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Constitutive P. pastoris strain and its construction method

A Pichia pastoris and compositional technology, applied in the field of genetically engineered bacteria and microbial fermentation, can solve the problems of complex condition control and long fermentation time, and achieve the effects of simple control of the fermentation process, shortened fermentation time, and low energy consumption

Inactive Publication Date: 2008-04-16
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Methanol-inducible Pichia can express DAO from T. It needs to be induced by adding methanol during the fermentation process, and the condition control of the induction process is relatively complicated. In addition, the fermentation time is relatively long, and it usually takes about 40 hours for the enzyme activity to reach the highest point.

Method used

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  • Constitutive P. pastoris strain and its construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1 , plasmid construction

[0019] 1. Materials:

[0020] Plasmid pPIC3.5k: product of Invitrogen Company, the size is 9.0kb, containing kanamycin resistance gene, ampicillin resistance gene, histidinol dehydrogenase gene, the promoter is alcohol oxidase gene, and has multiple restrictions restriction enzyme cleavage site.

[0021] Plasmid pGAPZA: a product of Invitrogen Company, with a size of 2.9kb, containing a Zeocin resistance gene, a promoter of glyceraldehyde triphosphate dehydrogenase gene, and multiple restriction enzyme cutting sites.

[0022] Plasmid pLHB-3: constructed according to the method of Liu HB., Jiang WH., Yang YL. Cloning, Sequencing and Expression of D-amino acid oxidase gene. Chinese Journal of Biotechnology 15: 337-342, 1999, size It is 6.4kb, contains DAO gene (HDAO) with histidine purification tag, kanamycin resistance gene, and the promoter is T7 lac.

[0023] 2. Method:

[0024] like figure 1 As shown, the plasmid pPIC3.5k was ...

Embodiment 2

[0026] Example 2 , plasmid amplification

[0027] Plasmid pMMZY03 was transformed into Escherichia coli host strain DH5α (Takara) using the conventional chemical transformation method "Reference Molecular Cloning", and a single transformant was picked and inoculated into LB liquid medium (1% peptone, 0.5% yeast extract, 1% chloride Sodium), cultivate and amplify the pMMZY03 plasmid at 37°C.

Embodiment 3

[0028] Example 3 , plasmid transformation

[0029] Referring to "Molecular Cloning", the plasmid pMMZY03 extracted and amplified from the Escherichia coli in Example 2 was extracted by alkaline lysis method, and the extracted plasmid was linearized by single enzyme digestion with BspE I, and about 10 μg of the linearized plasmid was recovered by ethanol precipitation, and mixed with Pichia Yeast host strain GS115 (his - mutate + ) 80 μl of competent cells were mixed and transferred to an electroporation cup, and placed in an ice bath for 5 minutes. Meanwhile, a copy of competent cells without the addition of linearized plasmid pMMZY03 was prepared as a negative control. The Bio-Rad electroporation instrument was used, the electroporation parameters were set to 1.5KV, 25μF, 200Ω, and the electroporation time was 4.6ms. Immediately after electroporation, 1ml of sorbitol in ice bath was added. Electroporation conversion products were coated on MD plates (1.34% YNB, 4×10 -5 %...

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Abstract

The invention discloses a constitutive Pichia pastoris recombinant bacterial strain for expressing D-amino acid oxidase, wherein the strain has recombination plasmid pMMZY03, the recombinant plasmid uses glyceraldehyde phosphate dehydrogenase gene as the promotor, and contains amino acid oxidase gene, histidinol dehydrogenase gene and kanamycin gene. The invention also discloses the method for constructing the constitutive Pichia pastoris recombinant bacterial strain.

Description

technical field [0001] The invention relates to the field of genetic engineering bacteria and microbial fermentation, in particular to a constitutive Pichia yeast strain expressing D-amino acid oxidase and a construction method thereof. Background technique [0002] D-Amino Acid Oxidase (DAO, EC 1.4.3.3) is a typical flavoproteinase with flavin adenine dinucleotide (FAD) as the prosthetic group, which catalyzes the oxidation of D-amino acid The deamination reaction generates the corresponding keto acid and ammonia, and releases hydrogen peroxide along with the reduction of a molecule of oxygen. In the two-step enzymatic production of 7-amino acid cephalosporanic acid (7-ACA), DAO is used in the first step reaction of converting cephalosporin C (CPC). [0003] Pichia pastoris has become a major eukaryotic microbial expression system (Cregg JM., Vedvick TS., Raschke WC. Bio / Technology. 11:905-910, 1993). Pichia pastoris can ferment at high density, and the promoter commonly ...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N15/53C12R1/84
Inventor 杨晟郑华宝陈军杨蕴刘姜卫红
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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