Notes

Findings Evidence Support Interest Statement Authors Interest
X.C., Y.M., F.T.

and Y.Z. were all employees of Abbott Laboratories Ltd. when Development of a high-throughput glycoanalysis method for the characterization of oligosaccharides in human milk utilizing multiplexed capillary gel electrophoresis with laser-induced fluorescence detection.During the last decade, enormous progress regarding knowledge about composition and properties of human milk (HM) has been made. Besides nutrition, the three macro-nutrients proteins, lipids, and carbohydrates combine a large variety of properties and functions. Especially, complex oligosaccharides emerge as important dietary factors during early life with multiple functions.

The characterization of these HM oligosaccharides (HMOS) within the total carbohydrate fraction is prerequisite to understand the relationship between milk composition and biological effects. Therefore, extended studies of large donor cohorts and thus, new high-throughput glycoanalytical methods are needed. The developed method comprises sample preparation, as well as analysis of HMOS by multiplexed CGE with LIF detection (xCGE-LIF). Via a respective database the generated fingerprints (normalized electropherograms) could be used for structural elucidation of HMOS. The method was tested on HM samples from five different donors, partly sampled as a series of lactation time points. HMOS could be easily identified and quantified. Consequently, secretor and Lewis status of the donors could be determined, milk typing could be performed and quantitative changes could be monitored along lactation time course.

The developed xCGE-LIF based real high-throughput HMOS analysis method enables qualitative and quantitative high-performance profiling of the total carbohydrate fraction composition of large sets of samples.An automated platform for the enzyme-mediated assembly of complex Li T(1), Liu L(1), Wei N(1), Yang JY(1), Chapla DG(1), Moremen KW(1)(2), Boons Pharmaceutical Sciences, and Bijvoet Center for Biomolecular Research, Utrecht An automated platform that can synthesize a wide range of complex carbohydrates will greatly increase their accessibility and should facilitate progress in glycoscience. Here we report a fully automated process for enzyme-mediated oligosaccharide synthesis that can give easy access to different classes of complex glycans including poly-N-acetyllactosamine derivatives, human milk oligosaccharides , gangliosides and N-glycans. Our automated platform uses a catch and release approach in which glycosyltransferase-catalysed reactions are performed in solution and product purification is accomplished by solid phase extraction. We developed a sulfonate tag that can easily be installed and enables highly efficient solid phase extraction and product release using a single set of washing conditions, regardless of the complexity of the glycan. Using this custom-built synthesizer, as many as 15 reaction cycles can be performed in an automated fashion without a need for lyophilization or buffer Conflict of interest statement Competing interests The authors declare no Production of a human milk oligosaccharide 2'-fucosyllactose by metabolically Urbana-Champaign, Urbana, IL, 611, USA.Urbana-Champaign, Urbana, IL, 611, USA.

BACKGROUND 2'-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.RESULTS Fucose 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 3 mgL were achieved from 48-h batch fermentation and 1-h fed-batch fermentation fed with ethanol as a carbon CONCLUSIONS This 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.
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