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System for codon optimization and pichia pastoris expression of genes of cellobiohydrolase II

An exo-glucanase and codon optimization technology, applied in the field of genetic engineering, can solve the problems of insufficient yield, different peak secretion periods, and inability to exert enzymatic hydrolysis effect, and achieve the effects of increasing protein expression and improving expression efficiency.

Inactive Publication Date: 2016-03-30
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these self-produced enzymes are born with certain proportioning defects, such as: the production of CBHII and β-glucosidase in the cellulase of Trichoderma reesei is insufficient, and the secretion peaks of each enzyme in Aspergillus niger are different. Cellulose hydrolysis is limited
In addition, the proportions of cellulose, hemicellulose and lignin in different lignocellulosic materials are significantly different, resulting in the same type of commercial cellulase preparations or self-produced enzymes cannot exert the best enzymatic hydrolysis effect

Method used

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  • System for codon optimization and pichia pastoris expression of genes of cellobiohydrolase II
  • System for codon optimization and pichia pastoris expression of genes of cellobiohydrolase II
  • System for codon optimization and pichia pastoris expression of genes of cellobiohydrolase II

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Trichoderma reesei exoglucanase II (CBHII) gene codon optimization and expression vector construction.

[0043] 1. The present invention utilizes GeneDesigner (DNA2.0, MenloPark, CA, USA) to realize CBHII (NCBIReferenceSequence: XM_006962518.1) nucleotide sequence codon bias optimization as shown in SEQIDNO.1, and the optimized nucleotide sequence is as follows: Shown in SEQ ID NO.3. The codon-optimized amino acid sequence is consistent with the original amino acid sequence, as shown in SEQ ID NO.2. Using Pichiapastoris GS115 as the host, the nucleotide sequence after codon bias optimization and the original nucleotide sequence are compared, see figure 1 , compare the amino acid sequence after codon bias optimization with the original amino acid sequence, see figure 2 .

[0044] 2. The restriction site at the 5' end is EcoRI, and the restriction site at the 3' end is NotI as the double restriction site, and inserted into the downstream of the AOX1 promote...

Embodiment 2

[0045] Example 2: Linearizing expression vectors and performing electrotransformation and screening high-yielding strains.

[0046] 1. Use SacI as the linearization site of the pPIC9K-cbh2 expression vector to realize the linearization of the expression vector, and verify the linearized plasmid by nucleic acid electrophoresis, see Figure 4. Transferred into Pichiapastoris GS115 by electroporation to obtain recombinant strains. Using colony PCR, colonies with clear amplified bands were fermented in shake flasks to obtain high-yield strains. Samples were taken at 120 hours and the CMC enzyme activity of the fermentation supernatant was determined by DNS method. See Figure 5 , wherein, 1 unit of enzyme activity is defined as the amount of enzyme that can convert 1 μmol of substrate in 1 minute under the condition of 50°C water bath.

Embodiment 3

[0047] Example 3: Optimizing the conditions for inducing enzyme production.

[0048] 1. Use BMMY as the enzyme-producing medium, methanol as the inducer, and add 0.5%, 1.0%, 1.5%, 2.0%, and 2.5% of methanol to produce enzymes, and use the DNS method to measure the fermentation supernatant at 192 hours CMC enzyme activity, and its bacterial concentration was measured, the optimization results are shown in Image 6 .

[0049] 2. Optimize the enzyme production by adjusting the pH of the potassium phosphate buffer in the BMMY medium to 5.0, 5.5, 6.0, 6.5, 7.0, and 7.5. At 192 hours, use the DNS method to measure the CMC enzyme activity of the fermentation supernatant, and measure its bacteria Concentration, optimization results see Figure 7 . For the comparison of CMC enzyme activity after optimization and before optimization, see Figure 8 .

[0050]

[0051]

[0052]

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Abstract

The invention relates to a system for codon optimization and pichia pastoris expression of genes of cellobiohydrolase II (CBH II) derived from trichoderma reesei. According to the system, a Gene Designer (DNA 2.0, Menlo Park, CA, USA) is used for carrying out codon directivity optimization on CBH II (NCBI Reference Sequence: XM_006962518.1) to construct a pPIC9K-cbh2 expression vector, and Pichia pastoris GS115 is taken as a host to obtain a transformant by virtue of an electro-transformation method. Detections show that the genes of CBH II subjected to codon optimization can be stably and efficiently expressed in pichia pastoris, the enzyme activity of a recombinant strain CMC is 0.14U / mL, and the protein content of a fermented supernatant is 0.15mg / mL.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to the codon optimization of Trichoderma reesei exoglucanase II (CBHII) gene and the construction of pPIC9K-cbh2 expression vector. Background technique [0002] Cellulase is a general term for a class of multicomponent enzymes, which can hydrolyze cellulose, which is difficult to be bioavailable, into glucose, which is convenient for bioavailability, so it is considered to be one of the enzyme preparations with great potential. Cellulase belongs to glycoside hydrolase, and the cellulase system capable of degrading cellulose includes at least three types of components: endo-β-glucanase (endo-1,4-β-D-glucanase, EC3.2.1 .4), exo-β-glucanase (exo-1,4-β-D-glucanase, EC3.2.1.91) and β-glucosidase (β-1,4-D-glucosidase, EC3.2.1.21). The main body of cellulose is composed of glucopyranose, which is connected by β-1,4 glycosidic bonds to form a macromolecular linear polymer. It is compo...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N15/10C12N15/81C12N9/42C12R1/84
Inventor 孙付保白仁惠张震宇张云博王春迪
Owner JIANGNAN UNIV
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