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Method for breeding high-producing strain of cellulase by gene knockout

A cellulase and high-yielding strain technology, applied in the field of enzyme engineering, can solve the problems of consuming cells, aggravating cell load, consuming large nutrients, etc., and achieving the effect of reducing cell load and improving enzyme activity

Active Publication Date: 2014-06-18
中农华威生物制药(湖北)有限公司
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AI Technical Summary

Problems solved by technology

Because the translation and secretion of these enzymes consume a lot of nutrients on the one hand, and on the other hand, their synthesis and secretion consume a lot of energy in cells, which increases the load on cells.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] A. Construction of Amylase Gene Knockout Plasmid

[0023] (1) Synthesis of recombinant fragments

[0024] The recombinant fragment consists of the first half of the amylase gene (SEQ ID NO.2), the hygromycin B resistance gene expression unit (SEQ ID NO.3), the second half of the amylase gene (SEQ ID NO.4), The synthetic recombinant fragment (SEQ ID NO.1) was digested with 2 μL of NcoI enzyme solution (purchased from TakaRa) at 30 °C for 16 h.

[0025] (2) Extraction and digestion of pCAMBIA1300

[0026] Escherichia coli E.coli DH5α containing the pCAMBIA1300 plasmid was cultured with shaking at 37°C for 12 h. Take 1.5 mL of bacterial cells in an EP tube, centrifuge at 4000 rpm for 3 min, and discard the supernatant. Add 0.1 mL of solution I (1% glucose, 50 mM EDTA pH 8.0, 25 mM Tris-HCl pH 8.0) and mix well. Add 0.2 mL solution II (0.2 mM NaOH, 1% SDS), gently invert to mix, and place in ice bath for 5 min. Add 0.15 mL of pre-cooled solution III (5 mol / L KAc, pH 4....

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Abstract

The invention discloses a method for breeding a high-producing strain of cellulase by gene knockout, and belongs to the field of enzyme engineering. The method for breeding the high-producing strain of cellulase by gene knockout comprises the step of knocking out an amylase gene of the cellulase producing strain. A high-producing strain of cellulase is aspergillus niger without the amylase gene. An amylase gene knockout plasmid is obtained by connecting the front half part of the recombinant segment amylase gene-hygromycin B-an expression unit of a resistant gene- the rear half part of the amylase gene to a pCAMBIA1300 plasmid. Then, the plasmid is converted into agrobacterium, and the amylase gene is knocked out by agrobacterium-mediated conversion of the aspergillus niger. By using the gene knockout technology, the amylase gene of the aspergillus niger is knocked out. After the amylase gene is knocked out, the cell load is reduced, thereby facilitating production of a lot of cellulase. The cellulase activity of the high-producing strain of cellulase obtained can reach 21U / g.

Description

[0001] technical field [0002] The invention belongs to the field of enzyme engineering, and in particular relates to a method for gene knockout and breeding of high-yield cellulase strains. Background technique [0003] Cellulose is the most abundant renewable resource on the earth. The cellulose produced by photosynthesis on the earth reaches about 10 billion tons every year. The use of cellulose to produce bioenergy is the key to alleviating the energy crisis and realizing the sustainable development of human beings. [0004] The key to cellulose utilization is to degrade cellulose into fermentable sugar-glucose. The degradation of cellulose requires the joint action of exonuclease, endonuclease and glucosidase. Among them, endonuclease degrades long fragments of cellulose into dimers, which is a key step in cellulose degradation. Degrading cellulose with cellulase has the characteristics of green, mild conditions and high conversion rate, but the high production cost...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/80C12N9/26C12N1/15C12R1/685
Inventor 薛栋升
Owner 中农华威生物制药(湖北)有限公司
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