In Vitro Synthesis of GDP-L-fucose by Multi-enzyme Cascade Catalysis
LING He
LIANG Shuli
LIN Ying
Abstract：GDP-L-fucose is recognized as an important nucleotide sugar and a critical glycosyl donor in fucosylation modification.To establish a new method to synthesize GDP-L-fucose,in this study,a biosynthetic pathway from starch to GDP-L-fucose was designed.The pathway is catalyzed by eight enzymes.After these eight enzymes were screened and identified,they were expressed in Escherichia coli and subsequently purified.Based on this,a multi-enzyme cascade system was constructed in vitro to synthesize GDP-L-fucose and the reaction conditions were optimized to enhance its synthesis.The eight enzymes,all derived from thermophilic bacteria,include alpha-glucan phosphorylase(αGP),phosphoglucosamine mutase(PGM),glucose-6-phosphate isomerase(PGI),class I mannose-6-phosphate isomerase(ManA),phosphomannomutase(ManB),mannose-1-phosphate guanylyltransferase(ManC),GDP-mannose 4,6-dehydratase(Gmd),and GDP-L-fucose synthetase(WcaG).After optimizing the reaction temperature,pH,enzyme ratio,Mg2+,NADPH concentrations,and phosphate buffer concentration,the reaction was conducted with 1 g/L starch in one vessel,resulting in a GDP-L-fucose yield of 53.23%and 0.53 g/L.This study is the first to report the biosynthesis of GDP-L-fucose from starch in vitro,providing a new method for the synthesis of GDP-L-fucose from starch-based renewable resources in vitro.
Keywords：multi-enzyme cascade in vitroGDP-L-fucoseEscherichia coli
Publication Date：2025-11-20
Online Publishing Date：2025-12-26(First online date of this platform, not the publication date of the document)
Pages：10( 47-56 )