Characterization of Thermotoga neapolitana Alcohol Dehydrogenases in the Ethanol Fermentation Pathway

Simple Summary Hyperthermophilic Thermotoga spp. are promising candidates for the metabolic engineering of cellulosic ethanol producing strains. However, Thermotoga spp. are hydrogen producers, and none of them have been found to produce significant amount of ethanol. In this study, we focused on the enzymes responsible for the part of the ethanol production pathway from ac-CoA to ethanol. Aldehyde dehydrogenases (Aldhs) and alcohol dehydrogenases (Adhs) are the vital enzymes involved in the metabolism of ethanol in Thermotoga spp.. However, the biochemical properties of a number of Adhs related to ethanol formation and consumption have not been identified until now. The aim of this study was to determine the role of putative Adhs in T. neapolitana (Tne) in the pathway from ac-CoA to ethanol. We found that the Fe-AAdh (encoded by CTN_0580) and Fe-Adh2 (encoded by CTN_1756) were the main enzymes responsible for ethanol production in Tne. While Zn-Adh (encoded by CTN_0257) was identified as the main protein responsible for ethanol consumption in the acid environment and Fe-Adh1 (encoded by CTN_1655) played a minor role in ethonal production and consumption in Tne. Basing on these results, it will be helpful to construct and assemble a novel hyperthermophilic strain to produce cellulosic ethanol. Hyperthermophilic Thermotoga spp. are candidates for cellulosic ethanol fermentation. A bifunctional iron-acetaldehyde/alcohol dehydrogenase (Fe-AAdh) has been revealed to catalyze the acetyl-CoA (Ac-CoA) reduction to form ethanol via an acetaldehyde intermediate in Thermotoga neapolitana (T. neapolitana). In this organism, there are three additional alcohol dehydrogenases, Zn-Adh, Fe-Adh1, and Fe-Adh2, encoded by genes CTN_0257, CTN_1655, and CTN_1756, respectively. This paper reports the properties and functions of these enzymes in the fermentation pathway from Ac-CoA to ethanol. It was determined that Zn-Adh only exhibited activity when oxidizing ethanol to acetaldehyde, and no detectable activity for the reaction from acetaldehyde to ethanol. Fe-Adh1 had specific activities of approximately 0.7 and 0.4 U/mg for the forward and reverse reactions between acetaldehyde and ethanol at a pH(opt) of 8.5 and T-opt of 95 degrees C. Catalyzing the reduction of acetaldehyde to produce ethanol, Fe-Adh2 exhibited the highest activity of approximately 3 U/mg at a pH(opt) of 7.0 and T-opt of 85 degrees C, which were close to the optimal growth conditions. These results indicate that Fe-Adh2 and Zn-Adh are the main enzymes that catalyze ethanol formation and consumption in the hyperthermophilic bacterium, respectively.