Engineering heterologous molybdenum-cofactor-biosynthesis and nitrate-assimilation pathways enables nitrate utilization by Saccharomyces cerevisiae.

Metabolic capabilities of cells are not only defined by their repertoire of enzymes and metabolites, but also by availability of enzyme cofactors. The molybdenum cofactor (Moco) is widespread among eukaryotes but absent from the industrial yeast Saccharomyces cerevisiae. No less than 50 Moco-dependent enzymes covering over 30 catalytic activities have been described to date, introduction of a functional Moco synthesis pathway offers interesting options to further broaden the biocatalytic repertoire of S. cerevisiae. In this study, we identified seven Moco biosynthesis genes in the non-conventional yeast Ogataea parapolymorpha by SpyCas9-mediated mutational analysis and expressed them in S. cerevisiae. Functionality of the heterologously expressed Moco biosynthesis pathway in S. cerevisiae was assessed by co-expressing O. parapolymorpha nitrate-assimilation enzymes, including the Moco-dependent nitrate reductase. Following two-weeks of incubation, growth of the engineered S. cerevisiae strain was observed on nitrate as sole nitrogen source. Relative to the rationally engineered strain, the evolved derivatives showed increased copy numbers of the heterologous genes, increased levels of the encoded proteins and a 5-fold higher nitrate-reductase activity in cell extracts. Growth at nM molybdate concentrations was enabled by co-expression of a Chlamydomonas reinhardtii high-affinity molybdate transporter. In serial batch cultures on nitrate-containing medium, a non-engineered S. cerevisiae strain was rapidly outcompeted by the spoilage yeast Brettanomyces bruxellensis. In contrast, an engineered and evolved nitrate-assimilating S. cerevisiae strain persisted during 35 generations of co-cultivation. This result indicates that the ability of engineered strains to use nitrate may be applicable to improve competitiveness of baker's yeast in industrial processes upon contamination with spoilage yeasts.

Class-II dihydroorotate dehydrogenases from three phylogenetically distant fungi support anaerobic pyrimidine biosynthesis.

BACKGROUND: In most fungi, quinone-dependent Class-II dihydroorotate dehydrogenases (DHODs) are essential for pyrimidine biosynthesis. Coupling of these Class-II DHODHs to mitochondrial respiration makes their in vivo activity dependent on oxygen availability. Saccharomyces cerevisiae and closely related yeast species harbor a cytosolic Class-I DHOD (Ura1) that uses fumarate as electron acceptor and thereby enables anaerobic pyrimidine synthesis. Here, we investigate DHODs from three fungi (the Neocallimastigomycete Anaeromyces robustus and the yeasts Schizosaccharomyces japonicus and Dekkera bruxellensis) that can grow anaerobically but, based on genome analysis, only harbor a Class-II DHOD. RESULTS: Heterologous expression of putative Class-II DHOD-encoding genes from fungi capable of anaerobic, pyrimidine-prototrophic growth (Arura9, SjURA9, DbURA9) in an S. cerevisiae ura1Δ strain supported aerobic as well as anaerobic pyrimidine prototrophy. A strain expressing DbURA9 showed delayed anaerobic growth without pyrimidine supplementation. Adapted faster growing DbURA9-expressing strains showed mutations in FUM1, which encodes fumarase. GFP-tagged SjUra9 and DbUra9 were localized to S. cerevisiae mitochondria, while ArUra9, whose sequence lacked a mitochondrial targeting sequence, was localized to the yeast cytosol. Experiments with cell extracts showed that ArUra9 used free FAD and FMN as electron acceptors. Expression of SjURA9 in S. cerevisiae reproducibly led to loss of respiratory competence and mitochondrial DNA. A cysteine residue (C265 in SjUra9) in the active sites of all three anaerobically active Ura9 orthologs was shown to be essential for anaerobic activity of SjUra9 but not of ArUra9. CONCLUSIONS: Activity of fungal Class-II DHODs was long thought to be dependent on an active respiratory chain, which in most fungi requires the presence of oxygen. By heterologous expression experiments in S. cerevisiae, this study shows that phylogenetically distant fungi independently evolved Class-II dihydroorotate dehydrogenases that enable anaerobic pyrimidine biosynthesis. Further structure-function studies are required to understand the mechanistic basis for the anaerobic activity of Class-II DHODs and an observed loss of respiratory competence in S. cerevisiae strains expressing an anaerobically active DHOD from Sch. japonicus.