Developing a Metabolic Flux Optimized <i>Escherichia coli</i> for Efficient Production of Difucosyllactose

Difucosyllactose (DFL) is a type of α-1,2/3-fucosylated oligosaccharide that confers significant growth and health benefits for developing infants. Although the synthesis of DFL has been established in engineered hosts, they still face problems, including a small number of matched glycosyltransferases and unsatisfactory catalytic capacity. Herein, a well-matched pair of fucosyltransferases, α-1,2-fucosyltransferase (SAMT) and α-1,3-fucosyltransferase (Fut3Bc), was first utilized for DFL production using an engineered Escherichia coli BL21 (DE3). For this, expression of key gene clusters in the GDP-fucose pathway was strengthened by using constitutive promoters Ptac70 and PJ23119. To better design artificial metabolic pathways, both 2′-fucosyllactose (2′-FL) and 3-fucosyllactose (3-FL) were utilized as substrates to verify the preference of fucosyltransferases. The regulator RcsAB was also integrated into the chromosome to replace the native gene galU, thereby supporting the maximized accumulation of GDP-fucose. Finally, the optimized strain produced 8.34 and 53.15 g/L of DFL in the shake-flask and a 5 L bioreactor through fed-batch fermentation, respectively, which is the highest titer achieved in E. coli to date. This work shows the immense potential of DFL production in engineered E. coli and provides a well-matched pair of fucosyltransferases for the sustainable synthesis of DFL.