Directed evolution of α1,2-fucosyltransferase via a scalable high-throughput screening platform

Abstract

α1,2-Fucosyltransferase (α1,2-FucT) catalyzes the constitution of 2′-fucosyllactose (2′-FL) via transferring the L-fucose from the donor GDP-L-fucose to the lactose acceptor, thus playing an essential role in the microbial biosynthesis of 2′-FL. However, the performance of natural α1,2-FucT catalytic activity is limited, and lack of efficient screening platforms is hampering its engineering. This study presents a whole-cell biosensor-based high-throughput screening platform to obtain α1,2-FucT with high catalytic activity. First, a whole-cell biosensor was designed to translate 2′-fucosyllactose, the catalytic product of α1,2-FucT, to a positively correlated fluorescence signal. Meanwhile, a thermosensitive lactose-degradation pathway was introduced to resolve the concern of lactose interference. Then, a cross-scale leap from microliter to picoliter was achieved through characterization and optimization, which allowed sorting of α1,2-FucT high-throughput droplets. We selected most widely utilized Helicobacter pylori α1,2-FucT and constructed a library of 0.1 million mutants, identifying V93I as the most optimal mutant. Its catalytic efficiency (k_cat/K_m) was 5.024 ± 0.702 min⁻¹ mM⁻¹, 2.31 times that of the wild-type α1,2-FucT. As far as we know, this study is the first to illustrate the high-throughput screening platform based on a 2′-FL whole-cell biosensor and droplet microfluidics, and this strategy can be extended to similar glycosyltransferase-screening applications.