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A method for metabolically engineering Escherichia coli to prepare α-ketoisovaleric acid

A recombinant Escherichia coli, heterogeneous technology, applied in the field of bioengineering

Active Publication Date: 2022-02-11
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In addition, there are very few studies on the production of α-ketoisovaleric acid by Escherichia coli fermentation. α-ketoisovaleric acid is only accumulated as a by-product of isobutanol production

Method used

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  • A method for metabolically engineering Escherichia coli to prepare α-ketoisovaleric acid
  • A method for metabolically engineering Escherichia coli to prepare α-ketoisovaleric acid
  • A method for metabolically engineering Escherichia coli to prepare α-ketoisovaleric acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Construction and optimization of acetyl lactate synthetase, an acetyl lactate isomeric reductase and dihydroxy acid dehydrat

[0035] 1) Amino acid sequence of acetyl lactic acid synthetase, such as SEQ ID NO.1, nucleotide sequence, such as SEQ ID NO.2; amino acid sequence of an acetyl lactate isomorbed, as shown in SEQ ID NO.3, The prime sequence is shown in SEQ ID NO.4. The amino acid sequence of dihydroxy acid dehydratase, as shown in SEQ ID NO.5, the nucleotide sequence such as SEQ ID NO.6, according to the purpose gene and the carrier, and is designed to design the primer (such as Table 1);

[0036] Table 1 Plasmid construct primer design

[0037]

[0038]

[0039] Note: Underline marking is an enzyme cleaning point

[0040] 2) The E.COLI MG1655 genome is a template, respectively, and cloned the gene BSALSS, ECILVC, ECILVD. The plasmid pETDUET-1 with cloned gene BSALSS, ECILVC, and ECILVD were double-cutted by the obtained enzyme digestion in Table 1, an...

Embodiment 2

[0053] Example 2: Method for the construction of alpha-ketoisukoic acid engineering strains of the knockout competitive metabolic pathway

[0054] 1) Departure strain is the main coli B0016-050 (ΔAck-PTA, ΔPFLB, ΔAck-PTA, ΔPFLB, ΔAdhe, ΔFrda, ΔLDHA) (disclosed in literature z et al. " Appl.biochem. Biotechnol., 2016,178: 324-37. In addition to the coding genes of by-product acetic acid, formic acid, ethanol, succinic acid, lactic acid synthesis, can avoid a lot of these metabolic by-products Synthesis, and provide sufficient pyruvate precursors for fermentation of α-ketoisuate. In order to ensure the normal exercise of the T7 promoter, the T7 RNA polymerase (T7 RNAP) gene is integrated on the genome, and also to block the further decomposition of α-keto ketoikoic acid, encoding the chromosome upper strand amino acid amino enzyme encoding gene ILVE Using the RED recombination method for knockout.

[0055] 2) The genome is a template in Escherichia coli E.COLI BL21 (DE3), and the P3...

Embodiment 3

[0076] Example 3: Construction Method for Coenzyme NADPH Circulating Regenerated α-ketoisuate

[0077] 1) Template is a template in Escherichia coli genomes and plasmid Pacycduet, P49 + P50, P51 + P52 in Table 3, and amplified PNTab fragment and plasmid skeleton PACYC, and the two will be subjected to Gibson assembled to obtain recombinant plasmid PACYC-PNTAB, respectively. .

[0078] 2) The plasmid pACYC-PNTAB and PKD13 are templates, P53 + P54, P55 + P56 in Table 3, and amplified to obtain the plasmid skeleton PACYC-PNTAB and KAN fragments, and the two will be subjected to Gibson assembly to obtain recombinant plasmid PACYC. -kan-pntab.

[0079] 3) Template with plasmid pacyc-kan-pntab, P57 + P58, P59 + P60, P61 + P62 in Table 3, to perform full-quality PCR, to obtain a T7 promoter of different strength, and delete the T7 promoter Laco gene to get recombinant plasmid PACYC-KAN-T7 100 PACYC-KAN-T7 92 PACYC-KAN-T7 16 .

[0080] 4) Particles PACYC-KAN-T7, respectively 100 PACYC-KAN...

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Abstract

The invention discloses a method for preparing α-ketoisovaleric acid by transforming Escherichia coli with metabolic engineering, and belongs to the field of bioengineering. The present invention constructs a recombinant bacterial strain coupling expressing acetolactate synthase, acetolactate isomerizing reductase and dihydroxyacid dehydratase, and transforms and optimizes the host strain to obtain efficient production of α-ketoisoamyl by fermentation Acid recombinant bacteria. The recombinant strain can use cheap glucose as a substrate to ferment α-ketoisovaleric acid with higher added value. After 36 hours of fermentation, the yield of α-ketoisovaleric acid can reach 22.91g / L, and the conversion rate of glucose can reach 80%.

Description

Technical field [0001] The present invention relates to a method of preparing alpha-ketoisua in metabolic engineering to prepare α-ketozoic acid, which belongs to the field of biological engineering. Background technique [0002] α-Ketoisovalerate is a kind of keto acid, which has a wide range of applications and larger market development potential in the fields of pharmaceutical, food, cosmetics. [0003] Currently, α-ketoisua can be synthesized by chemical method and biological conversion method, and mainly in chemical synthesis methods. However, the chemical process is cumbersome and the reaction conditions are harsh, accompanied by the production of poison by-products, and is not suitable for large-scale industrial production. The biochemical method is converted to α-ketoisua under the catalysis of L-proline as a substrate in amino acid decetonase and amino acid oxidase. The use of amino acid oxidase is an alpha-ketone has achieved industrialization in our country. However, t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/40C12N1/21C12R1/19
CPCC12P7/40C12N9/1022C12N9/90C12N9/88C12Y202/01006C12Y504/99003C12Y402/01009
Inventor 周哲敏周丽崔文璟刘中美郭军玲程中一李雅婷朱滢
Owner JIANGNAN UNIV
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