Use of an Escherichia coli recombinant producing thermostable polyphosphate kinase as an ATP regenerator to produce fructose 1,6-diphosphate

Abstract

Heat-treated Escherichia coli producing Thermus polyphosphate kinase regenerated ATP by using exogenous polyphosphate. This recombinant could be used as a platform to produce valuable compounds in combination with thermostable phosphorylating or energy-requiring enzymes. In this work, we demonstrated the production of fructose 1,6-diphosphate from fructose and polyphosphate.

FIG. 1.

Effect of temperature (A), pH (B), MgCl₂ (C), (NH₄)₂SO₄ (D), KCl (E), and the length of polyP (F) on the ATP synthesis activity of PPK_T. All data shown are representative of two replicates and were almost perfectly reproduced unless otherwise noted. Bars in panel F represent standard deviations in triplicate experiments.

FIG. 2.

ATP synthesis activity by E. coli producing PPK_T. After heat treatment, recombinant E. coli was precipitated. Heat-treated cells (filled circles) and the supernatant (open circles) were examined for the ability to synthesize ATP.

FIG. 3.

Synthesis of FDP from fructose and polyP75 by E. coli recombinants. FDP was synthesized using 4 mg of E. coli cells producing PPK_T, 0.5 mg of E. coli cells producing PFK, and 0.5 mg of E. coli cells producing FK (filled circles). The ratio of PPK to PFK to FK in terms of protein concentration was approximately 8:1:3. FDP was measured as previously reported (6). In a parallel reaction, ADP and polyP75 were replaced by 30 mM ATP as the phosphoryl donor (open circles). FDP was also synthesized using 5.8 mg of E. coli cells producing PPK_T, PFK, and FK simultaneously (filled triangles).