Cell survival enabled by leakage of a labile metabolic intermediate

Determining the molecular mechanisms responsible for the emergence and evolution of metabolic pathways is important for our understanding of how life has evolved. In that sense fundamental mechanisms of metabolic innovation may be revealed by experiments with cells in which a crucial enzyme has been deleted. The deletion blocks a metabolic pathway, makes the cells auxotrophic for its final product and allows evolutionary experiments on the restauration of prototrophy to be performed in the lab. A number of such evolutionary repair experiments have provided support for the proposed role of enzyme promiscuity in the emergence of new biochemical pathways. However, the enhancement upon a mutation may allow the organism to meet new challenges by patching novel pathways. Here, we report evolutionary repair experiments on E. coli cells in which an enzyme crucial for the biosynthesis of proline has been deleted. Remarkably, we find reversion to proline prototrophy through a mechanism that does not involve the recruitment of a low-level promiscuous activity, but the leakage to the intracellular milieu of a shielded metabolic intermediate which is metabolized due to a mutation in the glutamine synthetase gene.