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Trichoderma reesei recombinant strain containing artificial zinc finger transcription factor and application of Trichoderma reesei recombinant strain

A technology of transcription factor, Trichoderma reesei, applied in the field of microbial biology

Inactive Publication Date: 2015-08-26
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Artificial transcription factor technology has been successfully applied in many fields. Through this method, it has achieved success in improving protein expression yield, improving cell tolerance to solvents, osmotic pressure, temperature, and pH characteristics. In Saccharomyces cerevisiae and Escherichia coli strains It has been successfully applied in transformation (refer to Nature Biotechnology, 2003, 21:1208-1214; Applied Microbiology and Biotechnology, 2015, 99(5): 2441-9), but related work in filamentous fungi has not been reported

Method used

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  • Trichoderma reesei recombinant strain containing artificial zinc finger transcription factor and application of Trichoderma reesei recombinant strain
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  • Trichoderma reesei recombinant strain containing artificial zinc finger transcription factor and application of Trichoderma reesei recombinant strain

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Example 1: Trichoderma reesei zinc finger protein library construction

[0032]The artificial zinc finger involved in the present invention is a Cys2His2 type zinc finger. The construction method refers to Nature Biotechnology, 2003, 21: 1208-1214, which includes four zinc finger domains and is provided by ToolGen Company of South Korea. This zinc finger library is characterized by the conserved transcription factor activation domain Gal4 from Saccharomyces cerevisiae, and the variable C2H2 zinc finger binding domain, and the library capacity is about 10 6 The backbone of the binary expression vector is pCB303, which is composed of filamentous fungal expression vector pAN7-1 (reference Gene, 1987,56:117-124) and pCB301 (reference Plant Molecular Biology, 1999,40:711-717) It is modified, which contains the pki promoter and trpC terminator from Trichoderma reesei, and the nucleic acid sequence of the zinc finger protein library can be inserted between the two restriction ...

Embodiment 2

[0050] Example 2: Transformation of zinc finger protein Agrobacterium tumefaciens AGL-1 library into Trichoderma reesei C30

[0051] 1. The library of Agrobacterium tumefaciens AGL-1 containing zinc finger protein is inoculated in 5 milliliters of medium (100 micrograms per milliliter of Vic per milliliter of kanamycin) at 1% inoculum size, 28 degrees Celsius, 240 rpm in dark Cultivate for 48 hours; centrifuge at 5000 rpm for 10 minutes, collect Agrobacterium cells, and suspend in induction medium (IM) to adjust OD 660 It is about 0.2, and cultured for 6-12 hours according to the same conditions.

[0052] 2. Inoculate Trichoderma reesei C30 on a PDA plate, culture for 5-7 days under light at 28 degrees Celsius, wash the conidia with sterile water, and adjust to a final concentration of about 10 7 spores per milliliter.

[0053] 3. Take 100 microliters of activated AGL-1 and the same volume of C30 spore suspension and mix them evenly, and evenly spread them on the cellophane ...

Embodiment 3

[0055] Example 3: Cellulose Activity Screening of Trichoderma reesei Transformants Containing Zinc Finger Proteins

[0056] Transformants of Trichoderma reesei were identified by PCR and resistance screening, and the transformants were screened for mutants with improved cellulase activity by the cellulose liquid shake flask method. Add 50 milliliters of shaking flask screening medium to a 100 milliliter Erlenmeyer flask, and insert about 10 of Trichoderma reesei transformants activated for 7 days to produce spores. 8 per milliliter, at 150 rpm, cultured in a shaker at 28 degrees centigrade, and the culture solution was drawn to measure the enzyme activity of the cellulose filter paper. The wild-type Trichoderma reesei C30 was used as a control to compare the enzyme activity.

[0057] The strains with obvious changes in cellulase activity obtained from the primary screening were further subjected to liquid enzyme activity fermentation comparisons for further verification.

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Abstract

The invention discloses a Trichoderma reesei recombinant strain containing artificial zinc finger transcription factor and application of the Trichoderma reesei recombinant strain, and belongs to the field of microbial biotechnology. The Trichoderma reesei recombinant strain is stored in China General Microbiological Culture Collection Center on April 16, 2015, and the accession number is CGMCC No. 10649. The Trichoderma reesei recombinant strain is named Trichoderma reesei U5, which is a high-yield cellulose strain obtained by transferring an artificial zinc finger protein transcription factor into the starting strain of Trichoderma reesei C30, and then being subjected to cellulase activity screening. Under the condition of bran inducing, the cellulase activity of the high-yield cellulose strain is increased by about 35% as compared with that of a wild type Trichoderma reesei C30, reaching 8.4 enzyme-activity units per milliliter; beta-glucosidase of the Trichoderma reesei U5 is increased by 3.5 times as compared with that of the wild type Trichoderma reesei C30, reaching 8.8 enzyme-activity units per milliliter. The increase in cellulase secretion activity of the Trichoderma reesei U5 shows the potential application value of the Trichoderma reesei U5 in the field of renewable energy, and new microbial resources are provided for the efficient degradation of biomass.

Description

technical field [0001] The invention relates to a recombinant strain of Trichoderma reesei containing an artificial zinc finger transcription factor and an application thereof, belonging to the field of microbial biotechnology. Background technique [0002] In recent years, with the rapid development of modern social economy and the continuous growth of population, people's demand for energy is increasing day by day, and the development of renewable energy has attracted widespread attention at home and abroad. Cellulose and hemicellulose are the most abundant renewable resources in the world, which can be hydrolyzed into glucose and xylose by using cellulase and hemicellulase, and further fermented to produce renewable bioenergy (fuel ethanol and butanol, etc. ), and can also produce a variety of bio-based chemicals. Filamentous fungi and cellulolytic bacteria can produce extracellular cellulase. Filamentous fungal cellulase is mainly composed of three types of enzyme compo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12P19/14C12R1/885
CPCY02P20/59C07K14/37C12P19/14
Inventor 赵心清张飞白凤武
Owner DALIAN UNIV OF TECH
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