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

Abstract

Summary 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 VMAX 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.

Homoacetogenic Conversion of Mannitol by the Thermophilic Acetogenic Bacterium Thermoanaerobacter kivui Requires External CO2

“…Experiments were performed in biological duplicates and a representative growth curve is shown. electron acceptor increased the maximal OD 600 to 2.34 and decreased the doubling time to 2.0 ± 0.0 (Figure 3), which corresponds to the growth behavior observed before during growth on mannitol in the presence of CO 2 /HCO 3 − (Moon et al, 2019). Growth on formate as sole substrate contributed only little (Figure 3).…”

“…One option would be an additional reduced product, such as lactate, H 2 , ethanol or formate. Metabolite analyses in our recent experiments with T. kivui growing on mannitol (Moon et al, 2019), however, revealed no major other products. We are aware of only one other study in which products of mannitol utilization in an acetogen, Sporomusa termitida, were quantified; and in that organism, acetate was as well the major product, with a slightly lower ratio (2.6 mol per mol mannitol), and with minor amounts of some other products such as propionate or ethanol detected (Breznak et al, 1988).…”

“…Here, we describe the catabolism of the thermophilic acetogenic bacterium Thermoanaerobacter kivui growing on the sugar alcohol mannitol. We recently characterized the uptake of mannitol by a phosphotransferase system (PTS) and the subsequent conversion of mannitol-1-phosphate by a thermostable mannitol-1-phosphate dehydrogenase in T. kivui (Moon et al, 2019). By a variety of physiological experiments with growing cells and cell suspension, we now show unambiguously that T. kivui utilizes external CO 2 as additional electron acceptor during growth on and conversion of mannitol; the biochemical and eco-physiological consequences are discussed.…”

“…The wild type T. kivui strain LKT-1 (DSM2030) was cultivated under strict anoxic conditions at 65 • C in either complex or carbonate buffered defined medium as described previously (Moon et al, 2019). Carbonate free medium was prepared as carbonate buffered defined medium, but no KHCO 3 was added and the medium was flushed with 100% N 2 .…”

“…H 2 , alcohol and organic acid concentrations were determined by gas chromatography as described previously (Weghoff and Müller, 2016). The concentrations of glucose and mannitol were determined by high performance liquid as described previously (Moon et al, 2019). Lactic acid and formic acid concentrations were determined using test kits (R-Biopharm AG, Darmstadt, Germany) according to supplier's instruction.…”