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Recombinant genetically engineered bacterium of co-expressing olefine aldehyde reductase and glucose dehydrogenase and application of recombinant genetically engineered bacterium

A technology of glucose dehydrogenase and genetically engineered bacteria, applied in genetic engineering, oxidoreductase, applications, etc., can solve the problems that have not yet been seen in co-expressing enaldehyde reductase and glucose dehydrogenase recombinant E. Large-scale industrial production, lower production cost, high vitality effect

Inactive Publication Date: 2020-01-03
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there is no report on the construction of recombinant Escherichia coli co-expressing enaldehyde reductase and glucose dehydrogenase, and there is no report on the use of this recombinant cell to synthesize isopental by coupling enaldehyde reductase and glucose dehydrogenase to catalyze the reduction of prenyl aldehyde enol reports

Method used

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  • Recombinant genetically engineered bacterium of co-expressing olefine aldehyde reductase and glucose dehydrogenase and application of recombinant genetically engineered bacterium
  • Recombinant genetically engineered bacterium of co-expressing olefine aldehyde reductase and glucose dehydrogenase and application of recombinant genetically engineered bacterium
  • Recombinant genetically engineered bacterium of co-expressing olefine aldehyde reductase and glucose dehydrogenase and application of recombinant genetically engineered bacterium

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Example 1 Acquisition of the gene encoding alkenal reductase of Yokenella sp. WZY002

[0040] Using the published alkenal reductase encoding gene (GenBank accession number KF887947) derived from Yokenella sp. WZY002, after codon optimization, artificial synthesis (Suzhou Jinweizhi Biotechnology Co., Ltd. provides gene synthesis services ) The alkenal reductase encoding gene, the amino acid sequence and nucleotide sequence are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively.

Embodiment 2

[0041] Example 2 Acquisition of D-glucose dehydrogenase encoding gene derived from Exiguobacterium

[0042] Using the published gene encoding D-glucose dehydrogenase from Exiguobacterium sibiricum (GenBank accession number ACB59697.1), after codon optimization, artificial synthesis (Suzhou Jinweizhi Biotechnology Co., Ltd. provides gene synthesis service) The gene encoding D-glucose dehydrogenase derived from Exiguobacterium, the amino acid sequence and nucleotide sequence are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

Embodiment 3

[0043] Example 3 Construction of recombinant genetically engineered bacteria co-expressing alkenaldehyde reductase and D-glucose dehydrogenase

[0044] The alkenol reductase encoding gene (SEQ ID NO. 2) and the D-glucose dehydrogenase encoding gene (SEQ ID NO. 4) were inserted into Nco I, Hind III and Nde I, Xho on the pACYCDuet1 vector by "one-step cloning" Between two pairs of restriction sites, the recombinant plasmid pACYCDuet-1-YsADH-EsGDH ( figure 2 ). The recombinant plasmid pACYCDuet-1-YsADH-EsGDH was transformed into E.coli BL21(DE3) to obtain genetically engineered bacteria E.coli BL21(DE3) / pACYCDuet-1-YsADH-EsGDH.

[0045] The recombinant genetically engineered bacteria E.coliBL21(DE3) / pACYCDuet-1-YsADH-EsGDH was inoculated on LB solid medium containing 50 μg / mL chloramphenicol and streaked, and a single colony was picked and inoculated with 50 mL LB liquid medium. and add the final concentration of 50 μg / mL chloramphenicol, culture at 37 °C and 200 rpm constant ...

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Abstract

The invention discloses a recombinant genetically engineered bacterium of co-expressing olefine aldehyde reductase and glucose dehydrogenase and application of the recombinant genetically engineered bacterium to catalyzes the synthesis of allylic alcohols with methylcrotonaldehyde. The genetically engineered bacterium is obtained by co-introducing olefine aldehyde reductase gene and D- glucose dehydrogenase into a host bacteria. A method has the advantages of being high in regioselectivity and high in activity. The 500mM substrate methylcrotonaldehyde is completely converted into the product of the allylic alcohols in 3.5h, no by-product saturated alcohol is detected during the reaction, and it is indicated that the method is efficient and specific in catalyzing the C=O hydrogenation of methylcrotonaldehyde to obtain the corresponding allylic alcohols. Meanwhile, the recombinant cells induce the production of D-glucose dehydrogenase, with glucose as a co-substrate, the glucose dehydrogenase can continuously convert NADP+ to NADPH, additional coenzymes do not need to be added in the reaction process, thus the production cost is greatly lowered, and the method is more suitable for large-scale industrial production.

Description

[0001] (1) Technical field [0002] The present invention relates to a recombinant cell co-expressing alkenaldehyde reductase and glucose dehydrogenase and its application in the synthesis of prenol. [0003] (2) Background technology [0004] α,β-Unsaturated alcohols are very important intermediates in organic synthesis, which are widely used in the production of perfumes, pharmaceuticals and other fine chemicals. Prenol is one of the important α, β-unsaturated alcohols, its scientific name is 3-methyl-2-buten-1-ol, the relative molecular weight is 86.13, and the density is 0.848g·cm -3 , the boiling point is 140℃, the flash point is 43℃, it is a colorless transparent liquid, the solubility in water is 170g·L -1 (20°C). Prenol can be used for: (1) synthesizing the intermediate of high-efficiency and low-toxicity pesticide pyrethroid and its downstream products; (2) synthesizing the intermediate of flavor and fragrance (such as citral); (3) Thiogeraniol, which can be widely ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/53C12P7/04C12R1/19
CPCC12N9/0006C12P7/04
Inventor 应向贤乔艳汪钊
Owner ZHEJIANG UNIV OF TECH
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