Characterization of enzymes involved in the ethanol production of Moorella sp. HUC22-1.
Since the thermophilic bacterium Moorella sp. HUC22-1 produces 120 mM acetate and 5.2 mM ethanol from H(2)-CO(2), 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 microg of AdhA and 50 microg Aldh were added to the buffer solution (pH 8.0) containing NADPH, NADH and acetyl-CoA at 60 degrees 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 H(2)-CO(2) culture than that in the fructose culture, but levels of adhA, B and C were decreased.
Inokuma
Kentaro
K
Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Kagamiyama 1-3-1, Higashi-Hiroshima 739-8530, Japan.
Nakashimada
Yutaka
Y
Akahoshi
Takuya
T
Nishio
Naomichi
N
D000426:Alcohol Dehydrogenase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000445:Aldehyde Oxidoreductases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D000431:Ethanol / Q000378:metabolism*; D007700:Kinetics; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D046968:Thermoanaerobacter / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors
D001426:Bacterial Proteins; D000431:Ethanol; D000426:Alcohol Dehydrogenase; D000445:Aldehyde Oxidoreductases; C018699:aldehyde dehydrogenase (NAD(P)+)