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Saccharomyce engineering strain for expressing cbh2 gene and its construction method

A technology of genetically engineered strains and yeast, applied in the field of bioengineering, can solve problems such as increased product cost, high product cost, and poor thermal stability

Inactive Publication Date: 2006-04-12
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the main factors restricting the application of cellulase in the agricultural industry are: (1) long production cycle, high product cost, (2) poor thermal stability, and short shelf life
(3) High-temperature fermentation requires special equipment, which will increase production costs and energy consumption
For example, the culture conditions of thermophilic bacteria are relatively harsh, and the large-scale fermentation production of thermophilic enzymes requires special equipment, and the enzyme production efficiency is low, resulting in increased product costs, thus limiting its application

Method used

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  • Saccharomyce engineering strain for expressing cbh2 gene and its construction method
  • Saccharomyce engineering strain for expressing cbh2 gene and its construction method
  • Saccharomyce engineering strain for expressing cbh2 gene and its construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1. PCR amplification and cloning of the thermophilic Chaetomium cellobiohydrolase II gene

[0030] (1) Synthesis of amplification primers

[0031] The oligonucleotide primers were designed according to the cDNA sequence of Chaetomium thermophila cellobiohydrolase II cloned in the laboratory. The upstream primer is 26 nucleotides long, and the downstream primer is 27 nucleotides long. The primers were synthesized by Shanghai Sangon Biological Co., Ltd. and purified by PAGE. The sequence is as follows (EcoRI and Not I restriction sites were introduced in the upstream and downstream primers respectively, and the underlined part is the restriction site):

[0032] Forward: 5'---CC GAATTC GCCCCCTCTCCTTGAGGAG-----3'

[0033] Reverse: 5'----GG GCGGCCGC TCAGAGCGGAGGGTTGG--3'

[0034] (2) Extraction of total RNA

[0035]A thermophilic fungus Chaetomium thermophilum CT2 was isolated and identified from China, and total RNA was extracted according to the TRIZOL meth...

Embodiment 2

[0099] Example 2. Construction of Pichia pastoris genetic engineering strain expressing cellobiohydrolase II gene

[0100] (1) Construction of the shuttle expression plasmid:

[0101] The recombinant vector was extracted and digested with EcoR I and Not I to obtain the target fragment cbh2; the pPIC9K empty vector was also digested and dephosphorylated under the action of CIAP. The dephosphorylated linearized pPIC9K empty vector and the CBH II obtained after double enzyme digestion were placed under the action of T4 DNA ligase at 4°C overnight to obtain the recombinant expression vector pPIC9K-CBH II, and the recombinant vector was transferred into the large intestine Bacillus JM109 was amplified. Pick the white single colony and quickly extract the plasmid for enzyme digestion identification ( image 3 ).

[0102] (2) Construction of yeast engineering strain expressing cellobiohydrolase II gene:

[0103] a. Linearization of the shuttle expression plasmid:

[0104] The re...

Embodiment 3

[0113] Example 3. Induced expression of genetically engineered bacteria cellobiohydrolase II protein

[0114] (1) Pick a single colony, place it in a 250mL shake flask with 25mL BMGY medium, and culture it at 28-30°C / 250-300rpm until OD600=2-6 (about 16-18h);

[0115] (2) Centrifuge at 1500-3000g for 5 minutes at room temperature, collect the bacteria, and resuspend the bacteria in BMMY to make OD600=1.0 (about 200mL);

[0116] (3) Place the bacterial solution obtained in step 2 into a 1L shaker flask, seal it with double-layer gauze or cheesecloth, and place it on a shaker at 28-30°C / 250-300rpm to continue growing;

[0117] (4) Add 100% methanol to the medium every 24 hours to a final concentration of 0.5% (about 1 mL);

[0118] (5) Samples were taken at 24, 48, 72, 96, 120, 144, and 168 hours to detect the expression of the cbh2 gene in yeast.

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Abstract

A genetically engineered yeast Pichia pastoris GS-D is prepared through screening the thermophilic chaetomium CT2 by PCR method to obtain cellobiohydrolase II gene, cloning it, inserting it in the expression carrier of Pichia yeast, introducing it to Pichia yeast and screening said genetically engineered yeast. It can generate a great deal of active cbh2 protein to convert the rejected fiber material.

Description

(1) Technical field: [0001] The present invention relates to biological engineering, specifically a kind of Pichia pastoris engineering strain (Pichiapastoris GS-D) expressing cbh2 gene and its construction method (2) Technical background: [0002] Cellulose is a cheap renewable resource and the main component of the cell wall of higher plants. Its content reaches 35%-55% of the dry weight of the plant, and it exists widely in nature. Cellulose is hydrolyzed into glucose, and then fermented to produce organic chemical raw materials and fuels such as ethanol, acetone, butanol, as well as feed, food, and medicine. Cellulosic materials are the most promising resources to solve food problems, energy problems and environmental problems faced by human beings. Research and development of cellulose resources has far-reaching significance. Cellulase can cut the molecular chain of cellulose and convert it into glucose. In addition to being fermented and further converted into protei...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N15/56C12N15/09
Inventor 李多川刘守安
Owner SHANDONG AGRICULTURAL UNIVERSITY
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