Novel Trichoderma host cell and application thereof

A host cell, Trichoderma reesei technology, applied in the direction of enzymes, biochemical equipment and methods, microorganisms, etc., can solve problems in the initial stage, and achieve the effects of reducing stirring speed, dense mycelium, and reducing viscosity

Active Publication Date: 2018-07-06
QINGDAO VLAND BIOTECH GRP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are relatively many studies on the expression system of Trichoderma reesei, but the research

Method used

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  • Novel Trichoderma host cell and application thereof
  • Novel Trichoderma host cell and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0023] Example 1 Ultraviolet mutagenesis screening of Trichoderma reesei expression host

[0024] The applicant knocked out the four cellulase genes CBH1, CBH2, EG1 and EG2 in the wild-type Trichoderma reesei by means of gene knockout, and obtained a strain that does not secrete cellulase by itself. The strain of Trichoderma reesei was named Trichoderma reesei U (Trichoderma reesei U).

[0025] Inoculate Trichoderma reesei U on a fresh PDA plate and culture at 30°C for 5-7 days. When the surface of the colony turns white and a large number of spores are produced, draw 5ml of sterile water to elute to obtain the spore liquid, resuspend in sterile water after centrifugation, and count with a hemocytometer to make the spore concentration about 5×10 7 Pcs / ml. Put a rotor into a 90mm sterile petri dish, add 10ml of diluted spore suspension, and stir on a magnetic stirrer to make the spore liquid in a uniform state. In the aseptic ultra-clean workbench, the ultraviolet lamp with a powe...

Example Embodiment

[0029] Example 2 Screening of uracil auxotrophy in Trichoderma reesei expression host

[0030] 2.1 Principle:

[0031] 5-Fluororotate can induce the deletion of whey nucleotide transferase or orotidine monophosphate decarboxylase in the synthesis pathway of uracil nucleotides in bacteria, so that 5-fluoroorotic acid cannot form toxic substances 5 -Fluorouracil nucleotides, which produces resistance to 5-fluoroorotic acid, and its pyrimidine nucleotide nutrition can be supplemented by adding uracil to the medium, so the urine induced by 5-fluoroorotic acid Pyrimidine auxotrophic strains can grow in a medium containing 5-fluoroorotic acid and uracil; while wild-type strains are not resistant to 5-fluoroorotic acid and cannot Grow under different culture conditions. Therefore, 5-fluoroorotic acid is often used to screen uracil-deficient mutants.

[0032] 2.2 Screening method

[0033] The spores of 38 strains of Trichoderma reesei mutant strains screened in Example 1 were diluted to ab...

Example Embodiment

[0036] Example 3 Expression of Neutral Cellulase Gene NCE5 in Mutant Trichoderma reesei

[0037] In order to verify the expression efficiency of Trichoderma reesei U1, U2, U3, U4,..., U38 on the target gene, the applicant selected the neutral cellulase gene NCE5 (the nucleotide sequence of which is derived from Trichoderma reesei) SEQ ID NO: 1, the encoded amino acid sequence is SEQ ID NO: 2) were expressed in wild-type Trichoderma reesei U and the above mutant strains.

[0038] 3.1 Extract the total genomic DNA of Trichoderma reesei

[0039] Inoculate Trichoderma reesei U1 with shaking flask culture medium overnight, take an appropriate amount of bacteria into a centrifuge tube, centrifuge at 13000 rpm for 5 minutes, discard the supernatant; add 400μl extraction buffer (100mM TrisHCl, 100mM EDTA, 250mM NaCl, 1% SDS); then add 100mg quartz sand or glass beads, vigorously shake in the beading instrument for about 2min; add 200μl 10M NH4AC ice bath for 10min after 20min in the water b...

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Abstract

The invention provides a Trichoderma reesei host cell obtained by screening, and the preservation number is CCTCC NO:M2016726. The Trichoderma reesei host cell provided by the invention can be used for expressing one or more endogenous or heterologous proteins. On another aspect, the invention provides a Trichoderma reesei recombinant strain, which is obtained by transforming an expression vectorcarrying a target gene into the Trichoderma reesei host cell. The mutant strain Trichoderma reesei U11 obtained by mutagenesis has thick and short hypha and dense mycelia with more branches, can significantly lower the viscosity of the fermented bacteria liquid and reduce the purposes of lowering the stirring speed and increasing dissolved oxygen, and is more conducive to high-density fermentationof strains. The Trichoderma reesei U11 can be taken as a novel host cell and widely applied to recombinant expression of homologous or heterologous genes.

Description

technical field [0001] The invention relates to the technical field of genetic engineering microorganism screening, and specifically provides a novel Trichoderma host cell and application thereof. Background technique [0002] As a biocatalyst, enzymes can be used in many fields, and the yield, output, quality and function of enzymes are important determinants for the application of enzymes in industry. High-efficiency expression of enzymes is related to many factors: host cell growth characteristics, expression levels, intracellular / extracellular expression patterns, post-translational modifications, active proteins, etc. In order to achieve high-efficiency expression of enzymes, the choice of enzyme expression system is crucial. Recombinases have been successfully expressed in multiple hosts, such as Escherichia coli, Bacillus, yeast, filamentous fungi, lactic acid bacteria, etc. Due to the diversity of the properties and functions of exogenous genes or expression hosts ...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12N1/15C12N9/42C12N9/10C12R1/885
CPCC12N9/1051C12N9/2437C12N1/145C12R2001/885
Inventor 许丽红吴佳鹏黄亦钧
Owner QINGDAO VLAND BIOTECH GRP
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