Multistep synthesis of UDP-glucose using tailored, permeabilized cells of E. coli

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 Mn(2+) 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%.