Production of 3-Fucosyllactose in Engineered Escherichia coli with α-1,3-Fucosyltransferase from Helicobacter pylori

Abstract

3-Fucosyllactose (3-FL), one of the major oligosaccharides in human breast milk, is produced in engineered Escherichia coli. In order to search for a good α-1,3-fucosyltransferase, three bacterial α-1,3-fucosyltransferases are expressed in engineered E. coli deficient in β-galactosidase activity and expressing the essential enzymes for the production of guanosine 5'-diphosphate-l-fucose, the donor of fucose for 3-FL biosynthesis. Among the three enzymes tested, the fucT gene from Helicobacter pylori National Collection of Type Cultures 11637 gives the best 3-FL production in a simple batch fermentation process using glycerol as a carbon source and lactose as an acceptor. In order to use glucose as a carbon source, the chromosomal ptsG gene, considered the main regulator of the glucose repression mechanism, is disrupted. The resulting E. coli strain of ∆LP-YA+FT shows a much lower performance of 3-FL production (4.50 g L^-1 ) than the ∆L-YA+FT strain grown in a glycerol medium (10.7 g L^-1 ), suggesting that glycerol is a better carbon source than glucose. Finally, the engineered E. coli ∆LW-YA+FT expressing the essential genes for 3-FL production and blocking the colanic acid biosynthetic pathway (∆wcaJ) exhibits the highest concentration (11.5 g L^-1 ), yield (0.39 mol mol^-1 ), and productivity (0.22 g L^-1 h) of 3-FL in glycerol-limited fed-batch fermentation.

Keywords: 3-fucosyllactose; Escherichia coli; fed-batch fermentation; α-1,3-fucosyltransferase.