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Recombinant escherichia coli, preparation method and method for synthesizing 3,4-dihydroxybutyric acid

A technology for recombinant Escherichia coli, Escherichia coli, applied in microorganism-based methods, biochemical equipment and methods, recombinant DNA technology, etc., can solve problems such as unrealistic feasibility, no further research on yield or method optimization, etc.

Inactive Publication Date: 2017-11-03
BEIJING INSTITUTE OF TECHNOLOGYGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In the process of synthesizing 3,4-dihydroxybutyric acid using the biosynthetic method published by Prather's group in MIT's Department of Chemical Engineering in 2013 and 2014, there are many unavoidable by-products formed, which are largely shunted The carbon source of the substrate glucose, and these by-products are inevitable in this method, because the key enzymes for the synthesis of the by-product and the target product 3,4-dihydroxybutyric acid are the same, based on this, it is necessary to block the by-product It is not realistically feasible to increase the synthesis of the target product through the formation of the method, and the method has not been used for further research on the improvement of yield or method optimization

Method used

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  • Recombinant escherichia coli, preparation method and method for synthesizing 3,4-dihydroxybutyric acid
  • Recombinant escherichia coli, preparation method and method for synthesizing 3,4-dihydroxybutyric acid
  • Recombinant escherichia coli, preparation method and method for synthesizing 3,4-dihydroxybutyric acid

Examples

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Embodiment 1

[0174] A recombinant Escherichia coli prepared by the following method:

[0175] Step 1. Use Red homologous recombination technology to prepare recombinant E. coli strain A1 knocked out of xylose isomerase gene xylA

[0176] 1. Preparation of target fragment DNA for gene knockout

[0177] The xylA isomerase gene xylA-deficient Escherichia coli strain JW3537-1 purchased from the Yale University Culture Collection was used to activate the purchased strain and pick a single colony to cultivate overnight in LB liquid medium, and then take 2 mL to cultivate it. After the bacteria liquid is centrifuged at a high speed to obtain the bacteria, it is resuspended in sterile water of 1 / 3 volume of the bacteria liquid; the genome of E. coli strain JW3537-1 is extracted as a template, and PCR is used to amplify the homology arms. Use Takara’s PrimeSTAR Max DNA polymerase to amplify the gene fragments containing homology arms and separate them by gel electrophoresis to obtain the target fragment ...

Embodiment 2

[0289] A method for biosynthesizing 3,4-dihydroxybutyric acid by recombinant Escherichia coli prepared in Example 1. The method steps are as follows:

[0290] (1) Dip the inoculating loop of the recombinant E. coli solution prepared in Example 1 on the LB solid medium, repeat streaking overnight and culture three times to obtain a fully activated single colony of the recombinant E. coli; Example 1 The prepared recombinant Escherichia coli is the preferred recombinant Escherichia coli strains in Table 11, namely D1 to D6, I1, I2, and L1 to L3;

[0291] (2) Pick a single colony and inoculate it in 50mL LB liquid medium, culture it in a shaker with a rotating speed of 190 at 37°C for 18h until the cell OD 600nm Reach 5-6, used to inoculate the seeds of the fermentation medium;

[0292] (3) The fermentation medium adopts 1.5 times the concentration of liquid LB medium, add ampicillin or kanamycin, 10g / L CaCO 3 To adjust the pH during fermentation> 5; Transfer the cultivated seeds in st...

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Abstract

The invention relates to a recombinant escherichia coli, a preparation method and a method for synthesizing 3,4-dihydroxybutyric acid, and belongs to the field of biosynthesis. The recombinant escherichia coli is obtained through knocking out xylose isomerase gene xylA, 2-keto acid aldolase gene yjhH, 2-keto acid aldolase gene yagE and alcohol dehydrogenase gene in escherichia coli, overexpressing xylose dehydrogenase gene and / or 2-keto acid decarboxylase gene, and overexpressing aldehyde dehydrogenases gene. By taking xylose as a basic substrate, a compound substrate can be formed by adding glucose and / or glycerinum on the basis of the xylose, and the 3,4-dihydroxybutyric acid is biologically synthesized through the recombinant escherichia coli; by-products are extremely less, and the formation of the by-products can be interdicted through passage metabolism optimization. The recombinant escherichia coli, the preparation method and the method for synthesizing the 3,4-dihydroxybutyric acid have the advantages that the preparation method is simple, the production cycle is short, the cost is low, later continuous optimizing and transformation are realized, and the good industrial development and application prospect is realized.

Description

Technical field [0001] The invention relates to a recombinant Escherichia coli, a preparation method and a method for synthesizing 3,4-dihydroxybutyric acid, and belongs to the technical field of biosynthesis. Background technique [0002] 3,4-Dihydroxybutyric acid (3,4-dihydroxybutyric acid, 3,4-DHBA) is a general chiral compound that is easily soluble in water, ethanol and ether. It contains one molecule of carboxyl group and two molecules of hydroxyl group. The molecular structure allows it to be modified to form many useful derivatives, such as can be used to synthesize antibiotics, α-amino acids, β-amino acids and polypeptides, etc., or as a basic material for chiral synthesis. [0003] 3,4-Dihydroxybutyric acid can form a cyclic lactone substance by simple saponification reaction. 3-hydroxy-γ-butyrolactone (3HBL), 3-hydroxy-γ-butyrolactone Ester is also a very important chiral C4 compound, which can be used to synthesize various drugs, polymers and solvents. For example, th...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P7/42C12R1/19
CPCC12N9/0006C12N9/0008C12N9/1025C12N9/88C12N9/92C12N15/70C12P7/42C12Y102/0101C12Y401/01C12Y503/01005
Inventor 高海军高玉
Owner BEIJING INSTITUTE OF TECHNOLOGYGY
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