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Genetically engineered bacterium for producing alpha-ketobutyric acid and application of genetically engineered bacterium

A technology of genetically engineered bacteria and ketobutyric acid, applied in the biological field, can solve the problems of complex reaction conditions, high production cost, high energy consumption, etc., and achieve the effects of simple fermentation process, low production cost, and high acid production rate.

Active Publication Date: 2014-06-18
江苏澳创生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] The problem to be solved by the present invention is to overcome the deficiencies such as complex reaction conditions, high energy consumption, heavy pollution or high production cost and large pollution in the chemical synthesis method, enzymatic method or microbial transformation method, and to provide a method for producing α-ketobutyric acid Genetically engineered bacteria and method for fermenting and producing α-ketobutyric acid using the bacteria

Method used

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  • Genetically engineered bacterium for producing alpha-ketobutyric acid and application of genetically engineered bacterium
  • Genetically engineered bacterium for producing alpha-ketobutyric acid and application of genetically engineered bacterium
  • Genetically engineered bacterium for producing alpha-ketobutyric acid and application of genetically engineered bacterium

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Experimental program
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Effect test

Embodiment 1

[0063] Example 1: Knockout of the gene ilvB encoding the large subunit of E.coli MG1655 acylhydroxyacid synthase I

[0064] Design primers ilvB1-F, ilvB1-R, ilvB3-F and ilvB3 for the amplification of the upper and lower homology arms according to the 400 bp sequence of the 5' and 3' ends of the ilvB (GeneID: 948181) gene in Escherichia coli MG1655 in the NCBI database -R, and use the bacterial genomic DNA as a template to amplify the homology arm fragment.

[0065] The PCR conditions were 94°C for 5min for one cycle, 94°C for 30s, 55°C for 30s, 72°C for 40s for 30 cycles, and 72°C for 10min as one cycle, and the reaction system was 100 μL.

[0066] The PCR products were subjected to 1.5% agarose gel electrophoresis and then gel-cut to recover, and the obtained fragments were named ilvB1 and ilvB3, respectively.

[0067] Amplification primers ilvB2-F and ilvB2-R were designed according to the sequence of the chloramphenicol resistance gene cassette in the plasmid pKD3, and the...

Embodiment 2

[0077] Example 2: Knockout of the gene ilvI encoding the large subunit of acetohydroxyacid synthase III in E.coli MG1655 ilvB knockout strain

[0078] Homology arm amplification primers ilvI1-F, ilvI1-R, ilvI3-F and ilvI3-R were designed according to the 5' and 3' end 400bp sequences of the Escherichia coli (Escherichia coli) MG1655ilvI (GeneID: 947267) gene in the NCBI database. The genome DNA of the bacterium was used as a template to amplify the homology arm fragment.

[0079] The PCR conditions were 94°C for 5min for one cycle, 94°C for 30s, 55°C for 30s, 72°C for 40s for 30 cycles, and 72°C for 10min as one cycle, and the reaction system was 100 μL.

[0080] Amplification primers ilvI2-F and ilvI2-R were designed according to the sequence of the chloramphenicol resistance gene cassette in the plasmid pKD3, and the fragment of the chloramphenicol resistance gene cassette was amplified using the plasmid as a template. The PCR conditions were 94 ° C for 5 min for 1 cycle, 9...

Embodiment 3

[0086] Construction of embodiment 3THRZ bacterial strain

[0087] According to the 5' and 3' end 400bp sequences of thrL (GeneID: 948283) gene in E. coli MG1655 in the NCBI database, the homology arm amplification primers thrL1-F, thrL1-R, thrL3-F and thrL3-R were designed, and The homology arm fragment was amplified using the bacterial genome DNA as a template.

[0088] The PCR conditions were 94°C for 5min for one cycle, 94°C for 30s, 55°C for 30s, 72°C for 40s for 30 cycles, and 72°C for 10min as one cycle, and the reaction system was 100 μL.

[0089] The PCR products were electrophoresed on a 1.5% agarose gel and recovered by cutting the gel, and the obtained fragments were named thrL1 and thrL3, respectively.

[0090] Amplification primers thrL2-F and thrL2-R were designed according to the sequence of the chloramphenicol resistance gene cassette in the plasmid pKD3, and the fragment of the chloramphenicol resistance gene cassette was amplified using the plasmid as a temp...

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Abstract

The invention relates to a genetically engineered bacterium for producing alpha-ketobutyric acid as well as a construction method and application of the genetically engineered bacterium, belonging to the technical field of biology. The strain is relatively high in production efficiency when used for producing alpha-ketobutyric acid through fermentation. The genetically engineered bacterium is obtained through carrying out genetic engineering modification on Escherichia coli MG1655; the genetic engineering modification is realized by knocking out a large-subunit encoding gene ilvB of acetohydroxyacid synthase I, a large-subunit encoding gene ilvI of acetohydroxyacid synthase III and a threonine operon leading peptide encoding gene thrL in an over-expressive threonine dehydratase encoding gene ilvA. When the bacterium is used for producing alpha-ketobutyric acid by using a fermentation method, defects such as complex reaction conditions, high energy consumption, serious pollution or high production cost, serious pollution and the like existing in a chemical synthesis method, an enzyme method or a microbial conversion method can be overcome; after the bacterium is fermented for 20-24h, the yield of alpha-ketobutyric acid is up to 8.5-15.7g / L; aerobic fermentation is adopted in a fermentation process; the bacterium is rapid in growth, short in fermentation period and high in acid production rate; any reports for producing alpha-ketobutyric acid by using a direct fermentation method are not found at present.

Description

Technical field: [0001] The invention relates to a genetically engineered bacterium for producing α-ketobutyric acid and its construction method and application. Fermentation and production of α-ketobutyric acid by using the bacterial strain has relatively high production efficiency and belongs to the field of biotechnology. Background technique: [0002] α-ketobutyric acid (α-ketobutyric acid), also known as 2-oxobutyric acid (2-Oxobutyric acid), 2-ketobutyric acid (2-Ketobutyric acid), etc., is the synthesis of many important chemical substances Intermediates are widely used in medicine, food, chemistry and other fields. For example, α-ketobutyric acid can be used to synthesize L-isoleucine, food flavor, 1-propanol, furanone, baolomycin, L-α-aminobutyric acid and D-α-hydroxybutyric acid and other substances . Among them, L-α-aminobutyric acid is a precursor for synthesizing antiepileptic drugs such as levetiracetam, and D-α-hydroxybutyric acid is used to prepare anticanc...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P7/40C12R1/19
Inventor 陈宁张成林谢希贤徐庆阳刘淑云刘远刘宏亮
Owner 江苏澳创生物科技有限公司
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