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Method for improving cellulase expression capability of trichoderma reesei by regulating and controlling cell metabolism

A technology of Trichoderma reesei and cellulase, applied in the field of genetic engineering, to achieve the effect of improving cellulase enzyme activity and protein expression

Active Publication Date: 2022-03-01
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

[0005] Therefore, the existing methods of genetic engineering and the selection of regulatory genes have limitations, so it is very important to continue to discover new regulatory genes

Method used

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  • Method for improving cellulase expression capability of trichoderma reesei by regulating and controlling cell metabolism
  • Method for improving cellulase expression capability of trichoderma reesei by regulating and controlling cell metabolism
  • Method for improving cellulase expression capability of trichoderma reesei by regulating and controlling cell metabolism

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1. Overexpression of regulatory genes 72685 Plasmid construction

[0016] Build overexpression 72685 Plasmid: including cDNA1 promoter, 72685 gene, cDNA1 terminator and pyr4 Expression cassette plasmid. Using the APA-GOD plasmid as a template, the APA part was amplified ( AmpR-pyr4-AmpR ), the APA sequence ( AmpR-pyr4-AmpR ) contains the Trichoderma reesei auxotrophic selection marker pyr4 An expression cassette that can be used for lack of pyr4 Genetic transformation screening of Trichoderma reesei strains. exist pyr4 There are two tandem repeated ampicillin genes at both ends of the expression cassette, and the homologous recombination of this direct tandem sequence can be used to convert the Trichoderma reesei transformant pyr4 The gene is knocked out more rapidly, so that pyr4 Filter markers can be recycled. Using the Trichoderma reesei genome as a template to amplify the cDNA1 promoter, 72685 The gene (named as cDNA) and cDNA1 terminator, elec...

Embodiment 2

[0017] Example 2. Overexpression 72685 Plasmid introduction

[0018] (1) Overexpression 72685 Plasmid transformation of Trichoderma reesei SUS4-2

[0019] Trichoderma reesei SUS4-2 was inoculated on a potato medium (PDA) plate, and cultured statically at 28 ºC for 7 days until it produced spores, and the spores were scraped off and inoculated in 100 ml of PDB medium containing uracil, at 28 ºC , 160 rpm shaking culture overnight. Collect the germinated mycelium by filtering through a 200-mesh sieve, add 10 mg / ml cellulase and digest at 30°C for 2-3 hours. After collecting the protoplasts, the pCDNAP-72685 plasmid was cleaned with E wxya I digestion, transformation of Trichoderma reesei host cells.

[0020] (2) PCR verification of the introduction of the pCDNAP-72685 plasmid into the Trichoderma reesei genome

[0021] A single transformant was picked, inoculated on a 24-well plate containing MM-glucose medium, and cultured at 28°C for 5-7 days. Extract genomic DNA and v...

Embodiment 3

[0022] Example 3. Overexpression 72685 Effects on Secretion and Expression of Trichoderma reesei Cellulase

[0023] (1) Overexpression 72685 Shake flask induction of genetic transformants

[0024] Overexpression 72685 Gene transformants and starting strains were inoculated with 2×10 7 The spores were cultured in 50 ml MM-glucose medium at 28°C and 160 rpm for 2 days. Transfer to 50 ml MM+2% Avicel medium with 10% inoculum size to induce the expression of cellulase. From the third day, samples were taken every 24 h, and the samples were taken continuously for 7 days.

[0025] (2) Overexpression 72685 Determination of the protein concentration and cellulase of the transformant of the gene

[0026] Coomassie Brilliant Blue method was used for protein quantification. After adding 250 L of 1 × dye reagent and 10 µl of protein standard, reacted at room temperature for 10 minutes, and measured the absorbance at 595 nm. The results are shown in figure 2 . visible overexpress...

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Abstract

The invention relates to the field of gene engineering, in particular to a method for improving cellulase expression capacity of trichoderma reesei by regulating cell metabolism. The 6-phosphogluconate dehydrogenase gene 72685 of a pentose phosphate pathway of trichoderma reesei is subjected to overexpression, overexpression plasmids of the gene are constructed, and the overexpression plasmids are used for converting the trichoderma reesei, so that the protein expression quantity and the cellulase activity of the trichoderma reesei are improved.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for improving the ability of Trichoderma reesei to express cellulase by regulating cell metabolism. Background technique [0002] As an important industrial production strain, the filamentous fungus Trichoderma reesei is widely used in the production of industrial products such as feed enzymes, organic acids, antibiotics, etc. It has the characteristics of high expression and strong secretion ability. At the same time, it has a variety of post-translational processing methods similar to eukaryotes, such as glycosylation, protease cleavage and disulfide bond formation. In the mixed fermentation broth, the expression of cellobiohydrolase accounted for more than 50% of the total extracellular secreted protein. 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 improv...

Claims

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Application Information

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IPC IPC(8): C12N15/80C12N15/53C12N9/42C12R1/885
CPCC12N15/80C12N9/0006C12N9/2437C12Y101/01049
Inventor 苏小运孙先花姚斌罗会颖王晓璐秦星王苑涂涛张杰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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