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Method for synthesizing alpha-ketoglutaric acid by reinforcing Yarrowia lipolytica

A technology of Yarrowia lipolytica and ketoglutaric acid, which is applied in the field of metabolic engineering and can solve the problems of low concentration of target metabolites and low production intensity

Inactive Publication Date: 2015-02-18
JIANGNAN UNIV
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In the process of microbial production of α-ketoglutarate, there are unfavorable factors such as low concentration of target metabolites and low production intensity

Method used

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  • Method for synthesizing alpha-ketoglutaric acid by reinforcing Yarrowia lipolytica
  • Method for synthesizing alpha-ketoglutaric acid by reinforcing Yarrowia lipolytica
  • Method for synthesizing alpha-ketoglutaric acid by reinforcing Yarrowia lipolytica

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Experimental program
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Effect test

Embodiment 1

[0039] Example 1 Effect of overexpression of glutamate dehydrogenase on bacterial cell growth

[0040] (1) Construct the integrated expression vector used for expressing the target gene: amplify the hygromycin phosphotransferase hph gene, the nucleotide sequence of the hph gene is shown in SEQ ID NO.1, using the restriction endonuclease Stu I and Simultaneously process the amplified hph gene and p0 plasmid with Hind III, and connect the two digested products to obtain an integrated expression vector p0(hph) containing hygromycin transphosphoryl transferase as a selection marker; (2) construct a recombinant integrated expression vector Plasmid: According to the sequence published by NCBI (derived from Saccharomyces cerevisiae, GeneID: 851311), the open reading frame sequence encoding glutamic acid dehydrogenase encoding gene GDH2 was fully chemically synthesized, and the restriction endonucleases Sfi I and Not I were used to cut GDH2 The open reading frame and the integrated ex...

Embodiment 2

[0044] Example 2 Adding L-methionine imine to strengthen intracellular glutamate supply

[0045] Inoculate the wild-type Y.lipolytica WSH-Z06 cells and the recombinant strain Y.lipolytica-GDH2 into a 500ml Erlenmeyer flask containing 50ml of fermentation medium at the same time, culture at 28°C and 200rpm for 96h, collect the cells by centrifugation, and follow the previous method. The glutamic acid content in the cells of the two strains was determined by the method described above. Compared with wild-type Y.lipolytica WSH-Z06, the glutamic acid content in recombinant strain Y.lipolytica-GDH2 cells ranged from 0.53μmol / (mg·dry weight of bacteria) to 0.99μmol / (mg·dry weight of bacteria) ( image 3 ).

Embodiment 3

[0046] Example 3 Regulation of Intracellular Amino Acid Metabolism Enhances α-Ketoglutarate Accumulation

[0047] The wild-type Y.lipolytica WSH-Z06 strain and the recombinant strain Y.lipolytica-GDH2 were simultaneously inoculated in a 500ml Erlenmeyer flask containing 50ml fermentation medium according to the method for producing α-ketoglutarate as described before, and L-methionine was added Imine was fermented at 28°C and 200rpm for 144h to compare the accumulation of α-ketoglutarate accumulated outside the cells. It was found by comparison that the intracellular glutamate supply and glutamate metabolism were strengthened to produce α-ketoglutarate at the same time, the catalytic activity of glutamate dehydrogenase in the recombinant strain Y.lipolytica-GDH2 was significantly increased, and the extracellular accumulation α-ketoglutarate from 14.5g·L -1 Rise to 19.2g·L -1 ( Figure 4 ).

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Abstract

The invention discloses a method for synthesizing alpha-ketoglutaric acid by reinforcing Yarrowia lipolytica, belonging to the technical field of metabolism engineering. Glutamate dehydrogenase is overexpressed in wild type Y.lipolytica WSH-Z06 strain cells to construct a recombinant strain Y.lipolytica-GDH2, and the regulated cells metabolize glutamic acid to synthesize the alpha-ketoglutaric acid. In the fermentation process, in order to enhance the glutamic acid supply in the cells, L-methionine imine is added in the fermentation culture medium, thereby inhibiting the metabolism of the glutamic acid in the cells and the synthesis of the glutamine, and obviously enhancing the accumulation of the alpha-ketoglutaric acid. Therefore, the method for regulating metabolism of amino acid in the cells is an effective means for reinforcing accumulation of alpha-ketoglutaric acid.

Description

technical field [0001] The invention relates to a method for strengthening the synthesis of α-ketoglutarate by Yarrowia lipolytica, in particular to a method for strengthening the synthesis of α-ketoglutarate by regulating intracellular amino acid metabolism, which belongs to the field of metabolic engineering. Background technique [0002] α-ketoglutarate, as one of the important intermediates in the tricarboxylic acid cycle (TCA) pathway in microbial cells, participates in various metabolic activities of microbial cells. It is not only a key node in the tricarboxylic acid cycle, but also participates in the synthesis of amino acids, proteins, vitamins and energy metabolism in organisms. Compared with other metabolic intermediates in the tricarboxylic acid cycle, α-ketoglutarate is metabolized in microbial cells Accumulation is subject to multiple regulation. Therefore, it is of great research significance to reveal the metabolic accumulation and regulation mechanism of α-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12P7/50C12R1/645
CPCC12N9/0016C12P7/50C12Y104/01002
Inventor 陈坚周景文郭洪伟曾伟主堵国成
Owner JIANGNAN UNIV
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