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Co-expressing recombinant bacterium and application thereof in synthesizing furan carboxylic acid

A technology of recombinant bacteria and co-expression, which is applied in the field of genetic engineering technology and biocatalysis, can solve the problems of reducing the catalytic efficiency and reaction selectivity of recombinant bacteria, and achieve the effects of reducing production costs, good stability, and simple reaction process

Pending Publication Date: 2019-12-20
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, recombinant bacteria overexpressing these aldehyde dehydrogenases will produce a certain amount of by-product 2,5-bis(hydroxymethyl)furan (BHMF) in the synthesis of catalyzed HMFCA, thus reducing the catalytic efficiency and reaction selectivity of recombinant bacteria

Method used

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  • Co-expressing recombinant bacterium and application thereof in synthesizing furan carboxylic acid
  • Co-expressing recombinant bacterium and application thereof in synthesizing furan carboxylic acid
  • Co-expressing recombinant bacterium and application thereof in synthesizing furan carboxylic acid

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Example 1 Construction method of co-expressing aldehyde dehydrogenase and NOX recombinant bacteria

[0036] (1) Using C.testosteroniSC1588 genomic DNA as a template, amplify the full-length sequences of CtCALDH2, CtVDH1, CtVDH2 and CtSAPDH genes by designing specific primers (the nucleotide sequences are SEQ ID.1, SEQID.2, SEQ ID.3 respectively or SEQ ID.4);

[0037] (2) Using the recombinant plasmid pET28a-NOX carrying the NOX gene (the nucleotide sequence accession number on GenBank is CP021479.1) as a template, amplify the full-length sequence of the NOX gene by designing specific primers;

[0038] (3) connecting the aldehyde dehydrogenase gene and the NOX gene into the co-expression plasmid pETDuet-1 to obtain a co-expression recombinant plasmid, and verify by sequencing;

[0039] (4) Transform the above-mentioned co-expression recombinant plasmid into E.coliBL21(DE3) to obtain recombinant bacteria E.coli-CtCALDH2-NOX, E.coli-CtVDH1-NOX, E.coli-CtVDH1-NOX, E.coli- ...

Embodiment 2E

[0042] Example 2 Induced expression of E.coli-CtVDH1-NOX and E.coli-CtVDH2-NOX

[0043] The recombinant bacterium E.coli-CtVDH1-NOX (or E.coli-CtVDH2-NOX) obtained in Example 1 was inoculated into LB liquid medium containing 100 μg / mL ampicillin (tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, pH 7.2), cultured at 37°C and 180rpm for 12h. Then, transfer the above-mentioned bacterial suspension to LB liquid medium containing 100 μg / mL ampicillin according to the inoculum amount of 1%, cultivate it at 37°C and 180 rpm, when the OD of the bacterial solution 600 When it reaches 0.6-0.8, add 0.1mM isopropyl-β-D-thiogalactoside, place it at 20°C and 160rpm to induce culture for 20h, collect the bacterial cells after the culture, and wash the cells with 0.85% normal saline Twice, to obtain the induced expression recombinant bacteria E.coli-CtVDH1-NOX (or E.coli-CtVDH2-NOX).

Embodiment 3

[0045] Add 0.4mmol HMF (initial concentration of HMF is 100mM) in 4mL phosphate buffer (200mM, pH7.0), after mixing evenly, add the concentration obtained through Example 2 at a concentration of 50mg / mL (according to the wet weight of cells). The recombinant strain E. coli-CtVDH1-NOX was reacted at 30°C and 150rpm. The reaction was monitored by liquid chromatography ( image 3 ). After 2h, the conversion rate of HMF was 100%, and the yield of target product HMFCA was 96%.

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Abstract

The present invention belongs to the fields of genetic engineering technology and biocatalysis, and discloses a co-expressing recombinant bacterium and an application thereof in synthesizing furan carboxylic acid. A double-enzyme co-expression technology is used to construct a co-expressing system of an aldehyde dehydrogenase gene derived from comamonas testosteroni SC1588 respectively with 5-hydroxymethylfurfural oxidoreductase and NADH oxidase genes in Escherichia coli BL21 (DE3) to obtain the co-expressing recombinant bacterium. The co-expressing recombinant bacterium can catalyze oxidationof substrates with high concentration (150-250 mM) to synthesize target products with a yield of 90% or more and a spatiotemporal yield as high as 5.6 g / L h. The prepared biocatalyst has advantages of high-concentration substrate tolerance, high catalytic activity, and high selectivity. Besides, the furan carboxylic acid synthesis technology is mild in conditions, green and highly efficient.

Description

technical field [0001] The invention belongs to the field of genetic engineering technology and biocatalysis, and specifically relates to recombinant bacteria co-expressing aldehyde dehydrogenase and NADH oxidase, aldehyde dehydrogenase and 5-hydroxymethylfurfural oxidoreductase, and the ability of these co-expressing recombinant bacteria to catalyze furan aldehyde Applications in selective oxidation synthesis of furan carboxylic acids. Background technique [0002] Furan carboxylic acid such as 5-hydroxymethyl-2-furancarboxylic acid (5-hydroxymethyl-2-furancarboxylic acid, HMFCA), 2-furoic acid (2-furoic acid, FCA), 5-methoxymethyl-2-furfuran Acid (5-methoxymethyl-2-furancarboxylic acid, MMFCA) and 2,5-furandicarboxylic acid (2,5-furandicarboxylic acid, FDCA) is an important class of bio-based chemicals, in the fields of polymers, medicine and food have a broad vision of application. For example, HMFCA can be used as a monomer for synthetic polyester (Makromol.Chem., 1984...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/53C12P17/04C12R1/19
CPCC12N9/0036C12N9/0008C12Y106/99003C12Y102/01003C12N9/0006C12Y101/03C12P17/04
Inventor 李宁张雪莹王欣宗敏华
Owner SOUTH CHINA UNIV OF TECH
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