Uracil auxotroph Hansenula yeast, construction method thereof and application thereof

A technology of auxotrophic and Hansenula spp., applied in the direction of microorganism-based methods, biochemical equipment and methods, enzymes, etc., can solve the problem of low expression level of exogenous genes, low level of expressed protein secretion, low level of polypeptide chain sugar Over-transformation and other problems can be achieved to achieve the effect of low reverse mutation rate, short culture time and fast growth rate

Inactive Publication Date: 2009-10-21
元昊
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this expression system also has many shortcomings in the application process, such as low expression level of foreign genes, low secretion level of expressed proteins, excessive glycosylation of polypeptide chains, and unstable strains, etc.

Method used

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  • Uracil auxotroph Hansenula yeast, construction method thereof and application thereof
  • Uracil auxotroph Hansenula yeast, construction method thereof and application thereof
  • Uracil auxotroph Hansenula yeast, construction method thereof and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Construction of Hansenula

[0037] 1. Construction of the recombinant plasmid p18HURA containing the orotidine-5-phosphate decarboxylase gene (HURA3) of Hansenula spp. The construction process of the recombinant plasmid p18HURA is as follows:

[0038] 1. Acquisition of orotic acid-5-phosphate decarboxylase gene (HURA3) of Hansenula spp.

[0039] The primer sequences designed according to the reported nucleotide sequence of Hansenula HURA3 are as follows:

[0040] Primer 1: 5′-CCA GGATCC TCAACATTTCCCTGAATAAT-3' (sequence 1 in the sequence listing) (the bases underlined are BamH I recognition sites)

[0041] Primer 2: 5′-CGA GAATTC TCACTAGTATTC CCGCGACT-3' (sequence 2 in the sequence listing) (the underlined part of the base is the EcoR I recognition site)

[0042] Using the total DNA of Hansenula (Hansenula) JCM3621 (ATCC34438) as a template, PCR reaction was carried out to amplify HURA3 under the guidance of primers 1 and 2. The PCR reaction system was: template D...

Embodiment 2

[0064] Detection of biomass and biological activity of Hansenula YH-11CGMCCNO.2976, a uracil-auxotrophic Hansenula genetically engineered host strain:

[0065] Detection of Biomass of Uracil Auxotroph Hansenula YH-11

[0066] Inoculate wild-type Hansenula and uracil-auxotrophic Hansenula YH-11 on YEPD slant medium for activation, pick a ring of bacteria from the slant with an inoculation loop and inoculate 2ml of YEPD In liquid culture medium, shake culture at 37°C for 16 hours; transfer to 10ml YEPD liquid medium according to 10% inoculum size, shake culture at 37°C for 16 hours; collect bacterial cells by centrifugation, and wash the cells with sterile water for two Second, after weighing, the cells are divided into four equal parts, two parts of cells are one group, two groups of cells are respectively inoculated in 50ml YEPD liquid medium and 50ml in the liquid medium of glucose in YEPD with methanol, Then two copies of cells in each group were shaken and cultured at 37°C...

Embodiment 3

[0068] Genetic stability analysis of uracil-auxotrophic Hansenula genetically engineered host strain Hansenula YH-11CGMCCNO.2976.

[0069] Genetic stability analysis of uracil auxotroph Hansenula YH-11

[0070] Inoculate the uracil auxotrophic Hansenula YH-11 constructed above with blocked HURA3 gene function in YEPD liquid medium and subculture for 30 times, take 200 μl of diluted cells and spread them on the YEPD plate, Incubate at 37°C for 48 hours, randomly pick 100 single colonies and place them in 2ml sterile water, and starve them for 4-6 hours at room temperature; take the starved bacteria solution and inoculate them on YNB plates with glucose as the carbon source and add 35μg / ml uracil on the YNB plate, and on the YNB plate using methanol as a carbon source and the YNB plate adding 35 μg / ml uracil, cultured at 37°C for 48 hours, the results showed that single colonies were all on the YNB basic medium added uracil. growth, on the YNB basic medium without adding uracil,...

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Abstract

The invention discloses uracil auxotroph Hansenula yeast gene engineering host strain Hansenula yeast YH-11 CGMCC NO.2976 with stable heredity, and simultaneously provides a construction method for the Hansenula yeast. The construction method comprises the following steps: 1) constructing recombinant plasmid p18HURA containing orotidine-5-phosphate decarboxylase gene (HURA3) of the Hansenula yeast; and 2) constructing recombinant plasmid pHURK for the HURA3 with disrupted gene; and converting the plasmid pHURK of step 2) into the Hansenula yeast by using a yeast whole cell conversion method, and obtaining the uracil auxotroph strain through screening. The invention further discloses application of the constructed uracil auxotroph Hansenula yeast gene engineering host strain Hansenula yeast in producing foreign protein.

Description

technical field [0001] The invention relates to a recombinant Hansenula spp. and its construction method and its application in the production of exogenous proteins. More specifically, the present invention is a uracil-auxotrophic Hansenula spp. and its construction method and application. Background technique [0002] Using genetic engineering technology to obtain proteins with important biological activities is an important goal of modern biotechnology. When expressing proteins in vitro, it is important to keep the spatial structure and biological activity of the product consistent with the natural protein to the greatest extent while striving to increase the expression level. For this reason, four exogenous gene expression systems of Escherichia coli, baculovirus, mammalian cells and yeast have been established to adapt to the high-efficiency expression of proteins from various sources in vitro. Among them, the yeast expression system is a powerful tool for the study of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12P21/02C12N9/26C12N9/16C12R1/78
Inventor 元昊
Owner 元昊
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