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Construction and application of URA3 defective P. pastoris X-33 strain

A defect-type, strain-based technology, applied in the field of bioengineering, can solve problems such as the inability of yeast to survive, and achieve the effect of stabilizing genetic traits

Inactive Publication Date: 2011-07-13
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Yeast AMP decarboxylase (URA3), usually encoded by the ura3 gene, is an enzyme necessary for pyrimidine biosynthesis; if OMP decarboxylase function is lost, the pyrimidine biosynthetic pathway will be interrupted, and yeast cannot survive without exogenous uracil supply

Method used

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  • Construction and application of URA3 defective P. pastoris X-33 strain
  • Construction and application of URA3 defective P. pastoris X-33 strain

Examples

Experimental program
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Effect test

Embodiment 1

[0018] Cloning of URA3 gene and construction of homologous replacement DNA fragments: URA5', URA3' and fusion homologous arm fragments at both ends of URA3 gene PCR amplified from Pichia pastoris X-33 genome were 0.7kb, 0.7kb, 0.6kb and 1.3kb ( figure 1 ). Sequencing results showed that each sequence and fusion sequence were correct.

Embodiment 2

[0020] Knockout and identification of URA3 gene: After the URA5'-3' fusion homology arm fragment was electroporated into yeast, double-crossover homologous recombination occurred with the URA3 gene in the genome, replacing about 700 bp of the sequence in the coding frame of the URA3 gene. The strains without homologous recombination cannot grow on the medium containing 5-FOA because they carry the URA3 gene, so the ura3 deletion strain can be screened on the MD medium containing 5-FOA and uracil. Select strains that have been phenotype-identified and have stable traits, and use external primers URA5F and URA3R for genomic PCR identification. The size of the URA3 gene of wild-type X-33 bacteria is 2043bp, so the amplified product is about 2000bp; the URA3 gene encoding of ura3-deleted bacteria The sequence of about 700bp in the box was replaced, and the amplified product should be about 1300bp, and the electrophoresis results were consistent with expectations ( figure 2 ), ind...

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Abstract

The invention discloses construction and application of an orotidine-5'- phosphate decarboxylase (ura3) defective P. pastoris X-33 strain, belonging to the technical field of biologic engineering. The engineering strain P. pastoris X-33 (delta ura3) is obtained by a homologous recombination target gene knockout method. The strain can use the ura3 gene as a selected marker for constructing different kinds of engineering strains, and when the engineering strains are constructed, the carried ura3 genes are not required to be from the same microbial population. Simultaneously, the invention provides a better platform strain for performing subsequent engineering reconstruction such as glycosylation reconstruction on the X33, thus having great potential application prospect.

Description

technical field [0001] The construction and utilization of an orotidine-5'-phosphate decarboxylase (ura3)-deficient Pichia pastoris (P. pastoris) X-33 strain belongs to the technical field of bioengineering. Background technique [0002] The de novo biosynthesis of pyrimidine nucleotides in yeast uses glutamine as a raw material, undergoes multi-step biocatalysis to generate orotic acid (OMP), which is then catalyzed by OMP decarboxylase to decarboxylate to uridylic acid (UMP), which is further catalyzed to generate Uridine triphosphate (UTP) and cytidine triphosphate (CTP). Yeast AMP decarboxylase (URA3), usually encoded by the ura3 gene, is an enzyme necessary for pyrimidine biosynthesis; if OMP decarboxylase function is lost, the pyrimidine biosynthesis pathway will be interrupted, and yeast cannot survive without exogenous uracil supply. Orotic acid nucleoside-5'-phosphate decarboxylase encoding gene ura3 is used as a selectable marker gene, which is often used for gene...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/09C12N15/65C12R1/84
Inventor 朱瑞宇金坚张大成许永利雷楗勇陈蕴
Owner JIANGNAN UNIV
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