Developing\na Metabolic Flux Optimized Escherichia coli for Efficient Production of Difucosyllactose

Difucosyllactose (DFL) is a type of α-1,2/3-fucosylated\noligosaccharide\nthat confers significant growth and health benefits for developing\ninfants. Although the synthesis of DFL has been established in engineered\nhosts, they still face problems, including a small number of matched\nglycosyltransferases and unsatisfactory catalytic capacity. Herein,\na well-matched pair of fucosyltransferases, α-1,2-fucosyltransferase\n(SAMT) and α-1,3-fucosyltransferase (Fut3Bc), was first utilized\nfor DFL production using an engineered Escherichia\ncoli BL21 (DE3). For this, expression of key gene\nclusters in the GDP-fucose pathway was strengthened by using constitutive\npromoters P<sub>tac70</sub> and P<sub>J23119</sub>. To better design\nartificial metabolic pathways, both 2′-fucosyllactose (2′-FL)\nand 3-fucosyllactose (3-FL) were utilized as substrates to verify\nthe preference of fucosyltransferases. The regulator RcsAB was also\nintegrated into the chromosome to replace the native gene <i>galU</i>, thereby supporting the maximized accumulation of GDP-fucose.\nFinally, the optimized strain produced 8.34 and 53.15 g/L of DFL in\nthe shake-flask and a 5 L bioreactor through fed-batch fermentation,\nrespectively, which is the highest titer achieved in E. coli to date. This work shows the immense potential\nof DFL production in engineered E. coli and provides a well-matched pair of fucosyltransferases for the\nsustainable synthesis of DFL.