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Pichia pastoris engineering bacterium for heterologous expression of cellulase gene EGV and application of pichia pastoris engineering bacterium

A Pichia pastoris, heterologous expression technology, applied in the field of cellulase genetic engineering, to achieve the effect of improving enzyme production efficiency and industrial production potential

Pending Publication Date: 2020-11-06
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Finally, the strains were deposited in the General Microorganism Center (CGMCC) of the China Committee for the Collection of Microbial Cultures

Method used

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  • Pichia pastoris engineering bacterium for heterologous expression of cellulase gene EGV and application of pichia pastoris engineering bacterium
  • Pichia pastoris engineering bacterium for heterologous expression of cellulase gene EGV and application of pichia pastoris engineering bacterium
  • Pichia pastoris engineering bacterium for heterologous expression of cellulase gene EGV and application of pichia pastoris engineering bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment example 1

[0030] Implementation Case 1 Cloning of target gene

[0031] 1. Take the Trichoderma reesei QM9414 spore suspension stored in a glycerol tube out of the -80°C refrigerator and place it in ice (or 4°C refrigerator) to thaw until the spore suspension completely melts. Shake the spore suspension to mix evenly, dip the inoculation loop with a small amount of bacterial solution, and streak on the PDA medium plate for inoculation. After the plate was sealed with parafilm, it was placed in a 28°C incubator for cultivation. Cultivate for about 5-7 days until the surface of the medium is covered with green spores. The plates were taken out of the incubator and placed in a 4°C refrigerator for later use.

[0032] 2. Inoculate fresh spores on cellulase solid induction culture, spread sterilized cellophane on the surface, and let the culture stand until the hyphae grow out. The hyphae were scraped off from the cellophane and treated with a liquid nitrogen freezer grinder. RNA was extra...

Embodiment example 2

[0038] Implementation Case 2 Construction of Expression Vector Ppic9k-EGⅤ

[0039] 1. Use the restriction endonuclease EcoRI / Not I to double-digest Ppic9k and the target gene eg5, and prepare a double-digestion system as shown in the table below. The metal bath was reacted at 37°C for 50 minutes, and the product was recovered by the recovery kit. The product after double enzyme digestion was reacted with Taq Mix enzyme (volume 1:1) in a PCR instrument at 72°C for 30 min, and A-terminal ligation was carried out.

[0040] Double enzyme digestion system:

[0041]

[0042]2. Using EcoRI at the 5' end and NotI at the 3' end as the restriction site, insert the Ppic9k plasmid vector promoter AOX1 downstream to construct the expression vector Ppic9k-EGⅤ. For the expression vector map, see image 3 . Colony PCR verification see figure 2 . The expression vector was verified by single enzyme digestion to confirm that the expression vector Ppic9k-EGⅤ was successfully constructed...

Embodiment example 3

[0046] Implementation case 3 Construction of recombinant Pichia pastoris

[0047] 1. Use the restriction endonuclease bglⅡ to single-enzyme digest the recombinant expression plasmid Ppic9k-EGⅤ, prepare a linearization system, mix well, and incubate in a metal bath at 37°C for 1 hour to make the expression vector change from circular to linear, which is beneficial Later conversion. Nucleic acid electrophoresis was used to verify the linearized plasmid, see Figure 4 . Transformed into Pichia pastoris GS115 competent cells by electrotransformation, and plated to obtain transformants.

[0048] Linearization system:

[0049]

[0050] 2. Use the SDS pyrolysis method to roughly extract the genome of the recombinant yeast transformant obtained in 1, and verify its correctness and phenotype by PCR. The nucleic acid map of the genome PCR is shown in Figure 5 . Pick the transformants on the MM plate, spot in turn on the YPD medium plate containing different concentrations of G4...

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Abstract

The invention discloses a pichia pastoris engineering bacterium for heterologous expression of a cellulase gene EGV and application of the pichia pastoris engineering bacterium. The engineering bacterium is Pichia pastoris-eg5 and is preserved in China General Microbiological Culture Collection Center (CGMCC) in August 2019, with the preservation number of CGMCC No.18422. An endoglucanase gene (EGV) is obtained from trichoderma reesei through a PCR method, cloned and inserted into a pichia pastoris integrated expression vector pPIC9K, and a pPIC9K-eg5 expression vector is obtained. The vectoris introduced into pichia pastoris GS115 through electrotransformation to obtain recombinant pichia pastoris strains, and the recombinant pichia pastoris strains comprising high-copy integrated plasmids are screened by using antibiotics G418 with different concentrations. The optimal induction condition is determined through primary optimization of shake flask fermentation, and meanwhile, the enzymatic properties of the purified recombinant protease are analyzed.

Description

technical field [0001] The invention belongs to the technical field of cellulase gene engineering, and in particular relates to the construction and application of a Pichia yeast strain heterologously expressing cellulase gene EGV. Background technique [0002] Lignocellulose is an abundant renewable resource that can be converted into a variety of bioenergy and chemical products. The research on converting lignocellulose into fuel ethanol is receiving more and more attention. However, in the process of lignocellulose conversion to produce fuel ethanol, the cost of enzyme preparation and enzymatic hydrolysis process account for about 40% of its production cost, so to reduce the production cost of cellulase or integrate enzymatic hydrolysis and enzymatic hydrolysis through integrated bioprocessing (Consolidated bioprocessing, CBP) The fermentation process is an important means to improve the economics of lignocellulosic ethanol production. [0003] Cellulase is a complex en...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/81C12N1/19C12R1/84
CPCC12N9/244C12Y302/01006C12N15/815
Inventor 李文超王新光沈钰清贾士儒钟成
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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