Trichoderma reesei engineering strain with high yield of cellulase and construction method and application thereof

Abstract

The present application relates to the field of genetic engineering and microbial fermentation technology, and specifically discloses a high-yield cellulase Trichoderma reesei engineering strain, a construction method thereof and an application, wherein the Trichoderma reesei strain is used as a starting strain, a lactate-5'-phosphoric acid carboxylic acid enzyme coding gene in the genome of the Trichoderma reesei strain is replaced by a hygromycin resistance coding gene, and a Δ pyr4 intermediate strain is obtained pyr4 :: hph Then, a L-rhamnonate dehydrase-like enzyme gene in the genome of the Δ pyr4 :: hph intermediate strain is replaced by a lactate-5'-phosphoric acid carboxylic acid enzyme coding gene, and a Δ pyrG engineered strain is obtained Mesm1 :: pyrG , namely the high-yield cellulase Trichoderma reesei engineering strain. The Trichoderma reesei engineering strain provided by the present application significantly improves the enzyme activity, extracellular protein content and saccharified glucose yield compared with the wild-type starting strain.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and microbial fermentation technology, specifically to a high-yield cellulase-producing Trichoderma reesei engineered strain, its construction method, and its application. Background Technology

[0002] Cellulases are a class of complex enzyme systems that synergistically degrade cellulose, mainly comprising endo-β-1,4-glucanase EG, exo-β-1,4-glucanase CBH, and β-glucosidase BGL; the ratio and synergistic activity of these three enzymes directly determine the hydrolysis efficiency of cellulose. Cellulases are widely used in biofuel production, textile biopolishing, denim stone washing, paper deinking, detergent additives, and feed enzyme preparations, and their industrial applications can significantly reduce process energy consumption and environmental pollutant emissions.

[0003] Trichoderma reesei ( Trichodermareesei ) is a filamentous fungus, *Crassostrea rubrum* ( Hypocreajecorina The asexual form of *Trichoderma* belongs to the genus *Trichoderma* in the order Moniliales. Trichoderma Because it can secrete a highly efficient and stable cellulase system, it has become the core strain for industrial-grade cellulase production.

[0004] However, wild-type Trichoderma reesei strains isolated from nature generally suffer from low enzyme production capacity and imbalanced enzyme systems, such as insufficient β-glucosidase activity, which easily leads to cellobiose accumulation and inhibits EG / CBH activity, making it difficult to meet the cost and efficiency requirements of industrial production. Therefore, it is necessary to genetically engineer naturally isolated Trichoderma reesei to improve its enzyme production capacity. Summary of the Invention

[0005] To provide a genetic engineering method for improving the enzyme production capacity of *Trichoderma reesei* strains, this invention provides a high-cellulase-producing *Trichoderma reesei* engineered strain, its construction method, and its applications. The *Trichoderma reesei* engineered strain obtained by the genetic engineering method provided by this invention significantly improves enzyme activity, extracellular protein content, and saccharified glucose yield compared to the wild-type starting strain.

[0006] This invention also provides a method for constructing a high-cellulase-producing Trichoderma reesei engineered strain, using Trichoderma reesei as the starting strain and extracting the orotate-5'-phosphate carboxylase encoding gene from its genome. pyr4 Replace with hygromycin resistance encoding gene hph , obtain △ pyr4 :: hph Intermediate strain; then the △ pyr4 :: hph L-rhamnonate dehydratase-like enzyme encoding gene in the genome of intermediate strain mems1Replace with the orotic acid-5'-phosphatase encoding gene pyrG , obtain △ Mesm1 :: pyrG Engineered strains, namely Trichoderma reesei engineered strains that produce high levels of cellulase.

[0007] The engineered strain △ constructed by the construction method provided by this invention Mesm1 :: pyrG The activity of the filter paper enzyme produced during shake-flask fermentation was 123% higher than that of the original strain, and the extracellular protein concentration was 258% higher. The glucose yield of the engineered strain's enzyme-producing group was 45.2 g / L, while the glucose yield of the wild-type enzyme-producing group was 12.3 g / L; the saccharification efficiency of the engineered strain's enzyme production was 267% higher than that of the wild type. This indicates that the engineered Trichoderma reesei significantly improved enzyme activity, extracellular protein content, and saccharified glucose yield compared to the wild-type original strain. Therefore, the modification method provided by this invention improves the enzyme production capacity of Trichoderma reesei strains.

[0008] Furthermore, the gene encoding orotic acid-5'-phosphatase in its genome was further investigated. pyr4 Replace with hygromycin resistance encoding gene hph , obtain △ pyr4 :: hph The steps for intermediate strains are as follows: Fresh spores of the starting strain *Trichoderma reesei* were inoculated into MM medium supplemented with uracil to obtain mycelia. The mycelia were lysed using lysozyme and snailase to obtain lysates. After filtration and centrifugation, *Trichoderma reesei* protoplasts were obtained. The recombinant fragment I shown in SEQ ID NO. 20 was transformed into *Trichoderma reesei* protoplasts to obtain Δ... pyr4 :: hph Intermediate strain.

[0009] Furthermore, the △ pyr4 :: hph L-rhamnonate dehydratase-like enzyme encoding gene in the genome of intermediate strain mems1 Replace with the orotic acid-5'-phosphatase encoding gene pyrG , obtain △ Mesm1 :: pyrG The steps for engineered strains are as follows: Use △ pyr4 :: hph intermediate strain preparation to obtain △ pyr4 :: hph Intermediate strain protoplasts, transformed with recombinant fragment II shown in SEQ ID NO. 21 Δ pyr4 :: hph Intermediate strain protoplasts yielded engineered Trichoderma reesei strains with high cellulase production.

[0010] Furthermore, the cloning method for the recombinant fragment I is as follows: using Trichoderma reesei SDU1010 genomic DNA as a template, the gene encoding orotic acid-5'-phosphatase is amplified by PCR. pyr4 upstream homologous arm fragments and genes encoding orotate-5'-phosphatase. pyr4 The downstream homologous arm fragment; using plasmid pFC332 as a template, the hygromycin resistance encoding gene was obtained by PCR amplification. hph Fragment; Overlap extension PCR was used to target the gene encoding orotate-5'-phosphocarboxylase. pyr4 The upstream homologous arm fragment and the hygromycin resistance encoding gene hph After fragment ligation, it is then combined with the gene encoding orotate-5'-phosphocarboxylase. pyr4 The downstream homologous arm fragments are connected and assembled to obtain recombinant fragment I.

[0011] Furthermore, the cloning method for the recombinant fragment II is as follows: Using Trichoderma reesei SDU1010 genomic DNA as a template, the gene encoding the L-rhamnonate dehydrase-like enzyme was amplified by PCR. mems1 upstream homologous arm fragments and genes encoding L-rhamnonate dehydrase-like enzymes. mems1 The downstream homologous arm fragment was used as a template from the Clostridium thermocellum DSM1313 genome to amplify the orotate-5'-phosphatase encoding gene containing its own promoter via PCR. pyrG Fragment; The overlapping extension PCR technique was used to target the L-rhamnonate dehydrase-like enzyme encoding gene. mems1 The upstream homologous arm fragment is related to the orotate-5'-phosphatase encoding gene. pyrG After fragment ligation, it is then combined with the gene encoding the L-rhamnonate dehydrase-like enzyme. mems1 The downstream homologous arm fragments were connected and assembled to obtain recombinant fragment II.

[0012] Furthermore, the orotic acid-5'-phosphocarboxylase encoding gene pyr4 The nucleotide sequence is shown in SEQ ID NO.1, and the hygromycin resistance encoding gene... hph The nucleotide sequence is shown in SEQ ID NO.2, encoding the L-rhamnonate dehydratase-like enzyme gene. mems1 The nucleotide sequence is shown in SEQ ID NO.5, encoding the orotic acid-5'-phosphocarboxylase gene. pyrGThe nucleotide sequence is shown in SEQ ID NO.3.

[0013] Furthermore, the originating strain, *Trichoderma reesei*, was deposited on April 7, 2026, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 3.29531.

[0014] The present invention also provides a high-yield cellulase-producing Trichoderma reesei engineered strain, which is constructed by the above-described method.

[0015] The present invention also provides the application of the engineered Trichoderma reesei strain with high cellulase production in the production of cellulase.

[0016] Furthermore, the engineered Trichoderma reesei strain with high cellulase production has improved enzyme activity, extracellular protein production rate, and saccharification efficiency compared to the original strain Trichoderma reesei.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention utilizes the genome of Trichoderma reesei pyr4 Gene replacement hph Genes, and then the genome mems1 Gene replacement pyrG The gene significantly increased the filter paper enzyme activity and extracellular protein concentration of *Trichoderma reesei*. The engineered strain △ constructed in this invention... Mesm1 :: pyrG The activity of the filter paper enzyme produced during shake-flask fermentation was 123% higher than that of the original strain, and the extracellular protein concentration was 258% higher. The glucose yield of the engineered strain's enzyme-producing group was 45.2 g / L, while that of the wild-type enzyme-producing group was 12.3 g / L; the saccharification efficiency of the engineered strain's enzyme production was 267% higher than that of the wild-type. This indicates that the engineered Trichoderma reesei significantly improved enzyme activity, extracellular protein content, and saccharified glucose yield compared to the wild-type original strain.

[0018] This invention is the first to discover the negative regulatory effect of the L-rhamnonate dehydratase-like enzyme gene on cellulase synthesis in *Trichoderma reesei*. Knocking out this gene resulted in a 123% increase in filter paper enzyme activity and a 258% increase in extracellular protein concentration in the engineered strain compared to the wild-type SDU1010. This invention achieves gene replacement through precise two-step homologous recombination. Using the upstream and downstream sequences of the gene to be replaced in *Trichoderma reesei* as homologous arms, recombinant expression modules containing exogenous marker genes are constructed. Specific homologous recombination between the homologous arms and genomic target sites ensures stable expression of the exogenous marker gene while avoiding the genetic instability caused by random integration.

[0019] The cellulase produced by the engineered strain of this invention was applied to the saccharification process of pretreated sugarcane bagasse. After reacting at 50°C and pH 4.8 for 48 hours, the glucose yield reached 45.2 g / L, which is ≥267% higher than the saccharification effect of the enzyme produced by wild-type SDU1010. This provides an efficient enzyme source and strain support for the resource utilization of agricultural waste and the preparation of biomass energy.

[0020] Information on the preservation of biological materials: SDU1010, referred to in this application as Trichoderma reesei strain SDU1010, was deposited on April 7, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 3.29531. The address of the depository is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China. Its classification name is Trichoderma reesei. Trichoderma reesei . Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a schematic diagram illustrating the construction of the high-yield cellulase expression module of the present invention: recombinant fragment I and recombinant fragment II.

[0023] Figure 2 The enzyme activity, extracellular protein content, and saccharified glucose yield of the engineered Trichoderma reesei strain of this invention are shown. In the figure, A is a statistical chart comparing the enzyme activity of the engineered Trichoderma reesei strain of this invention with that of the starting strain; B is a statistical chart comparing the extracellular protein content of the engineered Trichoderma reesei strain of this invention with that of the starting strain; C is a statistical chart comparing the saccharified glucose production of the engineered Trichoderma reesei strain of this invention with that of the starting strain. Detailed Implementation

[0024] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0025] This invention uses Trichoderma reesei SDU1010 as the starting strain and examines the orotic acid-5'-phosphate carboxylase encoding gene in its genome. pyr4 Replace with hygromycin resistance encoding gene hph , obtain △ pyr4 :: hph Intermediate strain; then the △ pyr4 :: hph L-rhamnonate dehydratase-like enzyme encoding gene in the genome of intermediate strain mems1 Replace with the orotic acid-5'-phosphatase encoding gene pyrG , obtain △ Mems1 :: pyrG Engineered strains, namely Trichoderma reesei engineered strains that produce high levels of cellulase.

[0026] The orotic acid-5'-phosphatase encoding gene pyr4 The nucleotide sequence is shown in SEQ ID NO.1, and the hygromycin resistance encoding gene... hph The nucleotide sequence is shown in SEQ ID NO.2, and the gene encoding orotic acid-5'-phosphocarboxylase is... pyrG The nucleotide sequence is shown in SEQ ID NO.3; L-rhamnonate dehydratase-like enzyme encoding gene. mems1 The nucleotide sequence is shown in SEQ ID NO.5, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.4.

[0027] SEQ ID NO.1:

[0028] SEQ ID NO.2:

[0029] SEQ ID NO.3:

[0030] SEQ ID NO.4: MAPSRTDIETPSNGDNYSSRTPPASEVIITGFTAHDLRFPTSLDLSGSDAMNPGPNYSAAYIVLHTNTRHLSGHGFAFTIGRGNNLCVLAAEMVAERLVGKTLEQLTANMGQTWRHLVSDSQLRWVGPEKGVIHLGLSTCVNAVWDLWGRHLGKPVWEIVSDMTPEEFVRCIDFRYILDAITPEEAVELLREQAKTKKARLEEARLNRAVPAYTTQAGWLGFSDEKMVELIESNLAQGMDKFKFKVGGSLEDDKRRLQIARDTIGYDRLLMVDANQVWSVPEAIDYMLHLVKYKPLFIEEPTSPDDILGHAAIRKALKPYGVGVATGEHCQNRVMFKQLLQAGSIDYCQIDACRLGGVNEVMAVLLMAKKFNVPIVPHSGGIGLNEYTQHLALINYLCVSGEKSLLEHISSFREVLKTPCQTKDGHFVTPHDAGYSVEYKPEAIEKYTYPTGSFWRSEQGLAIINEPNI。

[0031] SEQ ID NO.5:

[0032] Example 1: A high-cellulase-producing Trichoderma reesei engineered strain, its construction method, and its application.

[0033] I. Experimental Reagents 1. Experimental reagents The lysozyme was purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number: S10083-1g.

[0034] Snail enzyme was purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number: S10107-250mg.

[0035] PDA medium formula: 200g potato, 20g glucose, 20g agar, distilled water to a final volume of 1L, natural pH.

[0036] 1L Trichoderma reesei enzyme-producing seed culture medium: glucose 10g, KH2PO4 15g, (NH4)2SO4 5g, CaCl2·2H2O 0.6g, MgSO4·7H2O 0.6g, 1x trace element stock solution 100μL, peptone 2g.

[0037] 1L Trichoderma reesei enzyme-producing medium: 20g microcrystalline cellulose, 5g KH2PO4, 5g (NH4)2SO4, 0.6g MgSO4·7H2O, 20g corn steep liquor, 1g CaCl2·2H2O, 100μL 1x trace element stock solution.

[0038] 1L Trichoderma reesei transformation supernatant medium: (NH4)2SO4 6g, KH2PO4 10g, MgSO4·7H2O 1g, trisodium citrate 3g, sorbitol 182g, trace element stock solution 100μL, agarose 6g.

[0039] 1L Trichoderma reesei transformation lower layer medium: (NH4)2SO4 6g, KH2PO4 10g, MgSO4·7H2O 1g, trisodium citrate 3g, trace element stock solution 100μL, agar powder 20g.

[0040] 1L basic culture medium (MM): 20g glucose, 15g KH2PO4, 5g (NH4)2SO4, 0.6g CaCl2·2H2O, 0.6g MgSO4·7H2O, 100μL trace element stock solution, 2g peptone. If preparing solid culture medium, add an additional 2% agar powder.

[0041] 10000x trace element stock solution: FeSO4·7H2O 50g, CoCl·6H2O 20g, ZnSO4·7H2O 14g, MnSO4·H2O 16g. Sterilize by vacuum filtration.

[0042] Solution I: 0.1M KH2PO4, 1.2M sorbitol, pH 5.6.

[0043] Solution II: 10 mM Tris-HCl, 50 mM CaCl2·2H2O, 1 M sorbitol, pH 7.5.

[0044] PEG solution: 25% PEG6000, 10mM Tris-HCl, 50mM CaCl2·2H2O, pH 7.5.

[0045] All of the above culture media require the addition of 10 mmol·L⁻¹ when culturing uracil-deficient uridines. -1 Uracil.

[0046] Plasmid extraction kits and gel extraction kits were purchased from OMEGA, seamless cloning kits were purchased from Novizan, DNA restriction endonucleases were purchased from Thermo, and ligases were purchased from NEB. Other companies' similar products can also be used instead.

[0047] This invention selects *Trichoderma reesei* SDU1010 as the starting strain, and the method for constructing the engineered strain of this invention is also applicable to other strains containing... pyr4 Genetically modified Trichoderma reesei strains, such as strain QM9414 with accession number ATCC26921, strain RUTC30 with accession number ATCC56765, or strain NG14 with accession number ATCC56767.

[0048] II. Construction of a high-cellulase-producing Trichoderma reesei engineered strain 1. Construction of Recombinant Fragment I and Recombinant Fragment II Recombination fragment I is denoted as pyr4up-hph-pyr4 down, and recombination fragment II is denoted as mems1up-pyrG-mems1down.

[0049] (1) Cloning of upstream and downstream fragments of the orotic acid-5'-phosphate carboxylase encoding gene and the Lrhamnonate dehydratase-like enzyme gene Using genomic DNA from *Trichoderma reesei* SDU1010 as a template, PCR amplification was performed using upstream primer P1 and downstream primer P2, yielding a 1368 bp pyr4up fragment. The 50 μL PCR reaction mixture consisted of: 25 μL 2×PhantaMax Buffer, 1 μL 10 mmol / L dNTP Mix, 2 μL 10 μmol / L upstream primer, 2 μL 10 μmol / L downstream primer, 1 μL template DNA, 1 μL PhantaMax Super-Fidelity DNA Polymerase, and 18 μL ddH2O. PCR conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 1 min 30 s, 35 cycles; final extension at 72℃ for 5 min. The amplified product was verified by 1% agarose gel electrophoresis and then recovered using a gel extraction kit to obtain the pyr4up fragment.

[0050] Using genomic DNA from *Trichoderma reesei* SDU1010 as a template, PCR amplification was performed using upstream primer P3 and downstream primer P4, yielding a 1368 bp pyr4down fragment. The 50 μL PCR reaction mixture consisted of: 25 μL 2×PhantaMax Buffer, 1 μL 10 mmol / L dNTP Mix, 2 μL 10 μmol / L upstream primer, 2 μL 10 μmol / L downstream primer, 1 μL template DNA, 1 μL PhantaMax Super-Fidelity DNA Polymerase, and 18 μL ddH2O. PCR conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 1 min 30 s, 35 cycles; final extension at 72℃ for 5 min. The amplified product was verified by 1% agarose gel electrophoresis and then recovered using a gel extraction kit to obtain the pyr4down fragment.

[0051] Using genomic DNA from *Trichoderma reesei* SDU1010 as a template, PCR amplification was performed using upstream primer P5 and downstream primer P6, yielding a 2000 bp mems1 up fragment. The 50 μL PCR reaction mixture consisted of: 25 μL 2×PhantaMax Buffer, 1 μL 10 mmol / L dNTP Mix, 2 μL 10 μmol / L upstream primer, 2 μL 10 μmol / L downstream primer, 1 μL template DNA, 1 μL PhantaMax Super-Fidelity DNA Polymerase, and 18 μL ddH2O. PCR conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 2 min, 35 cycles; and a final extension at 72℃ for 5 min. The amplified product was verified by agarose gel electrophoresis and then recovered using a gel extraction kit to obtain the mems1 up fragment.

[0052] Using genomic DNA from *Trichoderma reesei* SDU1010 as a template, PCR amplification was performed using upstream primer P7 and downstream primer P8, yielding a 2000 bp mems1 down fragment. The 50 μL PCR reaction mixture consisted of: 25 μL 2×PhantaMax Buffer, 1 μL 10 mmol / L dNTP Mix, 2 μL 10 μmol / L upstream primer, 2 μL 10 μmol / L downstream primer, 1 μL template DNA, 1 μL PhantaMax Super-Fidelity DNA Polymerase, and 18 μL ddH2O. PCR conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 2 min, 35 cycles; and a final extension at 72℃ for 5 min. The amplified product was verified by 1% agarose gel electrophoresis and then recovered using a gel extraction kit to obtain the mems1 down fragment.

[0053] Primer information for P1, P2, P3, P4, P5, P6, P7 and P8 is shown in Table 1.

[0054] Table 1 Primer Information

[0055] (2) Hygromycin resistance gene and pyrG Cloning of genes Using plasmid pFC332 as a template, PCR amplification was performed using upstream primer P9 and downstream primer P10, yielding a 1050 bp amplified sample. hphFragment. The PCR reaction system was the same as before. PCR reaction conditions: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 1 min, 35 cycles; 72℃ final extension for 5 min; after verification by 1% agarose gel electrophoresis, the amplified product was recovered using a gel extraction kit to obtain the hph fragment.

[0056] Using the Clostridium thermocellum DSM1313 genome as a template, pyrG-DSM1313-F / R amplified endogenous selection markers containing their own promoters. pyrG Subsequently, PCR amplification was performed using upstream primer P11 and downstream primer P12, yielding a sample of 1145 bp in length. pyrG Fragment. The PCR reaction system was the same as before. PCR reaction conditions: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 50 s, 35 cycles; 72℃ final extension for 5 min; the amplified product was verified by 1% agarose gel electrophoresis and then recovered using a gel extraction kit to obtain... pyrG Excerpt.

[0057] The primer sequences for P9, P10, pyrG-DSM1313-F, pyrG-DSM1313-R, P11, and P12 are shown in Table 1.

[0058] (3) Construction of recombinant fragment I and recombinant fragment II like Figure 1 As shown, recombinant fragment I and recombinant fragment II were constructed using overlap extension PCR technology: ①Recombined fragment I: 0) First round of overlap extension PCR: The pyr4 up fragment and hph fragment were mixed at a molar ratio of 1:1 as a mixed template. The PCR reaction system is shown below. PCR amplification was performed using primer P1 as the upstream primer and P10 as the downstream primer. The PCR reaction conditions were: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 60℃ annealing for 30 s, 72℃ extension for 2 min, for a total of 35 cycles; 72℃ final extension for 10 min. After amplification, the first round of products were obtained. The target band was verified by 1% agarose gel electrophoresis and then recovered for later use.

[0059] 0) The first-round product and the pyr4 down fragment were mixed at a molar ratio of 1:1 as a template. The PCR reaction system is shown below. PCR amplification was performed using primer P1 as the upstream primer and P4 as the downstream primer. The PCR reaction conditions were: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 60℃ annealing for 30 s, 72℃ extension for 3 min, for a total of 30 cycles; and a final extension at 72℃ for 10 min. The amplified recombinant fragment I, with an expected length of approximately 3738 bp, was obtained. The target band was verified by 1% agarose gel electrophoresis and then recovered for later use. The nucleotide sequence of recombinant fragment I is shown in SEQ ID NO. 20.

[0060] SEQ ID NO.20:

[0061] ②Recombined Fragment II: a) First round of overlap extension PCR: The mems1 up fragment and pyrG fragment were mixed at a molar ratio of 1:1 as a mixed template, with a total template mass of 50 ng. The PCR reaction system is shown below. PCR amplification was performed using primer P5 as the upstream primer and P12 as the downstream primer. The PCR reaction conditions were: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 60℃ annealing for 30 s, 72℃ extension for 3 min, for a total of 30 cycles; and 72℃ final extension for 10 min. After amplification, the first round of products were obtained. The target band was verified by 1% agarose gel electrophoresis and then recovered for later use.

[0062] b) Second-round overlap extension PCR: The first-round product and the mems1 down fragment were mixed at a molar ratio of 1:1 as a template. The PCR reaction system is shown below. PCR amplification was performed using primer P5 as the upstream primer and P8 as the downstream primer. The PCR reaction conditions were: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 60℃ annealing for 30 s, 72℃ extension for 4 min 30 s, for a total of 30 cycles; and 72℃ final extension for 10 min. The amplified recombinant fragment II, with an expected length of approximately 5145 bp, was obtained. After verifying the target band by 1% agarose gel electrophoresis, the fragment was recovered for later use. The nucleotide sequence of recombinant fragment II is shown in SEQ ID NO. 21.

[0063] SEQ ID NO.21:

[0064] The PCR reaction systems described above are the same, all using a 50 μL system: 50 μL PCR reaction system: 25 μL 2×Phanta Max Buffer, 1 μL 10 mmol / L dNTP Mix, 2 μL 10 μmol / L upstream primer, 2 μL 10 μmol / L downstream primer, 1 μL template DNA, 1 μL Phanta Max Super-Fidelity DNA Polymerase, 18 μL ddH2O.

[0065] Primers P2 and P9, P3 and P10, P6 and P11, and P7 and P12 each contain 15bp complementary overlapping sequences to ensure accurate fragment splicing.

[0066] 2. △ pyr4 :: hph Construction of intermediate strains (1) Preparation of Trichoderma reesei protoplasts: Take 100 μL of 10 8 Fresh spores of *Trichoderma reesei* SDU1010 per mL were inoculated into 50 mL of MM medium supplemented with 10 mmol / L uracil and cultured at 30 °C and 200 rpm for 20 h on a shaker. After culture, the bacterial suspension was centrifuged at 2500 rpm for 15 min, and the supernatant was discarded. The mycelia were gently resuspended in 20 mL of Solution I, and lysed with a final concentration of 0.5‰ lysozyme and a final concentration of 4‰ snailase. The suspension was incubated at 30 °C for 60 min to obtain the lysate. The lysate was filtered through sterile four-layer lens paper, and the filtrate was collected. The filtrate was centrifuged at 4 °C and 2500 rpm for 10 min, and the supernatant was discarded. The precipitate was resuspended in 4 mL of Solution II, centrifuged at 4 °C and 2500 rpm for 10 min, and washed twice. Finally, the protoplasts were resuspended in Solution II, counted under an optical microscope, and the concentration was adjusted to 10. 7 Protoplasts were obtained at a concentration of 1 protoplast per mL and placed on ice for later use. The entire procedure must be performed on ice, and the tip of the pipette used to aspirate the protoplasts must be cut off to avoid damaging the protoplasts.

[0067] (2) Transformation of protoplasts: Take 200 μL of a concentration of 10 7Add protoplast suspension of 100 ng / mL to a sterile centrifuge tube, then add 10 μL of recombinant fragment I (100 ng / μL) and 50 μL of PEG solution, mix gently, and incubate on ice for 20 min. Slowly add 2 mL of PEG solution to the tube and incubate at room temperature for 5 min. Add 4 mL of solution II and mix gently. Slowly add the mixture to 50 mL of Trichoderma reesei transformation upper layer medium pre-warmed at 45 °C, containing 100 μg / mL hygromycin B, 1.5 mg / mL 5-FOA, and 10 mmol / L uracil. Mix well and pour into a 9 cm diameter culture dish containing Trichoderma reesei transformation lower layer medium. After cooling and solidification, incubate at 30 °C for 4 days.

[0068] (3) Screening and verification of positive strains: Single colonies growing on the culture dish were picked and inoculated into PDA medium plates containing 100 μg / mL hygromycin B. The plates were cultured at 30℃ for 5 days and subjected to resistance re-screening. The strains that were positive in the re-screening were selected, and their genomic DNA was extracted. PCR verification was performed using the P9 and P10 primer pairs. The strains that were positive in the PCR verification were △ pyr4 :: hph Intermediate strain.

[0069] 3. △ Mesm1 :: pyrG Construction of engineered strains (1) Preparation of protoplasts: Take △ pyr4 :: hph Fresh spores of the intermediate strain were inoculated into MM medium containing 10 mmol / L uracil and prepared according to the aforementioned preparation steps for Trichoderma reesei protoplasts to obtain Δ pyr4 :: hph Protoplasts, adjust concentration to 10 7 pcs / mL, to obtain △ pyr4 :: hph Protoplast suspension, keep on ice for later use.

[0070] (2) Protoplast transformation: Take 200 μL △ pyr4 :: hph Protoplast suspension was mixed with 10 μL of recombinant fragment II (100 ng / μL) and 50 μL of PEG solution, and incubated on ice for 20 min. 2 mL of PEG solution was added and incubated at room temperature for 5 min. 4 mL of solution II was added and mixed well. The mixture was then added to 50 mL of pre-insulated uracil-free Trichoderma reesei transformation upper medium containing 1.5 mg / mL 5-FOA, mixed well, and poured into a culture dish containing Trichoderma reesei transformation lower medium. The mixture was incubated at 30 °C for 4 days.

[0071] (3) Screening and verification of positive strains: Single colonies of Trichoderma reesei that can grow on the supernatant medium containing 1.5 mg / mL 5-FOA and are sensitive to 5-FOA were selected and inoculated into MM solid medium without uracil and cultured at 30℃ for 5 days; genomic DNA of the strains was extracted and PCR verification was performed using primers P11 and P12; the strains that were positive by PCR verification were △ Mesm1 :: pyrG Engineered strains.

[0072] III. Engineered strains △ Mesm1 :: pyrG Filter paper enzyme activity assay 1. Filter paper enzyme activity assay method (1) Enzyme solution preparation: spore concentrations of 10 8 wild-type strain SDU1010, intermediate strain △ pyr4 :: hph Engineered strains △ Mesm1 :: pyrG Spores were inoculated into 50 mL of Trichoderma reesei enzyme-producing seed medium and cultured at 30℃ and 200 r / min for 36 h. Then, 100 mL of Trichoderma reesei enzyme-producing medium was inoculated at a 5% (v / v) inoculum and cultured at 30℃ and 200 r / min for 96 h to obtain the fermentation broth. The fermentation broth was centrifuged at 4℃ and 8000 r / min for 15 min, and the supernatant was filtered through a 0.22 μm filter membrane to obtain crude enzyme solution of wild-type strain SDU1010 and intermediate strain △. pyr4 :: hph Crude enzyme solution, engineered strain △ Mesm1 :: pyrG Crude enzyme solution.

[0073] (2) The method for determining the enzyme activity of filter paper was the DNS method: Take about 50 mg of Whatman No. 1 standard filter paper, cut it into strips of 1 cm × 6 cm, place it in a 5 mL centrifuge tube, add 1.5 mL of pH 4.8 citrate-sodium citrate buffer, and then add 0.5 mL of crude enzyme solution diluted 10 times with pH 4.8 citrate-sodium citrate buffer. Mix thoroughly and react at a constant temperature of 50 °C for 60 min. Then add 3 mL of DNS reagent to stop the reaction, heat in a boiling water bath for 10 min, cool quickly with distilled water and make up to 25 mL. After mixing by inversion, measure the OD value at a wavelength of 540 nm using a UV spectrophotometer. Control group: The crude enzyme solution diluted 10 times was inactivated by high temperature in a boiling water bath for 15 min. 1.5 mL of the above buffer and 0.5 mL of the inactivated crude enzyme solution diluted were added to the centrifuge tube in sequence. The subsequent operations of 50 °C water bath, addition of DNS reagent, boiling, cooling, making up to volume and OD value measurement were the same as those of the experimental group.

[0074] (3) Method for plotting the glucose standard curve: Prepare glucose standard solutions with concentrations of 0, 20, 40, 60, 80, and 100 μmol / mL. Take 0.5 mL of each of the above glucose standard solutions and add 1.5 mL of pH 4.8 citrate-sodium citrate buffer solution. Follow the DNS method described above: add DNS reagent, boil in a water bath, cool, make up to volume, and measure the OD value. Plot a standard curve with glucose concentration on the x-axis and OD value at 540 nm on the y-axis to calculate the glucose concentration in the reaction solution.

[0075] (4) Enzyme activity calculation Filter paper enzyme activity is defined as the amount of enzyme required to catalyze the hydrolysis of filter paper to produce 1 μmol of reducing sugar per minute (calculated as glucose) at 50°C and pH 4.8 in a citrate-sodium citrate buffer system. This amount is defined as one filter paper enzyme activity unit (IU).

[0076] Enzyme activity (IU / mL) = (C×V1×n) / (t×V2); In the formula, C is the glucose concentration calculated based on the standard curve, in μmol / mL. V1—The final volume of the reaction solution, in mL; n—Dilution factor of crude enzyme solution, in times; t — reaction time, in minutes; V2 — Volume of the enzyme solution to be tested added to the reaction: 10-fold dilution, unit: mL.

[0077] 2. Results of filter paper enzyme activity assay like Figure 2 As shown in the figure, it can be seen that as this modification process proceeds, the engineered strain △ Mesm1 :: pyrG The activity of the filter paper enzyme produced during shake-flask fermentation was 123% higher than that of the original strain, and the extracellular protein concentration was 258% higher than that of the original strain.

[0078] IV. Engineered strains △ Mesm1 :: pyrG Application of cellulase in sugarcane bagasse saccharification 1. Experimental Methods (1) Pretreatment of bagasse: Take dry bagasse, crush it and pass it through a 30-mesh sieve. Treat it with NaOH solution in a high-pressure cooker at 160°C for 1 hour. The amount of NaOH used is 10% of the dry weight of the substrate. Filter the reaction product through a 500-mesh filter bag, wash it with distilled water until the pH of the filtrate is neutral, and dry it at 60°C to constant weight to obtain pretreated bagasse.

[0079] (2) Saccharification reaction: Take 2.0 g of pretreated sugarcane bagasse, put it into a 50 mL Erlenmeyer flask, add 5 mL of 0.5 M pH 4.8 acetate-sodium acetate buffer and 13 mL of engineered strain Δ with an enzyme activity of 1.5 IU / mL. Mesm1 :: pyrG The crude enzyme solution was mixed and reacted in a shaker at 50℃ for 48 hours. 1 mL samples were taken at 24 and 48 hours, centrifuged at 12000 r / min for 5 min, and the supernatant was filtered through a 0.22 μm filter membrane to obtain the saccharified solution. The glucose concentration was determined by high performance liquid chromatography. The control group was "wild-type SDU1010 crude enzyme solution".

[0080] (3) High performance liquid chromatography determination: The saccharified solution was centrifuged at 12000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane. An Aminex HPX-87H column was used, the mobile phase was pure water containing 273 μL / L 98% concentrated sulfuric acid, the column temperature was 50℃, the flow rate was 0.6 mL / min, the injection volume was 20 μL, and the contents of glucose, xylose and arabinose were detected.

[0081] 2. Saccharification results Depend on Figure 2 As shown, after 48 hours of reaction, the glucose yield of the enzyme-producing group of the engineered strain was 45.2 g / L, while that of the wild-type enzyme-producing group was 12.3 g / L. The saccharification efficiency of the enzyme produced by the engineered strain was 267% higher than that of the wild-type strain, indicating that its saccharification adaptability to agricultural waste was significantly better than that of the wild-type strain.

[0082] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments.

[0083] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for constructing a high-cellulase-producing engineered Trichoderma reesei strain, characterized in that, Using Trichoderma reesei as the starting strain, the gene encoding orotic acid-5'-phosphate carboxylase in its genome was analyzed. pyr4 Replace with hygromycin resistance encoding gene hph , obtain △ pyr4 :: hph Intermediate strain; then the △ pyr4 :: hph L-rhamnonate dehydratase-like enzyme encoding gene in the genome of intermediate strain mems1 Replace with the orotic acid-5'-phosphatase encoding gene pyrG , obtain △ Mesm1 :: pyrG Engineered strains, namely Trichoderma reesei engineered strains that produce high levels of cellulase; The orotic acid-5'-phosphatase encoding gene pyr4 The nucleotide sequence is shown in SEQ ID NO.1, and the hygromycin resistance encoding gene... hph The nucleotide sequence is shown in SEQ ID NO.2, encoding the L-rhamnonate dehydratase-like enzyme gene. mems1 The nucleotide sequence is shown in SEQ ID NO.5, encoding the orotic acid-5'-phosphocarboxylase gene. pyrG The nucleotide sequence is shown in SEQ ID NO.

3.

2. The method for constructing a high-cellulase-producing Trichoderma reesei engineered strain according to claim 1, characterized in that, The gene encoding orotic acid-5'-phosphocarboxylase in its genome pyr4 Replace with hygromycin resistance encoding gene hph , obtain △ pyr4 :: hph The steps for intermediate strains are as follows: Fresh spores of the starting strain *Trichoderma reesei* were inoculated into MM medium supplemented with uracil to obtain mycelia. The mycelia were lysed using lysozyme and snailase to obtain lysates. After filtration and centrifugation, *Trichoderma reesei* protoplasts were obtained. The recombinant fragment I shown in SEQ ID NO. 20 was transformed into *Trichoderma reesei* protoplasts to obtain Δ... pyr4 :: hph Intermediate strain.

3. The method for constructing a high-cellulase-producing Trichoderma reesei engineered strain according to claim 2, characterized in that, The △ pyr4 :: hph L-rhamnonate dehydratase-like enzyme encoding gene in the genome of intermediate strain mems1 Replace with the orotic acid-5'-phosphatase encoding gene pyrG , obtain △ Mesm1 :: pyrG The steps for engineered strains are as follows: Use △ pyr4 :: hph intermediate strain preparation to obtain △ pyr4 :: hph Intermediate strain protoplasts were transformed with recombinant fragment II shown in SEQ ID NO.

21. pyr4 :: hph Intermediate strain protoplasts yielded engineered Trichoderma reesei strains with high cellulase production.

4. The method for constructing a high-cellulase-producing Trichoderma reesei engineered strain according to claim 3, characterized in that, The cloning method for the recombinant fragment I is as follows: using Trichoderma reesei SDU1010 genomic DNA as a template, the gene encoding orotic acid-5'-phosphatase was amplified by PCR. pyr4 upstream homologous arm fragments and genes encoding orotate-5'-phosphatase. pyr4 The downstream homologous arm fragment; using plasmid pFC332 as a template, the hygromycin resistance encoding gene was obtained by PCR amplification. hph Fragment; Overlap extension PCR was used to target the gene encoding orotate-5'-phosphocarboxylase. pyr4 The upstream homologous arm fragment and the hygromycin resistance encoding gene hph After fragment ligation, it is then combined with the gene encoding orotate-5'-phosphocarboxylase. pyr4 The downstream homologous arm fragments are connected and assembled to obtain recombinant fragment I.

5. The method for constructing a high-cellulase-producing Trichoderma reesei engineered strain according to claim 4, characterized in that, The cloning method for the recombinant fragment II is as follows: Using Trichoderma reesei SDU1010 genomic DNA as a template, the gene encoding the L-rhamnonate dehydrase-like enzyme was amplified by PCR. mems1 upstream homologous arm fragments and genes encoding L-rhamnonate dehydrase-like enzymes. mems1 The downstream homologous arm fragment was used as a template from the Clostridium thermocellum DSM1313 genome to amplify the orotate-5'-phosphatase encoding gene containing its own promoter via PCR. pyrG Fragment; The overlapping extension PCR technique was used to target the L-rhamnonate dehydrase-like enzyme encoding gene. mems1 The upstream homologous arm fragment is related to the orotate-5'-phosphatase encoding gene. pyrG After fragment ligation, it is then combined with the gene encoding the L-rhamnonate dehydrase-like enzyme. mems1 The downstream homologous arm fragments were connected and assembled to obtain recombinant fragment II.

6. The method for constructing a high-cellulase-producing Trichoderma reesei engineered strain according to claim 5, characterized in that, The orotic acid-5'-phosphatase encoding gene pyr4 The nucleotide sequence is shown in SEQ ID NO.1, and the hygromycin resistance encoding gene... hph The nucleotide sequence is shown in SEQ ID NO.2, encoding the L-rhamnonate dehydratase-like enzyme gene. mems1 The nucleotide sequence is shown in SEQ ID NO.5, encoding the orotic acid-5'-phosphocarboxylase gene. pyrG The nucleotide sequence is shown in SEQ ID NO.

3.

7. The method for constructing a high-cellulase-producing Trichoderma reesei engineered strain according to claim 6, characterized in that, The originating strain, *Trichoderma reesei*, was deposited on April 7, 2026, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 3.29531.

8. A highly cellulase-producing engineered Trichoderma reesei strain, characterized in that, It is constructed by the method described in any one of claims 1 to 7.

9. The application of the high-cellulase-producing Trichoderma reesei engineered strain as described in claim 8 in the production of cellulase.

10. The application according to claim 9, characterized in that, Compared with the original strain of *Trichoderma reesei*, the engineered strain with high cellulase production showed improved cellulase activity, extracellular protein production rate, and saccharification efficiency of cellulase production.

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