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Uracil auxotrophic hansenula polymorpha and preparation method and application thereof

A Hansenula polymorpha, auxotrophic technology, applied in the biological field, can solve problems affecting screening efficiency, genetic stability defects, industrial application limitations, etc.

Active Publication Date: 2017-12-01
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the auxotrophic strains of Hansenula polymorpha currently in use are derived from traditional physical or chemical mutagenesis, and defects in genetic stability affect the screening efficiency; moreover, the original strains are basically from the American type culture collection (American type culture collection, ATCC) model strain, limited in industrial application

Method used

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  • Uracil auxotrophic hansenula polymorpha and preparation method and application thereof
  • Uracil auxotrophic hansenula polymorpha and preparation method and application thereof
  • Uracil auxotrophic hansenula polymorpha and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1. Construction of uracil auxotrophic Hansenula polymorpha HP7.89-ura3

[0054] In this example, Hansenula polymorpha (Hansenula polymorpha) HP20110424 was used as the starting bacterium to construct uracil auxotrophic Hansenula polymorpha. The deposit number of the Microbiology Center (abbreviated as CGMCC) is CGMCC No.7.89, hereinafter referred to as Hansenula polymorpha CGMCC 7.89.

[0055] 1. Construction of a recombinant plasmid used to destroy orotidine-5'-phosphate decarboxylase encoding gene URA3

[0056] (1) Using primers URA3-2K-F and URA3-2K-R to carry out PCR amplification using the genomic DNA of Hansenula polymorpha CGMCC 7.89 as a template to obtain the URA3 gene shown in sequence 1 in the sequence table, the URA3 gene and The identity of URA3 and its flanking sequences in H. polymorpha DL-1 in NCBI is 95.93%. The URA3 gene was connected to the cloning vector pEASY-bluntsimple to obtain the recombinant plasmid pEASY-URA3. The 634th-1425th posit...

Embodiment 2

[0102] Example 2, Expression of Glucanase in Hansenula polymorpha (Hansenula polymorpha) HP7.89-ura3

[0103] 1. Construction of expression vector

[0104] Replace the DNA fragment between the EcoRI and XbaI recognition sequences of the pMOXZα-A (patent number: ZL200810101801.1) vector with the gene encoding the dextranase of Lipospora staratus (LSD1 gene, whose sequence is the 1798-3624th position of sequence 8) , to obtain a recombinant vector, which was named pMOXZα-LSD1, pMOXZα-LSD1 can express the LSD1 gene coded by the 1798-3624th position of Sequence 8. Lipospora glucanase.

[0105] Using the recombinant vector pMOXZα-LSD1 as a template, PCR amplification was performed using primers MOXp-F and AOX1TT-R, and the PCR product with the correct sequence was named DNA fragment 1, the sequence of DNA fragment 1 was sequence 8, and the first- Position 1511 is the methanol oxidase gene promoter MOXp sequence, position 1525-1791 of sequence 8 is the α signal peptide sequence, po...

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Abstract

The invention discloses uracil auxotrophic hansenula polymorpha and a preparation method and application thereof. The preparation method of the uracil auxotrophic hansenula polymorpha comprises the steps that orotidine-5'-phosphate decarboxylase genes in hansenula polymorpha original strains are knocked out, or the content of proteins coded by the orotidine-5'-phosphate decarboxylase genes are reduced, or the proteins coded by the orotidine-5'-phosphate decarboxylase genes are inactivated, and the uracil auxotrophic hansenula polymorpha is obtained, wherein knockout of the orotidine-5'-phosphate decarboxylase genes are conducted with the steps that M1, coding of the orotidine-5'-phosphate decarboxylase genes is early stopped; and M2, exogenous DNA fragments are inserted into the orotidine-5'-phosphate decarboxylase genes. The built uracil auxotrophic hansenula polymorpha is stable in heredity, has no obvious difference from wild type strains in other growth characteristics, and can be used as transformed chassis cells in metabolic engineering or synthetic biology, and cell factories of efficient expression recombinant proteins.

Description

technical field [0001] The invention relates to the uracil auxotrophic Hansenula polymorpha and its preparation method and application in the field of biotechnology. Background technique [0002] Yeast, as a single-celled eukaryote, is a widely used microorganism in the field of biotechnology, and plays a very important role in the production of bulk fermentation products, fine pharmaceutical chemicals and protein drugs. Hansenula polymorpha is a methanolotrophic yeast that can grow and induce high-efficiency protein expression with methanol as the only carbon source; the optimum growth temperature is 37°C, and it is resistant to heat stress, metal stress and oxygen stress, etc. It has good tolerance to various environmental stresses, so it has a wide range of adaptability to industrial environments; the stability and high-copy integration of foreign DNA on the chromosome ensures the high efficiency, stability and uniformity of protein expression; protein secretion The path...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N15/60C12N1/19C12N9/88C12R1/78
CPCC12N9/88C12N15/815C12Y401/01023
Inventor 何秀萍秦立春程艳飞郭雪娜
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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