Genetically engineered bacterium capable of secreting urate oxidase as well as construction method and application of genetically engineered bacterium

A technology of uric acid oxidase and genetically engineered bacteria, applied in the directions of oxidoreductase, microorganism-based methods, biochemical equipment and methods, etc., can solve the problem of low secretion, high cost of intracellular expression and secretion purification, and inability to realize industrialization, etc. problem, to achieve the effect of high extracellular enzyme activity, enhanced secretion, and enhanced secretion effect

Pending Publication Date: 2022-01-21
BEIJING UNIV OF CHEM TECH
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0006] At present, domestic and foreign studies on the heterologous expression of urate oxidase in yeast mainly focus on intracellular expression or the use of α-factor from S. As a signal peptide, the amount of extracellular secretion is low, and the final industrialization cannot be realized, while the cost of intracellular expression, secretion and purification is relatively high

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  • Genetically engineered bacterium capable of secreting urate oxidase as well as construction method and application of genetically engineered bacterium
  • Genetically engineered bacterium capable of secreting urate oxidase as well as construction method and application of genetically engineered bacterium
  • Genetically engineered bacterium capable of secreting urate oxidase as well as construction method and application of genetically engineered bacterium

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Embodiment approach

[0041] As mentioned above, domestic research on urate oxidase mainly focuses on direct extraction from natural microorganisms and expression in prokaryotic strains. Extraction from microorganisms is not conducive to purification, and the yield is low, which is not suitable for industrialization. At the same time, the existing genetically engineered bacteria that secrete urate oxidase have the problem that it is difficult to secrete it in Pichia pastoris after the gene expression of urate oxidase, and only the intracellular urate oxidase can be extracted, and the intracellular capacity is limited, so the yield cannot be increased. maximize. In view of this, the inventors have conducted a lot of research on the construction of genetically engineered bacteria that secrete urate oxidase, through the removal of urate oxidase targeting signals, and a large number of screening of endogenous signal peptides and exogenous signal peptides, obtained Three endogenous signal peptides that ...

Embodiment approach

[0044] According to some embodiments of the present invention, the exogenous gene encoding urate oxidase (UOX) is a gene exogenously encoding urate oxidase (UOX) with the peroxisome-targeted amino acid sequence removed; preferably , the gene exogenously encoding urate oxidase (UOX) with the peroxisome-targeted amino acid sequence removed is urate oxidase (UOX) exogenously encoding the last three amino acids of urate oxidase (UOX) ) gene, referred to in the present invention as "gene encoding UOX-3" or "UOX-3 gene".

[0045] Those skilled in the art should understand that the above urate oxidase gene is urate oxidase gene from any source, and the gene encoding the base sequence of the peroxisome targeting signal is removed.

[0046] In some specific preferred embodiments of the present invention, the gene encoding uric acid oxidase (UOX) is derived from Aspergillus flavus (Aspergillus flavus) (the deposit number is ATCC 16883), and its Genebank accession number is CAA43896.1, c...

Embodiment 1

[0094] Embodiment 1: Construction of recombinant bacterial strain

[0095] The uric acid oxidase gene derived from Aspergillus flavus was removed by primer design to remove the base sequence encoding the last three amino acids (300-302), and connected to the plasmid pPICZαA to construct the plasmid pPICZαA-UOX-3 , and then the signal peptides Fre2, Dse4, and Flo5 were replaced by the signal peptides Fre2, Dse4, and Flo5 in the plasmid pPICZαA-UOX-3, and the plasmids pPICZαA-Fre2-UOX-3, pPICZαA-Dse4-UOX-3, pPICZαA-Flo5-UOX-3. The constructed plasmid was digested and linearized at BstxI, and then the linearized fragment was integrated into the chromosome of Pichia pastoris X-33 by electroporation, so that the recombination of high-secretion urate oxidase was screened on the resistance plate Strains X-33-Fre2-UOX-3, X-33-Dse4-UOX-3, X-33-Flo5-UOX-3.

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Abstract

The invention relates to a genetically engineered bacterium capable of secreting urate oxidase. The genetically engineered bacterium is recombinant pichia pastoris containing a gene for endogenous coding and secreting signal peptide and a gene for exogenous coding of urate oxidase (UOX). The engineered bacterium is constructed by adopting pichia pastoris as a host cell, introducing the gene for coding the urate oxidase (UOX) or the gene for coding the urate oxidase (UOX) through codon optimization, and optimizing a secretion path, and the bacterium is the genetically engineered bacterium capable of efficiently secreting the urate oxidase. The invention also relates to a construction method of the genetically engineered bacterium capable of secreting urate oxidase. According to the method, by screening secretory signal peptides and removing urate oxidase peroxisome targeting signals, the secretory expression level of the urate oxidase is enhanced, operation is easy, and the method can be applied to construction of a high-yield urate oxidase strain and industrial preparation of the urate oxidase, and has good market application prospects.

Description

technical field [0001] The invention belongs to the technical field of gene recombination, and relates to a genetically engineered bacterium secreting uric acid oxidase and its construction method and application. Background technique [0002] Uric acid (also known as 2,6,8-trioxypurine) is the final product of purine metabolism in the human body. The chemical formula of blood uric acid is C5H4N4O3. It is slightly soluble in water and easy to form crystals. If the purine metabolism disorder in the human body produces excessive uric acid, or uric acid If the excretion is blocked, the concentration of uric acid in the blood will increase, forming hyperuricemia. In addition, urate is easily precipitated and deposited in joints, kidneys, heart valves and other tissues and organs, leading to gout. After chemotherapy in cancer patients, a large number of tumor cells die and dissolve rapidly, and the purine metabolites in the nucleus and cytoplasm are released into the blood, whic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12N15/62C12N9/06C12R1/84
CPCC12N9/0048C12N15/815C12Y107/03003C07K2319/02C12N2800/22
Inventor 谭天伟贺壮壮王峥
Owner BEIJING UNIV OF CHEM TECH
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