PT - JOURNAL ARTICLE AU - Encarnación Medina-Carmona AU - Luis I. Gutierrez-Rus AU - Fadia Manssour-Triedo AU - Matilda S. Newton AU - Gloria Gamiz-Arco AU - Antonio J. Mota AU - Pablo Reiné AU - Juan Manuel Cuerva AU - Mariano Ortega-Muñoz AU - Eduardo Andrés-León AU - Jose Luis Ortega-Roldan AU - Burckhard Seelig AU - Beatriz Ibarra-Molero AU - Jose M. Sanchez-Ruiz TI - Cell survival enabled by leakage of a labile metabolic intermediate AID - 10.1101/2022.05.20.492833 DP - 2022 Jan 01 TA - bioRxiv PG - 2022.05.20.492833 4099 - http://biorxiv.org/content/early/2022/05/20/2022.05.20.492833.short 4100 - http://biorxiv.org/content/early/2022/05/20/2022.05.20.492833.full AB - Many metabolic pathways are of ancient origin and have evolved over long periods of time (Noda-Garcia et al., 2018). Yet, new pathways can also emerge in short time scales in response, for instance, to the presence of anthropogenic chemicals in the environment (Copley, 2009). Models of metabolic pathway emergence and evolution often emphasize the acquisition of new reactions through horizontal gene transfer and promiscuous enzyme functionalities (Pál et al., 2005; Schulenburg & Miller, 2014; Copley, 2015; Noda-Garcia et al., 2018; Peracchi, 2018). A fundamentally different mechanism of metabolic innovation is revealed by the evolutionary repair experiments reported here. A block in the proline biosynthetic pathway that compromises cell survival is efficiently rescued by many single mutations (12 at least) in the gene of glutamine synthetase. The mutations cause the leakage to the intracellular milieu of a sequestered phosphorylated intermediate common to the biosynthetic pathways of proline and glutamine, thus generating a new route to proline. Metabolic intermediates may undergo a variety of chemical and enzymatic transformations, but are typically protected as shielded reaction intermediates or through channeling in multi-enzyme complexes and metabolons (Srere, 1987; Huang et al., 2001; Grunwald, 2018; Pareek et al., 2021). Our results show that intermediate leakage can readily occur and contribute to organismal adaptation. Enhanced availability of reactive molecules may enable the generation of new biochemical pathways. We therefore anticipate applications of mutation-induced leakage in metabolic engineering.Competing Interest StatementThe authors have declared no competing interest.