Production of perdeuterated fucose from glyco-engineered bacteria.

Lukas Gajdos,Eric Samain,Matthew P Blakeley,V Trevor Forsyth,Anne Imberty,Juliette M Devos,Michael Haertlein

doi:10.1093/glycob/cwaa059

Abstract

l-Fucose and l-fucose-containing polysaccharides, glycoproteins or glycolipids play an important role in a variety of biological processes. l-Fucose-containing glycoconjugates have been implicated in many diseases including cancer and rheumatoid arthritis. Interest in fucose and its derivatives is growing in cancer research, glyco-immunology, and the study of host–pathogen interactions. l-Fucose can be extracted from bacterial and algal polysaccharides or produced (bio)synthetically. While deuterated glucose and galactose are available, and are of high interest for metabolic studies and biophysical studies, deuterated fucose is not easily available. Here, we describe the production of perdeuterated l-fucose, using glyco-engineered Escherichia coli in a bioreactor with the use of a deuterium oxide-based growth medium and a deuterated carbon source. The final yield was 0.2 g L−1 of deuterated sugar, which was fully characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. We anticipate that the perdeuterated fucose produced in this way will have numerous applications in structural biology where techniques such as NMR, solution neutron scattering and neutron crystallography are widely used. In the case of neutron macromolecular crystallography, the availability of perdeuterated fucose can be exploited in identifying the details of its interaction with protein receptors and notably the hydrogen bonding network around the carbohydrate binding site.

Highlights

The deuteration of biomolecules by stable isotope labelling is of high interest for nuclear magnetic resonance (NMR) spectroscopy and neutron scattering studies of the structure and dynamics of biological macromolecules and is widely used (Haertlein et al 2016, Blakeley and Podjarny 2018)
FUC5 is a derivative of the E. coli K12 strain DH1, with genetic modifications for overexpression of genes involved in GDP-fucose production, introduction of 2'fucosyltransferase gene from Helicobacter pylori and fucosidase gene from Bifidobacterium bifidum strain JCM1254, and deletion of genes involved in the metabolism of lactose
For the production of deuterated fucose-d12, deuterated glycerol-d8 was used as the sole carbon source

Summary

Introduction

The deuteration of biomolecules by stable isotope labelling is of high interest for nuclear magnetic resonance (NMR) spectroscopy and neutron scattering studies of the structure and dynamics of biological macromolecules and is widely used (Haertlein et al 2016, Blakeley and Podjarny 2018). Through the use of appropriate protecting groups, direct H–D exchange reactions in the presence of an activated carbon-supported platinum group catalyst were used to produce a variety of deuterated monosaccharides (Koch and Stuart 1978, Sawama et al 2012). Using biotechnology approaches, it has been demonstrated more than 50 years ago that certain algae can be adapted to grow in 99.6% D2O, resulting in the large scale production of perdeuterated glucose and mannose (Crespi et al 1959, Chorney et al 1960). Polysaccharides have been produced in bacteria, in the form of perdeuterated cellulose (O'Neill et al 2015) and perdeuterated heparin (Cress et al 2019)

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