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Trichoderma reesei recombinant strain capable of highly producing cellulase and application thereof

A technology of Trichoderma reesei and recombinant strains, applied in the field of microbial biology, can solve the problems of high production cost of cellulase, unclear genetic stability and background, and unreported relationship between production and other issues

Active Publication Date: 2017-07-25
SHANGHAI JIAO TONG UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, in the bioconversion process of lignocellulosic biomass, the high production cost of cellulase is one of the main bottlenecks restricting its industrial application.
However, there is no research report on the use of vib1 overexpression to enhance cellulase production
Trichoderma reesei Rut-C30 also has a homologous gene vib1 of the vib1 gene tr , the gene name in the JGI database is TrireC30-125610, but the relationship between the gene and the production of cellulase by Trichoderma reesei has not been reported yet
[0005] Although scholars at home and abroad have obtained a variety of cellulase high-yielding strains through physical and chemical mutagenesis, their genetic stability and background are unclear; However, increasing the total extracellular protein secretion of cellulase-producing bacteria is more conducive to the large-scale economic production of cellulase

Method used

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  • Trichoderma reesei recombinant strain capable of highly producing cellulase and application thereof
  • Trichoderma reesei recombinant strain capable of highly producing cellulase and application thereof
  • Trichoderma reesei recombinant strain capable of highly producing cellulase and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1, Trichoderma reesei plasmid pCZF3-Vib1 construction

[0056] The vib1 gene involved in the present invention comes from Triremycetes reesei Rut-C30. The gene name in the JGI database is TrireC30-125610. The nucleic acid sequence is shown in SEQ ID NO.1. The protein sequence encoded by Trichoderma reesei vib1 has an NDT80 family DNA binding region , see SEQ ID NO.2 for the protein sequence code. In addition to the hygromycin resistance expression cassette and the necessary components required for the transformation of Agrobacterium tumefaciens, the binary plasmid vector pCZF3 also has the promoter of the pyruvate decarboxylase gene (pdc, pyruvate decarboxylase) of Trichoderma reesei - nesting Aspergillus tryptophan synthase gene (trpC, tryptophansynthase) terminator-driven target gene expression cassette, after double-digestion by Nco I and Xba I, the nucleic acid sequence of the target gene is connected to the large fragment after digestion, and the pdc ...

Embodiment 2

[0096] Example 2, Transformation of Agrobacterium tumefaciens AGL-1 containing pCZF3-Vib1 into Miriam reesei C30

[0097] 1. Agrobacterium tumefaciens AGL-1 containing pCZF3-Vib1 was inoculated in 5 milliliters of culture medium (100 micrograms per milliliter of kanamycin) by 1% inoculum size, 28 degrees Celsius, 240 revs per minute dark culture 48 hours; 5000 Spin per minute and centrifuge for 10 minutes, collect Agrobacterium cells, and suspend in induction medium (IM) to adjust OD 660 It is about 0.2, and cultivated under the same conditions for 6-12 hours.

[0098] 2. Inoculate Trichoderma reesei Rut-C30 on a PDA plate, incubate under light at 28 degrees Celsius for 5-7 days, wash the conidia with sterile water, and adjust to a final concentration of about 10 7 spores per milliliter.

[0099] 3. After taking 100 microliters of activated AGL-1 bacterial strain containing carrier pCZF3-Vib1 and the same volume of Trichoderma reesei Rut-C30 spore suspension, mix them even...

Embodiment 3

[0106] Example 3, Determination of the properties of cellulase secreted by pCZF3-Vib1 containing pCZF3-Vib1 Reeser's mildew transformants

[0107] Three random strains were identified by PCR and resistance screening to obtain the transformants of Trichoderma reesei Vib1 and the starting strain Trichoderma reesei Rut-C30 for sporulation culture, and the spores were added to 50 ml of germination medium with a final concentration of 10 6 After cultivating at 28 degrees Celsius and 150 rpm for 36 hours per milliliter, take 5 milliliters of mycelium and add it to 45 milliliters of cellulose fermentation medium, cultivate under the same conditions and take 1 milliliter of culture medium every 24 hours for preservation, continuously Sampling for 7 days. Using Trichoderma reesei C30 as a control, the enzyme activity of the cellulose filter paper was measured, and the enzyme activity of extracellular cellulase of two kinds of Trichoderma reesei was compared.

[0108] See the appendi...

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Abstract

The invention discloses a trichoderma reesei recombinant strain capable of highly producing cellulase and application thereof. Vlbltr which grows an incompatible repression protein gene is transformed into trichoderma reesei Rut-C30, so as to obtain the trichoderma reesei Vib1 (CCMCC No.13578). Compared with the strain Vib1 and the trichoderma reesei Rut-C30, the trichoderma reesei recombinant strain has the advantages that the excretion of extracellular protein and the production of cellulase are obviously improved; compared with the trichoderma reesei Rut-C30, the activity of cellulase in the Vib1 in cellulose and bran culture mediums is increased by about 200%, and reaches 3.3U / ml; the enzyme activities of cellobiohydrolase, endo-cellulase and beta-glucosidase are respectively improved; the excretion amount of the extracellular protein in the Vib1 strain is equal to 2.19 times of the excretion amount of trichoderma reesei Rut-30; proved by the results of hydrolysis reaction of pretreated corn straw, compared with the Rut-30 strain, the output of glucose in the Vib1 strain cellulase hydrolyte is increased by 20%.

Description

technical field [0001] The invention belongs to the field of microbial biotechnology; in particular, it relates to a recombinant bacterial strain of Trichoderma reesei with high cellulase production and application thereof. Background technique [0002] With the continuous consumption of fossil energy, the development of renewable biomass energy has attracted widespread attention at home and abroad. Lignocellulosic biomass resources are abundant and renewable, and are potential alternative energy sources for fossil energy. Cellulase and hemicellulase can be used to hydrolyze them into glucose and xylose, and further ferment them to produce renewable bioenergy and Wide range of bio-based chemicals. However, in the bioconversion process of lignocellulosic biomass, the high production cost of cellulase is one of the main bottlenecks restricting its industrial application. Therefore, it is very important to improve the production efficiency of strain cellulase and reduce the p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12N9/42C12N15/80C12R1/885
CPCC07K14/37C12N9/2437C12N15/80C12Y302/01004
Inventor 赵心清白凤武张飞
Owner SHANGHAI JIAO TONG UNIV
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