Crude glycerol was used as substrate for orotic acid production by Ashbya gossypii.
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Culture conditions optimization improved the production of orotic acid.
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Further improvements were obtained by engineering of the de novo pyrimidine pathway.
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A mitochondrial dihydroorotate (DHOD) dehydrogenase is crucial for this phenotype.
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AgDHOD enables orotic acid production by engineered Saccharomyces cerevisiae.
Abstract
Orotic acid is an intermediate of the de novo pyrimidine biosynthesis that plays a crucial role in several nutraceutical supplements. In some microorganisms, blockage of this pathway downstream the dihydroorotate dehydrogenase (DHOD) leads to orotic acid accumulation. Here, the Ashbya gossypii Agura3 pyrimidine auxotroph was shown to accumulate and excrete orotic acid (~1.5 g/L), and this trait was further explored for the production of orotic acid from raw substrates. Metabolic engineering of this strain combined with culture conditions optimization led to a 3.6-fold increase in orotic acid production from crude glycerol. The mitochondrial DHOD encoded by AgURA9 was shown to be determinant for this phenotype. Heterologous expression of AgURA9 in engineered S. cerevisiae enabled the accumulation and excretion of orotic acid (~0.5 g/L). This study demonstrates the potential of A. gossypii for the valorization of crude glycerol to orotic acid and discloses the molecular determinants for its biosynthesis in fungi.
