Abstract
Objectives
To investigate the translocation of nucleotide-activated sugars from the cytosol across a membrane into the endoplasmatic reticulum or the Golgi apparatus which is an important step in the synthesis of glycoproteins and glycolipids in eukaryotes.
Results
The heterologous expression of the recombinant and codon-adapted human GDP-l-fucose antiporter gene SLC35C1 (encoding an N-terminal OmpA-signal sequence) led to a functional transporter protein located in the cytoplasmic membrane of Escherichia coli. The in vitro transport was investigated using inverted membrane vesicles. SLC35C1 is an antiporter specific for GDP-l-fucose and depending on the concomitant reverse transport of GMP. The recombinant transporter FucT1 exhibited an activity for the transport of 3H-GDP-l-fucose with a Vmax of 8 pmol/min mg with a Km of 4 µM. The functional expression of SLC35C1 in GDP-l-fucose overproducing E. coli led to the export of GDP-l-fucose to the culture supernatant.
Conclusions
The export of GDP-l-fucose by E. coli provides the opportunity for the engineering of a periplasmatic fucosylation reaction in recombinant bacterial cells.
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Acknowledgments
The authors are thankful to Stephan Nußberger (Universtät Stuttgart) for his support with the electron microscope and to Josef Altenbuchner (Universtät Stuttgart) for providing us with plasmid pJOE2702. The authors acknowledge the financial support by a grant from the German Bundesministerium für Bildung und Forschung (BioChance Plus 0315170) to Jennewein Biotechnologie GmbH, Rheinbreitbach, Germany. We also thank Jennewein Biotechnologie GmbH for the supply of strains and plasmids.
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Parts of the presented results have been filed as a patent application.
Supporting information
Supplementary Table 1—Strains and plasmids used in this study.
Supplementary Fig. 1—DNA and sequence of the codon optimized fucT1 gene.
Supplementary Fig. 2—Kinetic data of the GDP-l-fucose transport by FucT1.
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Karin Förster-Fromme and Sarah Schneider have contributed equally to this work.
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Förster-Fromme, K., Schneider, S., Sprenger, G.A. et al. Functional expression of a human GDP-l-fucose transporter in Escherichia coli . Biotechnol Lett 39, 219–226 (2017). https://doi.org/10.1007/s10529-016-2233-x
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DOI: https://doi.org/10.1007/s10529-016-2233-x