We previously demonstrated the stoichiometric conversion of glycerol to glycerol-3-phosphate (G3P) using Escherichia coli recombinants producing the ATP-dependent glycerol kinase of the hyperthermophile Thermococcus kodakaraensis (TkGK) and the polyphosphate kinase of Therm us thermophilus HB27 (TtPPK). TtPPK was associated with the membrane fraction of E. coil recombinants, whereas TkGK was released from the cells during the reaction at 70 degrees C. In this study, TUCK was fused with either TtPPK or an E. coli membrane-intrinsic protein, YedZ, to minimize the heat-induced leakage of TkGK. When the E. coil recombinants having these fusion proteins were incubated at 70 degrees C for 2 h, more than 80% of TkGK activity was retained in the heated E. coil cells. However, the yields of G3P production by E. coil having the fusion proteins of TtPPK and TkGK were only less than 35%. Polyphosphate is a strong chelator for metal ions and has an inhibitory effect on TkGK which requires magnesium. Insufficient space between TtPPK and TkGK might enhance the inhibitory effect of polyphosphate on TkGK activity of the fusion protein. The mixture of E. con cells having TtPPK and those having TkGK fused with YedZ converted 80% of glycerol into G3P. These recombinant cells could be easily recovered from the reaction mixture by centrifugation and repeatedly used without a significant loss of enzyme activities. (C) 2011, The Society for Biotechnology, Japan. All rights reserved.