Genetically engineering bacterium capable of producing S-3-hydroxy-butanone as well as construction method and application thereof

Abstract

The invention discloses a genetically engineering bacterium capable of producing S-3-hydroxy-butanone as well as a construction method and application thereof. The bacterial strain is classified and named as paenibacillus polymyxa CGMCC 3044-Bud A<->. Bud A is a 2,3-butanediol dehydrogenase gene, and the Bud A gene in P. polymyxa CGMCC 3044 is knocked out through homologous double-crossover to obtain the bacterial strain p. polymyxa CGMCC 3044-Bud A<-1>. The recombinant bacterium constructed by the method can be used for fermenting and synthesizing optically pure S-3-hydroxy-butanone, the optical purity of which can reach 100%; reduction-state byproduct 2,3-butanediol of the 3-hydroxy-butanone is not generated; the bacterial strain can be produced by directly using substrates such as jerusalem artichoke and inulin, glucose and butanedione; the construction method has the advantages of being extensive in culture conditions, convenient and simple to operate and beneficial to large-scale industrial production; and the bacterial strain has high optical purity and is low in production cost.

Description

technical field [0001] The invention belongs to the field of biochemical industry, and in particular relates to a genetically engineered bacterium and its construction method and application. Background technique [0002] 3-Hydroxybutanone, also known as methyl acetylmethanol and acetoin, can be used as a platform compound and is widely used in many fields such as daily chemical food, pharmaceuticals, coatings, and liquid crystal materials. In 2004, the U.S. Department of Energy listed it as one of the 30 priority platform compounds for development and utilization. There are two optical isomers in 3-hydroxybutanone, which are R-type and S-type 3-hydroxybutanone, respectively, and S-3-hydroxybutanone can be used to synthesize 4-chloro-4,5-dimethyl-1, 3 Dioxolane-2-one (CDMDO), CDMDO is an important pharmaceutical intermediate for the synthesis of antibiotics such as penicillin and ampicillin; S-3-hydroxybutanone can also be used for the synthesis of antibacterial drug lempic...

AI Technical Summary

Problems solved by technology

There are many reaction steps in the chemical synthesis method, chiral resolution is required, and the process is cumbersome, resulting in high cost, low yield, serious environmental pollution, and most of the raw materials are non-renewable fossil resources—petroleum, and the source of raw materials is limited.

The biosynthesis method includes enzymatic method and microbial fermentation method. The enzymatic method uses butanedione or butanediol as raw material to produce S-3-hydroxybutanone under the catalytic specificity of the enzyme. The conversion rate and optical purity of this method are both It can reach 100%, but the source of raw materials is limited like the chemical method, and the output is low, so it is difficult to prepare S-3-hydroxybutanone in large quantities

[0004] The strains currently used for the production and research of 3-hydroxybutanone mainly include Klebsiella, Bacillus, Lactococcus, Enterobacter and Serratia Genus (Serratia), etc., but no wild strains have been found for the production of optically pure S-3-hydroxybutanone

At present, the use of genetic engineering methods to construct plasmids to transform Escherichia coli to obtain recombinant bacteria to produce S-3-hydroxybutanone reported in the literature, the researchers introduced the 2,3-butanediol dehydrogenase gene (Bud A) in Bacillus subtilis 168 into the large intestine In the bacillus Escherichia coli BL21 (DE3), construct the recombinant strain E.coli BL21-Bud A, and use meso-2,3-butanediol as the substrate to transform the recombinant strain into S-3-hydroxybutanone, although the obtained Successful, the yield is about 36.7g / L, but this method still has the disadvantages of substrate meso-2,3-butanediol, which is expensive and difficult to obtain, and the recombinant plasmid is unstable, low yield, and high cost.

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