Construction method of onion pseudomonas genetic engineering bacteria

Abstract

The present invention provides a method for constructing a genetically engineered bacterium from Pseudomonas cepacia, which replaces the lipase in the genome of Pseudomonas cepacia G63 and a chaperone gene with T7 RNA polymerase genes thereof, so that the T7 RNA polymerase genes are integrated into the genome of Pseudomonas cepacia and controlled by a lipase promoter. Afterwards, the Pseudomonas cepacia lipase and the chaperone gene thereof are cloned into expression plasmid containing T7 promoters and LacI repressor protein, and finally, the plasmid is delivered into the recombinant bacterium of Pseudomonas cepacia to obtain the super engineered bacterium of the Pseudomonas cepacia lipase. The method genetically modifies Pseudomonas cepacia G63, and the obtained super engineered bacterium has high expression and catalysis activity and good low-carbon alcohol tolerance and high-temperature resistance.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for constructing lipase genetically engineered bacteria. Background technique [0002] Today, when the energy crisis is intensifying, it is an extremely important and urgent task to find efficient and clean renewable energy, and it is undoubtedly the most effective and direct way to convert waste animal and vegetable oils into biodiesel. At present, there are mainly two kinds of biodiesel conversion methods: chemical method and enzymatic method. Among them, the chemical method has high energy consumption and high pollution; the enzymatic method has the advantages of mild reaction conditions and no pollution, and is an effective way to solve the energy crisis. [0003] In the process of enzymatic biodiesel preparation, the cost of lipase is the highest, which is one of the main bottlenecks restricting the further development of the process. The efficiency and ...

AI Technical Summary

Problems solved by technology

However, Pseudomonas cepacia lipase needs to be expressed correctly with the help of its own molecular chaperone, and its secretion is also closely related to more than a dozen proteins such as the intracellular Dsb complex. Due to the lack of corresponding expression and secretion mechanisms, Heterologous expression of Pseudomonas cepacia lipase is often inactive, and even with in vitro renaturation, the efficiency and yield are very low

However, lipase-producing bacteria also have problems such as unclear metabolic regulation mechanism, low enzyme production, and unstable expression.

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