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A method for improving the enzyme activity of Trichoderma reesei cellulase by integrating large fragment genes

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: 2019-09-10
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. Materials

[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 1-hygromycin resistance gene unit box-Trichoderma reesei genome fragment 1-Aspergillus niger gene The latter segment of the homologous fragment is 1-AAGCTTGG (HindIII restriction site sequence).

[0025] The sequence composition of the synthesized DNA fragment 1-2 is: GGCCGCGG (SacII restriction site sequence)-Aspergillus niger gene homologous fragment front segment 1-Trichoderma reesei genome segment 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 2-hygromycin resistance...

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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 in particular relates to a method for improving the enzyme activity of Trichoderma reesei cellulase by integrating a large fragment gene. Background technique [0002] Trichoderma reesei cellulase is a widely used enzyme; Trichoderma reesei has a good ability to express extracellular enzymes, and it is a strain widely used in food or industrial production. In the composition of cellulase enzyme system produced by Trichoderma reesei, the activity of glucosidase is relatively low. The existing method for improving the Trichoderma reesei cellulase enzyme system is to introduce the glucosidase gene by means of genetic engineering to improve the enzyme activity of the glucosidase; or knock out some 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 knocked out or the expression of...

Claims

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Application Information

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Patent Type & Authority Patents(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