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Multistep Synthesis of UDP-Glucose Using Tailored, Permeabilized Cells of E. coli

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Abstract

We constructed and applied a recombinant, permeabilized Escherichia coli strain for the multistep synthesis of UDP-glucose. Sucrose phosphorylase (E.C. 2.4.1.7) of Leuconostoc mesenteroides was over expressed and the pgm gene encoding for phosphoglucomutase (E.C. 5.4.2.2) was deleted in E. coli to yield the E. coli JW 0675-1 SP strain. The cells were permeabilized with the detergent Triton X-100 at 0.05 % v/v. The synthesis of UDP-glucose with permeabilized cells was then optimized with regard to pH, cell density during the synthesis and growth phase during cell harvest, metal cofactor, other media components, and temperature. In one configuration sucrose, phosphate, UMP, and ATP were used as substrates. At pH 7.8, 27 mg/ml cell dry weight, cell harvest during the early stationary phase of growth and Mn2+ as cofactor a yield of 37 % with respect to UMP was achieved at 33 °C. In a second step, ATP was regenerated by feeding glucose and using only catalytic amounts of ATP and NAD+. A UDP-glucose yield of 60 % with respect to UMP was obtained using this setup. With the same setup but without addition of external ATP, the yield was 54 %.

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Acknowledgments

We gratefully thank Christiane Goedl and Bernd Nidetzky for supplying pQE 30-LmSPase plasmid used for the creation of the E. coli JW 0675-1 SP strain. We acknowledge the support by BMBF (Federal Ministry of Education and Research, Project MECAT, FKZ 031P7238 within the initiative “Biotechnologie 2020+: Basistechnologien für eine nächste Generation biotechnologischer Verfahren.”

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  1. Biochemical Engineering Institute, Saarland University, Campus A1.5, 66123, Saarbrucken, Germany

    Christian Weyler & Elmar Heinzle

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  1. Christian Weyler
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  2. Elmar Heinzle
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Correspondence to Elmar Heinzle.

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Weyler, C., Heinzle, E. Multistep Synthesis of UDP-Glucose Using Tailored, Permeabilized Cells of E. coli . Appl Biochem Biotechnol 175, 3729–3736 (2015). https://doi.org/10.1007/s12010-015-1540-3

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