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Preparation method of succinic acid-producing recombinant escherichia coli cells

A technology of recombinant Escherichia coli and succinic acid production, which is applied in the field of bioengineering, can solve the problems of easy-to-contaminate bacteria, long time and trouble in recycling cells, etc.

Active Publication Date: 2015-10-14
SINOPEC YANGZI PETROCHEM +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the commonly used process for cell recovery is centrifugation or membrane separation. These two operations are more troublesome and prone to bacterial contamination. It takes a long time to recover cells, which is likely to cause a decrease in cell viability.

Method used

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  • Preparation method of succinic acid-producing recombinant escherichia coli cells

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The process of two-stage fermentation acid production adopts the description in the following technical scheme:

[0029] Strain activation: take a small amount of bacterial liquid from a glycerol cryopreservation tube, streak it on an LB plate, and culture it at 37°C for 12 hours; seed liquid preparation: take a single colony from an LB plate, inoculate it in LB liquid medium, and inoculate it in Shake the flask at 37°C and 200r / min until the cell density (OD 600 ) with a value of 0.6.

[0030] Two-stage fermentation: adding glucose to an initial concentration of 25g L -1 , the dissolved oxygen was maintained at 50% during the cultivation process; when the glucose was basically consumed, the CO 2 Gas, enter the stage of anaerobic fermentation acid production, the method of adding glucose at a constant rate during the fermentation process, and control the glucose concentration at 20g L -1 , while intermittently adding basic magnesium carbonate to provide carbon dioxid...

Embodiment 2

[0037] Strain activation: take a small amount of bacterial liquid from a glycerol cryopreservation tube, streak it on an LB plate, and culture it at 37°C for 12 hours; seed liquid preparation: take a single colony from an LB plate, inoculate it in LB liquid medium, and inoculate it in Shake the flask for 8 hours at 37°C and 200r / min until the cell density (OD 600 ) with a value of 0.7.

[0038] Two-stage fermentation: adding glucose to an initial concentration of 20g L -1 , the dissolved oxygen was maintained at 60% during the cultivation process; when the glucose was basically consumed, the CO 2 Gas, enter the acid production stage of anaerobic fermentation, adopt the method of adding glucose at a constant speed during the fermentation process, and control the glucose concentration at 5g L -1 , while intermittently adding basic magnesium carbonate to provide carbon dioxide donors, while adjusting the pH value at 6.4.

[0039] At the end of the fermentation, the bacterial c...

Embodiment 3

[0045] Strain activation: take a small amount of bacterial liquid from a glycerol cryopreservation tube, streak it on an LB plate, and culture it at 37°C for 12 hours; seed liquid preparation: take a single colony from an LB plate, inoculate it in LB liquid medium, and inoculate it in Shake the flask for 8 hours at 37°C and 200r / min until the cell density (OD 600 ) with a value of 0.8.

[0046] Two-stage fermentation: adding glucose to an initial concentration of 30g L -1 , the dissolved oxygen was maintained at 40% during the cultivation process; when the glucose was basically consumed, the CO 2 Gas, enter the stage of anaerobic fermentation acid production, the method of adding glucose at a constant rate during the fermentation process, and control the glucose concentration at 10g L -1 , while intermittently adding basic magnesium carbonate to provide carbon dioxide donors, while adjusting the pH value at 6.8.

[0047] At the end of the fermentation, the bacterial cells a...

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Abstract

The invention discloses a preparation method of succinic acid-producing recombinant escherichia coli cells and belongs to the technical field of bio-engineering. The preparation method includes following steps: (1) performing two-stage fermentation with the recombinant escherichia coli to produce succinic acid; (2) after the fermentation of the succinic acid is finished, collecting the microorganism in a sedimentation manner to recycle low-activity succinic acid-producing recombinant escherichia coli cells; and (3) moving the recycled low-activity succinic acid-producing recombinant escherichia coli cells to a fresh culture medium, feeing air or oxygen, maintaining dissolved oxygen to be 40-60%, and controlling the temperature to be 33-36 DEG C and the pH value of the fermentation liquid to be 6.8-7.5. According to the invention, the cell recycle process is carried out in the fermentation pot, so that probability of contamination is greatly reduced and damage on cells is reduced. The method can reach 91% maximumly in cell recycle rate. With the recycled low-activity succinic acid-producing recombinant escherichia coli as a catalyst and sodium succinate as a substrate, fumaric acid can be synthesized in an aerobic conversion manner with the conversion rate being higher than 92%.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a preparation method of succinic acid-producing recombinant Escherichia coli cells. Background technique [0002] The microorganisms involved in the current research on the production of succinic acid by microbial fermentation mainly include wild-type microorganisms and genetically engineered bacteria. The use of Escherichia coli to ferment and produce succinic acid utilizes many advantages such as fast growth of Escherichia coli, simple culture conditions, good safety, easy operation and easy transformation. At present, two-stage fermentation, pure anaerobic fermentation and pure aerobic fermentation are mainly adopted for the production of succinic acid by Escherichia coli fermentation. [0003] At present, the commonly used techniques for cell recovery are centrifugation or membrane separation. These two operations are cumbersome and prone to bacterial cont...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P7/46C12R1/19
Inventor 方晓江马江锋刘经伟李泽壮刘丽娟王英武陈韶辉杨爱武
Owner SINOPEC YANGZI PETROCHEM
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