Enzymes involved in alcohol production in the thermophilic bacterium Thermoanaerobacter sp. strain X514

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Thermoanaerobacter species are thermophilic saccharolytic bacteria with a temperature optimum at or above 60 °C. Ethanol is the major end product of sugar fermentation, besides acetate, hydrogen and lactate. The enzymes and pathways involved in ethanol formation have been studied in a few species, and the bifunctional aldehyde/alcohol dehydrogenase AdhE, reducing acetyl-CoA ultimately to ethanol with NAD(P)H as electron donor, has been suggested to be essential, while other alcohol dehydrogenases encoded in their genomes may or may not be involved. In conclusion, it is unclear, which enzymes are involved and the pathway may vary from species to species. Moreover, it is unclear how reduced ferredoxin, produced during pyruvate oxidation, is oxidized. Preliminary experiments with Thermoanaerobacter sp. strain X514 revealed that this species also produced an aldehyde ferredoxin oxidoreductase (AOR) during growth on cellobiose. Recently, I have published that an AOR is essential for ethanol production of a mutant strain of the archaeon Pyrococcus furiosus. The strain additionally produced the primary alcohol dehydrogenase AdhA from Thermoanaerobacter sp. strain X514. The resulting AOR-AdhA pathway lead to the production of ethanol from sugars and to the reduction of a variety of organic acids to their corresponding alcohols, with reducing equivalents derived from sugars. Interestingly, further preliminary experiments showed that Thermoanaerobacter sp. strain X514 produced isobutanol from isobutyrate in the presence of cellobiose. Based on these preliminary experiments, I hypothesize that AOR may play a role in alcohol formation in Thermoanaerobacter sp. strain X514. I intend to study the pathways and enzymes involved in ethanol formation from sugar and in reduction of organic acids to their corresponding alcohols in Thermoanaerobacter sp. strain X514. I intend to isolate and characterize the aldehyde and alcohol dehydrogenases involved. AOR and/or a ferredoxin:NAD(P)H oxidoreductase such as Rnf or Nfn may be essential for ferredoxin re-oxidation. Therefore, I intend to purify and characterize the ferredoxin oxidizing enzymes. A transcriptome analysis will be carried out in order to identify candidate genes / enzymes essential for alcohol formation. A genetic system for Thermoanaerobacter sp. strain X514 will be developed and used to delete genes coding for putatively essential enzymes such as the bifunctional aldehyde/alcohol dehydrogenase AdhE, the primary alcohol dehydrogenase AdhA, the secondary alcohol dehydrogenase AdhB, AOR, Rnf or Nfn. The phenotype of the deletion mutants will be studied in growth and cell suspension experiments. In conclusion, the combination of transcriptomic, genetic, physiological and biochemical approaches will lead to a comprehensive understanding of carbon and redox metabolism in Thermoanaerobacter sp. strain X514 during growth on sugars in the presence or absence of externally added organic acids.