fermentation under 20 g / L MGD induction conditions.

[0027] Figure 11 This describes the enzyme activity of the recombinant strain TriY120601 on filter paper under continuous feeding fermentation conditions in a 7L fermenter (4L fermentation broth) under MGD induction.

[0028] Figure 12 It is the recombinant strains TriY120601 and Δku70 Rut-C30 Comparison of glucose production effects of corn stalks after pretreatment with crude cellulase solution. Detailed Implementation

[0029] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Unless otherwise specified, the reagents and consumables used in the examples are readily available on the market. Where specific experimental methods or conditions are not specified, they shall be performed in accordance with conventional methods or conditions described in the literature in this field or in accordance with the product instructions.

[0030] Example 1: Construction of the recombinant Triyrus reesei strain TriY120601

[0031] The experimental materials and methods used are as follows:

[0032] 1. Culture medium

[0033] Potato glucose agar (PDA) medium for Trichoderma reesei spore production contains 6 g / L potato extract, 20 g / L glucose, 20 g / L agar, and pH 5.6 ± 0.2.

[0034] The protoplast transformation medium (TB3) contains 200 g / L sucrose, 3 g / L yeast extract, 3 g / L hydrolyzed casein, and 15 g / L agar.

[0035] The culture medium (CM) used for the passage growth of transformants contains 10 g / L sucrose, 3 g / L acid-hydrolyzed casein, 3 g / L yeast extract, and 15 g / L agar.

[0036] The shake-flask seed growth medium (MA) consists of: 2.8 g / L ammonium sulfate, 4 g / L potassium dihydrogen phosphate, 0.6 g / L magnesium sulfate heptahydrate, 0.8 g / L calcium chloride dihydrate, 0.0005 g / L ferrous sulfate heptahydrate, 0.0017 g / L manganese sulfate monohydrate, 0.0014 g / L zinc sulfate heptahydrate, 0.0002 g / L cobalt chloride, 1 g / L peptone, 0.2 M disodium hydrogen phosphate-citrate buffer (pH 5.0), and 20 g / L glucose.

[0037] For shake-flask fermentation medium: Replace glucose in MA medium with ACM, microcrystalline cellulose, lactose, or MGD.

[0038] 2. Strains and primers

[0039] Trichoderma reesei strain Δku70 Rut-C30 As the starting strain, the gene encoding the dynamin-related protein M419DRAFT_36429 was replaced with the gene encoding the small protein M419DRAFT_120601. The unfunctioning small protein M419DRAFT_120601 was overexpressed using the inducible promoter Pcbh1 to construct the recombinant strain. The plasmids and strains used are shown in Table 1, and the primers and fragments used for transformation are shown in Table 2. Strain Δ36429-Δku70 Rut-C30 For Δku70 Rut-C30 Blank control strain with the M419DRAFT_36429 coding gene knocked out.

[0040] Table 1. Information on the plasmids and strains used to construct strain TriY120601.

[0041]

[0042]

[0043] Table 2 Primers and fragments required for strain transformation

[0044]

[0045]

[0046] 3. Methods

[0047] (1) Amplification of the target fragment from recombinant strain TriY120601

[0048] Using genomic DNA from Trichoderma reesei strain Rut-C30 as a template, polymerase chain reaction (PCR) was used to amplify the upstream homologous arm of M419DRAFT_36429 with primers 36429-up-F / 36429-up-R, the Pcbh1 promoter with primers Pcbh1-F / Pcbh1-R, the open reading frame of M419DRAFT_120601 with primers 120601-F / 120601-R, and the downstream homologous arm of M419DRAFT_36429 with primers 36429-down-F / 36429-down-R.

[0049] Using the genomic DNA of plasmid pCB-Hyg as a template, the terminator Teg1 and the resistance gene Hyg were amplified by polymerase chain reaction (PCR) with primers Teg1-Hyg-F / Teg1-Hyg-R, and the new plasmid backbone was amplified with primers pCB-F / pCB-R.

[0050] The PCR reaction system and steps are shown in Tables 3-8. The PCR products were separated by 1% agarose gel electrophoresis (170V, 10min). The bands that met the target size were excised and recovered. The obtained DNA was stored at -20℃ for later use.

[0051] Table 3. PCR system for amplifying the upstream homologous arm of M419DRAFT_36429

[0052]

[0053] Table 4. PCR system for amplifying the Pcbh1 promoter

[0054]

[0055]

[0056] Table 5. PCR system for amplifying the open reading frame of M419DRAFT_120601

[0057]

[0058] Table 6. PCR system for amplifying the terminator Teg1 and the resistance gene Hyg.

[0059]

[0060]

[0061] Table 7. PCR system for amplifying the downstream homologous arm of M419DRAFT_36429

[0062]

[0063] Table 8. PCR system for amplifying the overexpression plasmid backbone

[0064]

[0065] (2) Strain Δ36429-Δku70 Rut-C30 Amplification of the target fragment

[0066] Using the genomic DNA of Trichoderma reesei strain Rut-C30 as a template, polymerase chain reaction (PCR) was used to amplify the upstream knockout homologous arm of M419DRAFT_36429 with primers 36429-delete-up-F / 36429-delete-up-R, and the downstream knockout homologous arm of M419DRAFT_36429 with primers 36429-down-F / 36429-down-R.

[0067] Using the genomic DNA of plasmid pCB-Hyg as a template, the resistance gene Hyg was amplified by polymerase chain reaction (PCR) with primers 36429-Hyg-F / 36429-Hyg-R, and the new plasmid backbone was amplified with primers pCB-F / pCB-R.

[0068] The PCR reaction system and steps are shown in Tables 9-11. The PCR products were separated by 1% agarose gel electrophoresis (170V, 10min). The bands that met the target size were excised and recovered. The obtained DNA was stored at -20℃ for later use.

[0069] Table 9. PCR system for amplifying the upstream knockout homologous arm of M419DRAFT_36429

[0070]

[0071] Table 10 PCR system for amplifying the M419DRAFT_36429 knockout antibiotic resistance gene Hyg.

[0072]

[0073] Table 11 PCR system for amplifying the downstream knockout homologous arm of M419DRAFT_36429

[0074]

[0075] (3) Recombinant ligation was used to construct the pCB-Hyg-TriY120601 plasmid expression cassette.

[0076] The obtained upstream homologous arm of M419DRAFT_36429, Pcbh1 promoter, M419DRAFT_120601 open reading frame, terminator Teg1, resistance gene Hyg, and downstream homologous arm fragment of M419DRAFT_36429 were mixed thoroughly. The optimal amount of linearized vector used in the recombination reaction is 0.03 pmol.

[0077] Optimal linearization carrier dosage (0.03 pmol) = [0.02 × number of base pairs] ng

[0078] Optimal insert size (0.06 pmol) = [0.02 × number of base pairs] ng

[0079] After calculation, different volume fragments were added for fusion and connection, and the connection reaction system is shown in Table 12.

[0080] Table 12 Recombination reaction system of pCB-Hyg-TriY120601 plasmid

[0081] Assembly Reaction Mixture Volume 36429-up 30ng Pcbh1 30ng 120601 6ng Teg1+Hyg 100ng 36429-down 30ng pCB 70ng 2×MultiF Seamless Assembly Mix 10μL Distilled water Up to 20μL

[0082] (4) Seamless cloning and ligation to construct the pCB-Hyg-delete-36429 plasmid expression cassette

[0083] The obtained upstream knockout homologous arm of M419DRAFT_36429, the resistance gene Hyg, and the downstream knockout homologous arm fragment of M419DRAFT_36429 were mixed evenly, and different volume fragments were added for fusion ligation after calculation. The ligation reaction system is shown in Table 13.

[0084] Table 13 pCB-Hyg-delete-36429 plasmid recombination reaction system

[0085] Assembly Reaction Mixture Volume 36429-delete-up 30ng 36429-Hyg 80ng 36429-down 30ng pCB 70ng 2×MultiF Seamless Assembly Mix 10μL Distilled water Up to 20μL

[0086] The mixture was incubated at 50℃ for 60 min. 5 μL of the ligation mixture was then transferred to *E. coli* DH5α competent cells. The cells were incubated on ice for 20 min, followed by heat shock at 42℃ for 45 s. 900 μL of LB broth was added, and the cells were incubated at 37℃ and 200 rpm for 1 h. After centrifugation at 7000 rpm for 2 min, 200 μL of the solution was plated on LB agar plates containing AMP resistance and incubated overnight at 37℃. Positive transformants were transferred to 5 mL of LB broth containing AMP resistance and incubated overnight. After collecting the bacterial cells, plasmids were extracted for sequencing verification and stored at -20℃ for later use.

[0087] (5) TriY120601 expression cassette amplification

[0088] Using the pCB-Hyg-TriY120601 plasmid expression cassette as a template, the TriY120601 expression cassette was amplified by PCR using primers All-120601-F / All-120601-R (PCR system is shown in Table 14). The amplification products were recovered and stored at -20℃ for later use.

[0089] Table 14 PCR system for amplifying the TriY120601 expression cassette

[0090]

[0091]

[0092] (6)Δ36429-Δku70 Rut-C30 Expression cassette amplification

[0093] Using the pCB-Hyg-delete-36429 plasmid expression cassette as a template, Δ36429-Δku70 was amplified by PCR using primers All-delete-36429-F / All-delete-36429-R. Rut-C30 The expression cassette (PCR system is shown in Table 15) was used to recover the amplification products, which were then stored at -20°C for later use.

[0094] Table 15 Amplification of Δ36429-Δku70 Rut-C30 PCR system of expression cassette

[0095]

[0096] (7) Preparation of Trichoderma reesei protoplasts

[0097] The starting strain Trichoderma reesei Δku70 was cultured on PDA solid medium at 28°C. Rut-C30 Fresh spores were obtained after 7 days. The collected spores were inoculated into CM liquid complete medium and cultured at 28℃ and 150 rpm for 36 h. Mycelia were then collected by filtration, pressed dry with sterile filter paper, and transferred to 50 mL sterile centrifuge tubes. A wall-lysing enzyme was prepared using 1 M sorbitol solution and added to the 50 mL centrifuge tubes containing mycelia. Lysis was carried out at 30℃ and 9100 rpm for 3.5-4.0 h. The lysed samples were examined under a microscope to confirm protoplast lysis. The lysed mycelia were filtered through a sterile filter membrane, and the filtrate was collected into a clean 50 mL centrifuge tube. The mycelia were washed with a small amount of 1 M sorbitol. The tubes were centrifuged at 4℃ and 5000 rpm for 10 min. The supernatant was discarded, and the precipitate was resuspended in an appropriate amount of STC solution (containing 0.01 M Tris-HCl, 1 M sorbitol, and 50 mM CaCl2, pH 7.5). The number of protoplasts was observed under a microscope, and the final concentration was controlled at 5 × 10⁻⁶ per mL. 7 -5×10 8 Aliquot 100 μL into 1.5 mL centrifuge tubes and store at -80 °C or use directly for conversion.

[0098] (8) Protoplast transformation of Trichoderma reesei

[0099] Add 3-5 μg of the target fragment (concentration above 300 ng / μL) to a 1.5 mL aliquot of protoplasts, gently tap to mix, and incubate on ice for 20-25 min. Add 625 μL of pre-chilled PTC (dissolve 8 g PEG 3350 in 20 mL STC) in two separate portions, gently tap to mix, and incubate for 20 min. Transfer the mixture from the 1.5 mL EP tube to a 50 mL centrifuge tube, add 5 mL of protoplast transformation liquid medium (TB3), seal the tube, tilt it, and fix it in a shaker. Incubate at 26℃ and 100 rpm for 4-6 h. For solid TB3 antibiotic-free medium, after cooling to a suitable temperature, pour the medium into a 50 mL container, invert to mix, and then pour into a petri dish. After the medium solidifies, pour in the top layer of solid TB3 medium containing 50 μg / mL hygromycin. After the medium solidifies, invert the dish and incubate at 28℃ for 2-4 days. Pick single colonies of transformants for validation culture.

[0100] (9) Transformer verification

[0101] The selected Trichoderma reesei transformants were transferred to CM medium containing 50 μg / mL hygromycin and cultured for 3-4 days. The bacterial cells were scraped off, and the Trichoderma reesei genome was extracted according to the following method:

[0102] Add 0.1 g of quartz sand and 400 μL of lysis solution (containing 1 M Tris-HCl, 0.5 M EDTA, 20% SDS and 5 M NaCl, pH 8.0), and disrupt at room temperature for 5 min at 60 Hz. Centrifuge at 12000 rpm for 5 min at room temperature. Transfer the supernatant to a new 1.5 mL centrifuge tube, add 0.6 times the volume of isopropanol, mix well, and incubate at -20℃ for at least 30 min. Centrifuge at 12000 rpm for 10 min at room temperature, discard the supernatant, wash the precipitate with 70% ethanol, centrifuge at 12000 rpm for 5 min, discard the supernatant, dry the precipitate in a 50℃ oven, add 50 μL of sterile single-distilled water to dissolve the precipitate, and store at -20℃ or perform direct verification.

[0103] 1 μL of genomic DNA was used as a template to amplify the TriY120601 anchor fragment using upstream anchoring primers TYZ-up-F / TYZ-up-R and downstream anchoring primers TYZ-down-F / TYZ-down-R for verification. The 36429 open reading frame was amplified by knocking out the verification primers TYZ-delete-36429-F / TYZ-delete-36429-R to confirm complete knockout. The PCR reaction system is shown in Tables 16-18. The PCR products were separated by 1% agarose gel electrophoresis (170V, 10 min). The correctly verified TriY120601 and Δ36429-Δku70 were then separated. Rut-C30After transformants were passaged 3-4 times on CM resistant plates, they were transferred to PDA-free medium for sporulation culture for 7 days. The sporulation solution was then collected, mixed with 60% glycerol at a volume ratio of 1:1, and stored at -80°C.

[0104] Table 16 Verification of the upstream anchoring location of the TriY120601 expression cassette in the transformant genome.

[0105]

[0106]

[0107] Table 17 Validation of the downstream anchoring location of the TriY120601 expression cassette in the transformant genome

[0108]

[0109] Table 18 Verification of the M419DRAFT_36429 open reading frame knockout location in the transformant genome.

[0110]

[0111] Example 2: Application of Trichoderma recombinant strain TriY120601

[0112] Using 20 g / L ACM, 20 g / L microcrystalline cellulose, 20 g / L lactose, and 20 g / L MGD as enzyme inducers, the recombinant strain TriY120601 obtained in Example 1 and the blank control strain Δ36429-Δku70 (with the M419DRAFT_36429 coding gene knocked out) were cultured under shake-flask fermentation conditions. Rut-C30 and the original starting strain Δku70 Rut-C30 .

[0113] Fermentation results showed that the blank control strain Δ36429-Δku70 Rut-C30 and the original starting strain Δku70 Rut-C30 Under shake-flask fermentation conditions with 20 g / L ACM inducer, there was no significant difference in filter paper enzyme activity, indicating that the knockout of gene M419DRAFT_36429 does not affect the growth of the strain or significantly affect cellulase production. Filter paper enzyme activity comparison data can be found in [link to relevant data]. Figure 1 .

[0114] Recombinant strain TriY120601 was compared with the blank control strain Δ36429-Δku70 under shake-flask fermentation conditions with 20 g / L ACM inducer. Rut-C30 The activity of filter paper enzymes, exonucleases, xylanases, and extracellular proteins increased by 76% after 96 hours. Figure 2 ), 245% Figure 3 ), 64% Figure 4 ) and 75% Figure 5 ), increased by 46% in 144 hours ( Figure 2 ), 90% Figure 3 ), 97% Figure 4 ) and 74% Figure 5 ), but β-glucosidase activity ( Figure 6 ) and endonuclease activity ( Figure 7 The improvement was not significant.

[0115] Under shake-flask fermentation conditions of 20 g / L lactose, 20 g / L microcrystalline cellulose, and 20 g / L MGD inducer, the recombinant strain TriY120601 was compared with the blank control strain Δ36429-Δku70 for 96 h. Rut-C30 The enzyme activity of filter paper increased by 34% ( Figure 8 ), 45% Figure 9 ) and 34% Figure 10 Under shake-flask fermentation conditions of 20 g / L microcrystalline cellulose and 20 g / L MGD inducer, the recombinant strain TriY120601 showed a relative improvement over the blank control strain Δ36429-Δku70 at 144 h. Rut-C30 The enzyme activity of filter paper increased by 26% ( Figure 9 ) and 57% Figure 10 Under conditions of 20 g / L MGD inducer, continuous fed-batch fermentation in a 7 L fermenter (4 L fermentation broth) ultimately resulted in a stable production of 62 IU / mL of cellulase. Figure 11 ).

[0116] The hydrolytic capacity of the fermentation broth of recombinant strain TriY120601 was evaluated. Under conditions of 50℃ and 200 rpm, the same amount of enzyme solution (8 FPU / g) was used to enzymatically hydrolyze corn stalks pretreated with dilute acid steam explosion (20% dry matter content) containing lignin 33.5% ± 0.98%, cellulose 37.83% ± 0.41%, and hemicellulose 4.73% ± 0.07%). The glucose content obtained from the enzymatic hydrolysis of the crude enzyme solution produced by fermentation of recombinant strain TriY120601 was compared with that of the original strain Δku70. Rut-C30 The glucose content of the crude enzyme solution produced by fermentation is increased by at least 29%. Figure 12 ).

[0117] In summary, this invention discovered the knockout strain Δku70 Rut-C30 The addition of the gene encoding the dynamin-related protein M419DRAFT_36429 did not affect the growth and cellulase production capacity of the strain, providing a suitable integration site for the successful ectopic overexpression of the small protein M419DRAFT_120601. The recombinant strain TriY120601 in this application, compared with the blank control strain Δ36429-Δku70...Rut-C30 It has superior enzyme production capacity, can significantly improve the activity of cellulase in Trichoderma reesei and optimize the cellulase enzyme system, and has good potential for industrial application.

[0118] The above description is merely a preferred embodiment of the present invention. It should be noted that those skilled in the art can make various improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing a recombinant strain of Trichoderma reesei, characterized in that: Trichoderma reesei strain Δku70 Rut-C30 Using the starting strain, the gene encoding the dynamin-related protein M419DRAFT_36429 was replaced with the gene encoding the small protein M419DRAFT_120601. The small protein M419DRAFT_120601 gene was overexpressed using the inducible promoter Pcbh1 to construct a recombinant strain that produces high levels of cellulase. The nucleotide sequence of the inducible promoter Pcbh1 is shown in SEQ ID No. 1, and the nucleotide sequence of the gene encoding the small protein M419DRAFT_120601 is shown in SEQ ID No.

2.

2. A recombinant strain of *Trichoderma reesei*, TriY120601, which produces high levels of cellulase, is characterized by: The strain TriY120601, obtained using the construction method described in claim 1, has the accession number CGMCCNo.41526 and is deposited at the China General Microbiological Culture Collection Center on September 24, 2024.

3. A microbial agent that produces high levels of cellulase-degrading enzymes, characterized in that: The bacterial agent contains the Trichoderma recombinant strain TriY120601 as described in claim 2.

4. The application of the Trichoderma recombinant strain TriY120601 of claim 2 or the inoculum of claim 3 in the fermentation production of cellulase.

5. The fermentation method for enzyme production from the recombinant Trichoderma reesei strain TriY120601 according to claim 2, characterized in that: Add enzyme-inducing agents such as microcrystalline cellulose, lactose, and soluble inducers such as ACM or MGD to the fermentation medium.

6. The fermentation method for enzyme production from the recombinant Trichoderma reesei strain TriY120601 according to claim 5, characterized in that: The amount of microcrystalline cellulose, lactose, and soluble inducers ACM or MGD used is 20 g / L.

7. The application of the Trichoderma recombinant strain TriY120601 of claim 2 or the microbial agent of claim 3 in the degradation of lignocellulosic biomass.

8. The application according to claim 7, characterized in that: The lignocellulosic biomass mentioned includes crop straw and forestry waste.

9. A crude cellulase solution, produced by the fermentation enzyme production method described in claim 5.

10. The application of the crude cellulase solution according to claim 9 in the degradation of lignocellulosic biomass.

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