Pichia pastoris engineering strain for heterologous expression of cellulase gene CBH II and application

A heterologous expression, Pichia pastoris technology, applied in the direction of microorganism-based methods, peptides containing His tags, enzymes, etc., can solve problems such as difficult manual control and low proportion of cellulase

Inactive Publication Date: 2020-10-30
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are a variety of proteins in the enzyme system of Trichoderma reesei, resulting in a low proportion of cellulase, and the proportion of each cellulase is mainly regulated by itself, which is difficult to artificially control

Method used

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  • Pichia pastoris engineering strain for heterologous expression of cellulase gene CBH II and application
  • Pichia pastoris engineering strain for heterologous expression of cellulase gene CBH II and application
  • Pichia pastoris engineering strain for heterologous expression of cellulase gene CBH II and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment example 1

[0029] Implementation Case 1 Cloning of target gene

[0030] 1. Take the Trichoderma reesei (Trichoderma reesei QM9414) spore suspension stored in a glycerol tube out of the -80°C refrigerator and thaw it in ice (or 4°C refrigerator) until the spore suspension completely melts. Shake the spore suspension to mix evenly, use an inoculation loop to dip a small amount of bacterial solution, and streak on the PDA medium plate for inoculation. After sealing the plate with parafilm, place it 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.

[0031] 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 ...

Embodiment example 2P

[0037] Implementation case 2 Construction of Ppic9k-CBH Ⅱ expression vector

[0038] 1. Use restriction endonuclease EcoR I / Not I to double-digest pUC18 and cbh2, and use the sticky ends of these two enzymes to connect the vector to the target gene. Prepare the double enzyme digestion system according to Table 2. The metal bath was reacted at 37°C for 50 minutes. After the enzyme digestion was completed, the enzyme digestion product was recovered using a purification recovery kit to remove residual enzymes and oligonucleotides in the enzyme digestion product. 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.

[0039] Table 2 Double Enzyme Digestion System

[0040]

[0041] 2. After adjusting the molar concentrations of the vector pUC18 and the target gene CBH 2 to roughly the same level, configure the connection system. See Table 3 for the connection system....

Embodiment example 3

[0044] Implementation case 3 Construction of recombinant Pichia pastoris

[0045] 1. Use the restriction endonuclease bglⅡ as the linearization site of the recombinant expression plasmid Ppic9k-CBHⅡ expression vector, perform single enzyme digestion, and prepare a linearization system (see Table 4 for the configuration of the linearization system), mix well, and place in a water bath at 37°C Incubate for 1 hour to achieve circular transformation of the expression vector and to lay the foundation for later transformation. Nucleic acid electrophoresis to verify the linearized plasmid, see Figure 4 . Transformed into Pichia pastoris GS115 competent cells by electrotransformation, and plated to obtain transformants.

[0046] Table 4 Linearization system:

[0047]

[0048] 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...

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Abstract

The invention discloses two pichia pastoris engineering bacteria for heterologous expression of a cellulase gene CBHII and application thereof. The engineering bacterium is Pichia pastoris-cbh2, and is preserved in the China General Microbiological Culture Collection Center in August 2019, and the preservation number of the engineering bacterium is CGMCC No. 18419. Glucose exonuclease II (CBHII) is obtained from trichoderma reesei through a PCR method, cloned and inserted into a pichia pastoris integration expression vector pPIC9K, and a pPIC9K-cbh2 expression vector is obtained. The vector isintroduced into pichia pastoris GS115 through electric conversion to obtain a recombinant pichia pastoris strain; antibiotic G418 with different concentrations is used for screening recombinant yeaststrains containing high-copy integration plasmids, the optimal induction condition is determined through shake-flask fermentation preliminary optimization, and meanwhile the enzymatic property of purified recombinant protease is analyzed.

Description

technical field [0001] The invention relates to the construction and application of a Pichia yeast strain heterologously expressing cellulase gene CBH II, and belongs to the technical field of cellulase gene engineering. 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 enzyme system...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/62C12N9/42C12R1/84
CPCC12N15/815C12N9/2437C07K2319/21
Inventor 钟成沈钰清王新光贾士儒李文超
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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