Construction method of 3-hydroxy propionate high-yielding strain recombinant plasmids

The technology of a hydroxypropionic acid bacteria and a recombinant plasmid is applied in the field of biochemical production and can solve the problems of weak start-up ability, influence on the yield of 3-hydroxypropionic acid, complex chemical synthesis method and the like

Active Publication Date: 2015-07-29
BEIJING UNIV OF CHEM TECH
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Problems solved by technology

[0002] 3-Hydroxypropionic acid (3-HP) has high commercial value in industrial applications, but its chemical synthesis method is complicated
The promoter has the disadvantages of weak starting ability, so that the protein expression is small, which affects the production of 3-hydroxypropionic acid

Method used

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  • Construction method of 3-hydroxy propionate high-yielding strain recombinant plasmids
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  • Construction method of 3-hydroxy propionate high-yielding strain recombinant plasmids

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Embodiment Construction

[0029] The following specific methods can enable those skilled in the art to understand the present invention more comprehensively, but do not limit the present invention in any way.

[0030] The concrete steps of the inventive method comprise:

[0031] 1. Cloning of tac promoter and aldehyde dehydrogenase puuc in Klebsiella pneumoniae

[0032] Bacterial strains and plasmids: Escherichia coli (E.coli) top10 was purchased from Beijing Bomed Company, Klebsiella pneumoniae (Klebsiella pneumoniae) DSM2026 was purchased from German DSM, and expression vector pET-28a was purchased from Novozymes.

[0033] Medium: LB medium (g / L) peptone 10, yeast powder 5, NaCl 10, add 1.5% (1.5g agar in 100ml liquid medium) agar in the solid medium. The resistance medium needs to be added with 50 μL / mL kanamycin sulfate.

[0034] Klebsiella pneumoniae fermentation medium (g / L): K 2 HPO 4 ·3H 2 O, 3.4; KH 2 PO 4 , 1.3; (NH 4 ) 2 SO 4 , 4; MgSO 4 ·7H 2 O, 0.5; CaCO 3 , 0.1; yeast powder,...

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Abstract

The invention discloses a construction method of 3-hydroxy propionate high-yielding strain recombinant plasmids and belongs to the production field of biochemical industry. In view of the fact that most conventional yields of 3-hydroxy propionate engineering bacteria are comparatively low, an effective tac promoter applicable to klebsiella pneumonia and puuc aldehyde dehydrogenase capable of converting 3-hydroxy propionaldehyde into 3-hydroxy propionate are discovered to construct and recombine the engineering bacteria. After the sequence of the encoding gene of the enzyme is analyzed, the gene is chemically synthesized; after sequencing confirmation, the promoter and enzyme are overexpressed in klebsiella pneumonia to obtain recombined klebsiella pneumonia pET (ptac-puuc) and the catalytic ability of the enzyme for 3-hydroxy propionaldehyde in vivo and in vitro is measured, wherein in vivo, the enzyme activity of the enzyme for 3-hydroxy propionaldehyde reaches 26.31 U / mg and is 6.9 times of the enzyme activity of the enzyme for original klebsiella pneumonia (3.82 U / mg). 48 hours after optimizing the medium components and IPTG concentration condition, the yield of 3-hydroxy propionate in the engineering bacteria cultivated and reformed in a 5L fermentation tank reaches 73 g / L.

Description

technical field [0001] The invention belongs to the field of biochemical production, and relates to a method for constructing a recombinant plasmid of a high-yielding 3-hydroxypropionic acid genetically engineered bacterium. Background technique [0002] 3-Hydroxypropionic acid (3-HP) has high commercial value in industrial applications, but its chemical synthesis method is complicated. In recent years, as synthetic biology has become a research hotspot in biochemical industry, the production of 3-hydroxypropionic acid by microbial synthesis is in urgent need of a breakthrough. Klebsiella pneumoniae (Klebsiella pneumoniae) has a strong ability to metabolize glycerol, and is very suitable as an expression host from the perspective of biomass accumulation. Klebsiella pneumoniae utilizes glycerol to produce the intermediate product 3-hydroxypropionaldehyde, which is then catalyzed by aldehyde dehydrogenase to produce 3-hydroxypropionate. [0003] In the method for producing 3...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N1/21C12P7/42C12R1/22
Inventor 田平芳陈柳霓李映
Owner BEIJING UNIV OF CHEM TECH
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