Unraveling the Leloir pathway of Bifidobacterium bifidum: significance of the uridylyltransferases

The GNB/LNB (galacto-N-biose/lacto-N-biose) pathway plays a crucial role in bifidobacteria during growth on human milk or mucin from epithelial cells. It is thought to be the major route for galactose utilization in Bifidobacterium longum as it is an energy-saving variant of the Leloir pathway. Both pathways are present in B. bifidum, and galactose 1-phosphate (gal1P) is considered to play a key role. Due to its toxic nature, gal1P is further converted into its activated UDP-sugar through the action of poorly characterized uridylyltransferases. In this study, three uridylyltransferases (galT1, galT2, and ugpA) from Bifidobacterium bifidum were cloned in an Escherichia coli mutant and screened for activity on the key intermediate gal1P. GalT1 and GalT2 showed UDP-glucose-hexose-1-phosphate uridylyltransferase activity (EC 2.7.7.12), whereas UgpA showed promiscuous UTP-hexose-1-phosphate uridylyltransferase activity (EC 2.7.7.10). The activity of UgpA toward glucose 1-phosphate was about 33-fold higher than that toward gal1P. GalT1, as part of the bifidobacterial Leloir pathway, was about 357-fold more active than GalT2, the functional analog in the GNB/LNB pathway. These results suggest that GalT1 plays a more significant role than previously thought and predominates when B. bifidum grows on lactose and human milk oligosaccharides. GalT2 activity is required only during growth on substrates with a GNB core such as mucin glycans

Creating a novel and versatile glycosylation platform in E. coli

Glycosyltransferases (GTs) are powerful enzymes for the regioselective glycosylation of various small molecules. Because addition of a sugar residue can greatly alter the solubility, stability or bioactivity, the demand for glycosides as nutraceuticals, therapeutics or cosmetics is steadily rising. However, the use of GTs in biocatalysis is often hampered by the need for activated sugar donors (often UDP-sugars) which are expensive and rarely available in large quantity. An efficient solution is the use of microbial whole-cell systems that overexpress GTs and form their own expensive UDP-sugars from cheap substrates. Unfortunately, these processes are frequently plagued by low production yields and rates or difficulties regarding scale-up. In this study, we present the creation of a novel glycosylation platform in Escherichia coli W through Metabolic Engineering. The resulting strain acts as a versatile host for the selective glycosylation of diverse small molecules such as oligosaccharides, phenylpropanoids, flavonoids or hydroxybenzoic acids, hereby using only sucrose as cheap and sustainable carbon source. By introducing a generic toolbox, these compounds can be efficiently glucosylated, galactosylated or rhamnosylated with up to 100 % conversion. Moreover, by exploiting a unique and novel engineering strategy, production is coupled to growth, yielding an easily scalable glycosylation strain with high specific rates and gram scale production