Cell survival enabled by leakage of a labile metabolic intermediate
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 (Pal 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.