Production of perdeuterated fucose from glyco-engineered bacteria

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.

Neutron crystallography reveals novel mechanisms used by Pseudomonas aeruginosa for host-cell binding

“…Recombinant perdeuterated LecB (D-LecB) was produced in an Escherichia coli high cell-density culture in the presence of D 2 O and glycerol-d 8 after adaptation of cells 23 . Perdeuterated fucose (Fuc-d 12 ) was obtained using recently described synthetic biology methods for which E. coli strains re-engineered for their carbohydrate metabolism were adapted for growth in deuterated media 24 . Co-crystals with volume of ~0.1 mm 3 were obtained over a period of approximately 4 weeks.…”

“…Perdeuterated fucose-d 12 was produced by glyco-enginereed E. coli in a high cell-density culture as reported previously 24 . Co-crystals of deuterated LecB with L-fucose-d 12 were obtained in the following conditions: 0.1 M Tris/DCl, pD 7.1, 20% (w/v) PEG 4000.…”

Visualization of hydrogen atoms in a perdeuterated lectin-fucose complex reveals key details of protein-carbohydrate interactions

“…To date, only a couple of neutron structures have used perdeuterated monosaccharides, which were all chemically synthetized (Kovalevsky et al, 2008;Langan et al, 2014). In our study, we used a perdeuterated sugar produced via biosynthetic pathways in glyco-engineered E. coli bacteria that was reported recently (Gajdos et al, 2020). The use of hydrogenated sugars can lead to cancellation effects in the neutron maps, mainly around CH 2 groups, which might make interpretation of maps challenging.…”

“…Very few neutron structures of proteins complexed with perdeuterated carbohydrate have been characterized (Kovalevsky et al, 2008(Kovalevsky et al, , 2010Langan et al, 2014). Recently, however, we reported the production of perdeuterated L-fucose-d 12 (Fucd 12 ) obtained using a glyco-engineered E. coli strain, genetically modified to produce high titers of L-fucose (Gajdos et al, 2020).…”

“…The fully-deuterated fucose (Fuc-d 12 ) was produced using glyco-engineered bacteria in a bioreactor as described previously (Gajdos et al 2020). The secreted fully-deuterated fucose was further purified and characterised before being used for the crystallisation experiments described in this work.…”