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Aldehyde dehydrogenases, gene thereof, construction of recombinant strain and application of recombinant strain in synthesis of furancarboxylic acid

A technology of aldehyde dehydrogenase and furan carboxylic acid, which is applied in the field of genetic engineering technology and biocatalysis, can solve the problems of reduced conversion efficiency and prolonged reaction time, and achieve the effects of simple production process, high production efficiency and mild production conditions

Active Publication Date: 2019-03-29
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, with the increase of the substrate concentration, the reaction time gradually prolongs and the conversion efficiency gradually decreases.

Method used

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  • Aldehyde dehydrogenases, gene thereof, construction of recombinant strain and application of recombinant strain in synthesis of furancarboxylic acid
  • Aldehyde dehydrogenases, gene thereof, construction of recombinant strain and application of recombinant strain in synthesis of furancarboxylic acid
  • Aldehyde dehydrogenases, gene thereof, construction of recombinant strain and application of recombinant strain in synthesis of furancarboxylic acid

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Mining of Aldehyde Dehydrogenase Encoding Genes

[0044] In order to dig out the key enzymes in the genome of C. testosteroni SC1588 that can catalyze the oxidation of bio-based furan to furan carboxylic acid, Shenzhen Huada Gene Technology Service Co., Ltd. was entrusted to sequence the draft genome of C. testosteroni SC1588. According to Functional annotation of the predicted coding sequence, mining five aldehyde dehydrogenase genes from the genome, namely CtCALDH1 gene, CtCALDH2 gene, CtVDH1 gene, CtVDH2 gene and CtSAPDH gene.

Embodiment 2

[0046] Construction of recombinant bacteria

[0047] (1) Using the genomic DNA of Comamonas testosteroni SC1588 as a template, the full-length sequences of CtCALDH1, CtCALDH2, CtVDH1, CtVDH2 and CtSAPDH genes were amplified by designing specific primers;

[0048] (2) connecting the aldehyde dehydrogenase gene into the expression vector pET-28a to obtain a recombinant plasmid;

[0049] (3) The recombinant plasmid verified by sequencing was transformed into the expression host E.coli BL21(DE3), and the recombinant bacteria E.coli BL21(DE3)_CtCALDH1, E.coli BL21(DE3)_CtCALDH2, E.coli BL21(DE3)_CtCALDH2, E .coli BL21(DE3)_CtVDH1, E.coli BL21(DE3)_CtVDH2 and E.coli BL21(DE3)_CtSAPDH.

[0050] Table 1 Primer Information

[0051] primer name

Embodiment 3

[0053] Inducible expression of aldehyde dehydrogenase

[0054] The recombinant bacteria obtained in Example 2 were inoculated into 30 mL of LB liquid medium containing 50 μg / mL kanamycin (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, pH 7.2) , incubated at 37°C and 180rpm for 12h. Then, 1% of the inoculum was inoculated into 100 mL of LB medium containing 50 μg / mL kanamycin, and cultured at 37° C. and 180 rpm. When the OD of the bacterial solution 600 When reaching 0.6 to 0.8, IPTG was added to make the final concentration 0.1 mM, and the cells were incubated at 18° C. and 160 rpm for 20 h. After the culture, the cells were centrifuged at 8000 rpm and 4°C for 5 min to collect the bacterial cells, and the cells were washed twice with 0.85% normal saline.

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Abstract

The present invention relates to the fields of genetic engineering technology and biocatalysis, and discloses aldehyde dehydrogenases, a gene thereof, construction of a recombinant strain and an application of the recombinant strain in synthesis of furancarboxylic acid. A gene mining technology is used to screen five aldehyde dehydrogenases from comamonas testosteroni SC1588, and amino acid sequences of the aldehyde dehydrogenases are shown in SEQ ID. 1, SEQ ID. 2, SEQ ID. 3, SEQ ID. 4 or SEQ ID. 5. The five aldehyde dehydrogenases can all efficiently catalyze a bio-based furan selective oxidation to synthetize the corresponding furancarboxylic acid. The yield of a target product is up to about 90%, the highest yield can even reach 100%, and a space-time yield is up to 1.9 g.L<-1>.h<-1>. The production process is simple, production conditions are mild and production efficiency is high.

Description

technical field [0001] The invention belongs to the field of genetic engineering technology and biocatalysis, and particularly relates to five kinds of aldehyde dehydrogenases derived from Comamonas testosteroni SC1588, coding genes, recombinant bacteria expressing aldehyde dehydrogenases, and their catalytic biological bases. Application of furan selective oxidation to furan carboxylic acid. Background technique [0002] Furan carboxylic acid such as 5-hydroxymethyl-2-furancarboxylic acid (5-hydroxymethyl-2-furancarboxylic acid, HMFCA), 2-furancarboxylic acid (2-furancarboxylic acid, FCA) and 2,5-furan dicarboxylic acid (2,5-furandicarboxylic acid, FDCA) is an important class of bio-based chemicals with important application value in the fields of polymers and medicine. For example, HMFCA is a key raw material for the synthesis of various bio-based polyesters (Makromol. Chem., 1984, 185, 2347) and bio-based terephthalic acid (ACS Catal., 2016, 6, 5052). Polyethylene terep...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N1/21C12P17/04C12R1/19
CPCC12N9/0008C12N15/70C12P17/04C12Y102/01067C12Y102/01068C12Y102/01083
Inventor 李宁张雪莹宗敏华
Owner SOUTH CHINA UNIV OF TECH
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