A kind of genetically engineered bacteria and its construction method and method for producing vanillin

Abstract

The invention belongs to the technical field of bioengineering, and discloses a genetically engineered bacterium, a construction method thereof and a method for producing vanillin. The genetically engineered bacteria of the present invention transform the metabolic pathway of 3-dehydroshikimic acid, delete the gene encoding shikimate dehydrogenase, and simultaneously delete the genes related to vanillin degradation, so that the bacterial strain accumulates a large amount of 3-dehydroshikimic acid , and enhanced the expression of 3-dehydroshikimate dehydratase gene, O-methyltransferase gene and vanillic acid reductase gene and 2 genes of 3-dehydroshikimate metabolic pathway, thereby increasing the production of vanillin, At the same time, the degradation of vanillin is reduced, the amount of vanillin in the fermented liquid is increased, and the ability of bacterial strains to produce vanillin by fermentation is improved. Experiments show that the genetically engineered bacterium of the present invention uses glucose or glycerin as a substrate to produce vanillin, and the yield of vanillin is obviously increased. Moreover, there is no iso-vanillin impurity in the vanillin fermented liquid produced by fermentation by the method of the present invention, which is convenient for subsequent separation.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a genetically engineered bacterium and its construction method and a method for producing vanillin, especially a genetically engineered bacterium for producing vanillin and its construction method and the use of the strain to produce A method for producing vanillin from cheap carbon sources such as glucose and glycerol. Background technique [0002] Vanillin, also known as vanillin, chemically named 3-methoxy-4-hydroxybenzaldehyde, is white or slightly yellow crystal, with vanilla aroma and strong milky aroma, and is generally loved by people. The main ingredient of creamy vanilla essence. Vanillin is currently the most produced synthetic fragrance in the world. The synthesis methods of vanillin mainly include chemical synthesis and microbial transformation. Among them, the chemical synthesis method has high yield and low cost, but there are problems such as...

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Problems solved by technology

[0004] In 1998, Li et al. used recombinant Escherichia coli to produce vanillic acid through the shikimic acid pathway with glucose as a substrate, and produced trace amounts of vanillin under the catalysis of aromatic acid reductase produced by Neurospora crassa in vitro, but the method was cumbersome.

In 2009, Hansen et al. modified Schizosaccharomyces pombe and Saccharomyces cerevisiae by metabolic engineering method. The modified strains can use glucose as substrate respectively, and can produce 65 mg / L and 45 mg / L vanillin. However, on the one hand, this method produces Isovanillin is a by-product that is difficult to separate. On the other hand, due to the strong ability of yeast to degrade vanillin and the toxicity of vanillin to bacteria, vanillin needs to be further derivatized into glucoside-vanillin. for subsequent applications

In 2015, Xu Ping used metabolic engineering methods to transform Escherichia coli, and the constructed strain produced 97.2 mg / L vanillin with tyrosine as a substrate, and 19.3 mg / L and 13.3 mg / L with glucose, xylose and glycerol as substrates, respectively. mg / L and 24.7mg / L vanillin, but this route introduces more foreign genes, the route is longer, and the efficiency needs to be improved

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