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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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, so far there has been no research report on the direct b

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

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Experimental program
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Example Embodiment

[0042] Example 1. Knock out genes to obtain succinic acid producing strains

[0043] The red recombination technology was used to knock out the genes sdhAB, iclR, arcA, ldhA, sfcA, maeB, sucABCD, poxB, adhE, fumC, and fadR. The specific operations are as follows:

[0044] For the knockout of gene sdhAB, primer 1 (F-sdhAB) with sequence SEQ ID NO: 1 and primer 2 (R-sdhAB) with sequence SEQ ID NO: 2 were first used to PCR clone a DNA fragment of about 1700 bp with pKD4 as a template. The host strain was transformed with calcium into plasmid pKD46, and the recombinant was screened with ampicillin. The recombinant bacteria introduced into pKD46 were cultured to OD at 30°C 600 At about 0.3, L-arabinose was added for induction for 1 hour, and then 10% glycerol was used to prepare the electrotransmission state. The electrotransformation was carried out in bacterial mode 1 (1.8KV, 5ms). Kanamycin was used to select transformants that undergo homologous recombination. Then use primer 3 ...

Example Embodiment

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

[0050] The citrate synthase gene gltA from Escherichia coli was overexpressed using the plasmid pTrc99a. First use primer 35 (pTrc99a-gltA-F(BamH1)) with the sequence of SEQ ID NO: 29 and primer 36 (pTrc99a-gltA-R-Hind3) with the sequence of SEQ ID NO: 30, using E. coli MG1655 as a template for PCR cloning with enzyme The gltA gene at the cut site. Then pTrc99a and the target fragment recovered by PCR were double digested with BamHI and HindIII. After the digested product was recovered, it was ligated with T4 ligase at 16°C. The calcium is then transformed into the target strain. Use ampicillin resistance to screen the successfully constructed recombinant bacteria. Protein electrophoresis determines whether the protein is expressed. Plasmids from strains with correct expression were drawn and sent for sequencing, which was recorded as pTrc99a-gltA.

[0051] The overexpressio...

Example Embodiment

[0063] Example 3. Succinic acid-producing strain complex medium shake flask fermentation

[0064] Taking E. coli MG1655 as the starting strain, knocking out the gene sdhAB and blocking the TCA cycle, the succinate-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 to obtain strains MG02, MG022, MG023, and MG024. After comparison, it was found that MG02 had the best effect, so the next step was to use MG02 as the starting strain.

[0066] Based on the double-deleted bacteria 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, MG033. The fermentation results show that MG032 has the best effect, so follow-up transformation is carried out based on MG032.

[0067] In order to understand the effect of the missing by-product generati...

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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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