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Method for improving trichoderma reesei cellulase enzyme activity through large-fragment gene integration

A technology of Trichoderma reesei and fragments, which is applied in the field of genetic engineering to achieve the effect of improving the enzymatic activity of cellulase

Active Publication Date: 2017-06-20
北京中农华威科技集团有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In these current methods, only one gene can be knocked out or the expression of one gene can be enhanced at a time

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] 1. Material

[0023] Aspergillus niger CBS 513.88, Trichoderma reesei QM6a, plasmid pCAMBIA1300 (Biovector), Agrobacterium EHA105, Escherichia coli DH5α, restriction endonuclease (TakaRA), etc.

[0024] Synthetic DNA fragment 1, its sequence composition is: GGCCGCGG (SacII restriction site sequence)-Aspergillus niger gene homologous fragment front section 1- Hygromycin resistance gene unit box-Trichoderma reesei genome fragment 1- Aspergillus niger gene Homologous fragment 1-AAGCTTGG (HindIII restriction site sequence).

[0025] Synthetic DNA fragment 1-2, its sequence composition is: GGCCGCGG (SacII restriction site sequence)-Aspergillus niger gene homologous fragment 1-Trichoderma reesei genome fragment 1-AAGCTTGG (HindIII restriction site sequence).

[0026] Synthetic DNA fragment 2, its sequence composition is: GGCCGCGG (SacII restriction site sequence)-Aspergillus niger gene homologous fragment front section 2- Hygromycin resistance gene unit box-Trichoderma reesei genome ...

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PUM

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Abstract

The invention discloses a method for improving trichoderma reesei cellulase enzyme activity through large-fragment gene integration and belongs to the field of gene engineering. In the method, gene large fragments relevant to an aspergillus niger cellulase system are integrated into a trichoderma reesei genome by one time through multiple times of homologous recombination; and the method concretely comprises the following steps: two trichoderma reesei genome fragments and a hygromycin resistance gene unit box are integrated into an aspergillus niger genome, and large fragments between two sites on the aspergillus niger genome are integrated into the trichoderma reesei genome by taking the two fragments as homologous recombination sites and the hygromycin resistance as a screening marker, so that the effect of cellulase gene and non-cellulase gene polyclone is achieved, metabolic network of trichoderma reesei is influenced, and the trichoderma reesei cellulase enzyme activity is improved. The filter paper enzyme activity of a strain obtained by the method provided by the invention is 2.28 times of that of an original strain.

Description

Technical field [0001] The invention belongs to the field of genetic engineering, and specifically relates to a method for improving the cellulase activity of Trichoderma reesei by integrating large-segment genes. Background technique [0002] Trichoderma reesei cellulase is a widely used enzyme; Trichoderma reesei has a good ability to express extracellular enzymes and is a widely used strain in food or industrial production. Among the cellulase enzyme system composition produced by Trichoderma reesei, the activity of glucosidase is relatively low. The existing method to improve the cellulase enzyme system of Trichoderma reesei is to introduce the glucosidase gene through genetic engineering to increase the enzyme activity of glucosidase; or to knock out certain exocrine proteins to reduce the secretion of exocrine proteins. Thereby increasing the secretion of cellulase protein. In these current methods, only one gene can be eliminated or the expression of one gene can be enha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/80C12N1/15C12R1/885
Inventor 薛栋升曾徐浩
Owner 北京中农华威科技集团有限公司