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Abstract
Background2′-Fucosyllactose (2-FL), one of the most abundant oligosaccharides in human milk, has potential applications in foods due to its health benefits such as the selective promotion of bifidobacterial growth and the inhibition of pathogenic microbial binding to the human gut. Owing to the limited amounts of 2-FL in human milk, alternative microbial production of 2-FL is considered promising. To date, microbial production of 2-FL has been studied mostly in Escherichia coli. In this study, 2-FL was produced alternatively by using a yeast Saccharomyces cerevisiae, which may have advantages over E. coli.ResultsFucose and lactose were used as the substrates for the salvage pathway which was constructed with fkp coding for a bifunctional enzyme exhibiting l-fucokinase and guanosine 5′-diphosphate-l-fucose phosphorylase activities, fucT2 coding for α-1,2-fucosyltransferase, and LAC12 coding for lactose permease. Production of 2-FL by the resulting engineered yeast was verified by mass spectrometry. 2-FL titers of 92 and 503 mg/L were achieved from 48-h batch fermentation and 120-h fed-batch fermentation fed with ethanol as a carbon source, respectively.ConclusionsThis is the first report on 2-FL production by using yeast S. cerevisiae. These results suggest that S. cerevisiae can be considered as a host engineered for producing 2-FL via the salvage pathway.
Highlights
Human milk oligosaccharides (HMOs) are important components of human milk that promote infant health [1]
To enable accumulation of GDP-l-fucose in the cytosol, genes coding for FKP originating from 3 different Bacteroides species were individually introduced into S. cerevisiae D452-2
When the engineered yeast D452-2_BF_FKP overexpressing FKP from B. fragilis 9343 was cultured in the presence of fucose, a significant peak at an approximate retention time of 15.1 min was detected in the sample (Fig. 2a)
Summary
Human milk oligosaccharides (HMOs) are important components of human milk that promote infant health [1]. 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 [2, 3]. Production of 2-FL using metabolically engineered microorganisms has been attempted [8, 9]. Production of 2-FL requires α-1,2-fucosyltransferase which transfers the fucosyl residue from guanosine 5′-diphosphate-l-fucose (GDPl-fucose) into lactose. 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
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