Strategies for Enhancing Microbial Production of 2′-Fucosyllactose, the Most Abundant Human Milk Oligosaccharide

Abstract

Human milk oligosaccharides (HMOs), a group of structurally diverse unconjugated glycans in breast milk, act as important prebiotics and have plenty of unique health effects for growing infants. 2′-Fucosyllactose (2′-FL) is the most abundant HMO, accounting for approximately 30%, among approximately 200 identified HMOs with different structures. 2′-FL can be enzymatically produced by α1,2-fucosyltransferase, using GDP-l-fucose as donor and lactose as acceptor. Metabolic engineering strategies have been widely used for enhancement of GDP-l-fucose supply and microbial production of 2′-FL with high productivity. GDP-l-fucose supply can be enhanced by two main pathways, including de novo and salvage pathways. 2′-FL-producing α1,2-fucosyltransferases have widely been identified from various microorganisms. Metabolic pathways for 2′-FL synthesis can be basically constructed by enhancing GDP-l-fucose supply and introducing α1,2-fucosyltransferase. Various strategies have been attempted to enhance 2′-FL production, such as acceptor enhancement, donor enhancement, and improvement of the functional expression of α1,2-fucosyltransferase. In this review, current progress in GDP-l-fucose synthesis and bacterial α1,2-fucosyltransferases is described in detail, various metabolic engineering strategies for enhancing 2′-FL production are comprehensively reviewed, and future research focuses in biotechnological production of 2′-FL are suggested.

Microbial Synthesis of l-Fucose with High Productivity by a Metabolically Engineered Escherichia coli

“…Currently, it has not been difficult to enhance 2′-FL production to more than 50 g/L using various metabolic engineering strategies . The more highly efficient 2′-FL-producing strains were used as the starting strains, the higher the l -fucose titers were likely to be produced .…”

“…Then, Gmd converts GDP-mannose to GDP-4-keto-6-deoxymannose and the latter is subsequently converted to GDP-L-fucose by WcaG. 22 GDP-L-fucose is an important intermediate for the biosynthesis of cell wall polysaccharide colanic acid containing L-fucose as a component in E. coli. Inactivation of wcaJ can prevent the flux toward colanic acid, thereby enhancing GDP-L-fucose accumulation.…”

“…The de novo biosynthesis of 2′-FL via the metabolic engineering approach has been widely studied, and the 2′-FL productivity has also been continuously increasing. 22 However, only two publications referred to L-fucose production based on the 2′-FL-producing engineered strains. Liu et al reported the de novo biosynthesis of 2′-FL using engineered Saccharomyces cerevisiae, with a titer of 0.51 g/L by shake-flask cultivation.…”

“…20 Compared to yeast hosts, E. coli as the host may produce much higher titers and yields of 2′-FL. 22 Liu et al further studied Lfucose synthesis by introducing X. manihotis α-L-fucosidase based on a 2′-FL-producing engineered E. coli. The final titers were enhanced to 3.67 and 16.7 g/L by shake-flask and fedbatch cultivation, respectively.…”

“…21 GDP-L-fucose is easily generated by the de novo synthesis pathway in both prokaryotes and eukaryotes. 22 When strengthening the expression of GDP-L-fucose pathway genes and introducing the specific α1,2-fucosyltransferase (EC 2.4.1.69), the microbial hosts can produce 2′-fucosyllactose (2′-FL) using exogenously added lactose as the glycosyl acceptor, the most abundant HMO with great commercial importance. 23 Based on the construction of a metabolically engineered Escherichia coli strain for 2′-FL synthesis, Liu et al further introduced a specific α-L-fucosidase (EC 3.2.1.51) for 2′-FL hydrolysis to liberate L-fucose.…”