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Construction method and applications of metabolic engineering escherichia coli strain for producing succinic acid by using acetic acid

A technology of Escherichia coli, metabolic engineering, applied in the field of bioengineering

Active Publication Date: 2016-05-04
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far there has been no research report on the direct biosynthesis of succinic acid using acetic acid as a carbon source.

Method used

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  • Construction method and applications of metabolic engineering escherichia coli strain for producing succinic acid by using acetic acid
  • Construction method and applications of metabolic engineering escherichia coli strain for producing succinic acid by using acetic acid
  • Construction method and applications of metabolic engineering escherichia coli strain for producing succinic acid by using acetic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1. Knockout of genes to obtain succinic acid producing strains

[0043] The genes sdhAB, iclR, arcA, ldhA, sfcA, maeB, sucABCD, poxB, adhE, fumC, fadR were knocked out by red recombination technology. The specific operation is as follows:

[0044] For the knockout of the gene sdhAB, first use the sequence of primer 1 (F-sdhAB) of SEQIDNO:1 and the sequence of primer 2 (R-sdhAB) of SEQIDNO:2, and use pKD4 as a template to PCR clone a DNA fragment of about 1700bp. The plasmid pKD46 was introduced into the host bacteria through calcium transformation, and recombinants were screened with ampicillin. Recombinant bacteria introduced with pKD46 were cultured at 30°C to OD 600 At about 0.3, L-arabinose was added for induction for 1 hour, and then 10% glycerol was used to prepare electroporation competence. Electroporation was performed using bacterial mode 1 (1.8KV, 5ms). Transformants were selected for homologous recombination using kanamycin. Then use sequence as...

Embodiment 2

[0049] Example 2. Overexpression of glyoxylate cycle key enzyme genes gltA, aceA and acnB.

[0050] The citrate synthase gene gltA derived from Escherichia coli was overexpressed using the plasmid pTrc99a. First use sequence is the primer 35 (pTrc99a-gltA-F (BamH1)) of SEQIDNO:29 and the sequence is the primer 36 (pTrc99a-gltA-R-Hind3) of SEQIDNO:30, with Escherichia coli MG1655 as template PCR clone has enzyme The gltA gene at the cleavage site. Then use BamHI and HindIII to double-enzyme digest pTrc99a and the target fragment recovered by PCR. After recovering the digested product, use T4 ligase to ligate at 16°C. Calcium is then converted into the strain of interest. Use ampicillin resistance to select successfully constructed recombinant bacteria. Protein electrophoresis to determine whether the protein is expressed. Extract the plasmid from the strain with correct expression, send it for sequencing, and record it as pTrc99a-gltA.

[0051] Plasmid pTrc99a was also use...

Embodiment 3

[0063] Embodiment 3. produce succinic acid strain compound medium shake flask fermentation

[0064] Using Escherichia coli MG1655 as the starting strain, the gene sdhAB was knocked out, the TCA cycle was blocked, and the succinic acid-producing basic strain MG01 was obtained.

[0065] On the basis of the single deletion strain MG01, the genes iclR, fadR, arcA and fumC were knocked out respectively to obtain strains MG02, MG022, MG023 and MG024. After comparison, it was found that MG02 had the best effect, so MG02 was used as the starting strain next.

[0066] On the basis of the double-deletion strain MG02, the effects of blocking the supply pathway and activating the TCA cycle on the fermentation of succinic acid were verified, so the genes arcA, maeB, and sfcA were knocked out to obtain strains MG03, MG032, and MG033. The fermentation results showed that MG032 had the best effect, so the follow-up transformation was carried out based on MG032.

[0067] In order to understa...

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Abstract

The present invention discloses a construction method and applications of a metabolic engineering escherichia coli strain for producing succinic acid by using acetic acid, wherein escherichia coli transformed through metabolic engineering uses acetic acid as a raw material to perform fermented production of succinic acid, and the transformation pathway comprises: blocking TCA cycle, and / or blocking succinic acid utilization pathway, and / or enhancing acetic acid uptake and oxaloacetic acid supply, strengthening glyoxylic acid cycle, and / or deleting by-product generation pathway, and / or reducing futile cycle caused by acetic acid production using pyruvic acid decarboxylation, and / or dredging acetyl CoA node metabolic flow. According to the present invention, through the analysis on the metabolic pathway and the regulation, the escherichia coli is transformed by using the genetic engineering way, the obtained strain can produce succinic acid in the culture medium adopting acetic acid as the carbon source, and no by-product is generated.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and more specifically relates to constructing a recombinant Escherichia coli strain using acetic acid to produce succinic acid, and using the metabolic engineering Escherichia coli to ferment and synthesize succinic acid by using acetic acid as a main raw material. Background technique [0002] Succinic acid, also known as succinic acid, is a four-carbon dicarboxylic acid widely used in agriculture, food and pharmaceutical industries. Succinic acid is a precursor of many important industrial chemicals, including adipic acid, 1,4-butanediol, tetrahydrofuran, N-methylpyrrolidone, 2-pyrrolidone, succinate and γ-butyrolactone. It can also be used to synthesize biodegradable polymer polybutylene succinate (PBS) and polyamide polymer ( x,4). At present, commercial succinic acid has been produced by chemical synthesis of liquefied petroleum gas or mineral oil, and converted to biological ferme...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N1/21C12P7/46C12R1/19
CPCC12P7/46
Inventor 李志敏李运杰吴辉叶勤
Owner EAST CHINA UNIV OF SCI & TECH
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