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Recombinant pichia pastoris engineering bacterium as well as construction method and application thereof

A technology of Pichia pastoris and its construction method, applied in the fields of application, genetic engineering, recombinant DNA technology, etc., can solve the problems of long growth and fermentation cycle, low productivity, potential safety hazards, etc., achieve broad application prospects and practical significance, and increase yield Effect

Pending Publication Date: 2022-05-13
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Nevertheless, the P. pastoris expression system also has some disadvantages, such as relatively long growth and fermentation cycle, and low productivity; when the expression gene is regulated by the AOX1 promoter, methanol needs to be used as an inducer, and methanol is flammable and explosive. The large amount of methanol used in the fermentation process has great potential safety hazards, and the toxicity of methanol makes it difficult for proteins induced by methanol to be used in industrial fields such as food or pharmaceuticals, etc.

Method used

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  • Recombinant pichia pastoris engineering bacterium as well as construction method and application thereof
  • Recombinant pichia pastoris engineering bacterium as well as construction method and application thereof
  • Recombinant pichia pastoris engineering bacterium as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Construction of pTEF1, pGCW14, pPGK1 expression vectors

[0035] (1) According to the Pichia pastoris translation elongation factor 1-α gene expression cassette (P TEF1 -tef1-T TEF1 ), Chr1-4_0586 gene expression cassette (P GCW14 -gcw14-T GCW14 ), glycolytic enzyme 3-phosphoglycerate kinase gene expression cassette (P PGK1 -pgk1-T PGK1 ) sequence and the characteristics of the genetic elements on the vector pGAPZB, using Oligo7.0 software to design primers, the specific primers are as follows

[0036]

[0037]

[0038] (2) Use the pGAPZB-F / pGAPZB-R primer pair to amplify the pGAPZB vector backbone (excluding 6×His tag), PCR reaction parameters: pre-denaturation, 98°C for 1min; denaturation, 98°C for 10s; annealing, 55°C for 10s; Extension, 72°C for 30s; stop extension, 72°C for 5min; pGAPZB vector backbone was obtained after 32 cycles.

[0039] Use TEF1-F / TEF1-R, GCW14-F / GCW14-R, PGK1-F / PGK1-R primers to amplify P TEF1 -tef1-T TEF1 ,P GCW14 -g...

Embodiment 2

[0042] Embodiment 2: the construction of 6-glucose phosphate dehydrogenase (G6PD) expression vector

[0043] (1) According to the sequence of Pichia pastoris coding glucose 6-phosphate dehydrogenase gene zwf1 and the characteristics of the gene elements on the vectors pGAPZB, pTEF1, pGCW14, the primers were designed using Oligo7.0 software, and the obtained primers were as follows:

[0044]

[0045]

[0046] (2) Use pGAP-F / pGAP-R, pTEF1-F / pTEF1-R, pGCW14-F / pGCW14-R primer pairs to PCR amplify pGAPZB vector backbone (excluding 6×His tag), pTEF1 vector backbone (excluding Containing tef1 gene), pGCW14 vector backbone (excluding gcwl4 gene). PCR reaction parameters: pre-denaturation, 98°C for 1min; denaturation, 98°C for 10s; annealing, 55°C for 10s; extension, 72°C for 30s; stop extension, 72°C for 5min; pGAPZB, pTEF1 and pGCW14 vector backbones were obtained after 32 cycles.

[0047] (3) Use the primer pair zwf1-FG / zwf1-RG to PCR amplify the zwf1 gene fragment (SEQ ID No...

Embodiment 3

[0050] Embodiment 3: the construction of 6-glucose phosphate dehydrogenase (G6PD) expression vector

[0051] (1) According to the gnd2 sequence of Pichia pastoris encoding 6-phosphogluconate dehydrogenase gene and the characteristics of the gene elements on the vectors pGAPZB, pTEF1, pGCW14, use Oligo7.0 software to design primers:

[0052]

[0053]

[0054] (2) Use pGAP-F / pGAP-R, pTEF1-F / pTEF1-R, pGCW14-F / pGCW14-R primer pairs to amplify pGAPZB vector backbone (excluding 6×His tag), pTEF1 vector backbone (excluding tef1 gene), pGCW14 vector backbone (excluding gcw14 gene), PCR reaction parameters: pre-denaturation, 98°C 1min; denaturation, 98°C 10s; annealing, 55°C 10s; extension, 72°C 30s; stop extension, 72°C 5min; After 32 cycles, the backbones of pGAPZB, pTEF1 and pGCW14 vectors were obtained.

[0055] (3) Use the primer pair gnd2-FG / gnd2-RG to PCR amplify the gnd2 gene fragment (SEQ ID No: 3), PCR reaction parameters: PCR reaction parameters: pre-denaturation, 98°...

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Abstract

The invention provides a recombinant pichia pastoris engineering bacterium as well as a construction method and application thereof, and belongs to the technical field of biological engineering. In the invention, a pichia pastoris engineering bacterium for cooperatively expressing 6-phosphoglucose dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (G6PDH) is constructed on the basis of P.pastoris GS115 by utilizing a genetic engineering means, the pichia pastoris engineering bacterium is recorded as P.pastoris GS115 / zwf1-gnd2, and a recombinant pichia pastoris engineering bacterium containing D-arabitol dehydrogenase (ArDH) genes with different copy numbers is further constructed on the basis of the pichia pastoris GS115 / zwf1-gnd2. The recombinant pichia pastoris engineering bacterium can be used for efficiently producing D-arabitol.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to a recombinant Pichia pastoris engineering bacterium and its construction method and application. Background technique [0002] D-arabitol is a five-carbon sugar alcohol that has potential uses in the food and pharmaceutical industries, and is also a high-quality raw material for some chemical products in this industry. Due to its sweet taste similar to sucrose, extremely low caloric value (0.2cal / g), and slow absorption and metabolism in the digestive tract, D-arabitol can be used as a potential low-energy sweetener and sugar substitute for diabetic patients. In addition, D-arabitol can be converted into arabic acid, propylene glycol, xylitol, xylonic acid, and other high value-added chemicals, and can also be used as adenosine, cytarabine and D-glucosidase inhibitors Drug intermediates. The general industrial production of D-arabitol involves the catalytic hy...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N15/53C12P7/18C12R1/84
CPCC12N15/815C12N9/0006C12Y101/01049C12Y101/01044C12Y101/01301C12P7/18
Inventor 齐向辉窦媛张宇飞翟彼得尤瓦赵梅孙雷
Owner JIANGSU UNIV