Title : Novel thermostable enzymes in alcohol fermentation at high temperatures
Hyperthermophiles are a group of microorganisms growing optimally at or above 80°C and can utilize carbohydrates and peptides to produce alcohols as end products. There are desirable advantages such as higher conversion rate and greater substrate solubility for alcohol fermentation at high temperatures. Two pathways are well-known for the alcohol production from pyruvate which is a central metabolic intermediate. In a two-step pathway, pyruvate is decarboxylated non-oxidatively by pyruvate decarboxylase (PDC) into acetaldehyde, which is then reduced to ethanol by alcohol dehydrogenase (ADH). In a three-step pathway, pyruvate is decarboxylated by either pyruvate ferredoxin oxidoreductase (POR) oxidatively or pyruvate formate lyase into acetyl-CoA that is then reduced to acetaldehyde by a CoAdependent acetaldehyde dehydrogenase (AcDH), which is then reduced to ethanol by ADH. To understand the pathways and key enzymes involved in alcohol production at high temperatures, a few hyperthermophilic archaea and bacteria were selected for our study. Both zinc- and iron-containing ADHs were characterized from hyperthermophilic archaeon Thermococcus guaymasensis (zinc-ADH) and bacterium Thermotoga hypogea (iron-ADH), and their physiological roles are proposed to be in the formation of alcohols such as ethanol. Both zinc- and iron-ADHs were successfully cloned and overexpressed in E. coli. The recombinant ADHs had indistinguishable properties from the native ones. It was more intriguing about the enzyme catalyzing the production of acetaldehyde from pyruvate at high temperatures. No homolog genes are found to encode commonly-known PDC and AcDH respectively. A novel bifunctional PDC is present in Pyrococcus furiosus, which catalyzes both Co-A-dependent oxidative (POR) and non-oxidative (PDC) decarboxylation of pyruvate, producing acetyl-CoA and acetaldehyde, respectively. We also found that such a PDC activity is present in hyperthermophilic archaeon T. guaymasensis and bacteria Thermotoga maritima and T. hypogea. Coenzyme A or desulfoCoA is required for these PDC activities. We further identified another PDC activity present in T. maritima, which showed acetohydroxyacid synthase activity as well. Recently, we characterized a bifunctional AdhE from T. maritima that catalyzes the reduction of acetyl-CoA to acetaldehyde (AcDH activity) and acetaldehyde to ethanol (ADH activity) respectively. It is concluded that thermostable PDCs, AcDHs and ADHs are present in hyperthermophilic archaea and bacteria, supporting that a new type of two-step and three-step pathways for alcohol production from pyruvate is operational. Further studies of these thermostable PDCs, AcDHs and ADHs are required for developing a more efficient alcohol fermentation process at high temperatures.