Efficient fermentative production of lactodifucotetraose by controlling sequential glycosyltransferase reactions in Escherichia coli

Abstract

Lactodifucotetraose (LDFT) is a human milk oligosaccharide (HMO) that might reduce inflammation in infants. In this study, we established a useful production process of LDFT by engineering two key enzymes, α1,2‐fucosyltransferase (α1,2‐FucT) and α1,3‐fucosyltransferase (α1,3‐FucT). First, we verified which of 2′‐fucosyllactose (2′‐FL) or 3‐fucosyllactose (3‐FL) (mostly unverified) was more useful. We searched for FucTs that functioned efficiently in vivo against the raw material lactose or the two intermediates 2′‐FL or 3‐FL by external substrate addition to culture medium. We found that α1,2‐ FucT (HMFT) from Helicobacter mustelae and the N‐terminal truncated form of α1,3‐FucT from Bacteroides fragilis (BfFucTΔN10) had high potential. 3‐FL was not efficiently converted to LDFT, which might be attributed to the low reactivity of HMFT to 3‐FL as well as the low uptake efficiency of 3‐FL by LacY, as revealed by a growth test with exogenously added FL as the sole carbon source and heterologously expressed intracellular fucosidase. Furthermore, because 3‐FL accumulation had a negative impact on cell growth, we avoided the route passing through 3‐FL. By adjusting the copy numbers of HMFT and BffucTΔN10, we produced LDFT from lactose predominantly via 2′‐FL. Finally, 17.5 g/L of LDFT (with 6.8 g/L 2′‐FL and no 3‐FL or residual lactose) accumulated in a 3‐L fed‐batch culture after 77 h. This study reports the detailed analysis of multiple pathways and shows the control of glycosyltransferases can improve the production efficiency of complex HMOs.