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Application of Trichoderma reesei cellulase transcription inhibition factor 70351 and method for improving cellulase expression quantity and enzyme activity

A technology of transcriptional repressor, Trichoderma reesei, applied in the field of agricultural biology, can solve problems such as high cost

Active Publication Date: 2022-07-01
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the high cost of cellulase is still one of the major bottlenecks in cellulose biorefinery, so it is necessary to continue to develop new methods to improve the expression of cellulase and reduce the application cost

Method used

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  • Application of Trichoderma reesei cellulase transcription inhibition factor 70351 and method for improving cellulase expression quantity and enzyme activity
  • Application of Trichoderma reesei cellulase transcription inhibition factor 70351 and method for improving cellulase expression quantity and enzyme activity
  • Application of Trichoderma reesei cellulase transcription inhibition factor 70351 and method for improving cellulase expression quantity and enzyme activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Construction of p70351 donor and p70351 sgRNA plasmid for transcriptional repressor

[0026] (1) Construction of transcription repressor p70351 donor plasmid

[0027] p70351 donor plasmid contains 70351 Upstream homology arms, pCre / Loxp-hph (hygromycin expression cassette and E. coli basic replication element) and 70351 Downstream homology arm.

[0028] Using the pCre / Loxp-hph plasmid stored in the laboratory as the template, the primers Loxp-hphF1 / Loxp-hphR1 amplify the pCre / Loxp-hph part; using the Trichoderma reesei TU-6 genome as the template, the primers 70351UPF1 / 70351UPR1 amplify Increase the length of 1.5kb 70351 Upstream homology arm, primer 70351downF1 / 70351downR1 amplified 1.5 kb length 70351 Downstream homology arm.

[0029] The PCR reaction system includes: 2 ng template, 1 µl forward primer, 1 µl reverse primer, 25 µl 2xPCR Mix, and make up to 50 µl with double distilled water.

[0030] The PCR reaction program was: 95°C, 5 min; 94°C, 30s, ...

Embodiment 2

[0039] Example 2. Transcriptional repressors 70351 knockout

[0040] (1) Knockout 70351 donor DNA and 70351 sgRNA transformation of Trichoderma reesei

[0041] Trichoderma reesei TU-6 was inoculated on a potato culture medium (PDA) plate, and cultured at 28 ºC for 7 days until the spores were produced. The spores were scraped and inoculated into 100 ml of PDB medium containing uracil at 28 ºC. , 160 rpm shaking culture overnight. The germinated mycelia were collected by mesh sieve, and 10 mg / ml cellulase was added to digest at 30ºC for 2-3 hours. After collecting the protoplasts, construct the knockout 70351 The donor DNA and p70351sgRNA were used Pac 1 and I- Ceu 1 Enzyme digestion, transform Trichoderma reesei host cells.

[0042] (2) PCR verification of transcriptional repressors 70351 Knockout in the Trichoderma reesei genome

[0043] Pick a single transformant, inoculate it in a 24-well plate containing MM-glucose medium, and culture at 28°C for 5-7 days. Gen...

Embodiment 3

[0046] Example 3. Knockout of transcriptional repressors 70351 effect on protein expression

[0047] (1) Knock out transcriptional repressors 70351 Shake flask induction of transformants

[0048] knockout transcriptional repressor 70351 The transformants and starting strains were inoculated with 2 × 10 7 Spores were cultured in 50 ml of MM-glucose medium at 28°C and 160 rpm for 2 days. 10% of the inoculum was transferred to 50 ml MM+2% Avicel medium to induce cellulase expression. From the 3rd day, samples were taken every 24 h, and the samples were continuously sampled for 6 days.

[0049] (2) Knock out transcriptional repressors 70351 The protein concentration of the transformants, determination of cellulase

[0050] Coomassie brilliant blue method was used for protein quantification. After adding 250 µl 1 × dye reagent and 10 µl protein standard, the absorbance at 595 nm was measured after 10 minutes of reaction at room temperature. See the results. Figure 4 . On ...

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Abstract

The invention relates to the technical field of agricultural biology, in particular to application of a Trichoderma reesei cellulase transcription inhibition factor 70351 and a method for improving cellulase expression quantity and enzyme activity. The transcriptional inhibition factor 70351 related to cellulase expression provided by the invention has a regulation effect on cellulase activity expression, a knockout plasmid of the gene is constructed and is used for converting host bacteria, and the expression of the transcriptional inhibition factor 70351 is knocked out from the host bacteria, so that the protein expression quantity of the host bacteria and the cellulase activity can be improved. The invention enriches the transcriptional regulation network of the trichoderma reesei cellulase, and provides a new way for improving the yield of the cellulase, reducing the cost of the cellulase and realizing the effective utilization of the cellulose.

Description

technical field [0001] The invention relates to the field of agricultural biotechnology, in particular to Trichoderma reesei cellulase transcription inhibitor 70351 application and method of improving cellulase expression and enzymatic activity. Background technique [0002] The filamentous fungus Trichoderma reesei has a strong ability to secrete cellulase. In its mixed fermentation broth, the expression of cellobiohydrolase accounts for more than 50% of the total extracellular secreted proteins. However, the high cost of cellulase is still one of the main bottlenecks in cellulose biorefinery, so it is necessary to continue to develop new methods to improve the expression of cellulase and reduce the cost of application. [0003] In T. reesei, cellulase expression is affected by multiple regulatory pathways. Among them, the regulation mainly occurs at the transcriptional level, including the main activators Xyr1, ACE2, ACE3, Vib1 and Hap2 / 3 / 5, and the co-regulation of mult...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/31C12N15/80C12N1/15C12R1/885
CPCC12N9/2437C07K14/37C12N15/80
Inventor 苏小运孙先花姚斌罗会颖王晓璐秦星王苑涂涛柏映国于会民黄火清张红莲王亚茹
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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