Trichoderma reesei engineering strain as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological energy and biology, in particular to a trichoderma reesei engineering strain and a preparation method and application thereof. The Trichoderma reesei engineering strain (Trichoderma reesei) TS-5 is preserved in the China Center for Type Culture Collection, the preservation number is CCTCC NO: M 2026207, the preservation date is January 23, 2026, the filter paper enzyme activity of the Trichoderma reesei engineering strain is remarkably improved, and the yield of glucose in enzymatic hydrolysate containing the Trichoderma reesei engineering strain is remarkably improved.

Description

Technical Field

[0001] This invention relates to the fields of bioenergy and biotechnology, specifically to an engineered strain of Trichoderma reesei, its preparation method, and its application. Background Technology

[0002] Cellulose degradation is achieved through the synergistic action of at least three enzymes: exo-β-1,4-glucanosidase, endo-β-1,4-glucanosidase, and β-glucosidase. During cellulose degradation, these three types of cellulase work synergistically to efficiently degrade cellulose. Exo-enzymes hydrolyze the crystalline regions of cellulose, breaking them down from both the reducing and non-reducing ends of the cellulose chain. Endo-enzymes hydrolyze the acrystalline regions of cellulose, randomly breaking down β-1,4-glycosidic bonds in the chain, breaking the long cellulose chain into shorter chains with varying degrees of polymerization, thus promoting the hydrolysis by exo-enzymes. β-glucosidase hydrolyzes cellobiose and soluble oligosaccharides. Through the synergistic action of these three enzymes, bioresistant cellulose is hydrolyzed into simple, fermentable glucose. With the deepening research on lignocellulose-degrading enzymes, researchers have discovered various lignocellulose-degrading accessory proteins. These accessory proteins themselves cannot degrade lignocellulose, but they can synergistically promote its degradation with cellulase. Plant expansins are a class of cell wall relaxants secreted by higher plants. They regulate cell wall extensibility through non-enzymatic physical processes and can disrupt the hydrogen bond network of cellulose during biomass degradation, thereby improving enzymatic hydrolysis efficiency. However, current research has revealed numerous difficulties and challenges in the heterologous expression of plant expansins in biomass. *Trichoderma reesei* is widely used in the industrial production of cellulase, producing exoglucanases, endoglucanases, and β-glucosidases required for cellulose degradation. However, the low content of expansins in *Trichoderma reesei* cell secretions limits the overall saccharification efficiency of the produced cellulase.

[0003] In summary, there is an urgent need for a strain and its preparation method that can improve the content of swelling hormones and enzyme production efficiency in the enzyme system produced by Trichoderma reesei. Summary of the Invention

[0004] This invention addresses the technical problem of providing a strain and its preparation method for increasing the content of swelling hormones and enzyme production efficiency in the enzyme system produced by Trichoderma reesei.

[0005] To achieve the above objectives, the first aspect of the present invention provides an engineered strain of Trichoderma reesei, TS-5, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2026207 and deposit date of January 23, 2026.

[0006] A second aspect of the present invention provides a method for preparing the above-mentioned engineered Trichoderma reesei strain TS-5, comprising the following steps:

[0007] S1. Preparation of spore suspension of Trichoderma reesei strain RUT-C30;

[0008] S2. Prepare recombinant plasmid pCZF8-TgSwol containing the target gene TgSwol;

[0009] S3. Transform the recombinant plasmid pCZF8-TgSwol into competent cells of Agrobacterium tumefaciens to obtain Agrobacterium tumefaciens AGL-1 containing the recombinant plasmid pCZF8-TgSwol;

[0010] S4. The Agrobacterium rhizogenes AGL-1 containing the recombinant plasmid pCZF8-TgSwol obtained in S3 was mixed evenly with the spore suspension obtained in S1, cultured, screened, and verified to obtain the Trichoderma reesei engineered strain TS-5.

[0011] A third aspect of the present invention provides the application of the above-mentioned Trichoderma reesei engineered strain TS-5 or the above-mentioned preparation method in straw enzyme saccharification, wherein the filter paper enzyme activity of the Trichoderma reesei engineered strain is 6.12-6.37 FPU / mL.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] This invention constructed an engineered Trichoderma reesei strain TS-5, fermented the strain in shake flasks, and measured the cellulase activity produced by the strain. The results showed that the cellulase activity in the enzyme system produced by the engineered Trichoderma reesei strain TS-5 was significantly improved. After batch fermentation in a fermenter for 132 h, the filter paper enzyme activity of the engineered Trichoderma reesei strain was 6.37 FPU / mL, which was 44% higher than that of the starting strain RUT-C30 (4.42 FPU / mL). Using the crude enzyme solution of cellulase with high swelling agent content produced by the engineered Trichoderma reesei strain TS-5 of this invention to hydrolyze corn straw, under the same enzyme dosage, the glucose yield in the enzymatic hydrolysate of the engineered Trichoderma reesei strain TS-5 was 24% higher than that of the starting strain RUT-C30. Attached Figure Description

[0014] Figure 1 The image shows the recombinant plasmid pCZF8-TgSwol obtained in Example 1.

[0015] Figure 2The results show the comparison of filter paper enzyme activity between the engineered Trichoderma reesei strains TS-1~TS-6 and the starting strain RUT-C30 in Example 3.

[0016] Figure 3 The comparison of enzyme activities and protein content between the starting strain RUT-C30 and the engineered strain TS-5 in Example 3 is shown below. (a) is filter paper enzyme activity; (b) is endoglucanase activity; (c) is exoglucanase activity; (d) is β-glucosidase activity; (e) is xylanase activity; and (f) is extracellular protein concentration.

[0017] Figure 4 This is a comparison of the filter paper enzyme activity of Trichoderma reesei RUT-C30 and engineered strain TS-5 fermented in fermenters in Example 4.

[0018] Figure 5 Example 5 compares the glucose yield of corn stalks pretreated with crude enzyme solution of Trichoderma reesei RUT-C30 and engineered strain TS-5. Detailed Implementation

[0019] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0020] Phylogenetic genomic analysis revealed that TgSwol shares homology with glucanase, GH5 family proteins, pectic acid lyase, galactosidase, CE16 family proteins, glucosidase, xylanase, expansin, and swelling pigment. TgSwol showed the highest homology with swelling pigment and expansin, and also exhibited similarities with cellulases (glucanase and glucosidase) and hemicellulases (galactosidase, CE16 family proteins, and xylanase). This suggests that TgSwol may play an important role in the degradation of lignocellulose. Swellsin can impair the tensile strength of filter paper without producing reducing sugars, and it exhibits a significant synergistic effect with different cellulases and hemicellulases in the degradation of lignocellulose. Overexpression of the swelling pigment gene in *Trichoderma reesei* leads to excessive production of swelling pigment, consuming large amounts of cellular resources (precursors, energy), creating a metabolic burden, and inhibiting the expression of the core cellulase gene. The TgSwol gene can promote the expression of glycoside hydrolases, especially glycoside hydrolases from the AA, CBM, and HFB families. These proteins can greatly improve the accessibility of cellulase to cellulose by targeting and depolymerizing lignocellulose biomass.

[0021] Therefore, the inventors discovered that by constructing an engineered strain of Trichoderma reesei that overexpresses the TgSwol gene, they could improve the enzyme production efficiency of Trichoderma reesei and regulate the enzyme system structure, thereby improving the overall saccharification efficiency of cellulase.

[0022] To achieve this goal, the inventors attempted a strain preparation method by overexpressing the intron-removed TgSwol gene from Trichoderma guizhouense to obtain an engineered Trichoderma reesei strain with significantly enhanced filter paper enzyme activity. The glucose yield in the enzymatic hydrolysate containing the engineered Trichoderma reesei strain was significantly increased.

[0023] The first aspect of this invention provides an engineered strain of Trichoderma reesei, TS-5, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2026207 and deposit date of January 23, 2026.

[0024] In this invention, the constructed Trichoderma reesei engineered strain TS-5 effectively increased the content of swelling hormones and enzyme production efficiency in the enzyme system produced by the Trichoderma reesei engineered strain, thereby improving its ability to degrade lignocellulosic biomass.

[0025] According to the present invention, the engineered Trichoderma reesei strain TS-5 is a Trichoderma reesei strain that overexpresses the gene TgSwol in RUT-C30, and the nucleotide sequence of the gene TgSwol is shown in SEQ ID NO.1.

[0026] In this invention, the TgSwol gene is randomly integrated into the chromosome of Trichoderma reesei RUT-C30.

[0027] According to the present invention, the filter paper enzyme activity of the engineered Trichoderma reesei strain TS-5 is increased by 38.4-44.1% compared with the original strain.

[0028] A second aspect of the present invention provides a method for preparing the above-mentioned engineered Trichoderma reesei strain TS-5, comprising the following steps:

[0029] S1. Preparation of spore suspension of Trichoderma reesei strain RUT-C30;

[0030] S2. Prepare recombinant plasmid pCZF8-TgSwol containing the TgSwol gene;

[0031] S3. Transform the recombinant plasmid pCZF8-TgSwol into competent cells of Agrobacterium tumefaciens to obtain Agrobacterium tumefaciens AGL-1 containing the recombinant plasmid pCZF8-TgSwol;

[0032] S4. The Agrobacterium rhizogenes AGL-1 containing the recombinant plasmid pCZF8-TgSwol obtained in S3 was mixed evenly with the spore suspension obtained in S1, cultured, screened, and verified to obtain the Trichoderma reesei engineered strain TS-5.

[0033] According to the present invention, in step S1, the method for preparing the spore suspension includes: inoculating Trichoderma reesei strain RUT-C30 into a plate culture medium, culturing at 25-30℃ for 4-10 days, washing with sterile water, and diluting and resuspending to a spore concentration of [missing information]. per L.

[0034] According to the present invention, in step S2, the method for preparing the recombinant plasmid includes: using an artificially synthesized TgSwol gene with introns removed as a template, using TgSwol-F as a forward primer and TgSwol-R as a reverse primer, amplifying the TgSwol gene fragment containing upstream and downstream homologous arms by PCR, digesting the plasmid pCZF8 with the restriction endonuclease XbaⅠ, and using seamless cloning technology, ligating the plasmid pCZF8 and the TgSwol gene fragment containing upstream and downstream homologous arms at 37℃ for 30-90 min to obtain the recombinant plasmid pCZF8-TgSwol;

[0035] The PCR reaction system includes: KOD FX DNA polymerase, 2×PCR Buffer for KOD FX dNTPs, DNA template, The forward primer, The reverse primer, add water to .

[0036] The PCR reaction conditions were as follows: pre-denaturation at 94℃ for 2 min, 1 cycle; denaturation at 98℃ for 10 s, annealing at 54℃ for 30 s, amplification at 68℃ for 1 KB / min, 30 cycles; amplification at 68℃ for 10 min, 1 cycle; storage at 4℃.

[0037] The nucleotide sequence of the gene TgSwol is shown in SEQ ID NO.1, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.3.

[0038] According to the present invention, in step S3, the method for preparing Agrobacterium rhizogenes AGL-1 containing the recombinant plasmid pCZF8-TgSwol includes: transforming the recombinant plasmid pCZF8-TgSwol obtained in S2 into Escherichia coli. The sample was spread on LB resistant plates containing Kan and cultured at 37°C for 12-16 h. The recombinant plasmid pCZF8-TgSwol was then extracted and chemically transformed into Agrobacterium rhizogenes competent cells AGL-1 to obtain Agrobacterium rhizogenes AGL-1 containing the recombinant plasmid pCZF8-TgSwol.

[0039] According to the present invention, in step S3, the method for preparing Agrobacterium rhizogenes AGL-1 containing the recombinant plasmid pCZF8-TgSwol includes: transforming the recombinant plasmid pCZF8-TgSwol obtained in S2 into Escherichia coli. The cells were plated on LB resistant plates containing Kan and cultured at 37°C for 12-16 h. The recombinant plasmid pCZF8-TgSwol was then extracted and chemically transformed into Agrobacterium rhizogenes competent cells AGL-1. The transformed cells were then plated on LB plates containing Kan and cultured at 28°C for 24-48 h to obtain Agrobacterium rhizogenes AGL-1 cells containing the recombinant plasmid pCZF8-TgSwol.

[0040] According to the present invention, the conditions for the chemical transformation include taking Agrobacterium competent cells were added to the pCZF8-TgSwol vector, mixed well, and then incubated sequentially on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min. Liquid culture medium was incubated at 28 °C with shaking for 3 h, centrifuged at 6000 rpm for 2 min, and the contents were aspirated and discarded. Liquid culture medium, reserving 100 μL LB at the end, was used to spread the evenly dispersed bacterial cells onto a plate containing... LB resistant plates were incubated at 28 °C for 3 days.

[0041] According to the present invention, step S4 specifically includes:

[0042] s1. Agrobacterium rhizogenes AGL-1 containing recombinant plasmid pCZF8-TgSwol was inoculated into LB liquid medium containing Kan, cultured at 28°C with shaking at 200 rpm, centrifuged and washed with distilled water. Agrobacterium rhizogenes AGL-1 containing recombinant plasmid pCZF8-TgSwol was collected, diluted with IM medium supplemented with 0.2 mM acetylsyleugenol, and detected by spectrophotometer. The value was 0.15-0.2. The diluted Agrobacterium tumefaciens culture was incubated in the dark at 28 °C and 200 rpm, and the values ​​were measured every 2 hours. ,when Culture medium of Agrobacterium rhizogenes AGL-1 containing recombinant plasmid pCZF8-TgSwol was obtained;

[0043] s2. Inoculate Trichoderma reesei RUT-C30 at a 10% inoculum onto activation medium. Spread the activated bacterial solution onto sporulation medium and incubate at 28°C for 7 days. Wash the spores off the plates with distilled water, filter to remove mycelia, centrifuge at 4000 rpm for 5 min, wash with distilled water, discard the supernatant, and resuspend in 1M medium containing 0.2 mM acetylsyleugenol until the spore concentration is [missing value]. Spores of Trichoderma reesei strain RUT-C30 were obtained by pre-germinating at 24℃ for 2-3 hours.

[0044] s3, take Culture medium of Agrobacterium rhizogenes AGL-1 containing recombinant plasmid pCZF8-TgSwol and The spore suspension of *Trichoderma reesei* strain RUT-C30 was mixed by pipetting and spreading onto an IM plate lined with a nitrocellulose membrane and containing 0.2 mM acetylsyleugenone. The plate was incubated at 24°C in the dark for 3 days. The nitrocellulose membrane from the IM plate was then flipped onto a plate containing 0.2 mM acetylsyleugenone. and On PDA plates, initial screening and killing of Agrobacterium rhizogenes were performed until transformants grew. The transformants were transferred to PDA plates containing Hyg for secondary screening. Mycelia were picked and activated for colony PCR verification. The successfully verified colonies were repeatedly passaged to obtain transformants that were stably passaged for 5 generations and had spores germinating. The transformants were transferred to sporulation medium and cultured at 28 ℃ for 3-5 days. The spores were then transferred to single-spore culture medium and cultured at 28 ℃ for 3-5 days to obtain single colonies with single spore growth. The single colonies were picked and cultured in sporulation medium at 28 ℃ for 3-5 days. The spores were collected and stored in 20% glycerol at -80 ℃.

[0045] s4. The spores obtained in s3 and the spores of the starting strain RUT-C30 were inoculated into the seed culture medium separately to achieve a final spore concentration of . The culture was carried out at 28℃ and 200 rpm for 36 h. 10 mL of seed culture was inoculated into 90 mL of fermentation medium and cultured at 28℃ and 200 rpm. 4 mL of fermentation broth was taken every 24 h and centrifuged at 12000 rpm for 10 min. The supernatant was saved and used to determine cellulase activity. Transformants with cellulase activity greater than that of the starting strain RUT-C30 at 168 h were screened to obtain the Trichoderma reesei engineered strain TS-5.

[0046] In this invention, in step S4, the seed culture medium contains 3-5 g / L glucose, 8-12 g / L corn steep liquor, water as the solvent, and natural pH.

[0047] The fermentation medium contains 15-25 g / L microcrystalline cellulose, 0.8-1.2 g / L peptone, and 0.2-0.4 g / L urea. , , , , 0.0016~0.0018 g / L MnSO 0.0013~0.0015 g / L ZnSO , The solvent is 0.2M pH 5.0. Citrate buffer.

[0048] The third aspect of the present invention provides the application of the above-mentioned Trichoderma reesei engineered strain or the above-mentioned preparation method in straw enzyme saccharification, wherein the filter paper enzyme activity of the Trichoderma reesei engineered strain TS-5 is 6.12-6.37 FPU / mL.

[0049] The present invention will be described in detail below through embodiments.

[0050] Example 1 Construction of recombinant plasmid pCZF8-TgSwol

[0051] The TgSwol gene of this invention was synthesized by Jiutian Company. The TgSwol gene fragment size is 831bp, and the gene sequence is shown in SEQ ID NO.1. Using forward primer P1 and reverse primer P2 as primers, the TgSwol gene fragment containing upstream and downstream homologous arms was amplified from the TgSwol gene by PCR. The PCR reaction conditions and system are shown in Table 1 and Table 2.

[0052] Forward primer P1:

[0053] 5′-ATGGCATGATGCTAGTCTAGATGTTGGGATCAACCTGTGATGCT-3′ (SEQ ID NO.2)

[0054] Reverse primer P2:

[0055] 5′-TACAGTTTAGTCAAAACTAAAGAATACACTTAACGTTACTGAAATCATCAAACAGCT-3′ (SEQID NO.3)

[0056] PCR reaction conditions are shown in Table 1:

[0057] Table 1 PCR reaction conditions

[0058]

[0059] The final 4℃ time of the PCR reaction is ∞, indicating that it is stored at 4℃ for later use.

[0060] Table 2 PCR reaction system

[0061]

[0062] The plasmid pCZF8 was digested with XbaⅠ at 37℃ for 120 min, and then purified. The digestion system is shown in Table 3.

[0063] Table 3 Enzyme digestion system

[0064]

[0065] Using seamless cloning technology, plasmid pCZF8 and the TgSwol gene fragment were ligated at 37℃ for 30 min. The reaction system is shown in Table 4. The recombinant plasmid pCZF8-TgSwol pattern is shown in the instruction manual. Figure 1 .

[0066] Table 4 Connection Reaction System

[0067]

[0068] Remove *E. coli* DH5α competent cells, add 10 μL of recombinant plasmid, gently shake to mix, and incubate on ice for 30 min; place in a 42℃ water bath, heat shock at 42℃ for 90 s, then quickly transfer to an ice bath to cool for 5 min; add LB medium preheated to 37℃, and incubate at 37℃ and 200 rpm for 50 min; spread the cultured cells on LB resistant plates containing Kan, and incubate at 37℃ for 14 h; pick a single colony and inoculate it into LB liquid medium containing Kan, and incubate at 37℃ and 200 rpm for 14 h; extract the plasmid, verify, and obtain the recombinant plasmid pCZF8-TgSwol.

[0069] Example 2: Construction of Agrobacterium tumefaciens AGL-1 strain containing plasmid pCZF8-TgSwol

[0070] Take the sample stored at -80℃ Agrobacterium competent cells were thawed in an ice box. In a clean bench, 500 ng of recombinant plasmid pCZF8-TgSwol was added to 100 μL of AGL-1 Agrobacterium competent cells and gently mixed by pipetting. The mixture of AGL-1 Agrobacterium competent cells and recombinant plasmid pCZF8-TgSwol was placed in an ice box and incubated for 5 minutes. Centrifuge tubes containing the mixture were then rapidly frozen in liquid nitrogen for 5 minutes. The centrifuge tubes were quickly removed from the liquid nitrogen and placed in a 37 ℃ water bath for 5 minutes, ensuring the water surface remained still and avoiding vigorous shaking. The centrifuge tubes were then placed in an ice box and incubated for 5 minutes. In a clean bench, 700 μL of antibiotic-free LB liquid medium was added to the centrifuge tubes, and the tubes were placed in a shaker operating at 28 ℃ and 200 rpm for 3 hours. The cells were collected by centrifugation at 6000 rpm for 2 minutes, and the culture was aspirated and discarded. Collect 100 μL of supernatant (LB) and finally retain 100 μL LB. Spread the evenly dispersed bacterial cells onto a plate containing... LB resistant plates were incubated at 28 °C for 3 days. Single colonies were picked and activated, and positive transformants were verified by PCR, yielding Agrobacterium rhizogenes AGL-1 containing the recombinant plasmid pCZF8-TgSwol.

[0071] Example 3 Construction and Validation of Trichoderma reesei Engineered Strain TS-5

[0072] Agrobacterium rhizogenes AGL-1 containing the recombinant plasmid pCZF8-TgSwol was inoculated into LB liquid medium containing Kan, cultured at 28°C with shaking at 200 rpm, centrifuged and washed with distilled water. Agrobacterium rhizogenes AGL-1 containing the recombinant plasmid pCZF8-TgSwol was collected, diluted with IM medium supplemented with 0.2 mM acetylsyleugenol, and detected using a spectrophotometer. The value was 0.15-0.2. The diluted Agrobacterium tumefaciens culture was incubated in the dark at 28 °C and 200 rpm, and the values ​​were measured every 2 hours. ,when Culture medium of Agrobacterium rhizogenes AGL-1 containing recombinant plasmid pCZF8-TgSwol was obtained.

[0073] Trichoderma reesei RUT-C30 inoculum was inoculated onto activation medium at a 10% inoculum rate. The activated bacterial solution was spread onto a sporulation medium plate and incubated at 28°C for 7 days. The spores on the plate were washed off with distilled water, filtered through six layers of gauze to remove the hyphae, centrifuged at 4000 rpm for 5 min, washed twice with distilled water, and the supernatant was discarded. The spores were resuspended in IM medium containing 0.2 mM acetylsyl syringone and placed in a 24°C incubator for pre-germination for 3 h.

[0074] Pick Cultured Agrobacterium rhizogenes AGL-1 and Trichoderma reesei conidia were mixed by pipetting and spreading onto IM plates lined with an NC membrane and containing 0.2 mM acetylsyleugenol, and incubated statically at 24°C for 3 days. The NC membrane from the IM plate was then flipped onto a plate containing 0.2 mM acetylsyleugenol. and On PDA plates, initial screening and killing of Agrobacterium were performed until transformants grew. The transformants were then transferred to PDA plates containing Hyg, and mycelia were picked and activated for colony PCR verification. Successfully verified colonies were repeatedly passaged to obtain transformants that had been stably passaged for 5 generations and had spores germinating. These transformants were then transferred to sporulation medium and incubated at 28 °C for 3-5 days. Spores were then transferred to single-spore culture medium and incubated at 28 °C for 3-5 days to obtain single colonies with individual spores. Mycelia were picked, activated, and colony PCR verified. Successfully verified colonies were picked and incubated at 28 °C for 3-5 days. Spores were collected and stored in 20% glycerol at -80 °C.

[0075] The obtained spores and the spores of the starting strain RUT-C30 were inoculated into seed culture medium separately to achieve a final spore concentration of [missing information]. The culture was carried out at 28℃ and 200 rpm for 36 h. 10 mL of seed culture was inoculated into 90 mL of fermentation medium and cultured at 28℃ and 200 rpm. 4 mL of fermentation broth was taken every 24 h and centrifuged at 12000 rpm for 10 min. The supernatant was saved for cellulase activity determination. Transformants with cellulase activity greater than that of the starting strain RUT-C30 at 168 h were screened and verified to obtain 6 Trichoderma reesei engineered strains, TS-1 to TS-6.

[0076] The above six engineered Trichoderma reesei strains and the starting strain RUT-C30 were inoculated into seed culture medium in equal amounts and cultured at 28℃ and 180 rpm for 24 h. 10 mL of seed culture was then inoculated into 90 mL of fermentation medium and cultured at 28℃ and 200 rpm. 4 mL of fermentation broth was taken every 24 h, centrifuged at 12000 rpm for 10 min, and the supernatant was saved for the determination of cellulase activity.

[0077] The seed culture medium contains 4 g / L glucose, 10 g / L corn steep liquor, and water as the solvent, and does not require pH adjustment.

[0078] The fermentation medium contains 20 g / L microcrystalline cellulose, 1 g / L peptone, and 0.3 g / L urea. , , , , , , , The solvent is 0.2M pH 5.0. Citrate buffer.

[0079] The results of the comparison of cellulase activity between the 6 engineered strains and the original strain RUT-C30 are shown in the figure. Figure 2 The filter paper enzyme activity of the starting strain RUT-C30 was 1.29 IU / mL, while the highest filter paper enzyme activity of the engineered Trichoderma reesei strain TS-5 was 1.50 IU / mL, representing a 16.29% increase compared to the starting strain RUT-C30. Other data are as per the instruction manual. Figure 3 As shown, it is related to the starting strain *Trichoderma reesei*. In comparison, the β-glucosidase activity, exocellulase activity, endocellulase activity, and xylanase activity of the engineered Trichoderma reesei strain TS-5 were significantly increased. After 7 days of fermentation, the β-glucosidase activity, exocellulase activity, endocellulase activity, xylanase activity, and extracellular protein concentration of the engineered Trichoderma reesei strain TS-5 reached 0.50 IU / mL, 0.47 IU / mL, 0.77 IU / mL, 120.31 IU / mL, and 3.91 mg / mL, respectively, which were 20.7%, 12.9%, 9.0%, 15.4%, and 11.8% higher than those of the starting strain RUT-C30. These data demonstrate that TgSwol can induce the expression of multiple lignocellulose degrading enzymes, thereby increasing substrate accessibility and promoting lignocellulose degradation.

[0080] Enzyme activity assay method

[0081] Filter paper enzyme activity assay:

[0082] Add 0.5 mL of diluted enzyme solution, 1.0 mL of citrate-sodium citrate buffer (50 mM, pH 4.8), and 50 mg of Whatman NO.1 filter paper to a colorimetric tube. Incubate the tube in a 50 ℃ water bath for 60 min. Immediately after the reaction, add 3 mL of DNS and incubate in a boiling water bath for 5 min to terminate the enzyme reaction. After cooling in cold water, measure the amount of reducing sugar produced. Calculate the enzyme activity of the filter paper based on the amount of reducing sugar produced.

[0083] Determination of xylanase activity:

[0084] Preheat the 1% xylan solution (prepared with 50mM citrate-sodium citrate buffer, pH 4.8) as the reaction substrate in a 50℃ water bath, and prepare boiling water at 100℃. First, add 0.5 mL of enzyme solution diluted appropriately to each colorimetric tube, then quickly add 1.0 mL of 1% xylan solution to the tube. Incubate at 50℃ for 5 min. Immediately after the enzymatic hydrolysis time is complete, add DNS solution to terminate the reaction, and place the tubes in boiling water for 10 min to allow the DNS to react with the reducing sugar and develop color. Then, cool the tubes in cold water. Once the tubes have cooled to room temperature, add water to a final volume of 25 mL, and measure the amount of reducing sugar released at 540 nm absorbance.

[0085] Glucosidase activity assay:

[0086] Preheat the 15 mM cellobiose solution (prepared with 50 mM citrate-sodium citrate buffer, pH 4.8) to a 50 ℃ water bath. Add 0.5 mL of appropriately diluted enzyme solution to each 1.5 mL centrifuge tube, then add... The cellobiose solution was preheated and reacted at 50 °C for 30 min. Ten minutes before the end of the reaction, boiling water at 100 °C was prepared. After the reaction, the solution was boiled in the water for 2 min to terminate the enzymatic hydrolysis. The amount of glucose released in the reaction system was measured using an SBA biosensor, and the β-glucosidase activity was calculated based on the glucose content. It is important to note that the dilution factor of the enzyme solution must be adjusted so that at least 1 mg of glucose is released in the reaction system before enzyme activity can be calculated.

[0087] Exoglucanase activity assay:

[0088] A 0.5% pNPC solution was prepared using 50 mM citrate-sodium citrate buffer at pH 4.8. 1% gluconolactone was added to the solution to inhibit the hydrolysis of cellobiose side chains by β-glucosidase. 50 μL of 0.5% pNPC solution and 100 μL of appropriately diluted enzyme solution were added to each PCR tube, and the reaction was carried out at 45 °C for 30 min. At the end of the enzymatic digestion time, [the following was added]... The reaction was terminated, and the amount of p-nitrophenol produced was determined at 420 nm.

[0089] Determination of endonuclease activity:

[0090] Preheat the 0.5% sodium carboxymethyl cellulose solution (prepared with 0.2 M acetate-sodium acetate buffer, pH 4.8) as the reaction substrate in a 50 °C water bath, and prepare boiling water at 100 °C. First, add 0.5 mL of enzyme solution diluted appropriately to each colorimetric tube, then add 1.0 mL of 0.5% sodium carboxymethyl cellulose solution to the colorimetric tube. React at 50 °C for 30 min. Immediately after the enzymatic hydrolysis time is completed, add DNS solution to terminate the enzymatic hydrolysis reaction, and place the colorimetric tube in boiling water for 10 min to allow DNS to react with reducing sugars and develop color. Then, cool the tubes in cold water. After the colorimetric tubes have cooled to room temperature, add water to a final volume of 25 mL, and measure the amount of reducing sugar released at 540 nm absorbance.

[0091] Extracellular protein concentration determination:

[0092] The BCA method is used to determine protein concentration because proteins can react under alkaline conditions. Restore to , It can react with BCA reagent to form a purple-blue substance. By measuring the absorbance of the complex at 562 nm and subtracting the absorbance value from the standard curve, the concentration of the protein to be tested can be calculated.

[0093] Example 4: Fermentation of cellulase by Trichoderma reesei starting strain RUT-C30 and engineered strain TS-5 in a fermenter

[0094] Trichoderma reesei originating strain RUT-C30 and engineered Trichoderma reesei strain TS-5 were inoculated into agar plates and cultured at 28℃ for 7 days. Spores were washed off with sterile water, filtered through gauze, and diluted to obtain a Trichoderma reesei spore suspension. Equal volumes of the spore suspensions of RUT-C30 and TS-5 were inoculated into seed culture medium and cultured at 28℃ and 180 rpm for 36 hours to obtain Trichoderma reesei seed culture. The engineered Trichoderma reesei seed culture was inoculated at a 10% inoculation ratio into fermentation medium containing a solid-liquid co-inducing agent. Fermentation conditions were controlled in multiple stages. The initial culture temperature was 28℃, the initial rotation speed was 200 rpm, and the initial aeration rate was 2 vvm. When the glucose concentration in the fermentation broth fell below 0.2 g / L, feeding was initiated to maintain the glucose concentration at 0.2 g / L for 144 hours. Centrifuge the fermentation broth at 10,000 rpm for 10 min, collect the supernatant, and the resulting liquid is the crude cellulase solution.

[0095] The multi-stage fermentation condition control method includes:

[0096] Temperature control: The culture temperature is 28℃ before feeding and 24℃ after feeding.

[0097] pH control: The pH of the fermentation broth is not controlled for about 12 hours before fermentation; after 12 hours of fermentation, the pH of the fermentation broth is maintained at about 4.2.

[0098] Dissolved oxygen control: During the first 12 hours of fermentation, the dissolved oxygen in the fermentation broth is not controlled; between 12 and 36 hours of fermentation, the dissolved oxygen in the fermentation broth is controlled at 40%; after 36 hours of fermentation, the dissolved oxygen in the fermentation broth is controlled at 20%.

[0099] The plate culture medium contains 30 g / L malt extract powder, 15 g / L agar, and water as the solvent, with a natural pH.

[0100] The seed culture medium contains 4 g / L glucose, 10 g / L corn steep liquor, and water as the solvent, with a natural pH.

[0101] The fermentation medium contains 10 g / L liquid inducer, 20 g / L solid inducer, 1 g / L peptone, and 0.3 g / L urea. , , , , , , , 0.2 ml / L Tween 80, 2 ml / L defoamer, solvent is water, pH is natural.

[0102] The preparation steps of the liquid inducer are as follows: using 0.2M at pH 4.8... Prepare an 850 g / L glucose solution with citrate buffer, add β-glucosidase at 25 CBU / g glucose, react at 55℃ and 150 rpm for 72 h, and then treat at 105℃ for 5 min to inactivate β-glucosidase to obtain the liquid inducer.

[0103] The solid inducer is wheat bran.

[0104] The cellulase activity assay results of Trichoderma reesei starting strain RUT-C30 and engineered strain TS-5 after fermentation for 132 h are shown in the appendix of the instruction manual. Figure 3 The results showed that the cellulase activity of the engineered strain TS-5 was significantly improved. After 132 h of fermentation, the filter paper enzyme activity of the engineered Trichoderma reesei strain TS-5 reached 6.37 FPU / mL, which was 44% higher than that of the original strain RUT-C30.

[0105] Example 5

[0106] Corn straw pretreated with crude enzyme solution of Trichoderma reesei origin strain RUT-C30 and engineered strain TS-5

[0107] Degradation of corn stalks: Pretreated corn stalks were resuspended in citric acid-sodium citrate (50 mM, pH 4.8) at a solid-liquid ratio of 10%. Crude enzyme solutions of the prepared *Trichoderma reesei* strain RUT-C30 and engineered strain TS-5 were added at a concentration of 4 FPU / g stalks, respectively. The mixture was placed in a water bath shaker and reacted at 55℃ and 180 rpm for 72 h. After the reaction, the glucose content was determined by high-performance liquid chromatography (HPLC).

[0108] The results are as shown in the attached instruction manual. Figure 5 As shown, the glucose yield in the enzymatic hydrolysate of the engineered Trichoderma reesei strain TS-5 was 24% higher than that of the starting strain RUT-C30. This is because the crude enzyme solution of the starting strain RUT-C30 contained low levels of swelling hormones, which limited the overall saccharification efficiency of cellulase; while the crude enzyme solution of the engineered strain TS-5 contained significantly higher levels of swelling hormones than the starting strain. Furthermore, the TgSwol gene also increased substrate accessibility and promoted lignocellulose degradation by inducing the expression of various lignocellulose-degrading enzymes.

[0109] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. An engineered strain of Trichoderma reesei, TS-5, is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2026207 and deposit date of January 23, 2026.

2. The engineered Trichoderma reesei strain TS-5 according to claim 1, characterized in that, The engineered Trichoderma reesei strain TS-5 is a Trichoderma reesei strain that overexpresses the gene TgSwol in RUT-C30. The nucleotide sequence of the gene TgSwol is shown in SEQ ID NO.

1.

3. The engineered Trichoderma reesei strain TS-5 according to claim 1, characterized in that, The filter paper enzyme activity of the engineered Trichoderma reesei strain TS-5 was increased by 38.4-44.1% compared with the original strain.

4. A method for preparing the engineered Trichoderma reesei strain TS-5 according to any one of claims 1-3, characterized in that, Includes the following steps: S1. Preparation of spore suspension of Trichoderma reesei strain RUT-C30; S2. Prepare recombinant plasmid pCZF8-TgSwol containing the TgSwol gene; S3. Transform the recombinant plasmid pCZF8-TgSwol into competent cells of Agrobacterium tumefaciens to obtain Agrobacterium tumefaciens AGL-1 containing the recombinant plasmid pCZF8-TgSwol; S4. The Agrobacterium rhizogenes AGL-1 containing the recombinant plasmid pCZF8-TgSwol obtained in S3 was mixed evenly with the spore suspension obtained in S1, cultured, screened, and verified to obtain the Trichoderma reesei engineered strain TS-5.

5. The preparation method according to claim 4, characterized in that, In step S1, the method for preparing the spore suspension includes: inoculating Trichoderma reesei strain RUT-C30 into a plate culture medium, culturing at 25-30℃ for 4-10 days, washing with sterile water, and diluting the spores with sterile water to a concentration that is within the specified range. A spore suspension was prepared by spores per L.

6. The preparation method according to claim 4, characterized in that, In step S2, the method for preparing the recombinant plasmid includes: using the artificially synthesized TgSwol gene with introns removed as a template, using TgSwol-F as a forward primer and TgSwol-R as a reverse primer, amplifying the TgSwol gene fragment containing upstream and downstream homologous arms by PCR, digesting the plasmid pCZF8 with the restriction endonuclease XbaⅠ, and using seamless cloning technology, ligating the plasmid pCZF8 and the TgSwol gene fragment containing upstream and downstream homologous arms at 37℃ for 30-90 min to obtain the recombinant plasmid pCZF8-TgSwol; The nucleotide sequence of the gene TgSwol is shown in SEQ ID NO.1, the nucleotide sequence of the forward primer is shown in SEQ ID NO.2, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.

3.

7. The preparation method according to claim 4, characterized in that, In step S3, the method for preparing Agrobacterium rhizogenes AGL-1 containing the recombinant plasmid pCZF8-TgSwol includes: transforming the recombinant plasmid pCZF8-TgSwol obtained in S2 into Escherichia coli. The cells were plated on LB resistant plates containing Kan and cultured at 37°C for 12-16 h. The recombinant plasmid pCZF8-TgSwol was then extracted and chemically transformed into Agrobacterium rhizogenes competent cells AGL-1. The transformed cells were then plated on LB plates containing Kan and cultured at 28°C for 24-48 h to obtain Agrobacterium rhizogenes AGL-1 cells containing the recombinant plasmid pCZF8-TgSwol.

8. The preparation method according to claim 7, characterized in that, The conditions for the chemical transformation include: taking Agrobacterium competent cells were added to the pCZF8-TgSwol vector, mixed well, and then incubated sequentially on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min. Liquid culture medium was incubated at 28 °C with shaking for 3 h, centrifuged at 6000 rpm for 2 min, and the contents were aspirated and discarded. Collect 100 μL of supernatant (LB) and finally retain 100 μL LB. Spread the evenly dispersed bacterial cells onto a plate containing... LB resistant plates were incubated at 28 °C for 3 days.

9. The preparation method according to claim 4, characterized in that, Step S4 specifically includes: s1. Agrobacterium rhizogenes AGL-1 containing recombinant plasmid pCZF8-TgSwol was inoculated into LB liquid medium containing Kan, cultured at 28°C with shaking at 200 rpm, centrifuged and washed with distilled water. Agrobacterium rhizogenes AGL-1 containing recombinant plasmid pCZF8-TgSwol was collected, diluted with IM medium supplemented with 0.2 mM acetylsyleugenol, and detected by spectrophotometer. The value was 0.15-0.

2. The diluted Agrobacterium tumefaciens culture was incubated in the dark at 28 °C and 200 rpm, and the values ​​were measured every 2 hours. ,when Culture medium of Agrobacterium rhizogenes AGL-1 containing recombinant plasmid pCZF8-TgSwol was obtained; s2. Inoculate Trichoderma reesei RUT-C30 at a 10% inoculum onto activation medium. Spread the activated bacterial solution onto sporulation medium and incubate at 28°C for 7 days. Wash the spores off the plates with distilled water, filter to remove mycelia, centrifuge at 4000 rpm for 5 min, wash with distilled water, discard the supernatant, and resuspend in 1M medium containing 0.2 mM acetylsyleugenol until the spore concentration is [missing value]. Spores of Trichoderma reesei strain RUT-C30 were obtained by pre-germinating at 24℃ for 2-3 hours. s3, take Culture medium of Agrobacterium rhizogenes AGL-1 containing recombinant plasmid pCZF8-TgSwol and The spore suspension of *Trichoderma reesei* strain RUT-C30 was mixed by pipetting and spreading onto an IM plate lined with a nitrocellulose membrane and containing 0.2 mM acetylsyleugenone. The plate was incubated at 24°C in the dark for 3 days. The nitrocellulose membrane from the IM plate was then flipped onto a plate containing 0.2 mM acetylsyleugenone. and On PDA plates, initial screening and killing of Agrobacterium rhizogenes were performed until transformants grew. The transformants were transferred to PDA plates containing Hyg for secondary screening. Mycelia were picked and activated for colony PCR verification. The successfully verified colonies were repeatedly passaged to obtain transformants that were stably passaged for 5 generations and had spores germinating. The transformants were transferred to sporulation medium and cultured at 28 ℃ for 3-5 days. The spores were then transferred to single-spore culture medium and cultured at 28 ℃ for 3-5 days to obtain single colonies with single spore growth. The single colonies were picked and cultured in sporulation medium at 28 ℃ for 3-5 days. The spores were collected and stored in 20% glycerol at -80 ℃. s4. The spores obtained in s3 and the spores of the starting strain RUT-C30 were inoculated into the seed culture medium separately to achieve a final spore concentration of . The culture was carried out at 28℃ and 200 rpm for 36 h. 10 mL of seed culture was inoculated into 90 mL of fermentation medium and cultured at 28℃ and 200 rpm. Every 24 h, 4 mL of fermentation broth was taken and centrifuged at 12000 rpm for 10 min. The supernatant was saved and used to determine the cellulase activity. Transformants with cellulase activity greater than that of the starting strain RUT-C30 at 168 h were screened to obtain the Trichoderma reesei engineered strain TS-5.

10. The application of the engineered Trichoderma reesei strain TS-5 according to any one of claims 1-3 or the preparation method according to any one of claims 4-9 in the enzymatic saccharification of straw, characterized in that, The filter paper enzyme activity of the engineered Trichoderma reesei strain TS-5 was 6.12-6.37 FPU / mL.

Images