High-throughput screening method for α-1,2-fucosyltransferase and mutant obtained by means of screening

WO2026138552A1PCT designated stage Publication Date: 2026-07-02JIANGNAN UNIV

Patent Information

Authority / Receiving Office: WO · WO
Patent Type: Applications
Current Assignee / Owner: JIANGNAN UNIV
Filing Date: 2025-12-15
Publication Date: 2026-07-02

AI Technical Summary

Technical Problem

The lack of efficient high-throughput screening methods in the current technology limits the catalytic performance and expression level of α-1,2-fucosyltransferase, affects the synthesis efficiency and cost of 2'-FL, and hinders its application in the food and pharmaceutical fields.

Method used

2'-fucosylation lactose indicator strains and chassis strains were constructed. α-1,2-fucosylation transferase mutants were screened using droplet microfluidics. The screening process was optimized by leveraging the positive correlation between fluorescent reporter genes and 2'-fucosylation lactose content, and the expression of β-galactosidase was controlled to improve screening efficiency.

Benefits of technology

Rapid screening of α-1,2-fucosyltransferase mutants was achieved, improving enzyme activity and the production efficiency of 2'-fucosyllactose. The screening efficiency was increased to 2.31 times that of FutC, showing significant industrial application potential.

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Abstract

Provided are a high-throughput screening method for α-1,2-fucosyltransferase and a mutant obtained by means of screening. The steps of the screening method comprise: constructing an indicator strain for 2'-fucosyllactose, such that the content of 2'-fucosyllactose is positively correlated with the fluorescence intensity, then expressing L-fucokinase and an α-1,2-fucosyltransferase mutant library in a chassis strain, encapsulating the chassis strain in water-in-oil droplets and fusing and co-culturing same along with the indicator strain, and obtaining a chassis strain comprising an α-1,2-fucosyltransferase forward mutant by means of droplet microfluidic sorting and on the basis of fluorescence detection results, wherein the enzyme activity of mutant FutCV93I is 2.31 times that of an original enzyme, and the enzyme activity is greatly improved. By using the provided method, high-throughput screening can be performed on α-1,2-fucosyltransferase, facilitating the industrial production of 2'-fucosyllactose and the large-scale application and promotion thereof in the fields of food and medicine.

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