Engineering <i>Escherichia coli</i> MG1655 for Highly Efficient Biosynthesis of 2′-Fucosyllactose by <i>De Novo</i> GDP-Fucose Pathway
Roulin Chen, Yingying Zhu, Hao Wang, Yuanlin Liu, Jiawei Meng, Yihan Chen, Wanmeng Mu
Abstract
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 P J23119 . 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 P J23119 promoter-based BKHT expression cassette (P J23119 - 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.