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

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...

AI Technical Summary

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

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