Corynebacterium glutamicum and production method of alpha-ketoglutarate through fermentation thereof

A technology of Corynebacterium glutamicum and ketoglutarate, applied in the field of bioengineering, can solve the problems of high production cost, long α-ketoglutarate fermentation cycle, low yield and the like, and achieves low production cost, easy control, The effect of simple fermentation process

Active Publication Date: 2012-03-28
江苏澳创生物科技有限公司
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

However, the production of α-ketoglutaric acid by Yarrowia lipolytica or Torulopsis glabrata fermentati

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment 1: the acquisition of Corynebacterium glutamicum mutant strain GKG-047

[0018] The present invention uses a glutamic acid producing bacterium Corynebacterium glutamicum GDK-9 as the starting bacterium [GDK-9 has ketomalonic acid, fluoroacetic acid, sulfaguanidine, glutamine resistance genetic markers, Its directional breeding research was published in Volume 34, Issue 7 of "Food and Fermentation Industry" in 2008, and it is now preserved in the Laboratory of Metabolic Engineering, Tianjin University of Science and Technology. A mutant strain sensitive to methotrexate was screened out through mutagenesis , named Corynebacterium glutamicum GKG-047, the strain can accumulate α-ketoglutarate under the condition of limited nitrogen supply.

[0019] Mutation and Breeding of α-Ketoglutarate-Producing Bacteria

[0020] Mutagenesis: take Corynebacterium glutamicum GDK-9 as the starting bacterium, and take fresh activated slant strains in complete liquid medium. 32°...

Embodiment 2

[0027] Embodiment 2: 5L fermentor fermentation of mutant strain GKG-047

[0028] 1) Seed culture: Pick a lawn of Corynebacterium glutamicum GKG-047 from a freshly activated slant and insert it into a 500 mL Erlenmeyer flask containing 30 mL of seed medium. The components in the seed medium are: glucose 20g / L, corn steep liquor 20mL / L, Na 2 HPO 4 3g / L, MgSO 4 ·7H 2 O 0.5g / L, urea 1g, and water as the balance, pH 7.0-7.2. Seal with 8 layers of gauze, place on a rotary shaker, 200r / min, and shake at 32°C for 8h.

[0029] 2) Fermentation in a fermenter: put the seed liquid into a 5L fully automatic fermenter with 3L of fermentation medium according to the inoculum size of 5%. The components in the fermentation medium are: glucose 60g / L, molasses 2g / L, corn steep liquor 20ml / L, Na 2 HPO 4 2g / L, KCl 2g / L, MgSO 4 ·7H 2 O 1g / L, MnSO 4 ·H 2 O 2mg / L, FeSO 4 2mg / L, VB 1 2mg / L, the balance is water, pH 7.0~7.2. The fermentation temperature is 34°C, the ventilation ratio is 1:...

Embodiment 3

[0031] Embodiment 3: 5L fermentor fermentation of mutant strain GKG-047

[0032] The α-ketoglutarate fermentation was carried out using a 5L fully automatic fermenter, and the seed culture medium, fermentation medium and seed culture method were the same as in Example 2. Put the seed liquid into a 5L fully automatic fermenter equipped with 3L fermentation medium according to 10% inoculation amount, the fermentation temperature is 36°C, the ventilation ratio is 1:0.5~1, the stirring speed is 300~700r / min, and the dissolved oxygen is maintained at 10 ~30%, adding glucose with a mass percentage concentration of 80% to maintain residual sugar at 1% ~ 2%. In the early stage of bacterial growth 0-8h, add 25% ammonia water to adjust the pH value of the fermentation broth to 7.0-7.2. After 8h, add NaOH with a mass percentage concentration of 20% to adjust the pH value of the fermentation broth to 7.0-7.2, and the fermentation time 35h.

[0033] The supernatant after centrifugation o...

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Abstract

Belonging to the technical field of bioengineering, the invention relates to corynebacterium glutamicum and a production method of alpha-ketoglutarate through fermentation of corynebacterium glutamicum. The bacterial strain of the invention is corynebacterium glutamicum GKG-047, with a preservation number of CGMCC No. 5481. With a strain of glutamic acid producing strain GDK-9 as a parent, the bacterial strain of the invention is a mutant strain that is screened out through mutagenesis and sensitive to methotrexate. Under the circumstance of a limited nitrogen source supply, the bacterial strain can accumulate alpha-ketoglutarate. During fermentation, the bacterial strain is characterized by aerobic fermentation, fast bacterium growth, and rapid acid production rate. After fermentation of the bacterial strain for 32h in a fermentation tank of 10L, the maximum output of alpha-ketoglutarate can be 47.2g/L. The method provided in the invention has the advantages of simple and easily controllable fermentation process, and low production cost of alpha-ketoglutarate, thus being in favor of the popularization and application of industrial production.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a strain of α-ketoglutarate-producing bacteria, in particular to a Corynebacterium glutamicum producing α-ketoglutarate and its screening method and the production of α-ketoglutarate by fermentation of the strain. Ketoglutarate. Background technique [0002] α-ketoglutarate (α-KG), also known as α-gluconic acid, 2-oxoglutarate, α-oxoglutarate, molecular formula: C 5 h 6 o 5 , molecular weight 146.1, appearance is white or off-white crystalline powder, easily soluble in water, plays an important role in cell metabolism, is one of the important intermediate products of tricarboxylic acid cycle, participates in the synthesis of amino acids, proteins and vitamins in vivo synthesis and energy metabolism. α-ketoglutaric acid is widely used in food, medicine, organic synthesis, cosmetics and feed industries. In the food industry, α-ketoglutaric acid can be used as the main com...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P7/50C12R1/15
Inventor 陈宁谢希贤徐庆阳刘淑云谢沛
Owner 江苏澳创生物科技有限公司
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