A novel bifunctional aldehyde/alcohol dehydrogenase mediating ethanol formation from acetyl-CoA in hyperthermophiles

Abstract

Background: Hyperthermophilic fermentation at temperatures above 80 °C allows in situ product removal to mitigate the ethanol toxicity, and reduces microbial contamination without autoclaving/cooling of feedstock. Many species of Thermotoga grow at temperatures up to 90 °C, and have enzymes to degrade and utilize lignocelluloses, which provide advantages for achieving consolidated processes of cellulosic ethanol production. However, no CoA-dependent aldehyde dehydrogenase (CoA-Aldh) from any hyperthermophiles has been documented in literature so far. The pyruvate ferredoxin oxidoreductases from hyperthermophiles have pyruvate decarboxylase activity, which convert about 2% and 98% of pyruvate to acetaldehyde and acetyl-CoA (ac-CoA), respectively. Acetyl-CoA can be converted to acetic acid, if there is no CoA-Aldh to convert ac-CoA to acetaldehyde and further to ethanol. Therefore, the current study aimed to identify and characterize a CoA-Aldh activity that mediates ethanol fermentation in hyperthermophiles.Results: In Thermotoga neapolitana (Tne), a hyperthermophilic iron-acetaldehyde/alcohol dehydrogenase (Fe-AAdh) was, for the first time, revealed to catalyze the ac-CoA reduction to form ethanol via an acetaldehyde intermediate, while the annotated aldh gene in Tne genome only encodes a CoA-independent Aldh that oxidizes aldehyde to acetic acid. Three other Tne alcohol dehydrogenases (Adh) exhibited specific physiological roles in ethanol formation and consumption: Fe-Adh2 mainly catalyzed the reduction of acetaldehyde to produce ethanol, and Fe-Adh1 showed significant activities only under extreme conditions, while Zn-Adh showed special activity in ethanol oxidation. In the in vitro formation of ethanol from ac-CoA, a strong synergy was observed between Fe-Adh1 and Fe-AAdh. The Fe-AAdh gene is highly conserved in Thermotoga spp. and in Pyrococus sp., which is probably responsible for ethanol metabolism in hyperthermophiles.Conclusions: Hyperthermophilic Thermotoga spp. are excellent candidates for biosynthesis of cellulosic ethanol fermentation strains. The finding of a novel hyperthermophilic CoA-Aldh activity of Tne Fe-AAdh revealed the existence of a hyperthermophilic fermentation pathway from ac-CoA to ethanol, which offers a basic frame for in vitro synthesis of a highly active AAdh for effective ethanol fermentation pathway in hyperthermophiles, which is a key element for the approach to the consolidated processes of cellulosic ethanol production.