Efficient production of lacto-N-fucopentaose III in engineered Escherichia coli using α1,3-fucosyltransferase from Parabacteroides goldsteinii.

Lacto-N-fucopentaose III (LNFP III) is a human milk oligosaccharide (HMO) with potential health benefits in infants, including in immune development and modulation of the intestinal environment. Low-cost fermentative production of various HMOs from lactose by engineered Escherichia coli has attracted attention, but few reports have investigated long-chain HMO production, such as of the pentasaccharide LNFP III. LNFP III is synthesized by α1,3-fucosyltransfer reaction to the glucosamine (GlcNAc) moiety in the lacto-N-neotetraose (LNnT) skeleton by α1,3-fucosyltransferase (α1,3-FucT). However, the known α1,3-FucTs also transfer fucose to the reducing terminal glucose moiety of LNnT or the starting material lactose, resulting in various byproducts. Here, we found a useful α1,3-FucT from Parabacteroides goldsteinii (PgsFucT), which is only reactive for GlcNAc in the N-acetyllactosamine (LacNAc) skeleton in vivo. On the basis of sequence alignment with a FucT of known structure, we also generated α1,3-FucT variants with altered reactivity for LacNAc or lactose. An E. coli strain heterologously expressing PgsFucT accumulated 3.84 g/L of LNFP III after 48 h of culture in a 3-L jar-fermenter. The amounts of various byproduct sugars were remarkably decreased compared with a strain expressing the previously characterized α1,3-fucT from Bacteroides fragilis.