Recombinant pichia pastoris genetically engineered bacterium as well as construction method and application thereof

A technology of genetically engineered bacteria and Pichia pastoris, applied in the fields of genetic engineering and fermentation engineering, can solve the problems of difficult immobilization fermentation and weak film-forming ability, and achieve the effect of shortening the fermentation period and enhancing the biological film-forming ability.

Active Publication Date: 2021-06-29
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the problem of weak biofilm-forming ability of Pichia pastoris and difficulty in immobilized fermentation needs to be solved urgent...

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Construction of an overexpression plasmid that overexpresses the HSF1 gene.

[0052] 1. Amplification of the target gene HSF1:

[0053] The gene HSF1 was amplified with primer 1 (SEQ ID NO.2) and primer 2 (SEQ ID NO.3) using the extracted P. Pastoris GS115 genome (purchased from Miaoling Biotech Co., Ltd.) as a template.

[0054] The PCR reaction system is shown in Table 1.

[0055] Table 1 Target gene HSF1 amplification PCR system

[0056]

[0057] Dispense into 25 μL per tube according to the above system.

[0058] PCR conditions: 1) pre-denaturation at 94°C, 5min; 2) denaturation at 98°C, 10s; 3) annealing at 55°C, 5s; 4) extension at 72°C, 15s, a total of 35 cycles; 5) complete extension at 72°C, 10min.

[0059] The size of the amplification product of the target gene HSF1 is 2441bp (SEQ ID NO.1), and the electropherogram is as attached figure 2 As shown in A. The PCR products were purified by TAKARA Gel Extraction Kit and used for subsequent exp...

Embodiment 2

[0077] Example 2: Construction of Pichia pastoris genetically engineered bacteria overexpressing the HSF1 gene.

[0078] 1. Transformation of recombinant plasmids

[0079] The plasmid pGAPZαA-HSF1 was digested and linearized with AvrII.

[0080] See Table 4 for the linearization system of the recombinant plasmid pGAPZαA-HSF1.

[0081] Table 4 Linearization system of recombinant plasmid pGAPZαA-HSF1

[0082] components Volume (μL) Manufacturer QuickCut AvrⅡ 1 TAKARA BIO INC. 10*QuickCut Buffer 5 TAKARA BIO INC. Recombinant plasmid pGAPZαA-HSF1 4 sterile water 40 total 50

[0083] The enzyme digestion condition was 37°C for 1 hour. After the enzyme digestion reaction, the enzyme digestion products were recovered from the gel and used for subsequent experiments.

[0084] The above-mentioned linearized recombinant plasmid pGAPZαA-HSF1 was introduced into Pichia pastoris P. Pastoris GS115 competent cells (purchased from M...

Embodiment 3

[0092] Example 3: Characterization and detection of biofilm formation.

[0093] Such as Figure 4 As shown, A and B are the results of the SEM electron microscopy experiments of the starting bacteria P. Pastoris GS115 and the recombinant strain GS115-HSF1*, respectively. It can be clearly seen that the film-forming effect of the recombinant strain is better than that of the starting bacteria, and the biofilm A large amount is formed.

[0094] Figure 5 It is a semi-quantitative biofilm measurement experiment conducted by crystal violet staining. Add 20 μL of starting bacteria and recombinant bacteria to a 96-well plate with 200 μL of YPD in each well, and culture it for 72-120 hours. After 12 hours, its OD570 was measured by crystal violet staining and microplate reader. It can be seen from the figure that the film-forming effect of the recombinant strain GS115-HSF1* is better than that of P. Pastoris GS115.

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Abstract

The invention discloses a recombinant pichia pastoris genetically engineered bacterium as well as a construction method and application thereof. The pichia pastoris genetically engineered bacterium is constructed by over-expressing a transcription inhibition factor and an activating factor HSF1 in pichia pastoris. The construction method comprises the following steps: (1) carrying out PCR (Polymerase Chain Reaction) amplification on a pichia pastoris genome to obtain an HSF1 gene segment; (2) cloning the HSF1 gene segment to an expression plasmid to obtain a recombinant plasmid; and (3) introducing the linearized recombinant plasmid into pichia pastoris, and screening to obtain the pichia pastoris genetically engineered bacterium. The pichia pastoris genetically engineered bacterium can effectively improve the biofilm forming ability of the pichia pastoris, so that the enzyme activity of the recombinant pichia pastoris under a single immobilized fermentation condition is improved by 28.3% compared with that of a free fermentation starting bacterium, and the fermentation period is shortened by 36.6%. The recombinant bacteria can be stably and continuously subjected to at least seven batches of immobilized fermentation enzyme production, and after seven batches of immobilized fermentation, the enzyme activity of the fermentation enzyme production of the recombinant bacteria is about 7 times higher than that of starting bacteria.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and fermentation engineering, and specifically relates to a recombinant Pichia pastoris genetically engineered bacterium, its construction method and its application in producing xylanase. Background technique [0002] Xylan is the main component of hemicellulose in plants. It is a polysaccharide that exists widely in nature and is the second largest renewable resource in the world. The structure of xylan is complex, and its complete hydrolysis requires the synergy of a series of enzymes, the most critical of which is xylanase. As far known, most microorganisms such as bacteria, fungi, actinomycetes, yeasts, algae and bacteria in the rumen of animals can produce xylanase. Engineering bacteria have become the main form of industrial production of xylanase, and genes encoding xylanase are cloned into homologous or heterologous hosts to be highly expressed. However, there are currently ...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N9/24C12R1/84
CPCC07K14/39C12N15/815C12N9/248Y02E50/10
Inventor 应汉杰牛欢青宋佳睿闵志迪陈勇刘庆国柳东
Owner NANJING UNIV OF TECH
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