Metabolic engineering of Saccharomyces cerevisiae for efficient production of 2’-fucosyllactose

Human milk oligosaccharides (HMOs) have been known for their abundance of nutrients for infants and its protection from pathogens and infectious diseases. 2’-fucosyllactose (2-FL) is the most abundant HMO, and it has a potential to be facilitated as nutraceutical and pharmaceutical purpose. Currently, many scientists are investigating the production of 2-FL in E.coli, but the research has not been optimized to produce 2-FL in yeast. Thus, the production of 2-FL in S. cerevisiae yeast strain D452-2 was proposed to be optimized for a similar level of 2-FL production in E. coli. For efficient 2-FL production, S. cerevisiae strain SK1, based on D452-2, was mutated for upper and lower glycolysis pathways, enhanced GDP-mannose synthetic pathway, and developed via transformation with the appropriate fucosyltransferase and transporter. The levels of GDP-mannose and GDP-fucose affect the production of 2-FL according to the previous studies in E. coli. Therefore, the overproductions of GPD-mannose and GDP-fucose were performed. These experiments found that engineered SK1 base strains had higher levels of GDP-mannose and GPD-fucose productions than developed D452-2 base strains. However, SK1P2L-gwf strain with widely used fucosyltransferase (FucT2) and transporter (LAC12) did not show the higher level of both intracellular and extracellular 2-FL productions than the D452L-gwf strain. The result of this experiment described a toxicity of lactose to produce 2-FL so that it was repeated with varied lactose concentrations. Repeating the experiment revealed that the low lactose concentration produced more 2-FL and the fucosytransferase and the transporter did not properly function because intracellular 2-FL was more highly accumulated than extracellular 2-FL. To be optimized the strain for efficient 2-FL production, SK1P2-w(b)gwh and D452-w(b)gwh were constructed with WbgL and mutant HXT2.4, as a fucosyltransferase and a lactose transporter, respectively, in the lower initial lactose concentration. It determined SK1P2 w(b)gwh had the highest productivity of 2-FL