Hansenula polymorpha with double selection marker and application thereof

A technology for yeast and screening medium, applied in application, fermentation, fungi and other directions, can solve the problem of difficult to meet the high-efficiency expression of yeast functional protein, and achieve the effect of being conducive to high-efficiency expression, increasing expression amount, and simple and rapid screening

Inactive Publication Date: 2011-10-26
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In recent years, the research on the genetic operating system of Hansenula has made great progress, but the current genetic operating system basically uses a single selection ...

Method used

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  • Hansenula polymorpha with double selection marker and application thereof
  • Hansenula polymorpha with double selection marker and application thereof
  • Hansenula polymorpha with double selection marker and application thereof

Examples

Experimental program
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Embodiment 1

[0040] Example 1. Construction and biological analysis of Hansenula polymorpha (Hansenula polymorpha) HUT-31

[0041] 1. Construction of Hansenula polymorpha double-deficient in uracil and tryptophan synthesis

[0042] 1. Cloning and sequence analysis of 5-phosphoribosylaminobenzoic acid isomerase gene HTRP1

[0043] According to the reported nucleotide sequence of HTRP1 of Hansenula polymorpha (GenBank Access No. AY795576, the sequence shown by the 1-1010 deoxyribonucleotides from the 5' end), the designed primer sequences are as follows:

[0044] Primer 1: 5′-ATA AGGCCT CGGAGGATCGCAGGAGACGG-3' (the underlined part of the base is the StuI recognition site)

[0045] Primer 2: 5′-TCA CATATG CGCCGAGGAGGTCATTGCTG-3' (the underlined part of the base is the NdeI recognition site)

[0046]Using the total DNA of Hansenula polymorpha (Hansenula polymorpha) JCM3621 (CGMCC 2.2498) (purchased from the General Microbiology Center of China Committee for Culture Collection of Microorg...

Embodiment 2

[0072] Example 2, Secreted expression of α-amylase in Hansenula polymorpha (Hansenula polymorpha) HUT-31

[0073] 1. Construction of α-amylase gene ALP1 expression plasmid

[0074] According to the reported nucleotide sequence of the α-amylase gene ALP1 of Saccharomycopsis fibuligera (GenBank Access No. X79051, the sequence shown by the 1530-3030th deoxyribonucleotide from the 5′ end) Primers were designed with the following sequences:

[0075] Primer 9: 5′-CC GAATTC AATATGCAAATTTCAAAAGC-3', (the bases underlined are EcoRI recognition sites);

[0076] Primer 10: 5′-CAT TCTAGA GCCATCATGAACAAATGTCAG-3', (the underlined part is the XbaI recognition site).

[0077] Using the chromosomal DNA of Saccharomycopsis fibuligera CGMCC No.2.1626 (purchased from China General Microorganisms Collection and Management Center CGMCC) as a template, the PCR reaction was carried out under the guidance of primers 9 and 10. The PCR reaction system was: template DNA 0.6 μg, primer 90.5 μmol / L...

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Abstract

The invention discloses hansenula polymorpha with a double selection marker and an application thereof. The hansenula polymorpha with a double selection marker provided by the invention is uracil-synthesized and tryptophan-synthesized double-deficient hansenula polymorpha HUT-31 with a preservation number of CGMCC No.4778. The strain has a uracil and tryptophan double-auxotrophic selection marker, maintains the physiological biochemical characteristics of wild-type strains, facilitates the culture of recombinant strains and the high-level expression of heterologous protein; not only recombinant strains can be selected easily and rapidly, but also the double selection marker can increase the copy number of target genes integrated on a chromosome, can increase the expression level of targetprotein, and has important industrial application value. Meanwhile, the double selection marker is also applicable to the polygene genetic modification of hansenula polymorpha in functional genomics,synthetic biology, and metabolic engineering.

Description

technical field [0001] The present invention relates to Hansenula polymorpha with dual screening markers and its application. Background technique [0002] Yeast, as a single-celled eukaryote, not only has the characteristics of fast growth and simple genetic manipulation of prokaryotes, but also has gene expression regulation and post-translational modification mechanisms similar to higher eukaryotes, so it is a very widely used microorganism in the field of biotechnology Cell factories play an important role in the production of bulk fermentation products, fine pharmaceutical chemical raw materials and protein drugs. Methanotrophic yeast is a type of yeast that has attracted much attention in recent years. Among them, Hansenula polymorpha has the following advantages: (1) It can use its own strong promoter to efficiently promote the expression of foreign genes; ( 2) The frequency of non-homologous recombination is high, which can make high-copy integration of the target g...

Claims

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Application Information

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IPC IPC(8): C12N1/16C12N1/19C12N15/81C12N15/61C12R1/78
Inventor 何秀萍郭雪娜张博润
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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