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Culture medium and fermentation method capable of improving yield of corynebacterium glutamicum ectoines

A technology of Corynebacterium glutamicum and tetrahydropyrimidine, applied in the field of bioengineering, can solve problems such as restricting application, and achieve the effects of shortening lag period and logarithmic growth period, improving acid-producing ability, and increasing plasmid copy number.

Active Publication Date: 2020-11-06
无锡晶扬生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, on the one hand, because the medium used in this program involves components with TSE risks, such as brain heart extract, its application in cosmetics and medicine is restricted; on the other hand, Corynebacterium glutamicum is a mature amino acid Product industrial microbial production strains, which produce valine, leucine and other amino acids and intermediate metabolites during their metabolism

Method used

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  • Culture medium and fermentation method capable of improving yield of corynebacterium glutamicum ectoines
  • Culture medium and fermentation method capable of improving yield of corynebacterium glutamicum ectoines
  • Culture medium and fermentation method capable of improving yield of corynebacterium glutamicum ectoines

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Corynebacterium glutamicum CG-ECT3 is cultivated according to the cultivation and fermentation conditions disclosed in CN110699310A, the inoculum size, and the way of supplementing sugar, and the seed medium and fermentation medium adopt the aforementioned culture medium of the present invention.

[0041] The medium is:

[0042] Seed medium (g / L): peptone 4.0; yeast powder 2.0; sodium chloride 5.0; disodium hydrogen phosphate 2.5; glucose 5.0; corn steep liquor 0.1. Adjust the pH to 7.0±0.3 with liquid ammonia.

[0043] Fermentation medium: (g / L): glucose 20; (NH4) 2 SO 4 15; KH 2 PO 4 1;K 2 HPO 4 1; MgSO 4 0.25; Biotin 0.2×10 -3 ; bubble enemy 0.25L; corn steep liquor 12.5; KCl 1.9; L-aspartic acid 3; compound trace elements 1mL (compound trace elements: FeSO 4 ·7H 2 O 16.4 g; MnSO 4 ·H 2 O 100mg; CuSO 4 200mg; ZnSO 4 ·7H 2 O 1g; unit: / L).

[0044] Seed culture: Corynebacterium glutamicum CG-ECT3 was cultured overnight on BHI plates. Pick a singl...

Embodiment 2

[0048] Primary seed medium (g / L): peptone 4.0, yeast powder 2.0, sodium chloride 5.0, disodium hydrogen phosphate 2.5, glucose 5.0, corn steep liquor 0.1. Adjust the pH to 7.0±0.3 with liquid ammonia.

[0049] Secondary seed medium (g / L): glucose 18; (NH 4 ) 2 SO 4 10; KH 2 PO 4 1;K 2 HPO 4 1; MgSO 4 0.25; Biotin 0.2×10 -3 ; corn steep liquor 15; KCl 1.9; compound trace elements 0.5mL (FeSO 4 ·7H 2 O 16.4 g; MnSO 4 ·H 2 O100mg; CuSO 4 200mg; ZnSO 4 ·7H 2 O 1g; unit: / L); adjust the pH to 7.0±0.3 with liquid ammonia.

[0050] Fermentation medium: (g / L): glucose 20; (NH 4 ) 2 SO 4 18; KH 2 PO 4 1;K 2 HPO 4 1; MgSO 4 0.25; Biotin 0.2×10 -3 ; bubble enemy 0.25L; corn steep liquor 12.5; KCl 1.9; L-aspartic acid 3; compound trace elements 1mL (compound trace elements: FeSO 4 ·7H 2 O 16.4 g; MnSO 4 ·H 2 O 100mg; CuSO 4 200mg; ZnSO 4 ·7H 2 O 1g; unit: / L). The concentration of glucose in feeding feed is 400g / L, and the pH is adjusted to 7.0±0....

Embodiment 3

[0058] The medium is the same as in Example 2 above.

[0059] The recombinant Corynebacterium glutamicum CG-ECT3 strain in the glycerol tube was inoculated into the primary seed medium at an inoculation amount of 0.5% for expansion cultivation, the liquid volume: 40%; temperature: 30.0°C; shaking frequency: 120rpm ;Cultivation time: 12h, obtain first-class species.

[0060] The first-class species was inoculated into the second-class seed medium with 1% inoculum amount for cultivation, the liquid volume: 40%; temperature: 30.0°C; shaking frequency: 120rpm; cultivation time: 12h, and the second-class species were obtained.

[0061] The secondary species is inoculated in the fermentation medium (10L fermenter) by 10% inoculum size and cultivated, liquid filling capacity: 60%; control temperature: 30.0°C; pressure 0.09MPa; maintain pH7.0 with liquid ammonia (in supplementary Adjust the pH after adding glucose); time 45h; volume 60M 3 / h; during the whole fermentation process, t...

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Abstract

The invention discloses a culture medium and fermentation method capable of improving yield of corynebacterium glutamicum ectoines, and belongs to the field of bioengineering. The invention provides aformula of a fermentation culture medium. The fermentation culture medium is rich in a carbon source, a nitrogen source, inorganic salt and compound trace elements, which are required for growth of corynebacterium glutamicum CG-ECT3; a strain is enabled to rapidly grow, reproduce and be cultured for 13 hours; OD600 can reach over 40; a plasmid copy number is increased; and time of a lag period and time of a logarithmic phase are shortened. Meanwhile, according to the invention, by controlling residual sugar and a precusor substance L-asparaginic acid in fermentation liquor to be replenished in the fermenting process, the acid-producing ability of the recombinant corynebacterium glutamicum CG-ECT3 is improved, a fermentation period is shortened, and after fermentation is carried out for 45h, yield of the ectoines can reach over 48g / L.

Description

technical field [0001] The invention relates to a culture medium and a fermentation method for increasing the output of Corynebacterium glutamicum ectoine, belonging to the field of bioengineering. Background technique [0002] Ectoines is a cyclic amino acid derivative, which is an osmotic pressure compensating solute synthesized by some microorganisms in response to environmental osmotic stress. Proteins and their cells have a stabilizing effect. It has a wide range of application value and application prospects in the fields of environmental restoration and protection agents, enzymes, proteins and nucleic acids and other biological macromolecules, cell protection agents, cosmetic additives and pharmaceutical preparations. [0003] At present, there are two main ideas for the fermentation of Ectoines by microorganisms: [0004] The first is to use halophilic microorganisms to achieve the intracellular accumulation and secretion of Ectoines through the "cell milking metho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P17/12C12N1/21C12N1/38C12R1/15
CPCC12N1/20C12N1/38C12P17/12
Inventor 董亮宁健飞蔡立明
Owner 无锡晶扬生物科技有限公司
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