Trichoderma reesei engineering bacterium for producing high-activity lysing polysaccharide monooxygenase as well as construction method and application of trichoderma reesei engineering bacterium

Abstract

The invention discloses a trichoderma reesei engineering bacterium for producing high-activity lytic polysaccharide monooxygenase as well as a construction method and application of the trichoderma reesei engineering bacterium, and belongs to the technical field of genetic engineering. According to the invention, a Trichoderma reesei endogenous constitutive strong promoter Pcdna1 is adopted to drive transcription of an LPMO gene, and the Trichoderma reesei endogenous constitutive strong promoter Pcdna1 is matched with a terminator Tpdc to construct a complete transcription unit (Pcdna1-lpmo-(GGGGS) 3-pdi2-Tpdc), so that the transcription efficiency of the LPMO is improved; the LPMO and a protein disulfide bond isomerase (PDI) encoding gene pdi2 are subjected to fusion expression through a (GGGGS) 3 flexible connecting peptide, and the PDI-mediated disulfide bond forming ability is utilized to enhance the enzyme activity and secretion efficiency of the LPMO. The activity of the expressed LPMO is remarkably improved, the LPMO and cellulase are cooperated to degrade microcrystalline cellulose, the glucose yield of the cellulase can be improved by 18.5%, and the glucose yield of the cellulase can be improved by 33.1% through fusion expression of LPMO-PDI. The invention provides an innovative technical scheme for producing high-activity LPMO, and is suitable for biomass conversion and industrial enzyme preparation development.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to an engineered bacterium of Trichoderma reesei producing highly active lytic polysaccharide monooxygenase, a construction method and an application thereof. Background Art

[0002] In the field of biomass energy utilization, lignocellulose, as the most abundant renewable resource on Earth, its effective degradation is key to achieving efficient biomass conversion. Currently, cellulose degradation mainly relies on a variety of cellulase enzymes, including endoglucanases, exoglucanases, and β-glucosidases. However, due to the hydrogen bonding between cellulose molecules in lignocellulose, it forms a highly crystalline structure, and hemicellulose and lignin, as intercellular matrix, fill the microfibrils, increasing the difficulty of cellulose degradation, making lignocellulose resources difficult 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 cellulose's crystalline structure, remove lignin, and loosen the cellulose molecules, increasing the contact area between cellulose and cellulase, thereby promoting cellulose saccharification. Lytic polysaccharide monooxygenase (LPMO), a novel oxidase from the AA9 family, can directly act on crystalline cellulose, breaking long cellulose chains into shorter ones, making them more susceptible to hydrolysis by cellulase. Therefore, the development and application of LPMOs has become an effective approach to improving the biodegradability of lignocellulose.

[0004] Currently used LPMO expression systems are mostly Escherichia coli or Pichia pastoris. As a host for recombinant protein production, E. coli offers advantages such as low cost, ease of operation, rapid growth, suitability for large-scale production, and ease of genetic modification. However, it suffers from issues such as a lack of eukaryotic post-translational modifications (such as glycosylation) and interference with the electron transport chain caused by its reducing cytoplasmic environment, all of which may affect LPMO enzyme activity. As a protein expression host, P. pastoris offers advantages such as high-density culture, high levels of secreted protein expression, and glycosylation capabilities. However, this system has significant limitations regarding post-expression modifications of LPMOs. For one thing, LPMOs are all composed of a single AA9 catalytic module, which begins with a typical conserved histidine residue shared by all LPMOs. To ensure the correct binding of the catalytic copper ion to the N-terminal histidine residue, the recombinant protein's signal peptide must be correctly cleaved, but cleavage errors often occur in the P. pastoris expression system. Furthermore, P. pastoris is unable to perform N-terminal τ-methylation on LPMO. LPMO produced by filamentous fungi have an N-terminal τ-methylation modification at histidine 1, which is associated with the correct binding of the copper ion and the stability of the active site. However, the P. pastoris expression system lacks this methylation modification, which can affect LPMO enzymatic activity. Therefore, in order to obtain recombinant proteins with a structure consistent with the native LPMO, it is essential to seek a new expression system.

[0005] Trichoderma reesei has demonstrated significant systemic advantages as an engineered bacterium. Compared to E. coli and P. pastoris, Trichoderma reesei not only has a very strong protein secretion capacity, but its eukaryotic glycosylation modification conferred by the endoplasmic reticulum-Golgi system can make the thermal stability and substrate affinity of the AA9 family LPMO reach the level of natural activity. In addition, Trichoderma reesei's powerful antioxidant system (catalase CAT1 and superoxide dismutase SOD) can efficiently remove H2O2 produced by LPMO catalysis, greatly improving cell survival rate and completely avoiding the growth inhibition of E. coli caused by the accumulation of reactive oxygen species (ROS). What is particularly outstanding is that Trichoderma reesei is adapted to solid-state fermentation technology, and the use of agricultural waste (such as corn cobs and sugarcane bagasse) can reduce the production cost of LPMO to US$50-80 / gram, which is 60%-80% lower than the methanol-induced liquid fermentation of yeast. Despite the numerous advantages of Trichoderma reesei for expressing LPMOs, target protein production in T. reesei expression systems is often limited by gene transcription efficiency. Improving the transcription efficiency of LPMO genes in T. reesei, and thereby increasing target protein production, remains a key challenge. Summary of the Invention

[0006] In view of the problem of enzyme activity influencing the existing LPMO expression systems (E. coli, P. pastoris), and the current technical status that although Trichoderma reesei has advantages, the target protein production is limited by the gene transcription efficiency, the purpose of the present invention is to provide a Trichoderma reesei engineered bacterium that produces highly active lytic polysaccharide monooxygenase and its construction method and application, utilize the Trichoderma reesei expression system to achieve correct post-translational modification of LPMO, adopt a constitutive strong promoter (Pcdna1) to break through the gene transcription bottleneck, and ensure correct folding and improve activity stability by co-expressing protein disulfide isomerase (PDI).

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention provides an engineered Trichoderma reesei strain that produces highly active lytic polysaccharide monooxygenase. The strain is deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms with a deposit number of CGMCC No. 41856 and a deposit date of March 27, 2025.

[0009] The engineering bacteria contains a Pcdna1-lpmo-(GGGGS)3-pdi2-Tpdc fusion expression vector.

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

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

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

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

[0014] Step 1: construct the lpmo-Tpdc transcription unit and connect it to the promoter Pcdna1 into the pUC19 plasmid to construct the recombinant vector pPLT2;

[0015] Step 2, using a connecting peptide to connect the protein disulfide isomerase encoding gene pdi2, and inserting it into the recombinant vector pPLT2 described in step 1 to construct the recombinant vector pPLT3;

[0016] Step 3: co-transform the recombinant vector pPLT3 and the helper plasmid from step 2 into Trichoderma reesei protoplasts, and screen to obtain engineered Trichoderma reesei that produces lytic polysaccharide monooxygenase.

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

[0018] The present invention provides a fermentation liquid of an engineered Trichoderma reesei. The fermentation liquid of the engineered Trichoderma reesei is obtained by fermenting the engineered Trichoderma reesei producing a lytic polysaccharide monooxygenase.

[0019] The method for preparing the fermentation broth of the engineered Trichoderma reesei comprises activating the engineered Trichoderma reesei producing lytic polysaccharide monooxygenase and inoculating the broth into a liquid fermentation medium for fermentation.

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

[0021] Furthermore, the fermentation medium is composed 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 powder, 5 mM urea, 0.02% (w / v) Tween80, 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 adjusted to pH 5.0.

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

[0023] Furthermore, the inoculation amount of the engineered Trichoderma reesei is 5%.

[0024] The application of the engineered Trichoderma reesei bacteria producing high-activity lytic polysaccharide monooxygenase or the fermentation liquid of the engineered Trichoderma reesei bacteria in degrading cellulose.

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

[0026] Furthermore, the composition of the cellulose degradation enzymatic hydrolysis system is (0.5 mL): cellulase (Celluclast 1.5 L, 10 FPU / g DM; Novozyme 188, 500 nkat / g DM); 5 μL of Trichoderma reesei engineered bacteria fermentation broth; 2 mM ascorbic acid; and 2% microcrystalline cellulose.

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

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

[0029] The present invention provides an engineered strain of Trichoderma reesei that produces a lytic polysaccharide monooxygenase. The fusion expression of lpmo and pdi2 utilizes the ability of the PDI protein encoded by the pdi2 gene to enhance disulfide bond formation, stabilizes the three-dimensional structure of the protein, and improves the activity of the lytic polysaccharide monooxygenase. The use of a flexible linker ensures the relative independence of the two proteins in space, avoids protein conformational changes and loss of function that may be caused by direct fusion, thereby improving the stability of the lytic polysaccharide monooxygenase in Trichoderma reesei and contributing to increasing the expression level and activity of the lytic polysaccharide monooxygenase.

[0030] Furthermore, by utilizing the endogenous constitutive strong promoter Pcdna1 of Trichoderma reesei and its natural adaptability to Trichoderma reesei RNA polymerase, it can efficiently drive the transcription of the LPMO gene and form a complete transcription unit in combination with the terminator Tpdc, ensuring the correctness and integrity of the LPMO gene transcription process, effectively improving the transcription efficiency of LPMO, and providing sufficient mRNA basis for the subsequent expression of highly active LPMO.

[0031] The present invention provides a method for constructing an engineered bacterium of Trichoderma reesei that produces a lytic polysaccharide monooxygenase, comprising constructing an lpmo-Tpdc transcription unit, connecting it to a pUC19 plasmid with a promoter Pcdna1 to construct a recombinant vector pPLT2, then inserting a pdi2 gene to construct a recombinant vector pPLT3, and finally co-transforming pPLT3 and an auxiliary plasmid into Trichoderma reesei protoplasts and screening to obtain the engineered bacterium. The construction method is simple to operate, has high repeatability, and is conducive to the large-scale construction and production of the engineered bacterium.

[0032] The fermentation liquid of the engineered Trichoderma reesei provided by the present invention and the product of the engineered bacteria expressing LPMO provide 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 the present invention is that LPMO can destroy the crystal structure of cellulose through oxidative cleavage, providing more action sites for cellulase, thereby enhancing the efficiency of cellulose degradation, helping 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 the present invention significantly improves the activity of LPMO expressed by the engineered bacteria of Trichoderma reesei, and synergistically degrades microcrystalline cellulose with cellulase, which can increase the glucose yield of cellulase by 18.5%, while the fusion expression of LPMO-PDI can increase the glucose yield of cellulase by 33.1%. This shows that this method has a significant effect in improving enzyme activity and can degrade cellulose more efficiently, providing an innovative technical solution for the production of highly active LPMO, and is suitable for biomass conversion and industrial enzyme preparation development. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a picture of the Trichoderma reesei GLPMO-01 colony of the present invention;

[0036] Figure 2 This is the OverlapPCR connection result of lpmo and Tpdc of the present invention;

[0037] Figure 3 The structures of the recombinant plasmids pPLT2 and pPLT3 of the present invention (with promoter, gene, terminator and restriction sites marked), wherein A is the recombinant plasmid pPLT2 and B is the recombinant plasmid pPLT3;

[0038] Figure 4 The PCR electrophoresis diagrams of transformant colonies of the present invention are shown, wherein (A) is the PCR electrophoresis diagram of the pPLT2 transformant, and (B) is the PCR electrophoresis diagram of the pPLT3 transformant;

[0039] Figure 5 It is the glucose yield of the enzymatic hydrolysis of crystalline cellulose of the present invention. DETAILED DESCRIPTION

[0040] The following will clearly and completely describe the concept of the present invention and the technical effects produced in conjunction with the embodiments, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention. The test methods used in the embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used, unless otherwise specified, can all be reagents and materials obtained from commercial channels.

[0041] The engineered Trichoderma reesei bacteria (Trichoderma reesei GLPMO-01) producing highly active lytic polysaccharide monooxygenase of the present invention was deposited on March 27, 2025 at the General Microbiology Center (CGMCC) of the China Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101, with a deposit number of CGMCC No. 41856 and a suggested classification name: Trichoderma reesei.

[0042] The Trichoderma reesei QM9414 (ATCC 26921) used in the present invention was purchased from Shanghai Baocang Microorganism Co., Ltd.

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

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

[0045] LB solid medium used: 2% agar powder was added to LB liquid medium.

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

[0047] The Mandels nutrient salt concentrate 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 make the volume to 1 L.

[0048] The Mandels trace element concentrate used was: FeSO4·7H2O: 5g / L, ZnCl2: 0.7g / L, CoCl2·6H2O: 3.7g / L, MnCl2: 1.67g / L, and water was added to make the volume to 1L.

[0049] 1 mol / L citric acid-sodium citrate buffer solution used: 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: Mandels nutrient salt concentrate 100 ml / L, Mandels trace element concentrate 1.0 mL / L, peptone 1.0 g / L, anhydrous glucose 20.0 g / L, 1 mol / L citric acid buffer 50 mL / L, Tween 80 1.0 mL / L, add water to make up to 1 L.

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

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

[0053] 60% (w / v) PEG-4000: Prepare 10 mmol / L Tris-HCl buffer (pH 7.5) containing 50 mmol / L CaCl2 in advance; dissolve 60 g of PEG-4000 in 10 mmol / L Tris-HCl buffer, stir to dissolve, and then add water to 1 L.

[0054] The fermentation medium comprises: 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; the trace element composition comprises 0.25 g / L FeSO4·7H2O, 0.08 g / L MnSO4·H2O, 0.07 g / L ZnSO4·7H2O, and 0.1 g / L CoCl2·2H2O, and the pH is 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 About 10 7 2 L flasks (Mandels liquid medium) were inoculated with 100 spores of Trichoderma reesei;

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

[0063] (3) Gene acquisition

[0064] ① Promoter and terminator:

[0065] Pcdna1, a strong constitutive promoter of an unknown protein gene; Tpdc, the terminator of the pdc gene. Primers were designed based on the promoter and terminator sequence information obtained from the Trichoderma reesei genome database (http: / / genome.jgi-psf.org / Trire2 / Trire2.home.html). Using genomic DNA from Trichoderma reesei QM9414 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, was artificially synthesized, and a 6×histidine tag was fused to the C-terminus of the target gene.

[0070] ③pdi2 gene:

[0071] The pdi2 gene sequence was obtained from the Trichoderma reesei genome database, and primers were designed based on this sequence. Using genomic DNA from Trichoderma reesei QM9414 as a template, PCR amplification was performed using primers pdi2-F and pdi2-R, respectively, 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, PCR amplification of the promoter Pcdna1 and terminator Tpdc sequences was performed. PCR amplification of the gene encoding the artificially synthesized THITE-170174 protein, lpmo, was also performed as a template.

[0074] Table 2: PCR reaction system

[0075]

[0076] PCR conditions: pre-denaturation: 95°C for 3 min; denaturation: 98°C for 10 s, annealing: 55°C for 15 s, extension: 72°C for 2 min, 30 cycles; final extension: 72°C for 10 min.

[0077] ② Overlap PCR to connect lpmo and the terminator Tpdc. PCR conditions: 95°C pre-denaturation for 3 minutes; 98°C for 10 seconds, 50°C for 15 seconds, 72°C for 2.5 minutes, 30 cycles; 72°C extension for 10 minutes. Incubation at 4°C. Figure 2 .lpmo and Tpdc OverlapPCR connection results).

[0078] (4) Construction of lpmo expression plasmid

[0079] The pUC19 vector was double-digested with NedI / EcoRI;

[0080] Purify the PCR products of Pcdna1 and lpmo-Tpdc and the enzyme digestion products of pUC19;

[0081] ③By using Gibson Master Mix (NEB) was used to connect Pcdna1 and lpmo-Tpdc to the NedI / EcoRI sites 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] ①Using the recombinant plasmid pPLT2 as a template, PCR amplification was performed using primers pPLT2-F / pPLT2-R, and the PCR product was purified;

[0084] ②By using Gibson The purified PCR product was ligated with the pdi2 gene fragment containing a flexible linker (GGGGS) 3 at the N-terminus by MasterMix (NEB) to construct the recombinant plasmid pPLT3 ( Figure 3 B. pPLT3 recombinant plasmid structure).

[0085] (6) Dual plasmid co-transformation

[0086] ① Protoplast preparation

[0087] The mycelia were treated with a filamentous fungal protoplast preparation kit (Solebo) to obtain protoplasts (concentration>10 8 cells / mL).

[0088] ②Transformation 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 minutes.

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

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

[0093] Regeneration culture: suspend the bacteria in regeneration medium, add 10 mL of regeneration medium and shake the suspended bacteria, regenerate at 30°C and 80 rpm for 24 hours.

[0094] Centrifuge and resuspend again: add 20mL STC solution into the centrifuge tube, centrifuge at 8000rpm for 8min, aspirate the supernatant with a pipette tip, and resuspend the bacteria with 1mL STC solution in preparation for coating.

[0095] Resistance screening: 200 μL of bacterial solution was spread on a PDA resistance plate containing 150 μg / mL hygromycin B, and cultured in a 28°C incubator for 5 days. Transformants were picked and inoculated on a hygromycin B-resistant PDA plate and cultured for 5 days. Successful transformed colonies were screened.

[0096] ③ Transformant verification

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

[0098] Through colony PCR and electrophoresis analysis, Trichoderma reesei transformants carrying the pPLT2 and pPLT3 recombinant plasmids were successfully screened and accurately numbered. These transformants provide important experimental material for subsequent studies on gene expression and protein function in Trichoderma reesei. The effectiveness of the transformation process was verified, indicating that the recombinant plasmids were successfully introduced into the Trichoderma reesei host strain, resulting in recombinant Trichoderma reesei GLPMO-01 (an engineered strain producing a highly active lytic polysaccharide monooxygenase) and recombinant Trichoderma reesei HLPMO-01.

[0099] The recombinant Trichoderma reesei GLPMO-01 (an engineered Trichoderma reesei that produces highly active lytic polysaccharide monooxygenase) was deposited on March 27, 2025, at the General Microbiology Center of the China Culture Collection Administration (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, with the deposit number: CGMCC No. 41856, and the recommended taxonomic name: Trichoderma reesei.

[0100] Enzyme activity verification: recombinant strain fermentation

[0101] The inoculum size of recombinant Trichoderma reesei HLPMO-01 and GLPMO-01 was as follows: a colony was dipped with an inoculation loop and inoculated into 50 mL of liquid minimal culture medium and cultured for 16 h. Then, a 5% inoculum size was inoculated into 500 mL of liquid fermentation medium; fermentation was carried out at 28°C, 200 rpm, and the supernatant was collected by centrifugation to obtain Trichoderma reesei engineered fermentation broth I and crude enzyme broth II, respectively, for subsequent enzyme activity determination;

[0102] The fermentation medium comprises: 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; the trace element composition comprises 0.25 g / L FeSO4·7H2O, 0.08 g / L MnSO4·H2O, 0.07 g / L ZnSO4·7H2O, and 0.1 g / L CoCl2·2H2O, and the pH is adjusted to 5.0 with citric acid.

[0103] Example 2

[0104] (1) Experimental materials

[0105] 1. 1.5L: Protein content: 127 mg / mL, filter paper enzyme activity (FPU): 62 FPU / mL (determined by IUPAC standard assay, reference: T. Ghose, Measurement of cellulase activities), β-glucosidase activity: 15 U / mL (determined according to the method of Bailey and Nevalainen, reference: M. Bailey, KJENevalainen, 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 degree of polymerization. It is an ideal substrate to simulate natural cellulose and can be used to evaluate the degradation ability of cellulase.

[0108] (2) Synergistic enzymatic hydrolysis reaction

[0109] 1.0.5 mL reaction system: cellulase (Celluclast 1.5 L, 10 FPU / g DM; Novozyme 188, 500 nkat / g DM); 5 μL fermentation broth of engineered Trichoderma reesei; 2 mM ascorbic acid; 2% microcrystalline cellulose.

[0110] 2. Reaction Step: The reaction was carried out in 50 mM sodium citrate buffer (pH 5.0) at 50°C with continuous shaking at 600 rpm on a thermomixer. After incubation for 24 hours, the sample was heated at 100°C for 10 minutes to stop enzyme activity. After cooling, the sample was centrifuged at 10,000 × g for 10 minutes, and the glucose content in the supernatant was determined by HPLC.

[0111] (3) Data processing

[0112] All experiments were set up in triplicate, and the average value was calculated. Glucose yield was calculated according to the yield formula, and significance analysis was performed using SPSS 19.0 analysis software. Glucose yield:

[0113]

[0114] (IV) Results of synergistic enzymatic hydrolysis reaction

[0115] See attached Figure 5 In the synergistic enzymatic hydrolysis reaction with microcrystalline cellulose as the substrate, the crude enzyme solution I can increase the glucose yield of cellulase by 18.5%, indicating that the HLPMO-01 engineered bacteria successfully expressed active LPMO and other components; these active ingredients can synergistically act with cellulase to oxidatively cleave cellulose chains, increase the accessibility of cellulose, thereby improving the efficiency of cellulase in cellulose degradation and increasing glucose production, reflecting the effectiveness of the engineered bacteria in expressing specific active ingredients. The HLPMO-01 engineered bacteria may have certain characteristics in gene expression regulation, protein folding and secretion, so that the active ingredients such as LPMO it produces can play a role in the synergistic enzymatic hydrolysis reaction.

[0116] Crude enzyme solution II can increase the activity of cellulase by 33.1%, indicating that the GLPMO-01 engineered bacteria is better at expressing active ingredients such as LPMO. This is because the GLPMO-01 engineered bacteria has undergone more sophisticated design in terms of 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 increase the yield of glucose.

[0117] The above content is only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. An engineered strain of Trichoderma reesei producing highly active lytic polysaccharide monooxygenase, characterized in that: The strain is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms, with the deposit number CGMCC No.41856 and the deposit date being March 27, 2025.

2. The engineered strain of Trichoderma reesei producing highly active lytic polysaccharide monooxygenase according to claim 1, characterized in that: The engineering bacteria contains a Pcdna1-lpmo-(GGGGS)3-pdi2-Tpdc fusion expression vector.

3. The engineered strain of Trichoderma reesei producing highly active lytic polysaccharide monooxygenase according to claim 2, characterized in that: In the fusion expression vector, the Pcdna1 promoter nucleotide sequence is shown as SEQ ID NO.1, and the lpmo encoding gene nucleotide sequence is shown as SEQ ID NO.

2.

4. The engineered strain of Trichoderma reesei producing highly active lytic polysaccharide monooxygenase according to claim 3, characterized in that: The upstream of the Pcdna1 promoter contains a NedI restriction enzyme site, and the downstream of the Tpdc terminator contains an EcoRI restriction enzyme site.

5. The method for constructing an engineered strain of Trichoderma reesei producing highly active lytic polysaccharide monooxygenase according to claim 1, characterized in that: include: Step 1: construct the lpmo-Tpdc transcription unit and connect it to the promoter Pcdna1 into the pUC19 plasmid to construct the recombinant vector pPLT2; Step 2, using a connecting peptide to connect the protein disulfide isomerase encoding gene pdi2, and inserting it into the recombinant vector pPLT2 described in step 1 to construct a recombinant plasmid pPLT3; Step 3: co-transform the recombinant plasmid pPLT3 and the auxiliary plasmid from step 2 into Trichoderma reesei protoplasts, and screen to obtain engineered Trichoderma reesei.

6. The method for constructing an engineered strain of Trichoderma reesei producing highly active lytic polysaccharide monooxygenase according to claim 5, characterized in that: The mass ratio of the recombinant plasmid to the auxiliary plasmid is 1:1, and the auxiliary plasmid is pAN7-1.

7. A fermentation broth of engineered Trichoderma reesei, characterized in that: The fermentation broth of the engineered Trichoderma reesei is obtained by fermenting the engineered Trichoderma reesei producing the highly active lytic polysaccharide monooxygenase according to any one of claims 1 to 4.

8. Use of the engineered Trichoderma reesei producing highly active lytic polysaccharide monooxygenase according to any one of claims 1 to 4 or the fermentation liquid of the engineered Trichoderma reesei according to claim 7 in cellulose degradation.

9. A method for degrading cellulose, characterized in that: The method comprises constructing an enzymatic hydrolysis reaction system using the fermentation liquid of the engineered Trichoderma reesei according to claim 7, cellulase, ascorbic acid and a cellulose sample to be degraded; and placing the enzymatic hydrolysis reaction system in a citric acid buffer for degradation and incubation.

10. The method for degrading cellulose according to claim 9, characterized in that: The degradation temperature is 40-60° C. and the degradation incubation is 20-28 hours.

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