PRJNA658462
Engineering Molybdenum cofactor biosynthesis enables the functional expression of a heterologous nitrate assimilation pathway in Saccharomyces cerevisiae
The metabolic state of a cell is not only defined by the presence of a specific set of enzymes and metabolites but also by the presence of cofactors within the cell. The Molybdenum cofactor (Moco) is found in nearly all organisms with Saccharomyces yeasts as a prominent eukaryotic exception. In this work, the Ogataea parapolymorpha Moco biosynthetic pathway was functionally characterised and then transferred in S. cerevisiae. The ability of S. cerevisiae to synthesize Moco was assessed by co-expressing the Ogataea nitrate assimilation pathway including the Moco-dependent nitrate reductase. After a rapid evolution that resulted in changes in gene dosage of the heterologous pathway, increased expression of the heterologous proteins, and a 5-fold increase in the nitrate reductase activity, the engineered strains were able to grow on nitrate as sole nitrogen source. Although not essential, growth on nM range of molybdate required the expression of a characterized Chlamydomonas reinhardtii high affinity molybdate transporter. Competition for alternative nitrogen source might be at the origin of fermentation contaminations at industrial scale, therefore nitrate assimilating S. cerevisiae strain was challenged in co-cultures together with the spoilage yeast Brettanomyces bruxellensis in medium containing nitrate. In contrast to a non-engineered S. cerevisiae that was totally outcompeted within few generations, the engineered strain IMS816 exhibited an improved robustness persisting in the co-culture for up to 35 generations. To date more than 50 Moco-dependent enzymes have been described and engineering Moco synthesis is a potential strategy to broaden the enzymatic repertoire of S. cerevisiae.