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Method for producing ethylene glycol and glycolic acid by using escherichia coli and genetically engineered bacteria

A technology of genetically engineered bacteria and Escherichia coli, which is applied in the field of production of ethylene glycol and glycolic acid, can solve the problems of poor growth and achieve the effects of low production cost, simple process route, good application value and economic benefits

Inactive Publication Date: 2021-05-11
SHANGHAI ADVANCED RES INST CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Xylose can also be used as a carbon source for the growth of some microorganisms. Escherichia coli can use xylose as a carbon source, but the growth is not as good as glucose.

Method used

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  • Method for producing ethylene glycol and glycolic acid by using escherichia coli and genetically engineered bacteria
  • Method for producing ethylene glycol and glycolic acid by using escherichia coli and genetically engineered bacteria

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preparation example Construction

[0033] Step 4, preparation of Escherichia coli electroshock competent cells and electrotransformation of homologous recombination fragments;

[0034] Step 5, identifying the Escherichia coli △aldA recombinant by PCR.

[0035] Obtaining the genetically engineered strain (taking aldA as an example) of gene overexpression is divided into the following steps:

[0036] Step 1, linearization of the high-level expression vector pDK6;

[0037] Step 2, obtaining the aldA gene fragment;

[0038] Step 3, constructing an overexpression plasmid by homologous recombination;

[0039] Step 4, transformation into Escherichia coli DH5α;

[0040] Step 5, identifying pDK6-aldA transformants by PCR;

[0041] Step 6, extracting the plasmid pDK6-aldA;

[0042] Step 7. Preparation of electroporated cells and electroporation of pDK6-aldA to target Escherichia coli;

[0043] Step 8: Identify pDK6-aldA transformants by PCR.

Embodiment 1

[0045] Production of ethylene glycol and / or glycolic acid by Escherichia coli fermentation using xylose as carbon source.

[0046] Escherichia coli used were wild type strains W3110, BW25113, S17-1, DH5α, BL21.

[0047] Escherichia coli was inoculated into a 250mL Erlenmeyer flask containing 50mL of seed culture medium, and the shaker cabinet was rotated at 200 rpm and kept at a constant temperature of 37°C for seed cultivation.

[0048] The components of the seed medium are: peptone 10g / L, yeast extract 5g / L, sodium chloride 5g / L.

[0049] The composition of the fermentation medium is: xylonic acid 15g / L, corn steep liquor 50g / L, potassium dihydrogen phosphate 3g / L, dipotassium hydrogen phosphate 2g / L, calcium chloride 1g / L, magnesium sulfate 4g / L.

[0050] The composition of the control medium was: xylose 15g / L, corn steep liquor 50g / L, potassium dihydrogen phosphate 3g / L, dipotassium hydrogen phosphate 2g / L, calcium chloride 1g / L, magnesium sulfate 4g / L.

[0051] The seed...

Embodiment 2

[0056]Production of ethylene glycol and / or glycolic acid by Escherichia coli fermentation using xylose as carbon source. The Escherichia coli used was wild-type strain W3110.

[0057] Escherichia coli was inoculated into a 250mL Erlenmeyer flask containing 50mL of seed culture medium, and the shaker cabinet was rotated at 200 rpm and kept at a constant temperature of 37°C for seed cultivation.

[0058] The composition of the fermentation medium is: xylose acid 30g / L, corn steep liquor 50g / L, potassium dihydrogen phosphate 3g / L, dipotassium hydrogen phosphate 2g / L, calcium chloride 1g / L, magnesium sulfate 4g / L.

[0059] The seeds were cultured for 12 hours, inoculated into a 5L fermenter, which contained 3L fermentation medium or control medium, kept ventilation and stirring during the fermentation process, and the fermentation temperature was 37°C. During the fermentation process, the pH of the fermentation liquid was adjusted by adding ammonia solution. Stabilized at 5.0 and...

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Abstract

The invention discloses a method for producing ethylene glycol and glycolic acid by using escherichia coli and genetically engineered bacteria. The method comprises the following steps: carrying out fermenting by using the escherichia coli or the escherichia coli genetically engineered bacteria by taking xylonic acid as a carbon source to produce the ethylene glycol and / or the glycolic acid, wherein the escherichia coli genetically engineered bacteria are obtained by inactivating an aldehyde oxidase gene aldA or an alcohol dehydrogenase gene yqhD by the escherichia coli, or are obtained by over-expressing the aldehyde oxidase gene aldA or the alcohol dehydrogenase gene yqhD by the escherichia coli. According to the method disclosed by the invention, when the escherichia coli is used and the xylonic acid is used as the carbon source to carry out fermentation culture, the xylonic acid is metabolized in cells to generate the glycolic acid and / or the ethylene glycol and is accumulated in fermentation liquid, so that the conversion rate is relatively high and the production strength is high. Furthermore, the escherichia coli is subjected to inactivation or over-expression of the aldehyde oxidase gene aldA and the alcohol dehydrogenase gene yqhD, so that the conversion rate and the production strength of the glycolic acid and / or the ethylene glycol can be further improved.

Description

technical field [0001] The invention belongs to the technical field of Escherichia coli genetically engineered bacteria, and in particular relates to a method for producing ethylene glycol and glycolic acid by using Escherichia coli and genetically engineered bacteria. Background technique [0002] Xylose is the second most abundant monosaccharide in nature after glucose, accounting for 18-30% of lignocellulose. The effective utilization of xylose is a key point in the utilization of lignocellulose resources. Xylose can also be used as a carbon source for the growth of some microorganisms. Escherichia coli can use xylose as a carbon source, but the growth is not as good as glucose. How to efficiently utilize xylose is a research hotspot in the world. Xylonic acid is an oxidation product of xylose, and its main application is as a raw material for the production of esters and as a chelating agent. [0003] Glycolic acid, also known as glycolic acid, is an alpha hydroxy acid...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P7/18C12P7/42C12R1/19
CPCC12N9/0006C12N9/0008C12P7/18C12P7/42C12Y102/03001
Inventor 郝健卢希阳史吉平
Owner SHANGHAI ADVANCED RES INST CHINESE ACADEMY OF SCI
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