Ongoing resolution of duplicate gene functions shapes the diversification of a metabolic network.

The evolutionary mechanisms leading to duplicate gene retention are well understood, but the long-term impacts of paralog differentiation on the regulation of metabolism remain underappreciated. Here we experimentally dissect the functions of two pairs of ancient paralogs of the actose sugar utilization network in two yeast species. We show that the network is more active, even as over-induction is prevented by a second co-repressor that the model yeast lacks. Surprisingly, removal of this repression system leads to a strong growth arrest, likely due to overly rapid galactose catabolism and metabolic overload. Alternative sugars, such as fructose, circumvent metabolic control systems and exacerbate this phenotype. We further show that experiences homologous metabolic constraints that are subtler due to how the paralogs have diversified. These results show how the functional differentiation of paralogs continues to shape regulatory network architectures and metabolic strategies long after initial preservation.