Engineered Escherichia coli and method of synthesis of catalyzing fumaric acid from maleic acid in presence of Engineered Escherichia coli

A technology of Escherichia coli and engineering bacteria, applied in microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve the problems of reducing the conversion rate and purity of fumaric acid, and achieve the effect of improving expression efficiency

Inactive Publication Date: 2016-12-14
JIANGNAN UNIV
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Problems solved by technology

[0004] When Escherichia coli containing maleic acid cis-trans isomerase derived from Serratia marcescens is used as the host for whole-cell catalytic production of fumaric acid, fumaric acid will be converted into L - Malic acid, which reduces the conversion and purity of fumaric acid

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  • Engineered Escherichia coli and method of synthesis of catalyzing fumaric acid from maleic acid in presence of Engineered Escherichia coli
  • Engineered Escherichia coli and method of synthesis of catalyzing fumaric acid from maleic acid in presence of Engineered Escherichia coli
  • Engineered Escherichia coli and method of synthesis of catalyzing fumaric acid from maleic acid in presence of Engineered Escherichia coli

Examples

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Embodiment 1

[0064] Using the pKD13 plasmid as a template, the primers FumCpKD3F and FumApKD3R of the fumA and fumC encoding genes of the target gene fumarase were used for polymerase chain reaction, and a linear line with the Kana resistance gene and FRT marker in the middle and short homology arms on both sides was amplified. Homologous recombination fragments. The sequences of FumCpKD3F and FumApKD3R are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0065] Taking the Escherichia coli Escherichia coli BL21 (DE3) preserved in the laboratory as the starting strain, the pKD46 plasmid carrying the Red recombinase (the pKD46 plasmid is reported in the article Datsenko et al., Proc Natl Acad Sci USA, 2000,97:6640-6645 ) into Escherichia coli, and screen the transformant in the Amp-resistant plate medium; insert the transformant into 50mL LB medium with Amp resistance and cultivate overnight at 30°C; insert the same LB medium, and added arabinose at a final concentration of 2mmol / L a...

Embodiment 2

[0069] This example illustrates the construction of strain BL21(DE3)△fumA-fumC / pET24a-maiA and the high-density fermentation expression method.

[0070] The pET24a-maiA plasmid that has been successfully constructed in the laboratory was electrotransformed into the gene knockout Escherichia coli BL21(DE3)△fumA-fumC in Example 1 to obtain the E. coli engineering strain BL21(DE3)△fumA-fumC / pET24a-maiA.

[0071] Streak the above-mentioned E. coli engineered strains stored at -80°C on an LB plate containing 50 μg / mL kanamycin for overnight activation, pick a single colony and inoculate it into a 5 mL LB test tube medium containing 50 μg / mL kanamycin resistance Incubate at 37°C for 8 hours, inoculate 120 mL of fermented seed medium containing 50 μg / mL kana resistance with 1% (v / v) inoculum, and cultivate at 37°C for 7.5 hours as the seed solution in the upper tank, and then inoculate with 7 % of the inoculum was inoculated in the fermentation medium. Cultured in fermentation med...

Embodiment 3

[0073] This example illustrates the ability of the strain BL21(DE3)△fumA-fumC / pET24a-maiA to convert maleic acid into fumaric acid.

[0074] Preparation of the substrate: Weigh 185.7 g of maleic acid solid and add it into 1 L of deionized water, and stir continuously with a magnetic stirrer, adjust the pH to 8.0 with potassium hydroxide, and prepare a 2 mol / L potassium maleate solution.

[0075] With the fermented bacterial liquid in embodiment 2 and the fermented bacterial liquid of original escherichia coli BL21 / pET24a-maiA, carry out the production of fumaric acid as biocatalyst, the ratio of 2mol / L potassium maleate solution and fermented bacterial liquid in the conversion system The conversion temperature was 4:1, and the conversion temperature was controlled at 37°C. After 1.2 hours of conversion, the conversion solution was taken to measure the concentration of maleic acid, fumaric acid and malic acid. It can be seen from Table 1 that during the production process of fu...

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Abstract

The invention relates to engineered Escherichia coli capable of high-yield production of fumaric acid; specifically, fumarase coded genes fumA and fumC of Escherichia coli are knocked off, and maleic cis-trans isomerase gene from Serratia marcescens is converted to obtain the engineered Escherichia coli. The invention also discloses a method of synthesis of catalyzing fumaric acid from maleic acid by using the engineered Escherichia coli. The method includes culturing the engineered Escherichia coli by fermenting, subjecting the engineered Escherichia coli to high-density inductive expression when OD600 reaches 40-80, and applying the obtained fermentation broth to biochemical catalysis of maleic acid in presence of Engineered Escherichia coli to obtain fumaric acid. The genes fumA and fumC in the Escherichia coli are knocked off by using Red homologous recombination, a metabolic pathway of its fumaric acid to L-malic acid is cut off, the maleic cis-trans isomerase from Serratia marcescens is then expressed, and the obtained genetically-engineered Escherichia coli can efficiently convert maleic matrix to synthesize high-purity fumaric acid, there is nearly no L-malic acid byproduct synthesized, and basis is provided for the industrialized production of fumaric acid by whole-cell process catalysis of maleic acid.

Description

technical field [0001] The invention relates to the fields of fermentation engineering and biocatalysis, in particular to an Escherichia coli engineering bacterium and a method for catalyzing the synthesis of fumaric acid from maleic acid. Background technique [0002] Fumaric acid, also known as fumaric acid and fumaric acid, is a naturally occurring organic acid. As an important four-carbon platform compound and fine chemical product, fumaric acid is widely used in food, medicine, chemical industry, coating, plasticizer and other fields. For example, in food, it can be used as sour agent and flavor enhancer with pure taste; in medicine, iron fumarate is widely used in the treatment of human microcytic anemia. [0003] At present, the industrial production of fumaric acid is mainly based on chemical synthesis, including furfural oxidation and maleic anhydride isomerization. However, the chemical synthesis method needs to use petroleum resources, and petroleum is a non-ren...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P7/46C12R1/19
CPCC12N9/88C12N9/90C12P7/46C12Y402/01002C12Y502/01001C12N1/205C12R2001/19
Inventor 周哲敏刘文茂周丽崔文璟刘中美
Owner JIANGNAN UNIV
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