iGEM 2021 - Jianhua
Production of a human milk oligosaccharide
by metabolically engineered Saccharomyces cerevisiae
Human milk oligosaccharides (HMOs) are important components of human milk that promote infant health . Fucosylated oligosaccharides, one of the most common HMOs, have been reported to offer health benefits, such as selectively enhancing bifidobacterial growth and effectively preventing binding of pathogens and toxins to the human gut. In particular, 2′-fucosyllactose (2-FL), the most abundant fucosylated oligosaccharide inhuman milk, attracted much interest as a functional food Substrate because of its nutraceutical and pharmaceutical properties.
- FKP (Produce GDP-l-fucose)
- α-1,2-fucosyltransferase (FucT2 or WcfB) (which catalyzes fucosylation of lactose into 2-FL using GDP-l-fucose.)
Previous studies of metabolic engineering to produce 2-FL have mostly been conducted using Escherichia coli as a host. However, it has several Faults:
- Low Yield (2-FL was produced at a concentration of 23.1 g/L with yields of 0.367 mol 2-FL/mol lactose and 0.355 mol 2-FL/mole fucose.)
- Possible endotoxin contamination in the produced 2-FL
- Bacteriophage infection in the fermentation process to produce 2-FL using engineered E. coli
1. Obtainning GDP-l-fucose
GDP-l-fucose can be generated through two distinct metabolic pathways: the de novo or salvage pathway. In the de novo pathway, GDP-l-fucose is synthesized from mannose-6-phosphate by GDPmannose 4,6-dehydratase and GDP-l-fucose synthase. The alternative salvage pathway requires l-fucose as the substrate for producing GDP-l-fucose. This pathway is catalyzed by a bifunctional enzyme, l-fucokinase/GDPl-fucose phosphorylase (FKP). The salvage pathway was assumed to exist only in eukaryotes until a bacterial FKP was discovered from Bacteroides fragilis 9343. An engineered S. cerevisiae strain was able to produce 0.13 mg of GDP-l-fucose from 25 mL culture containing 2.46 g/L of l-fucose via the salvage pathway, which is the method we are applying.
In order to produce intracellular GDP-l-fucose as a substrate of fucosyltransferase, FKP needs to be introduced. Intracellular GDP-l-fucose level has been considered as a bottleneck in 2-FL production. It was shown that the productivity of 2-FL increased by over 80% by introducing the salvage pathway in addition to the de novo pathway in engineered E. coli.
2. Transporting Lactose
Second, lactose, a fucose acceptor in 2-FL production, needs to be transported into the cytosol of S. cerevisiae cells. Lactose permease, such as Lac12 from Kluyveromyces lactics or CDT-1 from Neurospora crassa needs to be introduced into S. cerevisiae for transporting lactose into the cytosol because wild-type S. cerevisiae is incapable of transporting lactose into the cytosol.
3. Producing 2-FL
Finally, α-1,2-fucosyltransferase, which catalyzes fucosylation of lactose into 2-FL using GDP-l-fucose needs to be introduced into S. cerevisiae. Several α-1,2-fucosyltransferases have been verifed to facilitate the synthesis of 2-FL, which included FucT2 from Helicobacter pylori, WcfB from B. fragilis 9343, and WbgL from E. coli O126. Among those, FucT2 has been most frequently used for the microbial production of 2-FL.
Increase the tilter of 2-FL