Biological improvement synthesis method of glutaric acid

A synthetic method, glutaric acid technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problem of loss of yield and so on

Active Publication Date: 2017-02-15
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Glutaconic acid is unsaturated at the α and β positions, and can be further reduced to glutaric acid by chemical catalysis in industry, but there is no method for directly converting glutaconic acid to glutaric acid in cells
On the other hand, when lysine is used as a substrate to synthesize glutaric acid, one mole of carbon dioxide will be released per mole of substrate, thus losing the yield to carbon (sugar), while when α-ketoglutarate is used as a substrate does not release carbon dioxide

Method used

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  • Biological improvement synthesis method of glutaric acid
  • Biological improvement synthesis method of glutaric acid
  • Biological improvement synthesis method of glutaric acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034]The construction of the biosynthetic pathway in this example is based on the heterologous expression of expression plasmids. The strains, plasmids, enzymes and culture media used include: the expression plasmids are pTrc15C'-egTer-HgdH-GctAB and pZS*27-HgdABC ‐TesB; expression host is E. coli MG1655; cloning host is E. coli DH5α; genetic manipulation tools include: restriction endonuclease, DNA polymerase, T4 DNA ligase; LB medium: each liter contains pancreatic Peptone 10g, yeast extract 5g, sodium chloride 10g, chloramphenicol concentration is 50mg / L, and kanamycin concentration is 50mg / L, and its specific steps include:

[0035] 1) PCR amplification and construction of recombinant plasmids: design primer sequences, amplify with the whole genome sequence of the corresponding strain or whole gene synthetic DNA as a template, cut with endonuclease and connect to the expressed On the plasmid vector, wherein: egter, hgdH, gctA and gctB are expressed in the pTrc15C' plasmid...

Embodiment 2

[0054] Fermentative Production of Glutaric Acid Using Recombinant Strains

[0055] Strains and culture conditions:

[0056] The medium used by the E.coli MG1655 recombinant Escherichia coli is a fermentation medium, specifically pH 7.4 supplemented Stand I medium, and its components and contents are: 15g / L beef peptone, 3g / L yeast extract, 50mM 3‐(N‐morpholine)propanesulfonic acid, 3mM L‐cysteine, 10mM sodium glutamate, 0.2mM riboflavin, 2mM ferric citrate.

[0057] The E.coli MG1655 recombinant Escherichia coli was first activated by LB medium, and then the initial inoculum was OD 600 =0.1 into the supplemented Stand I medium, and the cells were cultured to OD at 37°C 600 When it was about 1.0, 100 μM IPTG was added and cultured at 30° C. for several days.

[0058] In the above cultivation process, according to the difference in oxygen supply, it is divided into aerobic, microaerobic and anaerobic cultivation. Under aerobic conditions, the cells were cultured in a 100ml s...

Embodiment 3

[0064] Catalytic ability of different thioesterases for the hydrolysis of glutaryl-CoA.

[0065] In Example 2, the last reaction step, ie, the hydrolysis of glutaryl-CoA to glutaric acid, was catalyzed by the thioesterase TesB of Escherichia coli. TesB originally catalyzes the hydrolysis of short- and medium-chain fatty acyl-CoA in E. coli, but it is also active on some other substrates, including glutaryl-CoA. On the other hand, there are many different thioesterases in organisms, and their substrate selectivity and activity are different. Therefore, it is necessary to screen enzymes with higher activity for hydrolysis of glutaryl-CoA.

[0066] Strains and culture conditions:

[0067] The enzymes in this example were obtained through exogenous expression and purification. The exogenous protein expression host is E.coli BL21(DE3), the expression vector is pET-28a(+), and the gene to be expressed is inserted into the expression vector by restriction endonuclease for expressi...

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Abstract

The invention relates to a biological improvement synthesis method of glutaric acid. According to the present invention, the method is achieved by expressing 2-hydroxyglutaric acid dehydrogenase, glutaryl-coenzyme A transferase, 2-hydroxyglutaryl coenzyme A dehydratase, trans-CoA reductase and thioesterase in a recombinant host Escherichia coli, the recombinant Escherichia coli expressing the genes is subjected to fermentation production, and the generation of the target product glutaric acid and the by-products such as glutaconic acid and 2-hydroxyglutaric acid is successfully detected in the fermentation broth; and the constructed biological synthesis approach provides the new method for the utilization of the renewable resources to synthesize the glutaric acid.

Description

technical field [0001] The present invention relates to a new glutaric acid synthesis pathway, that is, through 2-hydroxyglutarate dehydrogenase, glutaconate-CoA transferase, 2-hydroxyglutaryl-CoA dehydratase, enoyl-CoA Catalyzed by reductase and acyl-CoA thioesterase, the intracellular tricarboxylic acid cycle intermediate α-ketoglutaric acid is converted into glutaric acid, so as to achieve the purpose of using glucose or other carbon sources to ferment and produce glutaric acid. It specifically relates to the cloning and co-expression of genes encoding these enzymes, and the application of recombinant Escherichia coli to ferment and produce glutaric acid using the catalytic function of these enzymes. Background technique [0002] Glutaric acid is an important chemical raw material, which can be polymerized with diamines of different lengths of carbon chains to form polyamides. As an engineering plastic, polyamide has excellent properties and is widely used in various ind...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12P7/44C12R1/19
Inventor 钱志刚於佳乐夏小霞钟建江
Owner SHANGHAI JIAO TONG UNIV
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