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Preparation method of Trichoderma reesei gene engineering bacterium for highly yielding heat stability xylanase

A technology of genetically engineered bacteria and Trichoderma reesei, applied in the field of genetic engineering

Active Publication Date: 2017-05-17
深圳市鼎宏生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, many scholars are conducting research on the expression of xylanase in Trichoderma reesei, but for the xylanase obtained by transforming xyn-CDBFV to express in Trichoderma reesei to improve the expression yield Research not yet reported

Method used

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Examples

Experimental program
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Effect test

Embodiment

[0037] Embodiment: the preparation of the Trichoderma reesei genetically engineered bacterium that produces thermostable xylanase xyn-LVK

[0038] 1. Experimental conditions

[0039] (1) Strains and vectors

[0040] Escherichia coli E. coli Top10 was purchased from Invitrogen, Trichoderma reesei QM9414 was purchased from American Type Culture Collection (ATCC), and pUC19 and pMD19-T simple vector were purchased from TaKaRa. Plasmid pAN7-1 (with fungal selection marker hygromycin resistance gene hph and E. coli selection marker ampicillin resistance gene Ap).

[0041] (2) Enzymes and other biochemical reagents

[0042] Various DNA tool enzymes, DNA Marker, DNA Gel Recovery and Purification Kit (Agarose Gel DNA Purification Kit), DNA Purification Kit (Fragment Purification Kit Ver. 2.0) were purchased from TaKaRa. T4 DNA ligase was purchased from Fermetas. Other biochemical reagents were purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0043] (3)...

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Abstract

The invention discloses a preparation method of a Trichoderma reesei gene engineering bacterium for highly yielding heat stability xylanase. The method comprises the following steps: synthesizing an expression cassette sequence sequentially comprising a Trichoderma reesei glyceraldehyde-3-phosphate dehydrogenase promoter, a Trichoderma reesei cbh1 signal peptide sequence, an xylanase xyn-LVK gene sequence and a Trichoderma reesei glyceraldehyde-3-phosphate dehydrogenase terminator; cloning the expression cassette sequence into pUC19 in order to obtain a recombinant expression plasmid; and lightly uniformly mixing the recombinant expression plasmid with a pAN7-1 vector, co-transforming Trichoderma reesei protoplasts, and screening the Trichoderma reesei protoplasts to obtain the Trichoderma reesei gene engineering bacterium for highly yielding heat stability xylanase xyn-LVK. The content of the xylanase xyn-LVK expressed in the Trichoderma reesei is about 40% higher than the content of the xylanase xyn-LVK expressed in Pichia yeast.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a preparation method of a Trichoderma reesei genetic engineering bacterium that produces high thermostable xylanase. Background technique [0002] The filamentous fungus Trichoderma reesei ( Trichoderma reesei ) expression system is an important tool for homologous and heterologous gene recombination expression, which is superior to prokaryotic expression system and yeast expression system in many aspects. On the one hand, the E. coli expression system tends to form inclusion bodies when expressing heterologous proteins, and the expressed enzyme activity is relatively low and lacks glycosylation modifications. On the other hand, there are excessive glycosylation and protein folding problems in the yeast expression system, which will affect the activity of the expressed enzyme. In comparison, Trichoderma reesei is an ideal host for expressing homologous and heterolog...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/80C12N1/15
Inventor 白孟飞王剑英郭宏涛吴亚宁林智
Owner 深圳市鼎宏生物科技有限公司
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