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Yarrowia lipolytica capable of reinforcing precursor supply to enhance synthesis of alpha-oxoglutarate

A technology of Yarrowia lipolytica and ketoglutarate, which is applied in the field of metabolic engineering and can solve the problems of increasing the difficulty and cost of the separation and purification process

Inactive Publication Date: 2014-07-16
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Moreover, since pyruvate and α-ketoglutarate have similar physicochemical properties, the presence of large amounts of pyruvate increases the difficulty and cost of downstream separation and purification processes

Method used

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  • Yarrowia lipolytica capable of reinforcing precursor supply to enhance synthesis of alpha-oxoglutarate
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  • Yarrowia lipolytica capable of reinforcing precursor supply to enhance synthesis of alpha-oxoglutarate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Enhancing Intracellular Acetyl-CoA Supply

[0035] (1) Construct an integrated expression vector for expressing the target gene: design primers to amplify the hph gene according to the gene sequence of hygromycin phosphotransferase in the plasmid pUB4-CRE, and the nucleotide sequence of the hph gene is shown in SEQ ID NO. As shown in 1, use restriction endonucleases Stu I and Hind III to simultaneously treat the amplified hph gene and p0 plasmid, and connect the two digested products to obtain an integrated expression vector containing hygromycin phosphotransferase as a selection marker p0(hph). (2) Construction of a recombinant integrated expression plasmid: According to the sequence published by NCBI, the gene encoding the α subunit of the E1 component of pyruvate dehydrogenase was fully chemically synthesized, and the open reading frame obtained by simultaneous cutting with the restriction endonucleases NotI and EcoRI The part is connected with the integra...

Embodiment 2

[0039] Example 2 Effect of overexpression of PDA1 on pyruvate dehydrogenase

[0040] Wild-type Y.lipolytica WSH-Z06 cells and recombinant strain Y.lipolyticaT1 were inoculated in YPD medium at the same time, cultured at 28°C and 200rpm, and the cells in the exponential growth phase were separated by centrifugation. The two strains were tested according to the method described above. Intracellular pyruvate dehydrogenase E1 component catalytic activity and pyruvate dehydrogenase complex enzyme total catalytic activity. Compared with the wild-type Y.lipolytica WSH-Z06, the catalytic activity of the E1 component in the recombinant strain Y.lipolytica T1 cells increased from 278.5U / mg protein to 308.5U / mg protein, and the overexpression of PDA1 successfully made the recombinant strain Y. The catalytic activity of the E1 component in lipolytica T1 cells increased by 10.7% ( image 3 ). At the same time, comparing the total catalytic activity of the pyruvate dehydrogenase complex e...

Embodiment 3

[0041] Example 3 Overexpression of PDA1 enhances α-ketoglutarate accumulation

[0042] The wild-type Y.lipolytica WSH-Z06 cells and the recombinant strain Y.lipolyticaT1 were simultaneously inoculated in a 3L fermenter containing 1.5L fermentation medium according to the method for producing α-ketoglutarate as described before, at 28°C, 200rpm Under batch fermentation for 144h, the accumulation of extracellular pyruvate and α-ketoglutarate was compared. Comparison found that the extracellular accumulation of α-ketoglutarate in the recombinant strain Y.lipolyticaT1 increased from 37.6g·L -1 Rise to 43.3g·L -1 ; At this time, the extracellular metabolic intermediates from 34.2g L -1 Reduced to 20.1g·L -1 ( Figure 5 ).

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Abstract

The invention discloses Yarrowia lipolytica capable of reinforcing precursor supply to enhance synthesis of alpha-oxoglutarate, belonging to the technical field of metabolic engineering. According to the invention, catalytic activity of a pyruvate dehydrogenase E1 component in a cell is improved through overexpression of PDA1, so the accumulation of an intermediate metabolite pyruvic acid is decreased, and supply of intracellular acetyl-CoA is reinforced; moreover, supply of intracellular acetyl-CoA is increased for accumulation of alpha-oxoglutarate for cells, which enables extracellular accumulation of alpha-oxoglutarate to be obviously increased. Thus, overexpression of PDA1 enables the content of the intermediate metabolite pyruvic acid to be decreased and accumulation of alpha-oxoglutarate to be reinforced and provides novel thinking for accumulation of organic acid in TCA cycle for cells.

Description

technical field [0001] The invention relates to Yarrowia lipolytica capable of enhancing the supply of precursors and enhancing the synthesis of α-ketoglutarate, which belongs to the field of metabolic engineering. Background technique [0002] As one of the important intermediates in the tricarboxylic acid cycle (TCA) pathway, α-ketoglutarate plays an important role in the metabolism of microbial cells. It is not only a key node in the tricarboxylic acid cycle, but also participates in amino acid , protein, vitamin synthesis and energy metabolism; and it is an important node for microorganisms to regulate the balance of carbon and nitrogen metabolism, and plays an important role in the study of regulatory mechanisms related to microbial nitrogen metabolism. In industry, α-ketoglutaric acid is an important intermediate in fine chemical industry and pharmaceutical industry. It is widely used in the synthesis of various amino acids, vitamins and other small molecular substance...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12P7/50C12R1/645
Inventor 陈坚周景文郭洪伟堵国成
Owner JIANGNAN UNIV
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