Trichoderma reesei engineering strain for producing high-activity lytic polysaccharide monooxygenase and construction method and application thereof

Abstract

This invention discloses an engineered Trichoderma reesei strain producing a highly active polysaccharide monooxygenase, its construction method, and its applications, belonging to the field of genetic engineering technology. This strain uses the endogenous constitutive strong promoter Pcdna1 from Trichoderma reesei to drive the transcription of the LPMO gene, and combines it with the terminator Tpdc to construct a complete transcription unit (Pcdna1-lpmo-(GGGGS)3-pdi2-Tpdc), improving the transcription efficiency of LPMO. LPMO is fused with the protein disulfide isomerase (PDI) encoding gene pdi2 via a (GGGGS)3 flexible linker peptide, utilizing PDI-mediated disulfide bond formation to enhance LPMO enzyme activity and secretion efficiency. The LPMO expressed in this invention exhibits significantly increased activity, and in synergistic degradation of microcrystalline cellulose with cellulase, it can increase the glucose yield of cellulase by 18.5%, while the fused expression of LPMO-PDI can increase the glucose yield of cellulase by 33.1%. This invention is applicable to biomass conversion and the development of industrial enzyme preparations.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to an engineered Trichoderma reesei strain that produces highly active cleaving polysaccharide monooxygenase, its construction method, and its application. Background Technology

[0002] In the field of biomass energy utilization, lignocellulose, as the most abundant renewable resource on Earth, is crucial for efficient biomass conversion through effective degradation. Currently, cellulose degradation mainly relies on a combination of cellulases, including endoglucanase, exoglucanase, and β-glucosidase. However, the hydrogen bonding between cellulose molecules in lignocellulose creates a highly crystalline structure, and hemicellulose and lignin act as intercellular matrix filling the spaces between microfibrils, increasing the difficulty of cellulose degradation and making lignocellulose resources challenging to degrade and utilize.

[0003] Pretreatment of lignocellulose, such as straw, is a crucial step in improving its biodegradability. The primary goal is to disrupt the crystalline structure of cellulose, remove lignin, and loosen the cellulose molecular structure, thereby increasing the contact area between cellulose and cellulase and promoting saccharification. Lytic polysaccharide monooxygenase (LPMO), a novel oxidase from the AA9 family, can directly act on crystalline cellulose, breaking down long cellulose chains into shorter chains, making them more readily hydrolyzed by cellulase. Therefore, the development and application of LPMO is an effective way to improve the biodegradability of lignocellulose.

[0004] Currently used LPMO expression systems are mostly Escherichia coli or Pichia pastoris. E. coli, as a host for recombinant protein production, has advantages such as low cost, simple operation, rapid growth, suitability for large-scale production, and ease of genetic modification. However, it lacks eukaryotic post-translational modifications (such as glycosylation), and its reducing cytoplasmic environment interferes with the electron transport chain, all of which may affect LPMO enzyme activity. P. pastoris, as a protein expression host, has advantages such as high-density culture, high levels of secreted protein expression, and glycosylation capability. However, this system has significant shortcomings in LPMO post-expression modifications. On the one hand, LPMOs are all formed from a single AA9 catalytic module, which begins with a typical conserved histidine residue common to all LPMOs. To ensure the correct binding of copper ions to the N-terminal histidine residues, the signal peptide of the recombinant protein needs to be correctly cleaved. However, cleavage errors frequently occur in the *P. pastoris* expression system. Furthermore, *P. pastoris* cannot perform N-terminal τ-methylation on LPMO. LPMO produced by filamentous fungi has N-terminal τ-methylation at histidine 1, a modification related to the correct binding of copper ions and the stability of the active site. The absence of methylation in the *P. pastoris* expression system affects LPMO enzyme activity. Therefore, seeking a new expression system is essential to obtain a recombinant protein with a structure identical to that of natural LPMO.

[0005] Trichoderma reesei, as an engineered microorganism, has demonstrated significant systemic advantages. Compared to E. coli and P. pastoris, Trichoderma reesei not only possesses extremely strong protein secretion capabilities, but its endoplasmic reticulum-Golgi system-mediated eukaryotic glycosylation modification enables AA9 family LPMOs to achieve thermal stability and substrate affinity at natural activity levels. Furthermore, Trichoderma reesei's powerful antioxidant system (catalase CAT1 and superoxide dismutase SOD) efficiently scavenges H2O2 produced by LPMO catalysis, significantly improving cell viability and completely avoiding the growth inhibition caused by reactive oxygen species (ROS) accumulation in E. coli. Particularly noteworthy is that Trichoderma reesei is adapted to solid-state fermentation processes, utilizing agricultural waste (such as corn cobs and bagasse) to reduce LPMO production costs to $50-80 / gram, a 60%-80% reduction compared to methanol-induced liquid fermentation using yeast. Despite the numerous advantages of *Trichoderma reesei* in expressing LPMO, the yield of the target protein in *Trichoderma reesei* expression systems is often limited by the transcription efficiency of the gene. Improving the transcription efficiency of the LPMO gene in *Trichoderma reesei*, thereby increasing the yield of the target protein, remains a significant challenge. Summary of the Invention

[0006] Given the limitations of existing LPMO expression systems (E. coli, P. pastoris) in terms of enzyme activity, and the fact that Trichoderma reesei, while possessing advantages, has target protein yields limited by gene transcription efficiency, the present invention aims to provide an engineered Trichoderma reesei strain that produces highly active cleavable polysaccharide monooxygenase, along with its construction method and applications. This invention utilizes the Trichoderma reesei expression system to achieve correct post-translational modification of LPMO, employs a constitutive strong promoter (Pcdna1) to overcome gene transcription bottlenecks, and ensures correct folding and enhances activity stability through co-expression of protein disulfide isomerase (PDI).

[0007] To achieve the above objectives, the present invention employs the following technical solution:

[0008] This invention provides an engineered Trichoderma reesei strain that produces highly active lysin monooxygenase. The strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41856 and deposit date of March 27, 2025.

[0009] The engineered bacteria contain the Pcdna1-lpmo-(GGGGS)3-pdi2-Tpdc fusion expression vector.

[0010] In the fusion expression vector, the nucleotide sequence of the Pcdna1 promoter is shown in SEQ ID NO:1, and the nucleotide sequence of the lpmo encoding gene is shown in SEQ ID NO:2.

[0011] Furthermore, the Pcdna1 promoter is an endogenous constitutive promoter derived from Trichoderma reesei QM9414.

[0012] The Pcdna1 promoter contains a NedI restriction site upstream and the Tpdc terminator contains an EcoRI restriction site downstream.

[0013] This invention provides a method for constructing an engineered Trichoderma reesei strain that produces a highly active polysaccharide monooxygenase, comprising:

[0014] Step 1: Construct the lpmo-Tpdc transcription unit, link it to the pUC19 plasmid along with the promoter Pcdna1, and construct the recombinant vector pPLT2;

[0015] Step 2: The protein disulfide bond isomerase encoding gene pdi2 is linked using a linker peptide and inserted into the recombinant vector pPLT2 described in Step 1 to construct the recombinant vector pPLT3.

[0016] Step 3: The recombinant vector pPLT3 and the helper plasmid from Step 2 were co-transformed into Trichoderma reesei protoplasts, and the strains were screened to obtain engineered Trichoderma reesei strains that produce lytic polysaccharide monooxygenase.

[0017] The mass ratio of the recombinant plasmid to the helper plasmid is 1:1, and the helper plasmid is pAN7-1.

[0018] This invention provides a fermentation broth of engineered Trichoderma reesei, which is obtained by fermentation of the engineered Trichoderma reesei that produces lytic polysaccharide monooxygenase.

[0019] The above-mentioned method for preparing fermentation broth of Trichoderma reesei engineered bacteria involves activating the Trichoderma reesei engineered bacteria that produces lysin monooxygenase and then inoculating it into a liquid fermentation medium for fermentation.

[0020] The fermentation temperature is 26–30°C, and the fermentation time is 6–8 days.

[0021] Further, the fermentation medium consists of: 10 g / L glucose, 1.4 g / L (NH4)2SO4, 2.0 g / L KH2PO4, 0.3 g / L MgSO4·7H2O, 0.4 g / L CaCl2·2H2O, 5 g / L yeast extract, 5 mM urea, 0.02% (w / v) Tween 80, and 1 / 50 (v / v) trace element solution; wherein the trace element composition is 0.25 g / L FeSO4·7H2O, 0.08 g / L MnSO4·H2O, 0.07 g / L ZnSO4·7H2O, 0.1 g / L CoCl2·2H2O, and citric acid is used to adjust the pH to 5.0.

[0022] Furthermore, the inoculum amount of the engineered Trichoderma reesei is 4-6%.

[0023] Furthermore, the inoculum size of the engineered Trichoderma reesei is 5%.

[0024] The application of the *Trichoderma reesei* engineered strain that produces highly active cleaving polysaccharide monooxygenase or the fermentation broth of the *Trichoderma reesei* engineered strain in the degradation of cellulose.

[0025] The present invention provides a method for degrading cellulose, comprising constructing an enzymatic hydrolysis reaction system using the fermentation broth of the engineered Trichoderma reesei, cellulase, ascorbic acid and the cellulose sample to be degraded; and placing the enzymatic hydrolysis reaction system in a citrate buffer solution for degradation incubation.

[0026] Further, the enzymatic hydrolysis system for degrading cellulose consists of (0.5 mL): cellulase (Celluclast 1.5 L, 10 FPU / g DM; Novozyme 188, 500 kPa / g DM); 5 μL Trichoderma reesei engineered fermentation broth; 2 mM ascorbic acid; 2% microcrystalline cellulose.

[0027] Furthermore, the degradation temperature is 40–60°C, and the degradation incubation time is 20–28 hours.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The *Trichoderma reesei* strain producing cleaving polysaccharide monooxygenase provided by this invention utilizes the fusion expression of lpmo and pdi2. This leverages the ability of the PDI protein encoded by the pdi2 gene to enhance disulfide bond formation, stabilize the protein's three-dimensional structure, and improve the activity of the cleaving polysaccharide monooxygenase. The use of a flexible linker ensures the relative spatial independence of the two proteins, avoiding conformational changes and loss of function that may result from direct fusion. This improves the stability of the cleaving polysaccharide monooxygenase in *Trichoderma reesei* and helps to increase the expression level and activity of the cleaving polysaccharide monooxygenase.

[0030] Furthermore, by utilizing the endogenous constitutive strong promoter Pcdna1 of Trichoderma reesei, and leveraging its natural compatibility with Trichoderma reesei RNA polymerase, the transcription of the LPMO gene can be efficiently driven. Combined with the terminator Tpdc, a complete transcription unit is formed, ensuring the correctness and integrity of the LPMO gene transcription process, effectively improving the transcription efficiency of LPMO, and providing a sufficient mRNA basis for the subsequent expression of highly active LPMO.

[0031] The present invention provides a method for constructing a Trichoderma reesei engineered strain that produces cleaving polysaccharide monooxygenase, comprising constructing an lpmo-Tpdc transcription unit, ligating it with the promoter Pcdna1 to the pUC19 plasmid to construct the recombinant vector pPLT2, inserting the pdi2 gene to construct the recombinant vector pPLT3, and finally co-transforming pPLT3 with an helper plasmid into Trichoderma reesei protoplasts and screening to obtain engineered strains. This construction method is simple to operate, has high reproducibility, and helps to realize the large-scale construction and production of engineered strains.

[0032] The fermentation broth of Trichoderma reesei engineered bacteria provided by this invention expresses LPMO products, providing a material basis for subsequent research on the activity and application of LPMO and the development of industrial enzyme preparations.

[0033] The application provided by this invention is that LPMO can destroy the crystal structure of cellulose through oxidative cleavage, providing more action sites for cellulase, thereby enhancing the degradation efficiency of cellulose. This helps to solve the problem of cellulose degradation in biomass energy development and industrial waste treatment, and has broad market prospects and economic benefits.

[0034] The method for degrading cellulose provided by this invention utilizes engineered Trichoderma reesei strains to express LPMO, which significantly enhances its activity. This LPMO, in synergistic with cellulase, degrades microcrystalline cellulose, increasing the glucose yield of cellulase by 18.5%. Furthermore, the fusion expression of LPMO-PDI increases the glucose yield of cellulase by 33.1%. This demonstrates that the method has a significant effect on improving enzyme activity and can degrade cellulose more efficiently. It provides an innovative technical solution for the production of highly active LPMO and is applicable to biomass conversion and the development of industrial enzyme preparations. Attached Figure Description

[0035] Figure 1 This is a colony image of Trichoderma reesei GLPMO-01, the bacterial strain of this invention.

[0036] Figure 2 The results of the lpmo and Tpdc Overlap PCR ligation in this invention;

[0037] Figure 3 The structures of recombinant plasmids pPLT2 and pPLT3 of this invention are shown (with promoter, gene, terminator and restriction site labeled), wherein A is recombinant plasmid pPLT2 and B is recombinant plasmid pPLT3.

[0038] Figure 4 These are PCR electrophoresis images of transformant colonies of the present invention, wherein (A) is a PCR electrophoresis image of pPLT2 transformant, and (B) is a PCR electrophoresis image of pPLT3 transformant;

[0039] Figure 5 The glucose yield of enzymatic hydrolysis of crystalline cellulose in this invention is shown. Detailed Implementation

[0040] The following will describe the concept and technical effects of the present invention clearly and completely with reference to the embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0041] The engineered Trichoderma reesei strain (Trichoderma reesei GLPMO-01) of this invention, which produces highly active polysaccharide monooxygenase, was deposited on March 27, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode: 100101, with accession number CGMCC No. 41856. The suggested classification name is Trichoderma reesei.

[0042] The Trichoderma reesei strain used in this invention was purchased from Shanghai Preservation Microbial Co., Ltd.

[0043] The pUC19 vector used in this invention was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0044] The LB liquid culture medium used in this invention contains: 10 g / L peptone, 5 g / L yeast extract, and 10 g / L NaCl.

[0045] The LB solid medium used was LB liquid medium with 2% agar powder added.

[0046] The PDA solid culture medium used was purchased from Beijing Aoboxing Biotechnology Co., Ltd.

[0047] The Mandels nutrient solution used was: (NH4)2SO4: 14 g / L, urea: 3 g / L, KH2PO4: 20 g / L, CaCl2: 3 g / L, MgSO4·7H2O: 3 g / L, and water was added to a final volume of 1 L.

[0048] The Mandels trace element concentrate used was: FeSO4·7H2O: 5 g / L, ZnCl2: 0.7 g / L, CoCl2·6H2O: 3.7 g / L, MnCl2: 1.67 g / L, which was diluted with water to 1 L.

[0049] The 1 mol / L citric acid-sodium citrate buffer solution used was: citric acid: 210 g / L, NaOH: 78 g / L (after cooling, the volume was adjusted to 1 L, and the pH was measured to be 4.5).

[0050] The liquid basic culture medium used was: 100 mL / L Mandels nutrient concentrate, 1.0 mL / L Mandels trace element concentrate, 1.0 g / L peptone, 20.0 g / L anhydrous glucose, 50 mL / L 1 mol / L citrate buffer, 1.0 mL / L Tween 80, and water was added to a final volume of 1 L.

[0051] The protoplast regeneration medium used was: 4 mL of 2.5× basic medium (without glucose), 1 mL of 10× glucose (2 g / 10 mL), and 5 mL of 2× STC. All were sterilized separately and then mixed before use.

[0052] The STC used was: sorbitol: 218.6 g / L, 1 mol / L CaCl2: 50 ml / L, 1 mol / L Tris-HCl (pH 7.5) 10 ml / L, and water was added to bring the volume to 1 L.

[0053] The 60% (w / v) PEG-4000 used requires the following: a buffer solution containing 50 mmol / L CaCl2 and 10 mmol / L Tris-HCl (pH = 7.5) needs to be prepared in advance; 60 g of PEG-4000 is dissolved in the 10 mmol / L Tris-HCl buffer solution, stirred until dissolved, and then water is added to bring the volume to 1 L.

[0054] The fermentation medium consisted of: 10 g / L glucose, 1.4 g / L (NH4)2SO4, 2.0 g / L KH2PO4, 0.3 g / L MgSO4·7H2O, 0.4 g / L CaCl2·2H2O, 5 g / L yeast extract, 5 mM urea, 0.02% (w / v) Tween 80, and 1 / 50 (v / v) trace element solution; wherein the trace element composition was 0.25 g / L FeSO4·7H2O, 0.08 g / L MnSO4·H2O, 0.07 g / L ZnSO4·7H2O, 0.1 g / L CoCl2·2H2O, and the pH was adjusted to 5.0 with citric acid.

[0055] Example 1

[0056] (1) Design primers

[0057] Table 1: Primer Information

[0058]

[0059] (2) Obtaining the Trichoderma reesei genome

[0060] ① Collect Trichoderma reesei grown on PDA plates for 5 days and wash the spores;

[0061] ② Approximately 10 7 One Trichoderma reesei spore was inoculated into a 2L flask (Mandels liquid medium);

[0062] ③ Genomic DNA of Trichoderma reesei was extracted using the Ezup column-based fungal genomic DNA extraction kit (Sangon Biotech, Shanghai);

[0063] (3) Acquisition of genes

[0064] ① Promoter and terminator:

[0065] Pcdna1 is the promoter of an unknown protein gene, a strongly constitutive promoter; Tpdc is the terminator of the pdc gene. The promoter and terminator sequences were obtained from the *Trichoderma reesei* genome database (http: / / genome.jgi-psf.org / Trire2 / Trire2.home.html), and primers were designed based on these sequences. Using *Trichoderma reesei* QM9414 genomic DNA as a template, PCR amplification was performed using primers Pcdna1-F / Pcdna1-R and Tpdc-F / Tpdc-R, respectively, to obtain the Pcdna1 and Tpdc sequences.

[0066]

[0067]

[0068] ②lpmo gene:

[0069] The gene encoding the AA9 protein, lpmo (GenBank sequence number: THITE-170174), including its natural signal peptide sequence, is artificially synthesized and has a 6×histidine tag fused to the C-terminus of the target gene.

[0070] ③ pdi2 gene:

[0071] The pdi2 gene sequence information was obtained from the Trichoderma reesei genome database, and primers were designed based on this sequence information. Using the genomic DNA of Trichoderma reesei QM9414 as a template, PCR amplification was performed using primers pdi2-F / pdi2-R to obtain the pdi2 gene sequence containing a flexible linker (GGGGS)3 at the N-terminus.

[0072] (4) Overlap PCR:

[0073] ① Using the genomic DNA of *Trichoderma reesei* QM9414 as a template, the promoter Pcdna1 and terminator Tpdc sequences were amplified by PCR. The gene encoding the synthetically produced THITE-170174 protein, lpmo, was used as a template for PCR amplification.

[0074] Table 2: PCR reaction system

[0075]

[0076] PCR conditions: Pre-denaturation: 95℃ for 3 min; Denaturation: 98℃ for 10 s; Annealing: 55℃ for 15 s; Extension: 72℃ for 2 min, 30 cycles; Final extension: 72℃ for 10 min.

[0077] ②Overlap PCR ligation of lpmo and the terminator Tpdc. PCR conditions: 95℃ pre-denaturation for 3 min; 98℃ for 10 s, 50℃ for 15 s, 72℃ for 2.5 min, 30 cycles; 72℃ extension for 10 min. Incubation at 4℃. Figure 2 .lpmo and Tpdc Overlap PCR ligation results).

[0078] (4) Construction of lpmo expression plasmid

[0079] The pUC19 vector was double-digested using NedI / EcoRI.

[0080] The PCR products of Pcdna1 and lpmo-Tpdc, as well as the enzyme digestion product of pUC19, were purified.

[0081] ③ By using Gibson Master Mix (NEB) ligated Pcdna1 and lpmo-Tpdc to the NedI / EcoRI site of pUC19 to construct the recombinant plasmid pPLT2. Figure 3 (A.pPLT2 recombinant plasmid structure).

[0082] (5) Construction of lpmo-(GGGGS)3-pdi2 expression plasmid

[0083] ① Using recombinant plasmid pPLT2 as a template, PCR amplification was performed using primers pPLT2-F / pPLT2-R, and the PCR product was purified;

[0084] ② By using Gibson MasterMix (NEB) ligated the purified PCR product to the pdi2 gene fragment containing the N-terminus flexible linker (GGGGS)3, constructing the recombinant plasmid pPLT3. Figure 3 (B.pPLT3 recombinant plasmid structure).

[0085] (6) Co-transformation of two plasmids

[0086] ①Protoplast preparation

[0087] Trichoderma reesei hyphae were treated with a filamentous fungal protoplast preparation kit (Solepro) to obtain protoplasts (concentration >10). 8 (cells / mL).

[0088] ② Transformation and Screening

[0089] Plasmid mixing and ice bath: Add 10 μg of recombinant plasmid (linearized) and 10 μg of pAN7-1 plasmid to a centrifuge tube and react on ice for 30 min.

[0090] Metal bath treatment: 48℃ metal bath treatment for 2 min;

[0091] PEG4000-mediated transformation: Slowly add 450 μL of 60% PEG4000 and mix by pipetting, react at room temperature for 20 min; transfer the reaction solution to a new sterile 50 mL centrifuge tube, slowly add 2 mL of 60% PEG4000, mix by pipetting, react at room temperature for 5 min.

[0092] Centrifugation and washing: Add 20 mL of STC solution to a centrifuge tube, centrifuge at 8000 rpm for 8 min, and aspirate the supernatant with a pipette tip.

[0093] Regeneration culture: Suspend the bacterial cells in regeneration medium, add 10 mL of regeneration medium and shake to suspend the bacterial cells, then regenerate in a shaker at 30°C and 80 rpm for 24 hours.

[0094] Centrifuge and resuspend again: Add 20 mL of STC solution to a centrifuge tube, centrifuge at 8000 rpm for 8 min, aspirate the supernatant with a pipette tip, suspend the bacterial cells in 1 mL of STC solution, and prepare for plating.

[0095] Resistance screening: Spread 200 μL of bacterial culture onto a PDA plate containing 150 ug / mL hygromycin B and incubate at 28℃ for 5 days. Select transformants and inoculate them onto hygromycin B-resistant PDA plates and incubate for 5 days. Select successfully transformed colonies.

[0096] ③ Transformer verification

[0097] Colony PCR: Colonies were picked, and colony PCR was performed on the two recombinant bacteria using primers Pcdna1-F / Tpdc-R. The transformant that amplified the target band of 2930 bp was a successfully transformed recombinant Trichoderma reesei of pPLT2, numbered: Trichoderma reesei HLPMO-01. Figure 4 A. pPLT2 transformant PCR electrophoresis image); the transformant that amplified the target band of 4142bp was a recombinant Trichoderma reesei successfully transformed with pPLT3, numbered: Trichoderma reesei GLPMO-01 ( Figure 4 (PCR electrophoresis image of B.pPLT3 transformant).

[0098] Colony PCR and electrophoresis analysis successfully screened and accurately numbered *Trichoderma reesei* transformants transformed with pPLT2 and pPLT3 recombinant plasmids, respectively. These transformants provide important experimental materials for subsequent studies on the expression of related genes and protein functions in *Trichoderma reesei*, verifying the effectiveness of the transformation process and indicating that the recombinant plasmids have been successfully introduced into the *Trichoderma reesei* host bacteria, resulting in recombinant *Trichoderma reesei* GLPMO-01 (an engineered *Trichoderma reesei* strain producing highly active lysin monooxygenase) and recombinant *Trichoderma reesei* HLPMO-01.

[0099] Recombinant Trichoderma reesei GLPMO-01 (an engineered Trichoderma reesei strain producing highly active lysin monooxygenase) was deposited on March 27, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China, with accession number CGMCCNo.41856. The suggested classification name is Trichoderma reesei.

[0100] Enzyme activity verification: Fermentation of recombinant strains

[0101] The inoculation amounts of recombinant Trichoderma reesei HLPMO-01 and GLPMO-01 were as follows: colonies were picked up with an inoculation loop and inoculated into 50 mL of liquid basic medium and cultured for 16 h. Then, 5% of the colonies were inoculated into 500 mL of liquid fermentation medium. Fermentation was carried out at 28 °C and 200 rpm for 7 days. The supernatant was collected by centrifugation to obtain Trichoderma reesei engineered fermentation broth I and crude enzyme solution II, which were used for subsequent enzyme activity determination.

[0102] The fermentation medium consisted of: 10 g / L glucose, 1.4 g / L (NH4)2SO4, 2.0 g / L KH2PO4, 0.3 g / L MgSO4·7H2O, 0.4 g / L CaCl2·2H2O, 5 g / L yeast extract, 5 mM urea, 0.02% (w / v) Tween 80, and 1 / 50 (v / v) trace element solution; wherein the trace element composition was 0.25 g / L FeSO4·7H2O, 0.08 g / L MnSO4·H2O, 0.07 g / L ZnSO4·7H2O, 0.1 g / L CoCl2·2H2O, and the pH was adjusted to 5.0 with citric acid.

[0103] Example 2

[0104] (I) Experimental Materials

[0105] 1. 1.5L: Protein content: 127 mg / mL, Filter paper enzyme activity (FPU): 62 FPU / mL (determined by IUPAC standard method, reference: T. Ghose, Measurement of cellulase activities), β-glucosidase activity: 15 U / mL (determined according to Bailey and Nevalainen's method, reference: M. Bailey, KJNevalainen, M. Technology, Induction, isolation and testing of stable Trichoderma reesei mutants with improved production of solubilizing cellulase.);

[0106] 2. Novozyme 188: β-glucosidase activity: 231 U / mL;

[0107] 3. Microcrystalline cellulose, as a substrate for enzymatic hydrolysis, has high crystallinity and polymerization, making it an ideal substrate for simulating natural cellulose and can be used to evaluate the degradation ability of cellulase.

[0108] (II) Synergistic Enzymatic Hydrolysis Reaction

[0109] 1.05 mL reaction system: cellulase (Celluclast 1.5 L, 10 FPU / g DM; Novozyme 188, 500 kPa / g DM); 5 μ L Trichoderma reesei engineered fermentation broth; 2 mM ascorbic acid; 2% microcrystalline cellulose.

[0110] 2. Reaction Procedure: The reaction was carried out at 50°C in 50mM sodium citrate buffer (pH 5.0) with continuous shaking at 600 rpm on a hot mixer for 24 hours. After incubation, the sample was heated at 100°C for 10 minutes to stop enzyme activity. After cooling, the sample was centrifuged at 10000×g for 10 minutes, and the glucose content in the supernatant was determined by HPLC.

[0111] (III) Data Processing

[0112] All experiments were performed in triplicate. The average value was calculated, and the glucose yield was calculated using the yield formula. Significance analysis was performed using SPSS 19.0 software. Glucose yield:

[0113]

[0114] (iv) Results of the synergistic enzymatic hydrolysis reaction

[0115] See appendix Figure 5 In the synergistic enzymatic hydrolysis reaction using microcrystalline cellulose as a substrate, crude enzyme solution I increased the glucose yield of cellulase by 18.5%, indicating that the HLPMO-01 engineered bacteria successfully expressed active components such as LPMO. These active components can synergistically work with cellulase to increase the accessibility of cellulose by oxidatively cleaving cellulose chains, thereby improving the degradation efficiency of cellulase on cellulose and increasing glucose production. This demonstrates the effectiveness of the engineered bacteria in expressing specific active components. The HLPMO-01 engineered bacteria may have certain characteristics in gene expression regulation, protein folding, and secretion, enabling the active components such as LPMO produced by it to play a role in the synergistic enzymatic hydrolysis reaction.

[0116] Crude enzyme solution II can increase cellulase activity by 33.1%, indicating that the GLPMO-01 engineered bacteria is more outstanding in expressing active components such as LPMO. This is because the GLPMO-01 engineered bacteria has undergone more refined design in gene construction, promoter selection, and expression vector optimization, resulting in higher expression levels and stronger activity of LPMO. Its synergistic mechanism with cellulase is more efficient, which can more effectively promote the degradation of cellulose and further improve the glucose yield.

[0117] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A *Trichoderma reesei* engineered strain producing a highly active polysaccharide monooxygenase, characterized in that, The strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41856 and deposit date of March 27, 2025. The engineered strain contains the Pcdna1-lpmo-(GGGGS)3-pdi2-Tpdc fusion expression vector. In the fusion expression vector, the nucleotide sequence of the Pcdna1 promoter is shown in SEQ ID NO. 1, and the nucleotide sequence of the lpmo encoding gene is shown in SEQ ID NO.

2.

2. The engineered Trichoderma reesei strain producing highly active lysin-degrading polysaccharide monooxygenase according to claim 1, characterized in that, The Pcdna1 promoter contains a NedI restriction site upstream and the Tpdc terminator contains an EcoRI restriction site downstream.

3. The method for constructing an engineered Trichoderma reesei strain producing highly active lysin monooxygenase as described in claim 1, characterized in that, include: Step 1: Construct the lpmo-Tpdc transcription unit, link it to the pUC19 plasmid along with the promoter Pcdna1, and construct the recombinant vector pPLT2; Step 2: The protein disulfide bond isomerase encoding gene pdi2 is linked using a linker peptide and inserted into the recombinant vector pPLT2 described in Step 1 to construct the recombinant plasmid pPLT3. Step 3: The recombinant plasmid pPLT3 and the helper plasmid from Step 2 were co-transformed into Trichoderma reesei protoplasts, and the strains were screened to obtain engineered Trichoderma reesei strains.

4. The method for constructing the engineered Trichoderma reesei strain producing highly active lysin monooxygenase according to claim 3, characterized in that, The mass ratio of the recombinant plasmid to the helper plasmid is 1:1, and the helper plasmid is pAN7-1.

5. A fermentation broth of engineered Trichoderma reesei, characterized in that, The fermentation broth of *Trichoderma reesei* engineered bacteria is obtained by fermentation of *Trichoderma reesei* engineered bacteria that produce highly active cleaving polysaccharide monooxygenase as described in claim 1 or 2.

6. The application of the *Trichoderma reesei* engineered strain producing highly active cleaving polysaccharide monooxygenase as described in claim 1 or 2, or the fermentation broth of the *Trichoderma reesei* engineered strain as described in claim 5, in the degradation of cellulose.

7. A method for degrading cellulose, characterized in that, The method includes constructing an enzymatic hydrolysis reaction system using the fermentation broth of the engineered Trichoderma reesei as described in claim 5, cellulase, ascorbic acid, and the cellulose sample to be degraded; and placing the enzymatic hydrolysis reaction system in a citrate buffer solution for degradation incubation.

8. The method for degrading cellulose according to claim 7, characterized in that, The degradation temperature is 40-60℃, and the degradation incubation time is 20-28 hours.

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