A thermostable mannitol-1-phosphate dehydrogenase is required in mannitol metabolism of the thermophilic acetogenic bacterium Thermoanaerobacter kivui

Acetogenic bacteria recently attracted attention because they reduce carbon dioxide (CO₂) with hydrogen (H₂) to acetate or to other products such as ethanol. Besides gases, acetogens use a broad range of substrates, but conversion of the sugar alcohol mannitol has rarely been reported. We found that the thermophilic acetogenic bacterium Thermoanaerobacter kivui grew on mannitol with a specific growth rate of 0.33 h⁻¹ to a final optical density (OD₆₀₀) of 2.2. Acetate was the major product formed. A lag phase was observed only in cultures pre-grown on glucose, not in those pre-grown on mannitol, indicating that mannitol metabolism is regulated. Mannitol-1-phosphate dehydrogenase (MtlD) activity was observed in cell-free extracts of cells grown on mannitol only. A gene cluster (TKV_c02830–TKV_c02860) for mannitol uptake and conversion was identified in the T. kivui genome, and its involvement was confirmed by deleting the mtlD gene (TKV_c02860) encoding the key enzyme MtlD. Finally, we overexpressed mtlD, and the recombinant MtlD carried out the reduction of fructose-6-phosphate with NADH, at a high V_MAX of 1235 U mg⁻¹ at 65°C. The enzyme was thermostable for 40 min at 75°C, thereby representing the first characterized MtlD from a thermophile.