A method for constructing an engineered trichoderma reesei strain, a recombinant strain and application thereof

Abstract

This invention discloses a method for constructing engineered Trichoderma reesei strains, as well as the constructed recombinant strain TriY124993 and its applications. TriY124993 is based on the Trichoderma reesei strain Δku70. Rut‑C30 Using the starting strain, the M419DRAFT_36429 gene locus related to dynamin was replaced with an expression cassette containing the Pcbh1 promoter, the small protein M419DRAFT_124993 coding gene, and the terminator Teg1, to construct a recombinant strain producing high-yield cellulase. This strain has the accession number CGMCC No. 41527 at the China General Microbiological Culture Collection Center. This strain, TriY124993, can achieve highly efficient cellulase production using enzyme inducers microcrystalline cellulose, lactose, and soluble inducers, with enzyme activity higher than that of the starting strain.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, specifically to a method for constructing engineered Trichoderma reesei strains, recombinant strains, and their applications. Background Technology

[0002] Crop straw and forestry waste, rich in lignocellulose, are widely found in nature and possess advantages such as being renewable, abundant, and inexpensive. Traditional treatment methods, such as incineration and landfill, can lead to environmental pollution, soil pests, and waste renewable resources. Utilizing cellulase produced by filamentous fungi to degrade lignocellulose biomass can realize its resource utilization, hydrolyzing straw and other biomass into fermentable sugars, primarily glucose and xylose, which can then be used for fermentation to produce fuels and organic acids. This is crucial for national and societal sustainable development. Furthermore, cellulase has wide applications in the chemical, food, and energy sectors, demonstrating significant economic value.

[0003] *Trichoderma reesei* is currently a major cellulase-producing strain, producing a rich variety of cellulase-degrading enzymes, including exocellulase (CBH), endocellulase (EG), β-glucosidase (BGL), and hemicellulases that promote the hydrolysis of lignocellulose. The *Trichoderma reesei* Rut-C30 mutant strain is a high-yield cellulase strain. By knocking out the gene encoding ku70, which mediates non-homologous end joining, *Trichoderma reesei* Δku70 is produced. Rut-C30 This improves the efficiency of homologous recombination in strains (Guangtao Z, Hartl L, Schuster A, et al. Gene targeting in a nonhomologous endjoining deficient Hypocreajecorina. Journal of Biotechnology, 2009, 139(2): 146-151.).

[0004] Small proteins are short peptides or small proteins synthesized from small open reading frames (sORFs), generally no more than 100 amino acids in length. Currently, a large number of small proteins have been found in various organisms. Functional analysis of the identified small proteins has revealed their involvement in DNA replication and mRNA transcription, as well as protein synthesis and folding, substance transport, and responses to abiotic stress (Hassel KR, et al. Microproteins: Overlooked regulators of physiology and disease. iScience. 2023, 26(6): 106781.). However, due to their short sequences, difficulty in detection, and unknown structures, the functions of a considerable number of small proteins remain unknown. Currently, the functions of different small proteins in *Trichoderma reesei* and whether they regulate enzyme production are still unclear. Multi-omics analysis can provide possible mechanistic information and key modification targets when the mechanisms are unclear. Researchers have already employed multi-omics analysis, including genomics, transcriptomics, and proteomics, to study key enzyme production regulators under cellulase-induced synthesis conditions or in high-yielding strains. Currently, most efforts to construct high-yield cellulase-producing Trichoderma reesei strains are limited to identifying and modifying regulatory factors involved in cellulase synthesis at the transcriptional level. However, cellulase biosynthesis and secretion are regulated at multiple levels, including transcriptional, post-transcriptional, and post-translational regulation (Yang J, et al. Fungal strain improvement for efficient cellulase production and lignocellulosic biorefinery: current status and future prospects. Bioresource Technology 385: 129449.). On the other hand, cellulase synthesis is induced by cellulose, but during fermentation, soluble inducers containing sophorose are convenient for addition and have advantages such as low cost and the ability to achieve high-density culture (Li YH, et al. Overproduction of cellulase by Trichoderma reesei RUT C30 through batch-feeding of synthesized low-cost sugarmixture. Bioresource Technology 2016, 216: 503-510.). Therefore, studying the small proteins that change under soluble inducer-induced conditions through multi-omics analysis to explore the small proteins that regulate cellulase expression is a new approach to constructing high-yield cellulase strains.

[0005] In the process of constructing high-yielding strains, the selection of promoters is very important. Exocellulase CBH1 accounts for about 60% of the secreted proteins of Trichoderma reesei (Zhang YHP, Lynd LR. A functionally based model for hydrolysis of cellulose by fungal cellulase. Biotechnology and Bioengineering, 2006, 94(5): 888-898.). Its promoter Pcbhl has high promoter activity under the induction conditions with cellulose as the sole carbon source, so it is often used to construct strains with strong gene expression (Liu T'Wang T, Li X, et al. Improved heterologous gene expression in Trichoderma reesei by cellobiohydrolase I gene (cbh1) promoter optimization. Acta Biochimica et Biophysica Sinica, 2008, 40(2): 158-165.). Furthermore, since *Trichoderma reeseeris* lacks a stable free plasmid for expression, the expression of exogenous genes includes both random genomic integration and homologous recombination (Kuhls K, Lieckfeldt E, Samuels GJ, et al. Molecular evidence that the asexual industrial fungus *Trichoderma reeseeris* is a clonal derivative of the ascomycete *Hypocreajecorina*. Proceedings of the National Academy of Sciences, 1996, 93(15): 7755-7760.). Choosing a suitable integration site is important. Some gene knockouts have no significant effect on growth and metabolism. On the one hand, they do not interfere with the expression of host genes, and on the other hand, it is easier to distinguish the phenotypic effect of the target gene. Therefore, they can be used as neutral integration sites to express the target exogenous gene ( M, Nevalainen H, M, et al. A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei. Gene, 1987, 61(2): 155-164.). Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a method for constructing engineered Trichoderma reesei strains, recombinant strains, and their applications, achieved through the following technical solutions:

[0007] A method for constructing engineered Trichoderma reesei strains, using Trichoderma reesei strain Δku70 Rut-C30 For the starting strain, in the dynein-associated protein M419DRAFT The neutral site of the 36429 encoding gene was replaced with the Pcbh1 promoter and the small protein M419DRAFT. The gene encoding 124993 was used to overexpress the small protein M419DRAFT using the strong promoter Pcbh1. The 124993 gene was used to construct an engineered bacterium that produces high levels of cellulase. This recombinant strain was named TriY124993 in the laboratory. The nucleotide sequence of the promoter Pcbh1 is shown in SEQ ID No. 1, and the small protein M419DRAFT... _ The nucleotide sequence of the gene encoding 124993 is shown in SEQ ID No. 2, and the small protein M419DRAFT is also mentioned. _ The amino acid sequence of 124993 is shown in SEQ ID No. 3. The nucleotide sequence of the gene encoding 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_124993 is a very unique protein in ascomycetes, found only in *Trichoderma reesei* Rut-C30. M419DRAFT_124993 is a small protein of unknown function. This invention demonstrates that overexpression of M419DRAFT_124993 can increase cellulase production.

[0008] The recombinant strain TriY124993 has the accession number CGMCC No.41527 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, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0009] The recombinant strain TriY124993 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 by fermentation can be used to degrade lignocellulosic biomass.

[0010] 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.

[0011] The recombinant strain TriY124993 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.

[0012] The advantages of this invention are:

[0013] 1. This invention is the first to discover the regulatory function of an endogenous small protein from Trichoderma with unknown function in cellulase biosynthesis, demonstrating that overexpression of M419DRAFT using the inducible promoter Pcbh1 is effective. The 124993 gene can significantly enhance the cellulase production capacity of Trichoderma reesei, providing a new method for improving the cellulase production capacity of Trichoderma reesei, and also providing a recombinant Trichoderma strain TriY124993, which has good application potential.

[0014] 2. This invention discovered the knockout strain Δkku70 Rut-C30 The dynamin-related protein M419DRAFT The 36429 encoding gene does not affect the strain's growth or cellulase production ability; it is a small protein, M419DRAFT. The successful ectopic overexpression of the 124993 gene provides a suitable integration site.

[0015] 3. The addition of an enzyme inducer to the recombinant strain TriY124993 during fermentation significantly improves cellulase production. Compared with the control strain, the extracellular cellulase activity of the recombinant strain TriY124993 is increased by at least 39%. Attached Figure Description

[0016] Figure 1 It is strain Δ36429-Δku70 Rut-C30 and Δkuu70 Rut-C30 Comparison of filter paper enzyme activity during shake-flask fermentation under 20 g / L ACM induction conditions. (Strain Δkuu70) Rut-C30 It is the original strain, Δ36429-ΔΔkku70 Rut-C30 It is Δku70 Rut-C30 Control strain with the M419DRAFT_36429 coding gene knocked out.

[0017] Figure 2 It is the recombinant strains TriY124993 and Δ36429-Δku70 Rut-C30 Comparison of extracellular filter paper enzyme activity during shake-flask fermentation at 20 g / L ACM induction conditions for 96 and 144 h.

[0018] Figure 3 It is the recombinant strains TriY124993 and Δ36429-Δku70 Rut-C30 Comparison of growth on PDA, 20 g / L glucose, 20 g / L glycerol and 20 g / L lactose plates.

[0019] Figure 4 It is the recombinant strains TriY124993 and Δ36429-Δku70 Rut-C30 Comparison of exonuclease activity during shake-flask fermentation at 96 and 144 h under 20 g / L ACM induction conditions.

[0020] Figure 5 It is the recombinant strains TriY124993 and Δ36429-Δku70 Rut-C30 Comparison of extracellular endonuclease activity during shake-flask fermentation at 96 and 144 h under 20 g / L ACM induction conditions.

[0021] Figure 6 It is the recombinant strains TriY124993 and Δ36429-Δku70 Rut-C30 Comparison of extracellular protein content during shake-flask fermentation at 96 and 144 h under 20 g / L ACM induction conditions.

[0022] Figure 7 It is the recombinant strains TriY124993 and Δ36429-Δku70 Rut-C30 Comparison of extracellular β-glucosidase activity during shake-flask fermentation at 96 and 144 h under 20 g / L ACM induction conditions.

[0023] Figure 8 It is the recombinant strains TriY124993 and Δ36429-Δku70 Rut-C30Comparison of extracellular xylanase activity during shake-flask fermentation at 96 and 144 h under 20 g / L ACM induction conditions.

[0024] Figure 9 It is the recombinant strains TriY124993 and Δ36429-Δku70 Rut-C30 Comparison of extracellular filter paper enzyme activity during shake-flask fermentation at 96 and 144 h under 20 g / L lactose induction conditions.

[0025] Figure 10 It is the recombinant strains TriY124993 and Δ36429-Δku70 Rut-C30 Comparison of extracellular filter paper enzyme activity during shake-flask fermentation at 20 g / L microcrystalline cellulose induction conditions for 96 and 144 h.

[0026] Figure 11 It is the recombinant strains TriY124993 and Δ36429-Δku70 Rut-C30 Comparison of extracellular filter paper enzyme activity during shake-flask fermentation at 96 and 144 h under 20 g / L MGD induction conditions. Detailed Implementation

[0027] 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.

[0028] Example 1: Construction of the recombinant TriY124993 strain of Trichoderma reesei

[0029] The experimental materials and methods used are as follows:

[0030] 1. Culture medium

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] For shake-flask fermentation medium: Replace glucose in MA medium with ACM, microcrystalline cellulose, lactose, or MGD.

[0036] 2. Strains and primers

[0037] Trichoderma reesei strain Δku70 Rut-C30 As the starting strain, the neutral site of the gene encoding the dynamin-related protein M419DRAFT_36429 was replaced with an expression cassette containing the Pcbhl promoter, the gene encoding the small protein M419DRAFT_124993, and the terminator Tegl. The recombinant strain was constructed by overexpressing the unfunctional small protein M419DRAFT_124993 using the strong promoter Pcbhl. 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 Control strain with the M419DRAFT_36429 coding gene knocked out.

[0038] Table 1. Information on the plasmids and strains used to construct strain TriY124993.

[0039]

[0040] Table 2 Primers and fragments required for strain transformation

[0041]

[0042]

[0043]

[0044] 3. Methods

[0045] (1) Amplification of the target fragment from recombinant strain TriYl24993

[0046] 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 Pcbhl promoter with primers Pcbhl-F / Pcbhl-R, the open reading frame of M419DRAFT_124993 with primers 124993-F / 124993-R, and the downstream homologous arm of M419DRAFT_36429 with primers 36429-down-F / 36429-down-R.

[0047] Using the genomic DNA of plasmid pCB-Hyg as a template, the terminator Tegl and the hygromycin resistance gene Hyg were amplified by polymerase chain reaction (PCR) with primers Tegl-Hyg-F / Teg1-Hyg-R, and the new plasmid backbone was amplified with primers pCB-F / pCB-R.

[0048] 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.

[0049] Table 3. PCR system for amplifying the upstream homologous arm of M419DRAFT_36429

[0050]

[0051]

[0052] Table 4. PCR system for amplifying the Pcbh1 promoter

[0053]

[0054] Table 5. PCR system for amplifying the open reading frame of M419DRAFT 124993.

[0055]

[0056] Table 6. PCR system for amplifying the terminator Teg1 and the resistance gene Hyg.

[0057]

[0058]

[0059] Table 7. PCR system for amplifying the downstream homologous arm of M419DRAFT_36429

[0060]

[0061] Table 8. PCR system for amplifying the overexpression plasmid backbone

[0062]

[0063]

[0064] (2) Strain Δ36429-Δku70 Rut-C30 Amplification of the target fragment

[0065] 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.

[0066] 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.

[0067] 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.

[0068] Table 9. PCR system for amplifying the upstream knockout homologous arm of M419DRAFT_36429

[0069]

[0070] Table 10 PCR system for amplifying the M419DRAFT_36429 knockout antibiotic resistance gene Hyg.

[0071]

[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-TriY124993 plasmid expression cassette.

[0076] The obtained upstream homologous arm of M419DRAFT_36429, Pcbh1 promoter, M419DRAFT_124993 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 usage (0.03 pmol) = [0.02 x 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-TfiY124993 plasmid

[0081] Assembly Reaction Mixture Volume 36429-up 30ng Pcbhl 30ng 124993 6ng Tegl+Hyg 100ng 36429-down 30ng pCB 70ng 2xMultiF 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]

[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) TriY124993 expression cassette amplification

[0088] Using the pCB-Hyg-TriY124993 plasmid expression cassette as a template, the TriY124993 expression cassette was amplified by PCR using primers All-124993-F / All-124993-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 TriY124993 expression cassette

[0090]

[0091] (6)Δ36429-Δku70 Rut-C30 Expression cassette amplification

[0092] Using the pCB-Hyg-delete-36429 plasmid expression cassette as a template, Δ364293-Aku70 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.

[0093] Table 15 Amplification of Δ36429-Δku70 Rut-C30 PCR system of expression cassette

[0094]

[0095] (7) Preparation of Trichoderma reesei protoplasts

[0096] 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. The mycelia were then collected by filtration, pressed dry with sterile filter paper, and transferred to sterile 50 mL centrifuge tubes. A wall-lysing enzyme was prepared using 1 M sorbitol solution and added to the 50 mL centrifuge tubes containing the 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 8Aliquot 100 μL into 1.5 mL centrifuge tubes and store at -80 °C or use directly for conversion.

[0097] (8) Protoplast transformation of Trichoderma reesei

[0098] 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.

[0099] (9) Transformer verification

[0100] 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:

[0101] 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.

[0102] 1 μL of genomic DNA was used as a template to amplify the TriY124993 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 TriY124993 and Δ36429-Δku70 were then separated. Rut-C30 After 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.

[0103] Table 16 Verification of the upstream anchoring location of the TriY124993 expression cassette in the transformant genome.

[0104]

[0105] Table 17 Validation of the downstream anchoring location of the TriY124993 expression cassette in the transformant genome

[0106]

[0107] Table 18 M419DRAFT genome in transformants 36429 Open Reading Box Knockout Location Verification

[0108]

[0109]

[0110] Example 2: Application of Trichoderma recombinant strain TriY124993

[0111] 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 TriY124993 obtained in Example 1 and the 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 .

[0112] Fermentation results showed that the control strain Δ36429-Δku70 Rut-C30 and the original starting strain Δku70 Rut-C30Under 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 This indicates that the site is a neutral site that can be used to integrate and express exogenous genes in the host bacteria without affecting the growth of Trichoderma cells.

[0113] Under shake-flask fermentation conditions of 20 g / L ACM inducer, and control strain Δ36429-Δku70 Rut-C30 In comparison, the extracellular enzyme activity, exonuclease activity, and xylanase activity of recombinant strain TriY124993 increased by 39.1% after 144 h. Figure 2 ), 29.6% Figure 4 ) and 22.1% Figure 8 However, the activity of the endonuclease ( Figure 5 ), extracellular protein content ( Figure 6 ) and β-glucosidase activity ( Figure 7 No significant differences were observed. The recombinant strain TriY124993 showed no significant growth changes on PDA and 2% glucose, glycerol, and lactose plates. Figure 3 ).

[0114] Under shake-flask fermentation conditions of 20 g / L lactose, 20 g / L microcrystalline cellulose, and 20 g / L MGD inducer, the recombinant strain TriY124993 and the control strain Δ36429-Δku70 were compared after 144 h of fermentation. Rut-C30 Compared to the previous comparison, the extracellular filter paper enzyme activity increased by 66% ( Figure 9 ), 26% Figure 10 ) and 22% Figure 11 ).

[0115] In summary, this invention discovered the knockout strain Δku70 Rut-C30 The integration of the dynamin-related protein M419DRAFT_36429 gene did not affect the growth and cellulase production capacity of the strain, providing a suitable site for the successful integration and expression of the small protein M419DRAFT_124993. The recombinant strain TriY124993 in this application, compared to the 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 application potential.

[0116] 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 engineered Trichoderma reesei, characterized in that: Trichoderma reesei strain Δku70 Rut-C30 Using the starting strain, the neutral site of the gene encoding the dynamin-related protein M419DRAFT_36429 was replaced with the Pcbhl promoter and the gene encoding the small protein M419DRAFT_124993. The small protein M419DRAFT_124993 gene was overexpressed using the strong promoter Pcbhl to construct an engineered strain that produces high levels of cellulase. The nucleotide sequence of the promoter Pcbhl is shown in SEQ ID No. 1, and the nucleotide sequence of the gene encoding the small protein M419DRAFT_124993 is shown in SEQ ID No.

2.

2. A recombinant Trichoderma reesei strain TriY124993 with high production of cellulase, characterized by: The strain TriY124993, obtained using the construction method described in claim 1, has the accession number CGMCCNo.41527 and is deposited at the China General Microbiological Culture Collection Center on September 24, 2024.

3. A microbial agent that produces high levels of cellulose-degrading enzymes, characterized in that: The bacterial agent contains the Trichoderma recombinant strain TriY124993 as described in claim 2.

4. The application of the Trichoderma recombinant strain TriY124993 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 TriY124993 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 TriY124993 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 TriY124993 of claim 2 or the microbial agent of claim 3 in the degradation of lignocellulose 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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