The gene encoding polyphosphate kinase from Thermos thermophilus (TtPPK) was expressed in Escherichia coli Rosetta2 (DE3) pLysS. The E. coli recombinant cells were heated at 70 C to inactivate indigenous enzymes and used for regenerating adenosine triphosphate (ATP) from exogenous polyphosphate (polyp) and adenosine diphosphate (ADP). The heat-treated cells having TtPPK were able to regenerate ATP at rates similar to those detected in cell-free extracts, suggesting that exogenous polyP and ADP could freely access TtPPK through the heat-damaged cell envelope. More than 80% of TtPPK activity was retained in the heated cells after incubation for at least 40 min at 70 C. TtPPK in the heated cells could be easily recovered from the reaction mixture by centrifugation at 12,000 x g for 10 min. The gene encoding thermophilic ATP-dependent glycerol kinase from Thermococcus kodakaraensis KOD1 (TkGK) was expressed in E. coli Rosetta2 (DE3) pLysS. Using the mixture of E. coli recombinants expressing TkGK and TtPPK, the production of glycerol 3-phosphate (G3P) from glycerol was examined at 70 C as a model reaction. When polyP was added to the reaction mixture in a fed-batch mode, 100 mM glycerol was stoichiometrically converted to 80 mM G3P (a molar yield of 80%). (C) 2011 Elsevier Ltd. All rights reserved.