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Method for improving activity of trichoderma reesei cellulose hydrolase by using cellulose inducible promoter

A technology of cellulolytic enzyme and Trichoderma reesei, applied in the field of genetic engineering, can solve the problems such as the inconspicuous promotion effect of β-glucosidase, achieve good industrial development and application prospects, increase production, fermentation time cost and economy The effect of cost reduction

Active Publication Date: 2022-01-11
SHANDONG UNIV
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Problems solved by technology

By modifying the transcriptional regulatory network of Trichoderma reesei cellulolytic enzyme gene expression, it is expected to reduce the cost of biological enzyme preparations; -Increasing effect of glucosidase is not obvious
After searching, in the actual application of improving the production of cellulolytic enzymes, the literature or mature technical solutions for the transformation of transcription factors including Xyr1 and the specific use of cellulose-inducible promoters to improve the cellulolytic enzyme activity of Trichoderma reesei The method and its special engineering strain are also rarely reported

Method used

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  • Method for improving activity of trichoderma reesei cellulose hydrolase by using cellulose inducible promoter
  • Method for improving activity of trichoderma reesei cellulose hydrolase by using cellulose inducible promoter
  • Method for improving activity of trichoderma reesei cellulose hydrolase by using cellulose inducible promoter

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Embodiment 1

[0032] Example 1. Construction of an overexpression cassette for the transcriptional regulator xyr1.

[0033] Using the genomic DNA of Trichoderma reesei QEB4 as a template, use the primer pair Egl2-F / Egl2-R to amplify the cellulose inducible promoter Pegl2 (its nucleotide sequence is shown in SEQ ID NO.1), use the primer pair Xyr1-F / Xyr1-R amplifies the xyr1 gene and its terminator region, uses the primer pair Hph-UF / Hph-UR and Hph-DF / Hph-DR to amplify the upstream and downstream homology arms of the hph gene; and uses the primer pair ptrA-F1 / ptrA-R1 amplifies the ptrA expression cassette from the T-ptrA plasmid. Then, the upstream homology arm of the hph gene, the resistance genes prtA, Pegl2, xyr1 gene and terminator, and the downstream homology arm of the hph gene are fused. The fusion PCR program is: Buffer enzyme 25 μl, dNTP 1 μl, Phata enzyme 1 μl, fragment A total of 5 μg with ddH 2 Make up 50 μl with O; the fusion program is: pre-denaturation at 95°C for 5 minutes, ...

Embodiment 2

[0045] Embodiment 2. Utilize Pegl2-xyr1 overexpression cassette to transform Trichoderma reesei QEB4 strain to construct high cellulolytic enzyme active Trichoderma reesei engineering strain

[0046] Genetic transformation of Trichoderma reesei QEB4 using PEG / CaCl 2 Mediated protoplast transformation method, pyrithione resistance gene ptrA as selectable marker.

[0047] The purified Pegl2-xyr1 overexpression cassette was transformed into Trichoderma reesei QEB4 protoplasts to construct the engineering strain QE2X of Trichoderma reesei with high cellulolytic activity. Wherein the PEG / CaCl 2 The specific method of mediated protoplast transformation is as follows:

[0048] (1) Preparation of Trichoderma reesei QEB4 protoplast:

[0049] Prepare fresh Trichoderma reesei spores (within 2 weeks), spread on 5-6 PDA plates covered with cellophane, and spread 150 μL on each plate with a concentration of 10 8 individual / mL spore suspension. Incubate at 32°C for 14 hours. After the m...

Embodiment 3

[0058] Example 3. Transcription level analysis of the cellulase transcription regulator xyr1 gene of high cellulolytic enzyme activity Trichoderma reesei engineering strain

[0059] The high cellulolytic enzyme activity Trichoderma reesei engineering strain QE2X that embodiment 2 obtains and starting strain QEB4 press 10 8 Each / mL were inoculated into 50mL (250mL Erlenmeyer flask) seed medium respectively, and after 40 hours of shaking flask culture, 10mL were respectively inoculated into the fermentation medium, 32°C, 220rpm shaking flask cultured for 72h, and the mycelium was collected by suction filtration RNA was extracted and analyzed by reverse transcription qPCR. The result is as figure 2 As shown in A, it shows that the transcription of xyr1 in QE2X is significantly increased, which is 4 times higher than that of the starting strain. It further showed that the Pegl2-xyr1 overexpression cassette was successfully integrated into the genome and xyr1 was expressed at a ...

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Abstract

The invention discloses a method for improving the activity of trichoderma reesei cellulose hydrolase by using a cellulose inducible promoter, which comprises the following steps: preparing an expression cassette for overexpressing a transcriptional regulation factor xyr1 gene by using the cellulose inducible promoter Pegl2, transforming the expression cassette into a trichoderma reesei QEB4 strain to obtain a trichoderma reesei engineering strain QE2X with high cellulose hydrolase activity, and preparing the high-activity cellulose hydrolase by fermenting the engineering strain. Experiments prove that the activity of the cellulose hydrolase produced by the method disclosed by the invention is 34.9 IU / mg which is obviously higher than that of an original strain QEB4 (10IU / mg), each component of an enzyme system is greatly improved, and the enzyme activity of [beta]-glucosidase is 92.4 IU / mg which is 2.83 times that of the original strain. The method has good industrial development and application prospects.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a method for improving the activity of Trichoderma reesei cellulolytic enzyme by utilizing a cellulose-inducible promoter and a special engineering strain thereof. Background technique [0002] As one of the renewable resources on the earth, lignocellulosic biomass is mainly composed of cellulose, hemicellulose and lignin, and is rich in resources. Hydrolysis of biomass by lignocellulolytic enzymes is an efficient way to convert biomass into fermentable sugars for biofuel production. However, the high production cost of cellulolytic enzymes in practical applications greatly affects the price and competitiveness of the final product. In order to solve this problem, it is urgent to improve the production capacity of cellulolytic enzyme-producing strains. [0003] At present, the methods used to increase the production of cellulolytic enzymes mainly include:...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N15/113C12N15/80C12N1/15C12N9/42C12R1/885
CPCC12N9/2437C12N15/80Y02E50/10
Inventor 钟耀华沈林静王逸凡
Owner SHANDONG UNIV
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