Anaerobically evolved fungi that harbor Class II dihydroorotate dehydrogenase enzymes that function independently of the respiratory chain

The goal of this study was elucidate the respiratory chain dependency of the abovementioned Class II DHODs. The URA9 genes of Anaeromyces robustus, Sch. japonicus, D. bruxellensis, O. parapolymorpha and K. marxianus were expressed in a ura1 null mutant of S. cerevisiae and growth was studied under anaerobic conditions. The subcellular localization of the enzymes was determined by fluorescence microscopy of GFP-fused Ura9 proteins. Finally, alternative electron acceptors were determined for these proteins by enzyme assays using cell free extracts and mitochondrial extracts of S. cerevisiae ura1 null mutants expressing the URA9 genes from a multicopy plasmid.