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Method for realizing excessive accumulation of alpha-ketoglutarate acid by adding alpha-ketoglutarate acid dehydrogenase inhibitor

A technology for ketoglutarate dehydrogenase, ketoglutarate, applied in the directions of microorganism-based methods, biochemical equipment and methods, microorganisms, etc.

Inactive Publication Date: 2010-11-03
GUANGDONG HUANXI BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The inhibitory effects of hydrogen peroxide, methotrexate, sodium hypochlorite and hydroxythiamine on KGDH have been extensively studied in human neurological and cardiac diseases, but rarely reported in microorganisms, especially yeast

Method used

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  • Method for realizing excessive accumulation of alpha-ketoglutarate acid by adding alpha-ketoglutarate acid dehydrogenase inhibitor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Effect of adding hydrogen peroxide on acid production by fermentation of T.glabrata CCTCC NO.M 202019

[0026] Hydrogen peroxide was added at the initial stage of fermentation. With the increase of hydrogen peroxide addition, the pyruvic acid production showed an upward trend. When the hydrogen peroxide addition was 5mM, the KG production reached a maximum of 21.8g / L, an increase of 5.6% compared with the control , at this time C KG / C PYR The value is 0.73. Investigate the effect of adding 5mM hydrogen peroxide on acid production at different stages of fermentation, adding hydrogen peroxide at the initial stage of KG acid production (40h), the accumulation of KG increased the most, which was 23.2g / L, which was 12.2% higher than that of the control.

[0027] Thus, the optimal amount of hydrogen peroxide added is 5mM, and the added time is the initial stage of KG acid production (40h).

Embodiment 2

[0028] Example 2 Effect of adding methotrexate on acid production by fermentation of CCTCC NO.M 202019

[0029] Different concentrations of methotrexate (MTX) were added at the initial stage of fermentation. As the amount of MTX increased, the yields of PYR and KG both showed a trend of first increasing and then decreasing. Reached the maximum value, respectively 30.4g / L and 22.7g / L, respectively increased by 8.9% and 10.1% compared with the control, at this time C KG / C PYR The value is 0.75. Investigating the effect of adding MTX on acid production in different stages of fermentation, it was found that the effect of MTX on the production of KG in fermentation became smaller as the fermentation progressed.

[0030] Therefore, the optimum amount of MTX added was 0.08μM, and the added time was the initial stage of fermentation (0h).

Embodiment 3

[0031] Example 3 Effect of adding sodium hypochlorite on acid production by fermentation of CCTCC NO.M 202019

[0032] Adding sodium hypochlorite in the culture medium found that when the added concentration reached 0.4mM, the KG output was 22.2g / L, which was 7.5% more than the contrast increase, C pyr / C kg The value dropped by 6%. Investigating the addition of 0.4mM NaClO in different stages of fermentation 3 The effect on acid production found that the initial stage of fermentation (0h) was the best addition time.

[0033] The optimal amount of sodium hypochlorite added is thus 0.4mM, and the added time is the initial stage of fermentation (0h).

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Abstract

The invention relates to a method for adding alpha-ketoglutarate dehydrogenase inhibitor to realize excessive accumulation of alpha-ketoglutaric acid, which belongs to the technical field of the metabolic regulation optimized fermentation process of the protein level. The method of the invention comprises following steps: utilizing multi - vitamin - auxotrophic yeast of Torulopsis glabrata CCTCC M202019 as producing strains, regulating the activity of alpha-ketoglutarate dehydrogenase through adding the alpha-ketoglutarate dehydrogenase inhibitor: hydrogen peroxide, methotrexate, sodium hypochlorite or hydroxyamino in culture medium, purposively lowing the activity of the alpha-ketoglutarate dehydrogenase, reducing the degradation of the alpha-ketoglutaric acid in the metabolic process, and achieving the aim of the excessive accumulation of the alpha-ketoglutaric acid. The method of the invention cuts off carbon metabolism flow on a node of the alpha-ketoglutaric acid through regulating the stream distribution of carbon metabolism and the carbon metabolism flow, wherein the maximum output of the alpha-ketoglutaric acid reaches 23.2g / L, which is increased by 12.2% compared with thecontrol. The invention provides a new thought for the fermentation research of TCA cycle intermediate metabolite.

Description

technical field [0001] The invention relates to a method for adding an α-ketoglutarate dehydrogenase (KGDH) inhibitor to realize excessive accumulation of α-ketoglutarate (KG). By studying the effects of KGDH inhibitors on the direction of metabolic flow and the corresponding metabolic flux, a large accumulation of α-ketoglutarate was achieved. The invention belongs to the technical field of metabolic regulation and optimization fermentation process at protein level. Background technique [0002] α-ketoglutaric acid, also known as α-gluconic acid, 2-oxoglutaric acid or α-oxoglutaric acid, is one of the important intermediate products in the tricarboxylic acid (TCA) cycle, and is found in microbial cells. It plays an important role in metabolism and is also an important precursor substance for the synthesis of various amino acids and proteins. Its structural formula is: [0003] [0004] Alpha-ketoglutarate structure [0005] α-Ketoglutaric acid has important applicati...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/50C12R1/645
Inventor 陈坚张旦旦刘立明堵国成
Owner GUANGDONG HUANXI BIOLOGICAL TECH
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