Episomal expression vectors for Candida utilis, and construction method and application thereof

A technology for producing Candida utilis and an expression vector, which is applied in the field of genetic engineering, can solve the problems that chromosomes cannot be integrated successfully at the same time, cannot achieve stable inheritance, and reverse mutation, etc., and achieves the effects of convenient operation, stable inheritance and high-efficiency expression.

Active Publication Date: 2018-01-26
GUANGDONG QIZHI BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Candida utilis is a triploid single-celled microorganism, and there will be back mutations when the homologous recombination is carried out with the integration vector, resulting in the failure of all chromosomes to integrate successfully at the same time, which is not only inconvenient to operate, but also cannot ...

Method used

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  • Episomal expression vectors for Candida utilis, and construction method and application thereof
  • Episomal expression vectors for Candida utilis, and construction method and application thereof
  • Episomal expression vectors for Candida utilis, and construction method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Embodiment 1: The acquisition of each element required for vector construction

[0039] Step 1, the acquisition of G418 resistance gene fragment

[0040] By studying the sequence map of the Pichia pastoris vector pGAPZαA, in order to replace the bleomycin resistance gene on the original vector with the G418 resistance gene, the present invention first amplifies the coding region of the G418 resistance gene and a part of the CYC terminator respectively. Then use overlap extension PCR to connect the two into a complete fragment, and then carry out gene replacement by double enzyme digestion and ligation reaction.

[0041] (1) Amplification of the G418 resistance gene coding region and part of the CYC terminator fragment

[0042] According to the sequence of the G418 resistance gene coding region on the vector pScIKP and the sequence of pGAPZαA, respectively design the specific amplification primers for the G418 resistance gene coding region and part of the CYC terminator...

Embodiment 2

[0085] Example 2: Construction of Candida utilis episomal expression vector

[0086] The detailed construction flowchart of a kind of Candida utilis episomal expression vector of the present invention is as follows figure 1 As shown, the specific steps are as follows:

[0087] (1) Pichia pastoris expression vector pGAPZαA is digested with NcoI and EcoRV to obtain the backbone of removing the bleomycin resistance gene; simultaneously the pMD19 containing the overlapping G418 resistance gene fragment obtained in step 1 of Example 1 The -T plasmid was double digested with BbsI and EcoRV, and the overlapping G418 resistance gene fragment was recovered. Because in the design of the present invention, BbsI and NcoI can produce the same cohesive ends, therefore the overlapping G418 resistance gene fragments recovered and the Pichia expression vector pGAPZαA backbone that removes the bleomycin resistance gene can be used with T4 Ligase was connected to obtain expression vector 1, w...

Embodiment 3

[0093] Embodiment 3: Detection of green fluorescent protein gene expression performance

[0094] (1) if figure 2 As shown, the vector pEGFP-N1 and the expression vector 3 obtained in step (3) in Example 2 were simultaneously digested with Bsp119I and NotI to obtain the green fluorescent protein gene and the expression vector without the α signal peptide and the multiple cloning site respectively 3 skeletons. Then the two were ligated with T4 ligase to obtain expression vector 4, which was named pCuXGA-GFP (X represents each promoter fragment).

[0095] (2) The expression vector 4 obtained in the previous step was transferred into Candida utilis GIM2.176 by electroporation respectively, spread on the YPD agar plate containing G418 (300 μg / ml) and cultivated for 3-4 days, The colony with normal growth was picked, and identified by colony PCR as a transformant containing the above-mentioned recombinant plasmid.

[0096] (3) Inoculate the positive transformants of Candida util...

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Abstract

The invention discloses episomal expression vectors for Candida utilis, and a construction method and application thereof, belonging to the field of genetic engineering. The episomal expression vectors use a pichia yeast expressing vector pGAPZalphaA as a basic skeleton; the promoter of Candida utilis, the promoter of Saccharomyces cerevisiae or the promoter of Pichia pastoris is used for replacing a GAP promoter on the vector pGAPZalphaA; a G418 resistant gene is used for replacing a bleomycin gene on the vector pGAPZalphaA; and an autonomously replicating sequence of Candida utilis is inserted into the PscI site of the vector pGAPZalphaA. The expression vectors constructed in the invention contain different promoters. The objective of the invention is to construct the expression vectorsapplicable to Candida utilis, and the constructed expression vectors are applied to engineered Candida utilis strains for high-efficiency expression of foreign proteins.

Description

technical field [0001] The present invention relates to the field of genetic engineering, in particular to a Candida utilis free expression vector and its construction method and application. Background technique [0002] Yeast has a complete subcellular structure and strict eukaryotic gene regulation mechanism, so it is an ideal eukaryotic expression system. Candida utilis is a safe food yeast certified by the U.S. Food and Drug Administration (FDA). Compared with Saccharomyces cerevisiae and Pichia pastoris, it has the following advantages: [0003] ①Belongs to Crabtree-negative bacteria (Crabtree-negative), does not produce ethanol under aerobic conditions, and can grow rapidly; ②Can achieve high-density culture under effective continuous culture conditions; ③Can use cheap molasses as a nutrient for growth ; ④ can use pentose and hexose as carbon source at the same time; ⑤ adapt to a variety of carbon and nitrogen sources, and its secreted proteome does not contain any p...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N15/66
Inventor 刘泽寰林蒋海傅菁
Owner GUANGDONG QIZHI BIOTECHNOLOGY CO LTD
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