A Combinatorial Strategy of Semirational Screening and Rational Design for Engineering an α-1,2-Fucosyltransferase...

Area of Science:

• Synthetic biology
• Enzyme engineering
• Metabolic engineering

Background:

• Microbial production of 2 -fucosyllactose (2 -FL) is crucial for human milk oligosaccharides.
• Eliminating byproducts like difucosyllactose (DFL) is essential for efficient 2 -FL biosynthesis.

Purpose of the Study:

• To develop a synthetic biology platform for high-titer, byproduct-free 2 -FL production.
• To engineer the α-1,2-fucosyltransferase BKHT for enhanced regioselectivity and activity.

Main Methods:

• Dual protein engineering strategy combining rational computational design and semirational screening.
• Development of an l-fucose-responsive biosensor (FcsR-P_fcsK) for efficient mutant screening.
• Optimization of promoter and vector elements for improved biosensor performance and gene expression.

Main Results:

• An optimized quintuple mutant (M5) of BKHT was generated through iterative combinatorial assembly of beneficial mutations.
• The engineered Escherichia coli chassis expressing M5 achieved a record 2 -FL titer of 124.25 g/L from glucose in a 3 L bioreactor.
• No detectable difucosyllactose (DFL) byproduct was observed, indicating high purity of the produced 2 -FL.

Conclusions:

• The integrated workflow of rational design and biosensor-guided screening provides a robust framework for constructing advanced microbial cell factories.
• This approach enables efficient and byproduct-free biosynthesis of complex carbohydrates like 2 -FL.
• The developed platform holds significant potential for industrial-scale production of 2 -FL and other valuable glycans.