Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

A kind of novel Trichoderma host cell and its application

A host cell, Trichoderma reesei technology, applied in the direction of transferase, enzyme, microorganism, etc., can solve the problem of being in the initial stage, and achieve the effect of reducing the stirring speed, the mycelium is dense, and the viscosity is reduced

Active Publication Date: 2020-12-01
QINGDAO VLAND BIOTECH GRP
View PDF6 Cites 0 Cited by
  • 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 on the expression system of Trichoderma dark green and Trichoderma viride is still in its infancy

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A kind of novel Trichoderma host cell and its application
  • A kind of novel Trichoderma host cell and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] The ultraviolet mutagenesis screening of embodiment 1 Trichoderma reesei expression host

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

[0025] Trichoderma reesei U was inoculated on a fresh PDA plate and cultured 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 with sterile water after centrifugation, and count with a hemocytometer to make the spore concentration about 5×10 7 pieces / ml. Take a rotor and put it 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 a sterile...

Embodiment 2

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

[0030] 2.1 Principle:

[0031] 5-Fluoroorotic acid can induce the loss of orotate nucleotidyl transferase or orotidine monophosphate decarboxylase in the uracil nucleotide synthesis pathway, so that 5-fluoroorotic acid cannot form toxic substances5 -fluorouracil nucleotides, which confer resistance to 5-fluoroorotic acid, whose pyrimidine nucleotide nutrition can be supplemented by adding uracil to the medium, thus utilizing 5-fluoroorotic acid-induced formation of urine The pyrimidine auxotrophic strain can grow in the medium containing 5-fluoroorotic acid and uracil; while the wild-type strain cannot grow in the medium containing 5-fluoroorotic acid because it does not have resistance to 5-fluoroorotic acid. grown under the culture conditions. Therefore, 5-fluoroorotic acid is commonly used to screen for uracil-deficient mutants.

[0032] 2.2 Screening method

[0033]The spores of th...

Embodiment 3

[0036] Example 3 Expression of neutral cellulase gene NCE5 in Trichoderma reesei mutant

[0037] In order to verify the expression efficiency of Trichoderma reesei U1, U2, U3, U4, ..., U38 to the target gene, the applicant selects the neutral cellulase gene NCE5 (its nucleotide sequence being 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-mentioned mutant strains respectively.

[0038] 3.1 Extraction of Trichoderma reesei total genomic DNA

[0039] Trichoderma reesei U1 was inoculated with shake flask medium for overnight culture, and an appropriate amount of bacteria was placed in a centrifuge tube, centrifuged at 13,000 rpm for 5 min, and the supernatant was discarded; 400 μl of extraction buffer (100 mM TrisHCl, 100 mM EDTA, 250 mM NaCl, 1% SDS); then add 100mg of quartz sand or glass beads, shake vigorously in a bead beater for about 2min; add 200μl 10M NH4AC in...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides a Trichoderma reesei host cell obtained by screening, and its preservation number is CCTCC NO: M2016726. The T. reesei host cells of the invention can be used to express one or more endogenous or exogenous proteins. Another aspect of the present invention provides a recombinant Trichoderma reesei strain obtained by transforming an expression vector carrying a target gene into the above-mentioned Trichoderma reesei host cell. The mycelium of the mutant Trichoderma reesei U11 obtained by mutagenesis in the present invention is thick and short, with dense mycelium and more branches, which can significantly reduce the viscosity of the fermentation liquid, achieve the purpose of reducing the stirring speed and increasing the dissolved oxygen , which is more conducive to the high-density fermentation of strains. Trichoderma reesei U11 can be used as a new type of host cell, which is widely used in the 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com