Directed evolution of an α1,3-fucosyltransferase using a single-cell ultrahigh-throughput screening method

Abstract

Fucosylated glycoconjugates are involved in a variety of physiological and pathological processes. However, economical production of fucosylated drugs and prebiotic supplements has been hampered by the poor catalytic efficiency of fucosyltransferases. Here, we developed a fluorescence-activated cell sorting system that enables the ultrahigh-throughput screening (>107 mutants/hour) of such enzymes and designed a companion strategy to assess the screening performance of the system. After three rounds of directed evolution, a mutant M32 of the α1,3-FucT from Helicobacter pylori was identified with 6- and 14-fold increases in catalytic efficiency (kcat/Km) for the synthesis of Lewis x and 3′-fucosyllactose, respectively. The structure of the M32 mutant revealed that the S45F mutation generates a clamp-like structure that appears to improve binding of the galactopyranose ring of the acceptor substrate. Moreover, molecular dynamic simulations reveal that helix α5, is more mobile in the M32 mutant, possibly explaining its high fucosylation activity.

Highly Efficient Biosynthesis of Glycyrrhetinic Acid Glucosides by Coupling of Microbial Glycosyltransferase to Plant Sucrose Synthase

“…The glycosylation was analyzed using the high-performance liquid chromatography (HPLC) method ( Tan et al, 2019 ). The standard glycosylation reaction was performed in 250 μL reaction buffer, containing 20 mM Tris–HCl pH 8.0, 1 mM GA dissolved in DMSO, 5 mM UDPG, and 10 μg UGT109A3.…”

Cell-based high-throughput screening of polysaccharide biosynthesis hosts

“…In 2019, Tan et al . used the dichromatic-labeling FACS strategy to conduct high-throughput screening of a library of mutants of fucosyltransferase FutA (> 10 7 mutants) [ 49 ]. This enzyme can transfer fucose to the C3–OH site of the GlcNAc group of N -acetyllactosamine (LacNAc).…”

Recent advances and challenges in microbial production of human milk oligosaccharides

“…The only difference in the biosynthesis processes of 2′-FL and 3-FL is the FucT; therefore, screening and modification of the FucT3 is the focus of producing 3-FL. Modification strategies such as error-prone polymerase chain reaction (PCR) as well as truncation or elongation of the C-terminal regions have been performed on FucT3 from H. pylori [51][52][53]. Tan et al designed and synthesized several fluorescently labeled substrate derivatives for fucosylation reactions for high-throughput screening of FucT [53].…”

“…Modification strategies such as error-prone polymerase chain reaction (PCR) as well as truncation or elongation of the C-terminal regions have been performed on FucT3 from H. pylori [51][52][53]. Tan et al designed and synthesized several fluorescently labeled substrate derivatives for fucosylation reactions for high-throughput screening of FucT [53]. Overall, the intracellular content of GDP-L-fucose is usually sufficient for 2′-FL and 3-FL production in the improved strains; therefore, the low catalytic efficiency fucosyltransferease is the bottleneck.…”