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Method for expressing transketolase by applying pGAP regulation of Pichia pastoris

A Pichia pastoris and transketolase technology, applied in the biological field, can solve the problems of long production cycle, high production cost, and slow growth, and achieve the effects of short fermentation cycle, low production cost, and no toxic side effects

Inactive Publication Date: 2010-04-28
INST OF TROPICAL BIOSCI & BIOTECH CHINESE ACADEMY OF TROPICAL AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But, because the growth of natural transketolase bacterial strain is slow, the production period is long, and the intensity of the promotor of regulation gene is low and causes product amount to be few, makes production cost higher, is unfavorable for the large-scale production and utilization of transketolase

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] 1. First, amplify the pGAP promoter from Pichia pastoris;

[0021] 2. A His6 tag was fused to the 3' end of the transketolase sequence to construct a Pichia pastoris expression vector that regulated the transketolase gene by pGAP, and this vector was transformed into the Pichia pastoris genome;

[0022] 3. Screen high-copy recombinants as engineering strains according to the resistance genes carried on the vector;

[0023] 4. Inoculate the engineered bacterial strain in the liquid medium [the formula of the liquid medium (per liter): 26.0ml of 85% phosphoric acid, CaSO 4 2H 2 O 0.85g, K 2 SO 4 17.5g, MgSO 4 ·7H 2 O 13.9g, KOH 3.83g, Glycerin 40g, Peptone 19g, Yeast extract 9g, PTM4 trace elements 3ml] [PTM4 formula (per liter): CuSO 4 ·5H 2 O 2.0g, NaI 2H 2 O 0.1g, MnSO 4 ·H 2 O 3.0g, Na 2 MoO 4 2H 2 O 0.2g, H 3 BO 3 0.02g, CoCl 2 ·6H 2 O 1.05g, ZnCl 2 7.0g, Fe 2 (SO 4 ) 3 ·7H 2 O 22g, biotin 0.2g, sulfuric acid 1ml], ferment and express transket...

Embodiment 2

[0025] 1. First, amplify the pGAP promoter from Pichia pastoris;

[0026] 2. A His6 tag was fused to the 3' end of the transketolase gene sequence to construct a Pichia pastoris expression vector that regulated the transketolase gene by pGAP, and this vector was transformed into the Pichia pastoris genome;

[0027] 3. Screen high-copy recombinants as engineering strains according to the resistance genes carried on the vector;

[0028] 4. Inoculate the engineered bacterial strain in the liquid medium [the formula of the liquid medium (per liter): 26.7ml of 85% phosphoric acid, CaSO 4 2H 2 O 0.93g, K 2 SO 4 18.2g, MgSO 4 ·7H 2 O 14.9g, KOH 4.13g, oleic acid 20g, peptone 20g, Yeast extract 10g, PTM4 trace elements 4ml] (PTM4 formula (per liter): CuSO 4 ·5H 2 O 2.0g, NaI 2H 2 O 0.1g, MnSO 4 ·H 2 O 3.0g, Na 2 MoO 4 2H 2 O 0.2g, H 3 BO 3 0.02g, CoCl 2 ·6H 2 O 1.05g, ZnCl 2 7.0g, Fe 2 (SO 4 ) 3 ·7H 2 (22g, biotin 0.2g, sulfuric acid 1ml), ferment and express t...

Embodiment 3

[0030]1. First, amplify the pGAP promoter from Pichia pastoris;

[0031] 2. A His6 tag was fused to the 3' end of the transketolase gene sequence to construct a Pichia pastoris expression vector that regulated the transketolase gene by pGAP, and this vector was transformed into the Pichia pastoris genome;

[0032] 3. Screen high-copy recombinants as engineering strains according to the resistance genes carried on the vector;

[0033] 4. Inoculate the engineering bacterial strain bacterial liquid in the liquid medium [the formula of the liquid medium (per liter): 27.7ml of 85% phosphoric acid, CaSO 4 2H 2 O 1.05g, K 2 SO 4 18.2g, MgSO 4 ·7H 2 O 15.3g, KOH 4.33g, sorbitol 35g, peptone 21g, Yeast extract 12g, PTM4 trace element 4ml] [PTM4 formula (per liter): CuSO 4 ·5H 2 O 2.0g, NaI 2H 2 O 0.1g, MnSO 4 ·H 2 O 3.0g, Na 2 MoO 4 2H 2 O 0.2g, H 3 BO 3 0.02g, CoCl 2 ·6H 2 O 1.05g, ZnCl 2 7.0g, Fe 2 (SO 4 ) 3 ·7H 2 O 22g, biotin 0.2g, sulfuric acid 1ml], fermen...

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Abstract

The invention relates to a method for expressing transketolase by applying pGAP regulation of Pichia pastoris, belonging to the biotechnology field. Firstly, pGAP promoters are amplificated in Pichia pastoris, a Pichia pastoris expression vector of which xylose isomerase is regulated by the pGAP is built, and then the Pichia pastoris expression vector is transformed into Pichia pastoris genome. High copy recons are selected as engineering bacterial strain according to resistance genes on the Pichia pastoris expression vector, and the engineering bacterial strain is inoculated into liquid culture medium containing carbon source to ferment engineering bacteria for secreting and expressing the transketolase. The invention has the advantages of low production cost and short fermenting period. The using of the pGAP regulation of Pichia pastoris to express the transketolase is favorable to purifying the product, and the problems of high production cost and hard product purification caused by using natural bacterial strains produced transketolase to produce transketolase can be solved. The invention is favourable for mass production of transketolase.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for regulating and producing biological protein by using the pGAP promoter of Pichia pastoris, in particular to a method for regulating and expressing transketolase by using the pGAP of Pichia pastoris. Background technique [0002] Transketolase (transketolase EC 2.2.1.1), also known as transglycolase or ketolase. Transketolase widely exists in the microbial community, and it is known that the microorganisms producing transketolase include certain bacteria, fungi, and Vibrio. Xylose is the main component of hemicellulose, accounting for about 30% in the hydrolyzate of plant fiber materials, and is the most abundant sugar in nature after glucose. The production of ethanol from xylose by microbial fermentation is one of the current research hotspots. Microorganisms that can make wine efficiently (such as Saccharomyces cerevisiae and Zymomonas mobilis) do not have the ability ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/81C12R1/84
Inventor 屈直张爱联张添元尹慧祥罗进贤
Owner INST OF TROPICAL BIOSCI & BIOTECH CHINESE ACADEMY OF TROPICAL AGRI SCI
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