Method used for high throughput screening of bacterial strains capable of generating 1-deoxynojirimycin

Abstract

The invention discloses a method used for high throughput screening of bacterial strains capable of generating 1-deoxynojirimycin (DNJ). The method comprises following steps: 1, recombinant vectors used for expressing DNJ biosynthetic pathway and original strains used for expressing beta-galactosidase are constructed; 2) error-prone PCR products of corresponding DNJ synthetic genes are obtained; 3, the error-prone PCR products of corresponding DNJ synthetic genes are taken as primers, the recombinant vectors are taken as templates, and MEGAWHOP PCR is carried out so as to obtain an MEGAWHOP PCR plasmid mutant library; and 4, the MEGAWHOP PCR plasmid mutant library is inserted into the original strains, plate coating is carried out so as to obtain a mutant library plate, bacterial colonies on the mutant library plate with color lighter than that of bacterial colonies on a wild bacterial plate are collected, and the collected bacterial colonies are high DNJ yield bacteria. The method can be used for screening bacteria strains high in DNJ yield, low in DNJ yield, or producing no DNJ; screening efficiency is increased; time is saved; and screening steps are simplified.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for high-throughput screening of strains producing deoxynojirimycin. Background technique [0002] 1-Deoxynojirimycin (DNJ) ( figure 1 ) is a piperidine polyhydroxyalkaloid that can competitively bind to the substrate-binding pockets of sucrase, trehalase, α-glucosidase, etc., thereby inhibiting its activity, reducing carbohydrate digestion and glucose production, So as to achieve the effect of preventing and treating diabetes. In addition, DNJ also has anti-virus and anti-tumor metastasis effects, which has very important academic significance and practical value for the development of new drugs for diabetes treatment and the development of anti-viral drugs. [0003] Natural DNJ can be produced in plants (mainly Moraceae), animals (mainly silkworms and insects that eat mulberry leaves), and microorganisms (mainly Streptomyces and Bacillus). The current method of synthesi...

AI Technical Summary

Problems solved by technology

Streptomyces and Bacillus can synthesize DNJ using glucose as a substrate through isomerization, transamination, oxidation, reduction and other steps, but the content of DNJ isolated from them is extremely low

Although the yield of DNJ can be increased by means of genetically engineered bacteria and directed evolution, it is difficult to screen wild-type and genetically engineered mutants that efficiently synthesize DNJ due to the lack of effective screening methods.

[0004] At present, the detection of DNJ mainly adopts reversed-phase high-performance liquid chromatography-ultraviolet detection method, adopts methyl 9-fluorenyl chloroformate (FMOC-Cl) to derivatize the structure of DNJ, and detects the generated fluorescent products with a fluorescence detector. Although this method It has the advantages of good repeatability and high stability, but the operation is cumbersome, the sensitivity is low, and the product obtained after derivatization is not stable, etc. It is not suitable for the detection of low concentration and small sample volume, and the price of the detector is relatively expensive. It is limited in many respects and is not suitable for high-throughput screening of DNJ high-efficiency synthetic strains.

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