Development of a pyruvate decarboxylase for use in thermophilic bacteria

Bioethanol produced from organic waste, as a second generation biofuel, is an important renewable energy source. Yeast and a limited range of bacteria use the homoethanol fermentation pathway, which employs pyruvate decarboxylase (PDC) in conjunction with alcohol dehydrogenase (ADH), to convert pyruvate to ethanol. PDCs are widely distributed in plants and fungi, but are rare in bacteria. Of the known bacterial PDCs, that from Zymomonas mobilis is the most extensively characterized. The pdc and adh genes of Z. mobilis have been successfully cloned and expressed in E. coli and a number of other mesophiles thus creating highly effective ethanologenic strains.

Despite extensive screening no PDC has yet been identified in a thermophilic organism. The most thermostable PDC, from the mesophile Zymobacter palmae, is active at 55°C (when sourced from its native host or a recombinant mesophile) but is not expressed in its active form at temperatures above 60°C. We believe that this is not simply a problem of protein thermostability, but also one of protein folding at high temperatures.

Using Geobacillus thermoglucosidasius as a host platform, we are endeavoring to develop a PDC that both folds and functions at high temperatures in order to create a thermophilic version of the homoethanol pathway for use in both this host and others. In this poster we will describe strategies to improve PDC expression of active PDC at high temperatures, including codon harmonization and creation of ancestral and hybrid enzymes.