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Acetic acid CoA transferase gene-deficient engineered Escherichia coli strain and application thereof

A technology of Escherichia coli and transferase, applied in the direction of transferase, enzyme, bacteria, etc., can solve the problem of synthesis of unfavorable metabolite isopropanol

Active Publication Date: 2018-11-13
扬州麦塔瑞思生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, acetoacetate transferase prefers the degradation of acetoacetate rather than its synthesis, which is not conducive to the synthesis of the metabolite isopropanol

Method used

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  • Acetic acid CoA transferase gene-deficient engineered Escherichia coli strain and application thereof
  • Acetic acid CoA transferase gene-deficient engineered Escherichia coli strain and application thereof
  • Acetic acid CoA transferase gene-deficient engineered Escherichia coli strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] 1. Extraction of Escherichia coli MG1655 genomic DNA

[0034] The whole genome DNA of MG1655 was extracted from 1ml of wild-type Escherichia coli MG1655 (purchased from the American Type Culture Collection) culture using a commercial bacterial genomic DNA rapid extraction kit (Sangon Bioengineering (Shanghai) Co., Ltd.), dissolved In 0.1mL ultrapure water.

[0035] 2. Extraction of Enterococcus faecalis genomic DNA

[0036] The whole genome DNA was extracted from the culture of Enterococcus faecalis ATCC 29212 (purchased from the American Type Culture Collection) using a commercial bacterial genomic DNA rapid extraction kit (Sangon Bioengineering (Shanghai) Co., Ltd.). Dissolve in 0.1mL ultrapure water.

[0037] 3. Preparation of acetoacetyl-CoA synthetase gene atoB

[0038] The atoB gene can be prepared by the following method, or can be obtained by artificial synthesis.

[0039]Using Escherichia coli MG1655 genomic DNA as a template, PCR amplification was carried ...

Embodiment 2

[0061] Embodiment 2: Construction of expression vector pT-ISOP1

[0062] The genes related to isopropanol synthetic metabolic pathway are constructed on expression vectors, such as pTrc99A vector. All relevant genes can be arranged in any order on the expression vector. In this example, the sequence of 5'-SADH-ADC-HMGL-HMGS-atoB-3' was arranged at the multiple cloning restriction site on the pTrc99A vector to form pT-ISOP1. The specific implementation steps are as follows:

[0063] Purified PCR products (each PCR product prepared in Example 1 steps 3, 4, 5, and 6) and vector plasmid pTrc99A were respectively introduced by PCR with restriction endonuclease Pme I / Xho I corresponding to the enzyme cutting site, Double digestion with Pac I / Pme I, SalI / Pac I, and Xba I / Sal I; agarose electrophoresis to recover enzyme-cut PCR and large vector fragments, and DNA ligase to ligate the digested gene fragments at 16°C The corresponding digestion product of pTrc99A. Escherichia coli D...

Embodiment 3

[0064] Example 3 Construction of Acetate CoA Transferase Gene Deletion Mutant Strain

[0065] The host Escherichia coli used in the isopropanol-producing Escherichia coli engineering bacteria of the present invention is a mutant strain of acetate CoA transferase gene deletion. The host Escherichia coli can be Escherichia coli BL21(DE3), BL21(DE3)pLysS, JM109, DH5α, TOP10, HB101, DH10B or wild type Escherichia coli. The genes encoding acetate CoA transferases missing in the genome are atoD and atoA. The original host bacteria used in this example is wild-type Escherichia coli MG1655, and the genes atoD and atoA encoding acetate CoA transferase were knocked out from its genome by homologous recombination, and the mutant strain was named MG1655ΔatoDA. The specific operation method is as follows:

[0066] The target genes atoD and atoA are arranged adjacent to each other on the genome, using the pKD13 plasmid (purchased from China Plasmid Vector Strain Cell Line Gene Collection ...

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Abstract

The invention discloses an acetic acid CoA transferase gene-deficient engineered Escherichia coli strain and application thereof. The engineered Escherichia coli strain constructs an engineering strain of an isopropanol synthesis pathway composed of a plurality of genes, and is obtained by knocking out encoding genes of acetic acid CoA transferase of host escherichia coli. The isopropanol synthesis pathway provided by the invention consists of acetoacetyl ketase CoA synthetase, hydroxymethylglutaryl CoA synthetase, hydroxymethylglutaryl CoA lyase, acetoacetate decarboxylase and secondary ethanol dehydrogenase, and can achieve the transformation from acetyl-CoA as starting metabolite to isopropanol in vivo. The mutant strain provided by the invention is obtained by expressing a gene encoding all enzymes constituting the isopropanol anabolic pathway. The recombinant escherichia coli strain constructed in the invention can synthesize 364mg / L isopropanol from a rich medium-containing glucose or glycerol under facultative anaerobic conditions.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to acetate CoA transferase-deficient Escherichia coli engineering bacteria and applications thereof. Background technique [0002] Isopropanol is a secondary alcohol. In traditional industries, it is mainly used as dehydrating agent and cleaning agent in pharmaceutical, cosmetics, plastics, spices, coatings and electronic industries. With the increasing energy demand in today's society, its application in energy has been paid attention to and developed. Isopropanol can be used as a biofuel to partially replace petrochemical gasoline, and can also replace methanol to esterify fats and oils to synthesize biodiesel, and can also be dehydrated to produce propylene, which can only be obtained from petroleum at present. [0003] At present, the production of isopropanol is mainly based on the chemical route of propylene or acetone as raw material. The chemical synthesis method ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P7/04C12R1/19
CPCC12N9/0006C12N9/13C12N9/88C12N9/93C12P7/04C12Y101/01001C12Y208/03008C12Y401/01004C12Y401/03004C12Y602/01C12Y602/01001
Inventor 周佳田宝霞李相前
Owner 扬州麦塔瑞思生物科技有限公司
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