A phylogeny-based directed evolution approach to boost the synthetic applications of glycosyltransferases

Abstract

Enzymatic catalysis in mild conditions plays a pivotal role in green chemistry, and the mining and the directed evolution of novel enzymes are crucial for the utilization of efficient biocatalysts. Glycosyltransferases (GTs) hold great promise in the synthesis of many valuable glycosylated products; however the narrow substrate scope and low catalytic efficiency of GTs often limit their broader applications. Phylogenetic mining and directed evolution of GTs are well-established methodologies to generate powerful enzymes. The combination of these established methodologies, termed PhyloDirect, integrates the identification of an excellent performing enzyme with its engineering by directed evolution to rapidly match the demands in biocatalysis. Herein, we report and validate a combined phylogeny-based directed evolution (PhyloDirect) approach to improve the performance of novel GT BarGT-3. A single GT high-throughput screening system based on UDP-glucose recycling was established. Through one-round of directed evolution, a promising variant I63P/H108G was obtained with significantly improved catalytic efficiency (28-fold for acetaminophen) and increased conversions toward a broad range of synthetically valuable substrates (phenols, coumarins, and flavonoids, up to 5-fold). The success of this PhyloDirect approach offers new opportunities for the development and application of efficient biocatalysts in green chemistry and other industrial processes.