A high-throughput screening method for glycosaminoglycan backbone synthases

By constructing recombinant Bacillus subtilis BS168ASS and expressing the tuaD and glycosaminoglycan backbone synthase genes, the problem of high-throughput screening of glycosaminoglycan backbone synthases was solved by using luciferin click chemistry and fluorescence-activated cell sorting, thus achieving efficient screening and discovery of new enzymes.

CN117004635BActive Publication Date: 2026-07-24SHANDONG UNIV
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Patent Information

Application Number
CN202210468072.3
Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
Filing Date
2022-04-29
Publication Date
2026-07-24
Estimated Expiration
2042-04-29

AI Technical Summary

Technical Problem

Existing technologies lack high-throughput screening methods for glycosaminoglycan backbone synthases, making it difficult to effectively broaden the substrate range, change substrate specificity, and improve GTs activity. Furthermore, the lack of efficient methods for measuring fluorescence or absorbance changes makes the screening process difficult.

Method used

Recombinant Bacillus subtilis BS168ASS was constructed, expressing the uridine diphosphate-glucose-6-dehydrogenase gene tuaD and the glycosaminoglycan backbone synthase gene. The strain surface was made fluorescent by the Cyanine5 DBCO click chemical reaction, and high-throughput screening was achieved by combining fluorescence-activated cell sorting.

Benefits of technology

This method enables efficient and accurate screening of glycosaminoglycan backbone synthases, improving screening efficiency and providing a new starting point for the discovery of enzymes from novel sources. It is applicable to the discovery of novel glycosaminoglycan backbone synthases in metagenomic libraries.

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Abstract

The application relates to a high-throughput screening method of a glycosaminoglycan backbone synthase. The method comprises the following steps: (1) constructing a glycosaminoglycan backbone synthase library; (2) constructing a recombinant vector; (3) constructing a recombinant bacterium for screening the glycosaminoglycan backbone synthase; (4) culturing the recombinant bacterium; (5) after the bacterial liquid of the recombinant bacterium is analyzed or sorted through flow, the fluorescence is measured, and the target glycosaminoglycan backbone synthase is screened. In the application, the Bacillus subtilis BS168ASS is used as a host bacterium, the uridine diphosphate-glucose-6-dehydrogenase gene and the glycosaminoglycan backbone synthase gene are overexpressed, and the recombinant bacterium for screening the glycosaminoglycan backbone synthase is constructed. Then, the fluorescence of the strain is measured, the glycosaminoglycan backbone synthase strain is accurately screened in a high-throughput mode, the screening efficiency is improved, and a new starting point is provided for the discovery of new glycosaminoglycan backbone synthases in microorganism groups of new sources, especially in the microorganism groups that cannot be cultured in a metagenome library.
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