LAUSR

2'‐Fucosyllactose (2'‐FL) is the most abundant oligosaccharide in human milk and one of the most actively studied human milk oligosaccharides (HMOs). When 2'‐FL is produced through biological production using a microorganism, like Escherichia coli, d‐lactose is often externally fed as an acceptor substrate for fucosyltransferase (FT). When d‐glucose is used as a carbon source for the cell growth and d‐lactose is transported by lactose permease (LacY) in lac operon, d‐lactose transport is under the control of catabolite repression (CR), limiting the supply of d‐lactose for FT reaction in the cell, hence decreasing the production of 2'‐FL. In this study, a remarkable increase of 2'‐FL production was achieved by relieving the CR from the lac operon of the host E. coli BL21 and introducing adequate site‐specific mutations into α‐1,2‐FT (FutC) for enhancement of catalytic activity and solubility. For the host engineering, the native lac promoter (Plac) was substituted for tac promoter (Ptac), so that the lac operon could be turned on, but not subjected to CR by high d‐glucose concentration. Next, for protein engineering of FutC, family multiple sequence analysis for conserved amino acid sequences and protein–ligand substrate docking analysis led us to find several mutation sites, which could increase the solubility of FutC and its activity. As a result, a combination of four mutation sites (F40S/Q150H/C151R/Q239S) was identified as the best candidate, and the quadruple mutant of FutC enhanced 2'‐FL titer by 2.4‐fold. When the above‐mentioned E. coli mutant host transformed with the quadruple mutant of futC was subjected to fed‐batch culture, 40 g l−1 of 2'‐FL titer was achieved with the productivity of 0.55 g l−1 h−1 and the specific 2'‐FL yield of 1.0 g g−1 dry cell weight.