Synthesis of fucosylated lacto-N-tetraose using whole-cell biotransformation

Baumgärtner, Florian; Jurzitza, Lukas; Conrad, Jürgen; Beifuß, Uwe; Sprenger, Georg A.; Albermann, Christoph

doi:10.1016/j.bmc.2015.10.005

Cited by 48 publications

(35 citation statements)

References 44 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Nevertheless, the enzymatic in vitro synthesis of linear LNT and LNnT HMOS was demonstrated only for tetra and hexaose structures before . Whole cell biotransformation, which can reach multigram scales easily, seems to be actually very limited to reach mainly LNnT tetraoses, low levels of hexa‐ and octaoses . With our synthesis strategy we achieved large linear HMOS like structures far beyond so far published.…”

Section: Discussionmentioning

confidence: 91%

See 1 more Smart Citation

Enzymatic Cascade Synthesis Provides Novel Linear Human Milk Oligosaccharides as Reference Standards for xCGE-LIF Based High-Throughput Analysis

et al. 2018

View full text Add to dashboard Cite

A rising amount of known health benefits leads to an increased attention of science and nutrient industry to human milk oligosaccharides (HMOS). The unique diversity of HMOS includes several rare, complex, and high molecular weight structures. Therefore, identification and elucidation of complex structures, which may occur only in traces, poses a daunting analytical challenge, further complicated by the limited access to suitable standards. Regarding this, inherent diversity of HMOS and their structural complexity make them difficult to synthesize. The use of recombinant Leloir-glycosyltransferases offers a common strategy to overcome the latter issues. In this study, linear long-chained Lacto-N-biose-type (LNT) and Lacto-N-neo-type (LNnT) HMOS are tailored far beyond the known naturally occurring length. Thereby novel well-defined reference standards for screening HMOS composition by high performance and high throughput analytics are provided. It is shown here for the first time the synthesis of LNT oligomers up to 26 and LNnT oligomers up to 30 sugar units in a semi-sequential one-pot synthesis as analyzed by high performance multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (xCGE-LIF). While being a high-throughput method, xCGE-LIF can also handle long chained linkage isomers of challenging similarity, some of them even present only in trace amounts.

show abstract

Section: Discussionmentioning

confidence: 91%

“…The enzymatic synthesis of HMOS is a common strategy . Several in vivo and in vitro systems can be found in the literature . In that respect in vitro systems provide the benefit to deliver well‐defined products or product distributions and can be directed to form high molecular structures.…”

Section: Introductionmentioning

confidence: 99%

Enzymatic Cascade Synthesis Provides Novel Linear Human Milk Oligosaccharides as Reference Standards for xCGE-LIF Based High-Throughput Analysis

et al. 2018

View full text Add to dashboard Cite

show abstract

“…2′-FL was an obvious starting point for HMO production as it is the most abundant HMO [11,12] and has a simple structure. In contrast, LNnT is less abundant both on its own and as an HMO core, and is also present in human milk in lower levels than, e.g., lacto- N -tetraose (LNT) [11,12], but it appears that LNnT is easier to synthesize in large scale and was therefore marketed first [9,13,14]. Indeed, for more complex and larger structures, fermentation yields are often low [13,15].…”

Section: Introductionmentioning

confidence: 99%

Synthesis of Human Milk Oligosaccharides: Protein Engineering Strategies for Improved Enzymatic Transglycosylation

et al. 2019

View full text Add to dashboard Cite

Human milk oligosaccharides (HMOs) signify a unique group of oligosaccharides in breast milk, which is of major importance for infant health and development. The functional benefits of HMOs create an enormous impetus for biosynthetic production of HMOs for use as additives in infant formula and other products. HMO molecules can be synthesized chemically, via fermentation, and by enzymatic synthesis. This treatise discusses these different techniques, with particular focus on harnessing enzymes for controlled enzymatic synthesis of HMO molecules. In order to foster precise and high-yield enzymatic synthesis, several novel protein engineering approaches have been reported, mainly concerning changing glycoside hydrolases to catalyze relevant transglycosylations. The protein engineering strategies for these enzymes range from rationally modifying specific catalytic residues, over targeted subsite −1 mutations, to unique and novel transplantations of designed peptide sequences near the active site, so-called loop engineering. These strategies have proven useful to foster enhanced transglycosylation to promote different types of HMO synthesis reactions. The rationale of subsite −1 modification, acceptor binding site matching, and loop engineering, including changes that may alter the spatial arrangement of water in the enzyme active site region, may prove useful for novel enzyme-catalyzed carbohydrate design in general.

show abstract

“…Beside chemical routes for the HMOS synthesis [20,21], the enzyme-mediated HMOS tailoring is a very straightforward strategy [7,21,22,23,24,25]. Leloir -glycosyltransferases are especially known for their quantitative substrate conversion, a very high specificity, and high regioselectivity by utilizing activated nucleotide sugars [26].…”

Section: Introductionmentioning

confidence: 99%

Enzymatic Cascades for Tailored 13C6 and 15N Enriched Human Milk Oligosaccharides

et al. 2019

View full text Add to dashboard Cite

Several health benefits, associated with human milk oligosaccharides (HMOS), have been revealed in the last decades. Further progress, however, requires not only the establishment of a simple “routine” method for absolute quantification of complex HMOS mixtures but also the development of novel synthesis strategies to improve access to tailored HMOS. Here, we introduce a combination of salvage-like nucleotide sugar-producing enzyme cascades with Leloir-glycosyltransferases in a sequential pattern for the convenient tailoring of stable isotope-labeled HMOS. We demonstrate the assembly of [13C6]galactose into lacto-N- and lacto-N-neo-type HMOS structures up to octaoses. Further, we present the enzymatic production of UDP-[15N]GlcNAc and its application for the enzymatic synthesis of [13C6/15N]lacto-N-neo-tetraose for the first time. An exemplary application was selected—analysis of tetraose in complex biological mixtures—to show the potential of tailored stable isotope reference standards for the mass spectrometry-based quantification, using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) as a fast and straightforward method for absolute quantification of HMOS. Together with the newly available well-defined tailored isotopic HMOS, this can make a crucial contribution to prospective research aiming for a more profound understanding of HMOS structure-function relations.

show abstract

Synthesis of fucosylated lacto-N-tetraose using whole-cell biotransformation

Cited by 48 publications

References 44 publications

Enzymatic Cascade Synthesis Provides Novel Linear Human Milk Oligosaccharides as Reference Standards for xCGE-LIF Based High-Throughput Analysis

Enzymatic Cascade Synthesis Provides Novel Linear Human Milk Oligosaccharides as Reference Standards for xCGE-LIF Based High-Throughput Analysis

Synthesis of Human Milk Oligosaccharides: Protein Engineering Strategies for Improved Enzymatic Transglycosylation

Enzymatic Cascades for Tailored 13C6 and 15N Enriched Human Milk Oligosaccharides

Contact Info

Product

Resources

About