LAUSR

L‐Fucose (6‐deoxy‐L‐galactose) is a major constituent of glycans and glycolipids in mammals. Fucosylation of glycans can confer unique functional properties and may be an economical way to manufacture L‐fucose. Research can extract L‐fucose directly from brown algae, or by enzymatic hydrolysis of L‐fucose‐rich microbial exopolysaccharides. However, these L‐fucose production methods are not economical or scalable for various applications. We engineered an Escherichia coli strain to produce L‐fucose. Specifically, we modified the strain genome to eliminate endogenous L‐fucose and lactose metabolism, produce 2′‐fucosyllactose (2′‐FL), and to liberate L‐fucose from 2′‐FL. This E. coli strain produced 16.7 g/L of L‐fucose with productivity of 0.1 g·L−1·h−1 in a fed‐batch fermentation. This study presents an efficient one‐pot biosynthesis strategy to produce a monomeric form of L‐fucose by microbial fermentation, making large‐scale industrial production of L‐fucose feasible.