Construction method of trichoderma reesei strain with high yield of cellulose degrading enzyme, recombinant strain and application

Abstract

The invention discloses a construction method of a trichoderma reesei strain with high yield of cellulose degrading enzyme, a recombinant strain Pcbh-OEDlc1-1 and application of the recombinant strain Pcbh-OEDlc1-1. The preservation number of the strain Pcbh-OEDlc1-1 is CGMCC (China General Microbiological Culture Collection Center) No.40648, the Trichoderma reesei strain deltaku70Rut-C30 is used as a starting strain, a promoter of a dynamic protein light chain Dlc1 is replaced by an inducible promoter Pcbh1, and a recombinant strain for overexpressing Dlc1 is constructed, so that a novel method for improving the cellulase production capacity of the Trichoderma reesei is provided. The invention also provides a fermentation method of the Pcbh-OEDlc1-1. According to the fermentation method, an enzyme-producing inducer microcrystalline cellulose, lactose or an acid-catalyzed inducer ACM is added into a fermentation medium. The recombinant strain and the crude cellulase liquid produced by the recombinant strain can be effectively applied to degradation of lignocellulose biomass to produce fuels and chemicals, and have good industrial application potential.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a construction method, a recombinant strain and an application of a Trichoderma reesei strain capable of producing high-yield cellulose-degrading enzymes. Background Art

[0002] The bioconversion and utilization of lignocellulosic biomass is crucial for sustainable development. This process generally involves three main steps: 1) feedstock pretreatment, 2) cellulose hydrolysis to obtain sugars, and 3) fermentation of hydrolyzed sugars to produce products. Step 2 is the core, and cellulose hydrolysis can be achieved through two different technical routes: chemical catalysis and enzymatic catalysis. Chemical methods, generally based on acid-catalyzed reactions, have disadvantages such as harsh process conditions, special requirements for equipment materials, and the need for post-hydrolysis neutralization, resulting in high acid and alkali consumption. Enzymatic hydrolysis, on the other hand, offers advantages such as mild reaction conditions, environmental friendliness, and high hydrolyzed sugar yields. Therefore, it shows better application prospects in industrial production. However, the high cost of cellulase production is the main bottleneck.

[0003] Filamentous fungi play a key role in the degradation of lignocellulosic biomass. These fungi, from diverse groups of microorganisms such as Trichoderma, Aspergillus, and Penicillium, produce a rich suite of hydrolytic enzymes to decompose cellulose and hemicellulose in lignocellulosic biomass, converting them into oligosaccharides and even monosaccharides. These enzyme-producing microorganisms have evolved sophisticated mechanisms to sense complex substrates and regulate the expression of these hydrolytic enzyme systems. Cellulase biosynthesis requires induction. Microcrystalline cellulose and lactose are commonly used inducers for cellulase synthesis, but the cost of using these two inducers in industrial production is high. The development and application of efficient soluble inducers for cellulase, such as glucose (syrup)-based, enzyme- or acid-catalyzed synthesis of mixed sugar inducers containing β-disaccharides and oligosaccharides, can achieve efficient cellulase production while reducing the production cost of cellulase. The extracellular cellulase secreted by filamentous fungi has high production, a comprehensive enzyme system, and is easy to collect, so it has great application potential. Among them, the research and application of cellulase production by Trichoderma reesei in the genus Trichoderma are the most in-depth and extensive. However, the enzyme production capacity of T. reesei strain cannot currently meet the requirements for industrial application in reducing hydrolyzed sugar preparation, and it needs to be modified.

[0004] Reducing the production cost of cellulase requires the selection of strains with strong enzyme production capacity, so genetic modification of filamentous fungi has attracted widespread attention. The Trichoderma reesei Rut-C30 mutant strain is a high-producing cellulase strain. The gene cre1 that regulates carbon catabolic repression (CCR) in this strain has been modified, which weakens the CCR effect to a certain extent, thereby improving cellulase production capacity. However, further improvement in enzyme production requires a combination of genetic engineering and fermentation process improvements. Rut-C30 It is a genetically modified engineered bacterium that uses Rut-C30 as the starting strain and knocks out the coding gene ku70 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 dmm2inDNA repair and cellulase production. Biotechnology for Biofuels and Bioproducts. 2022, 15(1): 34).

[0005] At present, the construction of high-yield cellulase-producing Trichoderma reesei strains is mostly limited to exploring and modifying transcription factors involved in regulating cellulase synthesis-related genes from the transcriptional regulatory level, including transcription activators and transcription repressors. However, cellulase biosynthesis and secretion are regulated at multiple levels. In many eukaryotic cells, the microtubule cytoskeleton is responsible for the long-distance movement and spatial organization of intracellular vesicles, organelles, and large complexes containing proteins and RNA. Microtubules are polarized structures with a negative pole and a positive pole. Minus-end-directed dynein and plus-end-directed kinesin participate in many microtubule-based biological functions, among which dynein participates in various basic cellular processes, including nuclear migration, mitotic spindle organization, chromosome segregation in mitosis, and the position and function of many intracellular organelles (Reck-Peterson et al. The cytoplasmic dynein transport machinery and its many cargoes. Nature Reviews Molecular Cell Biology. 2018, 9(6): 382-398).

[0006] Dyneins can be divided into two types: axonal dyneins and cytoplasmic dyneins. Axonal dyneins are responsible for transporting substances from the axon to the cell body, while cytoplasmic dyneins are responsible for negatively transporting specific substances within the cell to microtubules, including organelles, proteins, mRNA, endosomes and viruses. Cytoplasmic dyneins are multi-subunit complexes, including cytoplasmic dynein 1 (Dynein1) and cytoplasmic dynein 2 (Dynein2). These protein complexes contain four subunits: dynein heavy chain (DHC), dynein intermediate chain (DIC), dynein light intermediate chain (DLIC) and dynein light chain (DLC). Among them, Roadblock, LC8 (Dlc1) and TCTEX are light chains shared by dynein 1 and dynein 2 (Toropova et al. Structure of the dynein-2 complex and its assembly with intraflagellar transport trains. Nature Structural and Molecular Biology. 2019, 26(9): 823-829).

[0007] Dynein plays an important role in the transport of different substances within cells of different organisms. However, there are no reports on the effects of different subunits of cytoplasmic dynein on the synthesis and secretion of enzyme proteins, especially cellulase, and its function has not been studied in filamentous fungi. Summary of the Invention

[0008] The present invention aims to address the shortcomings of the prior art in that the effect of dynein on enzyme production has not been explored, and to provide a method for constructing a Trichoderma reesei strain that produces high cellulose-degrading enzymes, a recombinant strain, and its application, which are achieved through the following technical solutions:

[0009] A method for constructing a high-yield cellulose-degrading enzyme-producing Trichoderma reesei strain: using Trichoderma reesei strain Δku70 Rut-C30 For the starting strain, the dynein light chain 1 (Dlc1) promoter, Pdlc1, was replaced with the inducible promoter, Pcbh1, to construct a strain that overexpresses Dlc1. The nucleotide sequence of the inducible promoter, Pcbh1, is shown in SEQ ID No. 1. Dlc1 is the light chain of dynein. Because the protein is relatively small, it is easier to engineer. The present invention demonstrates that overexpressing Dlc1 can increase cellulase production. The nucleotide sequence of Dlc1 is shown in SEQ ID No. 2, and the amino acid sequence it encodes is shown in SEQ ID No. 3. The nucleotide sequence of the Dlc1 promoter, Pdlc1, is shown in SEQ ID No. 4.

[0010] A strain of Trichoderma reesei with high production of cellulose degrading enzymes, named Pcbh-OEDlc1-1, has a deposit number of CGMCC No. 40648 and is deposited in the General Microbiology Center of the China Culture Collection Administration Committee on June 5, 2023. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The Pcbh-OED1c1-1 is a strain of Trichoderma reesei strain Δku70. Rut-C30 As a starting strain, the promoter Pdlc1 of the dynein light chain Dlc1 was replaced with the inducible promoter Pcbh1 to construct a recombinant strain overexpressing Dlc1. The nucleotide sequence of the inducible promoter Pcbh1 is shown in SEQ ID No. 1.

[0011] The present invention also provides a high-yield cellulose-degrading enzyme bacterial agent, comprising the recombinant Trichoderma reesei strain Pcbh-OEDlc1-1. Also provided is the use of the Trichoderma reesei strain Pcbh-OEDlc1-1 or a bacterial agent containing the strain in the fermentation production of cellulase.

[0012] Use of the recombinant Trichoderma reesei strain Pcbh-OEDlc1-1 or a bacterial agent containing the strain in the degradation of lignocellulosic biomass. The lignocellulosic biomass includes, but is not limited to, various types of crop straw (such as corn straw), forestry waste, cellulose-containing food (processing) waste, and domestic and industrial waste.

[0013] The present invention also provides a method for culturing and fermenting a recombinant strain of Trichoderma reesei Pcbh-OEDlc1-1 with a high yield of cellulose-degrading enzymes to produce cellulase, which comprises adding an enzyme-producing inducer to the fermentation medium. The enzyme-producing inducer includes microcrystalline cellulose, lactose, or an enzyme (acid)-catalyzed glucose (syrup) transglycoside reaction to synthesize a mixed sugar inducer containing β-disaccharides and oligosaccharides, etc., preferably an inducer synthesized by an acid-catalytic reaction (Acid-catalytic synthesized mixture, ACM). The preparation of the ACM refers to the patent "A High-Efficiency Cellulase Inducer and Its Preparation and Application Method", application number 202210201654.5. The addition of the enzyme-producing inducer enables the strain to efficiently produce cellulase, increase enzyme activity and enzymatic performance on cellulose.

[0014] Furthermore, the amount of the microcrystalline cellulose is preferably 2 g / L, the amount of the lactose is preferably 2 g / L, and the amount of the acid-catalyzed inducer ACM is preferably 1 g / L.

[0015] The present invention also provides a crude cellulase enzyme solution produced by the above fermentation enzyme production method. The crude cellulase enzyme solution can be used in degrading lignocellulose biomass.

[0016] The advantages of the present invention are:

[0017] 1. This paper first discovered the regulation of dynein light chain on the production of cellulase in Trichoderma reesei, and proved that overexpression of Dlc1 can significantly improve the production of cellulase in Trichoderma reesei. Rut-C30 As the starting strain, the promoter of the dynein light chain encoding gene Dlc1 was replaced with the inducible strong promoter Pcbh1, and a new strain Pcbh-OEDlc1-1 with a high Dlc1 expression level was obtained. This revealed for the first time the regulatory function of dynein in cellulase biosynthesis, providing a new method to improve the cellulase production ability of Trichoderma reesei, and has good industrial application potential.

[0018] 2. The addition of an enzyme inducer during fermentation significantly increased cellulase production. Compared to the control strain, the recombinant strain Pcbh-OEDlcl-1 showed at least a 50% increase in cellulase production. Furthermore, continuous fed-batch fermentation of the strain Pcbh-OEDlcl-1 under ACM induction conditions in a 7 L fermentor (4 L fermentation broth) consistently produced 46 IU / mL of cellulase.

[0019] 3. Hydrolysis of pretreated corn straw using cellulase produced by the recombinant strain Pcbh-OED1c1-1 showed an approximately 16% increase in glucose yield compared to the starting strain under equivalent enzyme activity conditions, and the hydrolysis time was shortened by 24 hours. This suggests that the recombinant strain Pcbh-OEDlc1-1 and the cellulase it produces can be used to degrade lignocellulosic biomass to produce reducing sugars, which can then be further fermented to produce biofuels and bio-based chemicals, demonstrating promising industrial application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Conservative phylogenetic tree of Trichoderma reesei Rut-C30 among ascomycete fungi.

[0021] Figure 2 strains Pcbh-OEDlc1-1 and Δku70 Rut-C30 Comparison of filter paper enzyme activities in shake flask fermentation under 2 g / L microcrystalline cellulose induction conditions.

[0022] Figure 3 strains Pcbh-OEDlc1-1 and Δku70 Rut-C30 Comparison of exonuclease activity in shake flask fermentation under 2 g / L microcrystalline cellulose induction conditions.

[0023] Figure 4strains Pcbh-OEDlc1-1 and Δku70 Rut-C30 Comparison of endo-enzyme activities in shake flask fermentation under 2g / L microcrystalline cellulose induction conditions.

[0024] Figure 5 strains Pcbh-OEDlc1-1 and Δku70 Rut-C30 Comparison of β-glucosidase activity in shake flask fermentation under 2 g / L microcrystalline cellulose induction conditions.

[0025] Figure 6 strains Pcbh-OEDlc1-1 and Δku70 Rut-C30 Comparison of xylanase activity in shake flask fermentation under 2 g / L microcrystalline cellulose induction conditions.

[0026] Figure 7 strains Pcbh-OEDlc1-1 and Δku70 Rut-C30 Comparison of filter paper enzyme activities in shake flask fermentation under 2 g / L lactose induction conditions.

[0027] Figure 8 strains Pcbh-OEDlc1-1 and Δku70 Rut-C30 Comparison of filter paper enzyme activities in shake flask fermentation under 2 g / L ACM induction conditions.

[0028] Figure 9 strains Pcbh-OEDlc1-1 and Δku70 Rut-C30 Comparison of enzyme activities in continuous fed-batch fermentation in a 7L fermentor (4L fermentation broth) under ACM induction conditions.

[0029] Figure 10 strains Pcbh-OEDlc1-1 and Δku70 Rut-C30 Comparison of the glucose production effect of corn straw after hydrolysis pretreatment of the produced crude cellulase liquid. DETAILED DESCRIPTION

[0030] The specific embodiments of the present invention are 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. If no specific experimental methods or conditions are specified, they are performed according to conventional methods or conditions described in the literature in this field or according to the product instructions.

[0031] Example 1 Construction of Trichoderma reesei strain Pcbh-OEDlc1-1

[0032] The experimental materials and methods used are as follows:

[0033] 1. Culture medium

[0034] The potato dextrose agar (PDA) medium used for the sporulation of Trichoderma reesei contained 6 g / / L potato extract, 20 g / L glucose, 20 g / L agar, and a pH value of 5.6±0.2.

[0035] 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.

[0036] The culture medium (CM) used for subculture growth of transformants contained 10 g / L sucrose, 3 g / L acid hydrolyzed casein, 3 g / L yeast powder, and 1.5% agar.

[0037] Shake flask seed growth medium (MA): 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 sodium dihydrogen phosphate-citrate buffer (pH 5.0), 2% glucose.

[0038] For shake flask fermentation medium: replace glucose in MA medium with microcrystalline cellulose, lactose or ACM.

[0039] The culture medium for fermentation tank seed growth contains 25g / L glucose, 10g / L corn steep liquor, 0.66g / L calcium chloride dihydrate, 10g / L potassium dihydrogen phosphate, 5g / L ammonium sulfate, 1g / L magnesium sulfate, 0.0005g / L ferrous sulfate, 0.0017g / L manganese sulfate, 0.0014g / L zinc sulfate, 0.0002g / L cobalt chloride, and 0.076g / L defoamer.

[0040] The fermentation medium used in a 7 L fermentor contained 5 g / L glucose, 25 g / L lactose, 23 g / L corn steep liquor (Com extract liquid, Tianjin Lifalong Chemical Technology Co., Ltd.), 0.4 g / L calcium chloride, 5 g / L potassium dihydrogen phosphate, 4.8 g / L ammonium sulfate, 0.85 g / L magnesium sulfate heptahydrate, 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, and 0.28 g / L defoamer.

[0041] 2. Strains and Primers

[0042] Trichoderma reesei strain Δku70 Rut-C30As the starting strain, the promoter Pdlc1 of the dynein light chain Dlc1 was replaced with the inducible promoter Pcbh1 to obtain the strain Pcbh-OEDlc1-1. The plasmids and strains used are shown in Table 1, and the primers and fragments used for strain transformation are shown in Table 2.

[0043] Table 1 Plasmid and strain information used to construct strain Pcbh-OEDlc1-1

[0044]

[0045] Table 2 Primers and fragments required for strain transformation

[0046]

[0047]

[0048] 3. Methods

[0049] (1) Dlc1 promoter prediction

[0050] To replace the endogenous promoter, the Dlc1 promoter, Pdlc1, was analyzed. The upstream sequence of the D1c1 open reading frame (1500-2000 bp) was used for promoter prediction using Promoter 2.0 - DTU Health Tech-Bioinformatic Services. The predicted promoter, Pdlc1 (SEQ ID No. 4, 1711 bp upstream of the gene), was deleted and replaced with the strong inducible promoter, Pcbh1 (SEQ ID No. 1).

[0051] (2) Amplification of target fragment

[0052] Using genomic DNA from Trichoderma reesei strain Rut-C30 as a template, polymerase chain reaction (PCR) was performed using primers Pcbh1-F / Pcbh1-R for Pcbh1 to amplify the Pcbh1 promoter. The upstream fragment of the Dlc1 promoter was amplified using primers Pdlc-up-F / Pdlc-up-R. The Dlc1 open reading frame and its downstream sequence were amplified using primers Dlc1-dn-F / Dlc1-dn-R. The PCR reaction system and steps are shown in Tables 3, 4, and 5. PCR products were separated by 1% agarose gel electrophoresis (140 V, 20 min). Bands of the target size were excised and recovered, and the resulting DNA was stored at -20°C until further use.

[0053] Table 3 PCR system for amplifying the Pcbh1 promoter

[0054]

[0055] Table 4 PCR system for amplifying the upstream fragment of the Dlc1 promoter

[0056]

[0057]

[0058] Table 5: PCR system for amplifying the Dlc1 open reading frame and its downstream sequences

[0059]

[0060] (3) Homologous recombination to connect the Pcbh1 promoter, Dlc1 open reading frame and its downstream sequence

[0061] The obtained Pcbh1 promoter fragment, Dlc1 open reading frame and its downstream sequence fragment were added to the fusion system with the same mass fragments for fusion connection and amplification and recovery. The PCR reaction system is shown in Table 6.

[0062] Table 6 PCR system for amplifying the Dlcl open reading frame and its downstream sequences

[0063]

[0064]

[0065] (4) Enzyme digestion and ligation to construct the pBS-Hyg-Pcbh-dlc1 plasmid expression cassette

[0066] The plasmid pBS-Hyg was digested at the Kpn I and Cla I restriction sites to obtain a linear backbone. The resulting linear backbone (120 ng) was mixed with the upstream fragment of the Dlc1 promoter (20 ng). 5 μL of homologous recombination enzyme was added, and the reaction mixture was made up to 10 μL with double-distilled water. The reaction was incubated at 50°C for 30 min. The entire ligation system was removed and transformed into Escherichia coli DH5α. Positive transformants were transferred to 5 mL of LB liquid medium (containing AMP) and cultured overnight. The cells were harvested and the plasmid was extracted. The extracted plasmid was further digested with BamHI and Xba I. The recovered backbone was then ligated with the Dlc1 open reading frame and its downstream sequence fragments through the same homologous recombination ligation and E. coli transformation. Positive transformants were harvested and the pBS-Hyg-Pcbh-dlc1 plasmid was extracted. The plasmid was sequenced and stored at -20°C until further use.

[0067] (5) Amplification of the Pcbh-OEdlc1 expression cassette

[0068] Using the pBS-Hyg-Pcbh-dlc1 plasmid expression cassette as a template, the Pcbh-OEdlc1 expression cassette was amplified by PCR with the Pcbh1 primers Pdlc-up-F / Dlc1-dn-R (Table 7). The amplified product was recovered and stored in a -20°C refrigerator for future use.

[0069] Table 7 PCR system for amplifying the Pcbh-OEdlc1 expression cassette

[0070]

[0071] (6) Preparation of Trichoderma reesei protoplasts

[0072] The starting strain Trichoderma reesei Δku70 was cultured on PDA solid medium at 28°C. Rut-C30 After 7 days, fresh spores were obtained. The collected spores were inoculated into CM liquid complete medium, cultured at 28°C and 150rpm for 36 minutes, and then the mycelia were collected by filtration. The mycelia were pressed dry with sterile filter paper and transferred to an empty sterile 50mL centrifuge tube. A 1M sorbitol solution was used to prepare a wall-lytic enzyme, which was added to a 50mL centrifuge tube containing mycelia. The mycelia were lysed at 30°C and 90-100rpm for 3.5-4.0 hours. The lysed samples were examined under a microscope to confirm the protoplast lysis. The lysed mycelia were filtered with a sterile filter membrane, and the filtrate was collected into a clean 50mL centrifuge tube. The mycelia were washed with a small amount of 1M sorbitol. Centrifuged at 4°C and 5000rpm for 10 minutes, the supernatant was discarded, and an appropriate amount of STC solution (containing 0.01M Tris-HCl, 1M sorbitol and 50mM CaCl2, pH 7.5) was added to resuspend the precipitate. The number of protoplasts was observed under a microscope to control the final concentration to 5×10 per milliliter. 7 -5×10 8 Aliquot 100 μL of the aliquot into 1.5 mL centrifuge tubes and store at -80°C or use directly for transformation.

[0073] (7) Protoplast transformation of Trichoderma reesei

[0074] Add 2-3 μg of the target fragment (concentration 200 ng / μL or higher) to the aliquoted protoplasts, gently flick to mix, and let stand on ice for 20-25 minutes. Add 625 μL of PTC (8g PEG 3350 dissolved in 20 mL of STC) in two separate batches, gently shake to mix, and let stand for 20 minutes. Transfer the mixture from the 1.5 mL EP tube to a 50 mL centrifuge tube and add 5 mL of protoplast transformation liquid medium (TB3). Seal the tube and tilt it in a shaker. Let it recover at 26°C and 100 rpm for 4-6 hours. Cool the tube to the desired temperature and pour 50 mL of solid TB3 medium (antibiotic-free) into the tube. Invert to mix thoroughly, then pour into a 15 cm culture dish. Once the medium solidifies, add a layer of solid TB3 medium containing 50 μg / mL hygromycin. Once the medium solidifies, invert the tube and incubate it at 28°C for 2-4 days. Select a single colony of transformants for verification culture.

[0075] (8) Transformant Verification

[0076] Randomly selected Trichoderma reesei transformants were transferred to CM medium containing 50 μg / mL hygromycin and cultured for 1-2 days. The cells were scraped and the Trichoderma reesei genome was extracted according to the following method:

[0077] 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), crush at 60 Hz at room temperature for 5 min, centrifuge at 12000 rpm at room temperature for 5 min, aspirate the supernatant and transfer it to a new 1.5 mL centrifuge tube, add 0.6 times the volume of isopropanol, mix well and place at -20 ° C for at least 30 min, centrifuge at 12000 rpm at room temperature for 10 min, discard the supernatant and wash the precipitate with 70% ethanol, centrifuge at 12000 rpm for 5 min, discard the supernatant, dry the precipitate in a 50 ° C oven, add 50 μL of sterile distilled water to dissolve the precipitate, store at -20 ° C or directly use for verification.

[0078] 1 μL of the genome was used as a template to amplify the Pcbh-OEdlc1 anchor fragment using the upstream anchor primers dlc-proex-UA-F / HY-R for verification. The PCR reaction system is shown in Table 8. PCR products were separated by 1% agarose electrophoresis (140 V, 20 min). Correctly identified Pcbh-OEDlc1 transformants were serially passaged 3-4 times on CM resistance plates and then transferred to PDA-free medium for conidia production. After 7 days of culture, the spore fluid was collected, mixed with 60% glycerol at a volume ratio of 1:1, and stored in a -80°C freezer.

[0079] Table 8 Verification of the anchor position of the Pcbh-OEdlc1 expression cassette in the transformant genome

[0080]

[0081] Example 2 Application of Trichoderma reesei strain Pcbh-OEDlc1-1

[0082] Dynein light chain Dlc1 is a relatively conserved protein in ascomycetes, and the identity of its homologous proteins in filamentous fungi is at least 70% ( Figure 1 ).

[0083] The Pcbh-OEDlc1-1 strain obtained in Example 1 was cultured under shake flask fermentation conditions with 2 g / L microcrystalline cellulose, 2 g / L lactose or 1 g / L ACM induction, and the Pcbh-OEDlc1-1 strain obtained in Example 1 was cultured in a 7 L fermentor (4 L fermentation broth) under ACM induction conditions. The same conditions were used to culture the Pcbh-OEDlc1-1 strain in Example 1 with the Trichoderma reesei strain Δku70. Rut-C30 As the control strain, the fermentation enzyme production performance test results are as follows Figure 2-10 shown.

[0084] The fermentation results showed that the recombinant strain Pcbh-OED1c1-1 increased the activity of filter paper enzyme, endo-enzyme and exo-enzyme by 60% ( Figure 2 )、14%( Figure 3 ) and 25% ( Figure 4 ), and the activity of hemicellulase xylanase also increased significantly by 34% ( Figure 6 ), but the β-glucosidase activity was not significantly increased ( Figure 5 ).

[0085] Under the conditions of 2g / L lactose induction and 1g / L ACM induction, the strain Pcbh-OEDlc1-1 had a relative Δku70% relative to the starting strain at 96h. Rut-C30 The enzyme activity of filter paper increased by 48% ( Figure 7 ) and 51% ( Figure 8 Under ACM induction conditions, continuous fed-batch fermentation in a 7-L fermenter (4-L fermentation broth) could stably produce 46 IU / mL of cellulase, which was significantly higher than that of the starting strain Δku70. Rut-C30 Increase by 135% ( Figure 9 ).

[0086] At 50°C and 200 rpm, corn stover (33.5%±0.98% lignin, 37.83%±0.41% cellulose, 4.73±0.07%) pretreated with dilute acid steam explosion and a dry matter content of 20% was enzymatically hydrolyzed with the same enzyme amount (6 FPU / g). The glucose obtained by enzymatic hydrolysis of the enzyme produced by the recombinant strain Pcbh-OEDlc1-1 was higher than that of the starting strain Δku70.Rut-C30 The glucose content of the enzyme solution produced by the fermentation of the recombinant strain Pcbh-OEDlc1-1 increased by at least 16%, and the glucose content of the enzyme solution produced by the fermentation of the recombinant strain Pcbh-OEDlc1-1 exceeded 70g / L after 72h of enzymatic hydrolysis. The results showed that under the conditions of equal enzyme activity, the hydrolysis time of the enzyme solution produced by the fermentation of the recombinant strain Pcbh-OEDlc1-1 was shortened by 24h, showing better enzymatic hydrolysis ability. Using the same experimental method, wheat straw, rice straw, sugarcane straw, bark, fallen leaves, and branches were used instead of corn straw, and after pretreatment, they were treated with Pcbh-OEDlc1-1 and Δku70 Rut-C30 The enzyme solution produced by fermentation was enzymatically hydrolyzed, and the results showed that the enzyme solution produced by the recombinant strain could obtain a higher reducing sugar content.

[0087] In summary, the recombinant strain Pcbh-OEDlc1-1 of the present application is relatively Rut-C30 The enzyme liquid produced by Pcbh-OED1c1-1 fermentation has better enzyme production capacity and has better enzymatic hydrolysis performance on pretreated straw and other lignocellulosic biomass. It is proved that overexpression of Dlc1 can significantly improve the enzyme activity of Trichoderma reesei cellulase and optimize the enzyme system of cellulase, which has good industrial potential.

[0088] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and supplements without departing from the method of the present invention, and these improvements and supplements should also be considered as the scope of protection of the present invention.

Claims

1. A method for constructing a high-yield cellulose-degrading enzyme Trichoderma reesei strain, characterized in that: Trichoderma reesei strain Δku70 Rut-C30 As a starting strain, the promoter of the dynein light chain Dlc1 was replaced with the inducible promoter Pcbh1 to construct a strain overexpressing Dlc1. The nucleotide sequence of the inducible promoter Pcbh1 is shown in SEQ ID No.

1.

2. A Trichoderma reesei strain Pcbh-OEDlc1-1 that produces high levels of cellulose-degrading enzymes, characterized in that: The deposit number of the strain is CGMCC No.40648, which is deposited in the General Microbiology Center of China Culture Collection Administration Committee, and the deposit date is June 5, 2023; the strain is based on the Trichoderma reesei strain Δku70 Rut-C30 As a starting strain, the promoter of the dynein light chain Dlc1 was replaced with the inducible promoter Pcbh1 to construct a recombinant strain overexpressing Dlc1. The nucleotide sequence of the inducible promoter Pcbh1 is shown in SEQ ID No.

1.

3. A bacterial agent for high-yield cellulose-degrading enzyme, characterized in that: The bacterial agent contains the Trichoderma reesei strain Pcbh-OEDlc1-1 according to claim 2.

4. Use of the Trichoderma reesei strain Pcbh-OEDlc1-1 according to claim 2 or the bacterial agent according to claim 3 in fermentation production of cellulase.

5. Use of the Trichoderma reesei strain Pcbh-OEDlc1-1 according to claim 2 or the bacterial agent according to claim 3 in degrading lignocellulosic biomass.

6. The use according to claim 5, characterized in that The lignocellulosic biomass includes crop straw and forestry waste.

7. The method for producing high-yield cellulose-degrading enzyme by fermentation of the Trichoderma reesei strain Pcbh-OEDlc1-1 according to claim 2, characterized in that: An enzyme production inducer is added to the fermentation medium, wherein the enzyme production inducer is microcrystalline cellulose, lactose or acid-catalyzed reaction synthetic inducer ACM.

8. A crude cellulase solution produced by the fermentation enzyme production method according to claim 7.

9. Use of the crude cellulase solution according to claim 8 in degrading lignocellulosic biomass.

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