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A genetically engineered bacterium producing α-ketobutyric acid and its application

A technology of genetically engineered bacteria and ketobutyric acid, applied in the biological field, can solve problems such as complex reaction conditions, high production costs, and heavy pollution, and achieve the effects of simple fermentation process, low production cost, and high acid production rate

Active Publication Date: 2017-01-11
江苏澳创生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] 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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  • A genetically engineered bacterium producing α-ketobutyric acid and its application
  • A genetically engineered bacterium producing α-ketobutyric acid and its application
  • A genetically engineered bacterium producing α-ketobutyric acid and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1: E.coli MG1655 acylhydroxy acid synthase I large subunit coding gene ilvB knockout

[0068] According to the 5'and 3'400bp sequence of the ilvB (GeneID:948181) gene of Escherichia coli MG1655 in the NCBI database, the upper and downstream homology arm amplification primers ilvB1-F, ilvB1-R, ilvB3-F and ilvB3 were designed -R, and use the genomic DNA of the bacteria as a template to amplify homologous arm fragments.

[0069] The PCR conditions were 94°C for 5min, 1 cycle, 94°C for 30s, 55°C for 30s, 72°C for 40s, 30 cycles, 72°C for 10min, 1 cycle, and the reaction system was 100μL.

[0070] The PCR product was subjected to 1.5% agarose gel electrophoresis and then gelled and recovered, and the obtained fragments were named ilvB1 and ilvB3.

[0071] The amplification primers ilvB2-F and ilvB2-R were designed according to the chloramphenicol resistance gene cassette sequence in the plasmid pKD3, and the chloramphenicol resistance gene cassette fragment was amplified usi...

Embodiment 2

[0081] Example 2: E.coli MG1655ilvB knockout strain acetohydroxy acid synthase III large subunit coding gene ilvI knockout

[0082] Design homology arm amplification primers ilvI1-F, ilvI1-R, ilvI3-F and ilvI3-R according to the 5'and 3'400bp sequences of Escherichia coli MG1655ilvI (GeneID:947267) gene in NCBI database, and The genomic DNA of the bacteria is used as a template to amplify homologous arm fragments.

[0083] The PCR conditions were 94°C for 5 minutes, 1 cycle, 94°C for 30s, 55°C for 30s, 72°C for 40s, 30 cycles, and 72°C for 10 minutes, and the reaction system was 100 μL.

[0084] The 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 plasmid was used as a template to amplify the chloramphenicol resistance gene cassette fragment. The PCR conditions were 94°C for 5 min and 1 cycle. 30 cycles of 94°C for 30s, 55°C for 30s, 72°C for 70s, and 72°C for 10min for 1 cy...

Embodiment 3

[0090] Example 3 Construction of THRZ strain

[0091] Design homology arm amplification primers thrL1-F, thrL1-R, thrL3-F and thrL3-R according to the 5'and 3'end 400bp sequences of thrL (GeneID:948283) gene in E.coli MG1655 in NCBI database, and The genomic DNA of the bacteria was used as a template to amplify homologous arm fragments.

[0092] The PCR conditions were 94°C for 5 minutes, 1 cycle, 94°C for 30s, 55°C for 30s, 72°C for 40s, 30 cycles, and 72°C for 10 minutes, and the reaction system was 100 μL.

[0093] The PCR products were subjected to 1.5% agarose gel electrophoresis and then gelled and recovered. The obtained fragments were named thrL1 and thrL3.

[0094] The amplification primers thrL2-F and thrL2-R were designed according to the chloramphenicol resistance gene cassette sequence in the plasmid pKD3, and the chloramphenicol resistance gene cassette fragment was amplified using the plasmid as a template.

[0095] The PCR conditions were 94°C for 5 minutes, 1 cycle, 94...

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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

[0001] Technical field: [0002] The invention relates to a genetically engineered bacteria for producing α-ketobutyric acid, and a construction method and application thereof. The fermentation production of α-ketobutyric acid by using the strain has high production efficiency and belongs to the field of biotechnology. [0003] Background technique: [0004] α-ketobutyric acid, also known as 2-Oxobutyric 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 flavors, 1-propanol, furanone, paulomycin, L-α-aminobutyric acid and D-α-hydroxybutyric acid, etc. . Among them, L-α-aminobutyric acid is a precursor for the synthesis of antiepileptic drugs such as levetiracetam, and D-α-hydroxybutyric acid is used to prepare azinothricin family anticancer drugs. [0005] The current production methods of α-keto...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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