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Construction method and application of metabolic engineering escherichia coli strain for producing acetone or isopropanol by virtue of acetic acid

A technology of Escherichia coli and metabolic engineering, which is applied in the field of bioengineering to achieve the effect of increasing production and improving absorption rate

Active Publication Date: 2018-04-03
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Escherichia coli can aerobically metabolize acetic acid to grow. Studies by Lourdes et al. have shown that adding a small amount of acetic acid to a medium with glucose as a carbon source is conducive to the accumulation of acetone (Appl.Environ.Mircrobiol.1998,64,1079-1085 ), but the production of acetone and isopropanol using acetic acid as the sole carbon source has not been reported

Method used

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  • Construction method and application of metabolic engineering escherichia coli strain for producing acetone or isopropanol by virtue of acetic acid
  • Construction method and application of metabolic engineering escherichia coli strain for producing acetone or isopropanol by virtue of acetic acid
  • Construction method and application of metabolic engineering escherichia coli strain for producing acetone or isopropanol by virtue of acetic acid

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Embodiment 1

[0037] Embodiment 1. The construction of acetone production pathway to obtain the bacterial strain of acetone production

[0038] Using the pTrc99a plasmid as a vector, a heterologous expression pathway for acetone production was constructed by overexpressing key enzymes from the acetone production pathway in E.coli or C.acetobutylicum. The enzymes that synthesize acetone from acetyl-CoA are thiolase, CoA transferase, and acetoacetate decarboxylase, and CoA transferase is coded by two different genes for its different subunits. However, there is no gene encoding acetoacetate decarboxylase in E.coli. Three different recombinant plasmids were obtained by combining and expressing the above four genes from E.coli and C.acetobutylicum in pTrc99a. They are pTrc99a-thl-RBS-adc-Trc-ctfAB (the above genes are all from C. acetobutylicum), pTrc99a-thl-RBS-adc-Trc-atoDA (thl, adc are from C. acetobutylicum, atoDA is from E. coli), pTrc99a-atoB-RBS-adc-Trc-atoDA (adc comes from C.acetobu...

Embodiment 2

[0049] Example 2. Enhancing the transport pathway of acetic acid

[0050] Replace the promoter of the gene (ack, pta) of the acetyl-CoA pathway of acetic acid production by Red recombination. In E.coli, ack and pta share a promoter, and the constitutive expression through the mutation of the trc promoter is stronger The promoter of the gene replaces the ack and pta promoters on the genome to enhance its expression. The strain with successful promoter replacement was named HY01, and the specific operation of promoter replacement was as follows:

[0051] First, primers were designed to amplify the Kana fragment with the target promoter, and the template plasmid was digested with Dpn I to remove methylation. 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, ...

Embodiment 3

[0053] Embodiment 3. Knock out gene to further improve the output and yield of acetone

[0054] Since the gene expression of the gluconeogenesis pathway is up-regulated in the medium with acetic acid as the carbon source, excess carbon source flows to the gluconeogenesis pathway. Therefore, the red recombination method was used to knock out the genes maeB, pckA, icdA, adhE and sfcA. The strain with maeB knockout on the basis of HY 01 was named HY 02, and the strain with pckA knockout on the basis of HY 02 was named HY 03. The specific operation of gene knockout is as follows:

[0055] For the knockout of the gene maeB, use the E.coli MG1655 single-deletion maeB strain preserved in our laboratory as a template, first design primers (primer sequences are shown in the table below), and clone a DNA fragment of about 1700bp with kana resistance by PCR . The fragment was transferred into the electroporation-competent host bacteria by electroporation. The preparation of the electr...

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Abstract

The invention discloses a construction method of a metabolic engineering escherichia coli strain for producing acetone or isopropanol by virtue of acetic acid. Through metabolic engineering transformed escherichia coli, the acetone or isopropanol is produced by conducting fermentation with the acetic acid as a carbon source; and transforming pathways include the follows: constructing a metabolic pathway of producing the acetone or isopropanol through acetyl CoA, and / or promoting over-expression of acetone intake pathway related genes so as to enhance the transport rate of the acetone, and / or blocking off TCA circulation or down-regulating the TCA circulation so as to increase an acetyl CoA metabolic flux flowing to a target metabolic product, and / or relieving a decarboxylic reaction between malic acid and oxaloacetic acid to delete a by-product generating pathway, and / or deleting key genes in an ethanol producing pathway to regulate an acetyl CoA node metabolic flux. According to the construction method provided by the invention, through analysis on the metabolic pathway as well as regulation and control, the escherichia coli is transformed by virtue of genetic engineering means; and with the application of the produced strain, the acetone and the isopropanol can be produced in a culture medium that the acetic acid is taken as the carbon source.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and more specifically relates to a construction method and application of a metabolic engineering Escherichia coli strain for producing acetone or isopropanol using acetic acid. Background technique [0002] Acetone, also known as dimethyl ketone, has a wide range of uses, mainly as solvents for various polymers, diluents, detergents for degreasing electronic products, extractants for various vitamins, hormones, and petroleum dewaxing, etc. ; In addition, acetone, as a chemical raw material, can also be used to produce methyl methacrylate and bisphenol A, and then used to synthesize polycarbonate plastics. The production methods of acetone mainly include direct oxidation of propylene, isopropanol method, cumene method and fermentation method. At present, about 83% of acetone is produced from non-renewable fossil raw materials. Due to the disadvantages of chemical synthesis, the use of ren...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/21C12P7/28C12P7/04C12R1/19
CPCC12N9/0006C12N9/1029C12N9/13C12N9/88C12N15/70C12P7/04C12P7/28C12Y101/01001C12Y208/03C12Y401/01004
Inventor 吴辉杨昊李志敏黄兵叶勤
Owner EAST CHINA UNIV OF SCI & TECH
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