Pathway Optimization of 2'-Fucosyllactose Production in Engineered .

2'-Fucosyllactose (2'-FL), one of the most valuable oligosaccharides in human milk, is used as an emerging food ingredient in the nutraceutical and food industries due to its numerous health benefits. Herein, the and salvage pathways for GDP-fucose synthesis were engineered and optimized in BL21 (DE3) to improve the production of 2'-FL. The pathway genes encoding phosphomannomutase (ManB), mannose-1-phosphate guanyltransferase (ManC), GDP-d-mannose-4,6-dehydratase (Gmd), and GDP-l-fucose synthase (WcaG) combined with the gene from the salvage pathway encoding fucose kinase/fucose-1-phosphate guanylyltransferase (Fkp) were reconstructed in two vectors to evaluate the GDP-fucose biosynthesis. Then, the gene, encoding α1,2-fucosyltransferase, was introduced into the GDP-fucose-overproducing strains to realize 2'-FL biosynthesis. Furthermore, the genes in bypass pathways, including , , , and , were inactivated to improve 2'-FL production. In addition, the two GDP-fucose synthesis pathways, along with , were transcriptionally fine-tuned to efficiently increase 2'-FL production. The final metabolically engineered produced 2.62 and 14.1 g/L in shake-flask and fed-batch cultivations, respectively.