Highly-efficient in vivo production of lacto-N-fucopentaose V by a regio-specific α1,3/4-fucosyltransferase from Bacteroides fragilis NCTC 9343

Lacto-N-fucopentaose V (LNFP V) is a typical human milk pentasaccharide. Multi-enzymatic in vitro synthesis of LNFP V from lactose was reported, however, microbial cell factory approach to LNFP V production has not been reported yet. In this study, the biosynthetic pathway of LNFP V was examined in Escherichia coli. The previously constructed E. coli efficiently producing lacto-N-tetraose was used as the starting strain. GDP-fucose pathway module and a regio-specific glycosyltransferase with α1,3-fucosylation activity were introduced to realize the efficient synthesis of LNFP V. The α1,3/4-fucosyltransferase from Bacteroides fragilis was selected as the best enzyme for in vivo biosynthesis of LNFP V from nine candidates, with the highest titer and the lowest by-product accumulation. A beneficial variant K128D was obtained to further enhance LNFP V titer using computer-assisted site-directed mutagenesis. The final strain EW10 could produce 25.68 g/L LNFP V by fed-batch cultivation, with the productivity of 0.56 g/L·h.