Engineering Escherichia coli MG1655 for Highly Efficient Biosynthesis of 2′-Fucosyllactose by De Novo GDP-Fucose Pathway

2'-Fucosyllactose (2'-FL), the most typical human milk oligosaccharide, is used as an additive in premium infant formula. Herein, we constructed two highly effective 2'-FL synthesis producers via a de novo GDP-fucose pathway from engineered Escherichia coli MG1655. First, lacZ and wcaJ, two competitive pathway genes, were disrupted to block the invalid consumption of lactose and GDP-fucose, respectively. Next, the lacY gene was strengthened by switching its native promoter to PJ23119. To enhance the supply of endogenous GDP-fucose, the promoters of gene clusters manC-manB and gmd-fcl were strengthened individually or in combination. Subsequently, chromosomal integration of a constitutive PJ23119 promoter-based BKHT expression cassette (PJ23119-BKHT) was performed in the arsB and recA loci. The most productive plasmid-based and plasmid-free strains produced 76.9 and 50.1 g/L 2'-FL by fed-batch cultivation, respectively. Neither of them generated difucosyl lactose nor 3-fucosyllactose as byproducts.