A Combinatorial Strategy of Semirational Screening and Rational Design for Engineering an α-1,2-Fucosyltransferase toward Efficient 2′-Fucosyllactose Biosynthesis

Developing microbial platforms for 2′-fucosyllactose (2′-FL) production requires meticulous enzyme engineering to eliminate byproducts such as difucosyllactose (DFL). Here, we establish a synthetic biology platform for high-titer, byproduct-free 2′-FL biosynthesis using the strictly regioselective α-1,2-fucosyltransferase BKHT. We employed a dual protein engineering strategy: rational computational design (incorporating structural, evolutionary, and stability predictions) and semirational screening powered by an engineered l -fucose-responsive biosensor (FcsR-P fcsK ). Through promoter and vector optimization, this low-noise biosensor enabled efficient screening of saturation mutagenesis libraries. Iterative combinatorial assembly of the identified beneficial mutations yielded an optimized quintuple mutant, M5 (L36P–I58V–E261G–S317D–K324H). In a 3 L bioreactor, an Escherichia coli chassis expressing M5 achieved a record 2′-FL titer of 124.25 g/L from glucose, with no detectable DFL. This integrated workflow combining rational design and biosensor-guided screening provides a robust framework for constructing advanced microbial cell factories.