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Trichoderma reesei engineering bacterium with high cellulase yield as well as construction method and application of trichoderma reesei engineering bacterium

A technology of Trichoderma reesei and cellulase, which is applied in the field of bioengineering to achieve the effects of increasing expression, good genetic stability and reducing production costs

Pending Publication Date: 2022-03-29
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Nevertheless, the high production cost of cellulase is still a bottleneck restricting its industrialization in the field of bioenergy and bulk biobased products

Method used

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  • Trichoderma reesei engineering bacterium with high cellulase yield as well as construction method and application of trichoderma reesei engineering bacterium
  • Trichoderma reesei engineering bacterium with high cellulase yield as well as construction method and application of trichoderma reesei engineering bacterium
  • Trichoderma reesei engineering bacterium with high cellulase yield as well as construction method and application of trichoderma reesei engineering bacterium

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1: the construction of Trichoderma reesei OEtup1 engineering bacteria

[0049] 1. Construction of tup1 promoter replacement vector

[0050] 1) According to the promoter P of Trichoderma reesei tcu1 gene tcu1 The principle of the replacement system is designed to amplify the upstream and downstream homology arm amplification primers of the tup1 gene. The upstream homology arm amplification primers are tup1-up-F and tup1-up-R, and the downstream homology arm amplification primers are tup1-down- F and tup1-down-R. Wherein, the primer sequences of the tup1-up-F, tup1-up-R, tup1-down-F and tup1-down-R are as follows:

[0051] tup1-up-F: 5′-CCCAAGCTTTGGTAGCTTATTAGCTGGATTTGG-3′,

[0052] tup1-up-R: 5′-AGGCGCGCCCGTGGCTCTATCGAGGCCGTAA-3′,

[0053] tup1-down-F: 5′-AAGGAAAAAAGCGGCCGCATGTCCATGTATTCCCATCGCGGCA-3′, tup1-down-R: 5′-GACTAGTCCCAGACACGAACGCTCTTATCCAA-3′,

[0054] 2) Extract the genomic DNA of Trichoderma reesei QM9414, use the extracted genomic DNA as a t...

Embodiment 2

[0082] Example 2: Analysis of tup1 transcription and extracellular cellulase secretion level in Trichoderma reesei OEtup1 engineering bacteria

[0083] 1. Use qRT-PCR method to analyze the tup1 transcription level in Trichoderma reesei OEtup1 engineering bacteria, the steps are as follows:

[0084] 1) The control strain QM9414 and the constructed Trichoderma reesei OEtup1 engineering bacteria were inoculated on MA medium with 1% (v / v) glycerol as carbon source for pre-cultivation, at 30° C., 200 rpm for 48 h; collected mycelia, Then transfer to a fermentation medium with 1% (w / v, g / mL) microcrystalline cellulose as a carbon source, and continue to cultivate at 30°C and 200rpm;

[0085] Wherein, the MA medium components are as follows: 17.907g / L Na 2 HPO 4 12H 2 O,2g / L KH 2 PO 4 ,1.4g / L(NH4) 2 SO 4 , 0.3g / L urea, 0.5ml / L Tween-80, adjust the pH to 5.0 with anhydrous citric acid. During pre-cultivation, 1% (v / v) glycerol was added as a carbon source, and 2 g / L peptone wa...

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Abstract

The invention relates to a Trichoderma reesei engineering bacterium for high yield of cellulase and a construction method and application thereof, a promoter of a tup1 gene is replaced by a promoter Ptcu1 of a tcu1 gene in chromatin in situ by adopting a promoter replacement technology, a Trichoderma reesei OEtup1 engineering bacterium is constructed, and overexpression of tup1 is realized. Compared with the original strain, the trichoderma reesei OEtup1 engineering strain has the advantages that the cellulase expression under the induction condition is improved by about 30%, and the production cost is reduced when the trichoderma reesei OEtup1 engineering strain is applied to cellulase production. The obtained Trichoderma reesei engineering bacterium has good hereditary stability, can significantly improve the expression quantity of cellulase under the cellulose induction condition, and has important application value in industrial production of cellulase.

Description

technical field [0001] The invention relates to a Trichoderma reesei engineering bacterium with high cellulase production and a construction method and application thereof, belonging to the technical field of bioengineering. Background technique [0002] Lignocellulosic biomass resources are the most abundant renewable resources on earth and have broad application prospects in the production of bulk bio-based products. The effective use of biomass resources can not only solve the increasingly serious energy crisis faced by human society, but also turn waste into treasure to solve the environmental pollution caused by straw burning. Trichoderma reesei is a model fungus for degrading lignocellulosic biomass, and it is also the main industrial strain of cellulase. The cellulase produced by it is widely used in energy, textile, pulp and paper and other fields. Nevertheless, the high production cost of cellulase is still a bottleneck restricting its industrialization in the fiel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12N15/80C12R1/885
CPCC12N9/2437C12N15/80
Inventor 刘巍峰张伟欣王磊
Owner SHANDONG UNIV
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