Since the thermophilic bacterium Moorella sp. HUC22-1 produces 120 mM acetate and 5.2 mM ethanol from H2–CO2, several candidate genes, which were predicted to code for three alcohol dehydrogenases (AdhA, B, C) and one acetaldehyde dehydrogenase (Aldh), were cloned from HUC22-1. The cloned genes were subcloned into a His-tagged expression vector and expressed in Escherichia coli. Recombinant AdhA and B were both dependent on NADP(H) but independent of NAD(H), and their reduction activities from aldehyde to alcohol were higher than their oxidation activities. In contrast with AdhA and B, no activity of AdhC was observed in either reaction. On the other hand, Aldh was active toward both NADP(H) and NAD(H). The enzyme activity of Aldh was directed toward the thioester cleavage and the thioester condensation. When 50 μg of AdhA and 50 μg Aldh were added to the buffer solution (pH 8.0) containing NADPH, NADH and acetyl-CoA at 60°C, 1.6 mM ethanol was produced from 3 mM acetyl-CoA after 90 min. Expression analysis of the mRNAs revealed that the expression level of aldh was threefold higher in the H2–CO2 culture than that in the fructose culture, but levels of adhA, B and C were decreased.
Ethanol production H2–CO2Thermophile Anaerobe Alcohol dehydrogenase Acetaldehyde dehydrogenase
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We appreciate T. K. Hikosaka for DNA sequence determination service at Natural Science Center for Basic Research and Development, Hiroshima University.
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