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A method for improving cellulase activity of Aspergillus niger by integrating large fragment genes

A technology of Aspergillus niger 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
中农华威生物制药(湖北)有限公司
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  • 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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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] 1. Material

[0023] Aspergillus niger CBS 513.88, Trichoderma reesei RUT C-30, 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)-Trichoderma reesei gene homologous fragment front segment 1- Hygromycin resistance gene unit box-Aspergillus niger genome fragment 1- Reesei wood The homologous fragment of mold gene is 1-AAGCTTGG (HindIII restriction site sequence).

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

[0026] Synthetic DNA fragment 2, its sequence composition is: GGCCGCGG (SacII restriction site sequence)-Trichoderma reesei gene homologous fragment front segment 2- Hygromycin resistance gene unit cassette-Aspergi...

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PUM

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Abstract

The invention discloses a method for improving the activity of aspergillus niger cellulase by large fragment gene integration, and belongs to the field of gene engineering. According to the method, a large gene segment related to a trichoderma reesei cellulase system is integrated into an aspergillus niger genome once through multiple homologous recombination. The method specifically comprises the following steps of integrating two aspergillus niger genome segments and a hygromycin resistant gene unit box onto a trichoderma reesei genome, afterwards, using the two segments as homologous recombination sites and hygromycin resistance as a screening marker to integrate a large fragment between the two sites on the trichoderma reesei genome onto the aspergillus niger genome. The polyclonal purposes of a cellulase gene and a non-cellulase gene are achieved; therefore, a metabolic network of aspergillus niger is influenced; the activity of the cellulase of the aspergillus niger is improved. The filter paper activity of a strain obtained through the method provided by the invention is 2.17 times the filter paper activity 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 Aspergillus niger by integrating large-segment genes. Background technique [0002] Aspergillus niger cellulase is a widely used enzyme; Aspergillus niger has good food safety and is a widely used strain in food or industrial production. Among the cellulase enzyme system composition produced by Aspergillus niger, the activity of exonuclease is relatively low. The existing method to improve the cellulase enzyme system of Aspergillus niger is to introduce the exonuclease gene through genetic engineering to increase the enzyme activity of the exonuclease; or 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 enhanced at a time. [0003] In fact, no...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/80C12N1/15C12R1/685
Inventor 薛栋升曾徐浩
Owner 中农华威生物制药(湖北)有限公司