Many metabolites are generated in one step of a biochemical pathway and consumed in a subsequent step. Such metabolic intermediates are often reactive molecules which, if allowed to freely diffuse in the intracellular milieu, could lead to undesirable side reactions and even become toxic to the cell. Therefore, metabolic intermediates are often protected as protein-bound species and directly transferred between enzyme active sites in multi-functional enzymes, multi-enzyme complexes, and metabolons. Sequestration of reactive metabolic intermediates thus contributes to metabolic efficiency. It is not known, however, whether this evolutionary adaptation can be relaxed in response to challenges to organismal survival. Here, we report evolutionary repair experiments on Escherichia coli cells in which an enzyme crucial for the biosynthesis of proline has been deleted. The deletion makes cells unable to grow in a culture medium lacking proline. Remarkably, however, cell growth is efficiently restored by many single mutations (12 at least) in the gene of glutamine synthetase. The mutations cause the leakage to the intracellular milieu of a highly reactive phosphorylated intermediate common to the biosynthetic pathways of glutamine and proline. This intermediate is generally assumed to exist only as a protein-bound species. Nevertheless, its diffusion upon mutation-induced leakage enables a new route to proline biosynthesis. Our results support that leakage of sequestered metabolic intermediates can readily occur and contribute to organismal adaptation in some scenarios. Enhanced availability of reactive molecules may enable the generation of new biochemical pathways and the potential of mutation-induced leakage in metabolic engineering is noted.
Bibliographical noteFunding Information:
This work was supported by Human Frontier Science Program Grant RGP0041/2017 (J.M.S.R. and B.S.), Spanish Ministry of Science and Innovation FEDER Funds Grants RTI2018-097142-B-100 and PID2021-124534OB-100 (J.M.S.R.), National Aeronautics and Space Administration (NASA) Grant 80NSSC18K1277 (B.S.) Grant RYC2021- 031155-I (E.M.C.) from Spanish Ministry of Science and Innovation and NextGenerationEU/PRTR and Grant E-BIO-464-UGR-20 (E.M.C.) from FEDER Funds and Consejería de Economía, Conocimiento, Empresas y Universidad de la Junta de Andalucia. E.A.L. was a recipient of a postdoctoral fellowship from the regional Andalusian Government (2020-DOC-00541). We thank the "Centro de Supercomputacion" (ALHAMBRA-CSIRC) of the University of Granada for providing computational resources and Dr Valeria A. Risso for useful discussions, comments on the manuscript and help with the evolutionary and structural analyses of the rescuing mutations.
© 2023 The Author(s).
- auxotrophy rescue
- evolutionary repair experiments
- labile metabolic intermediates
- laboratory evolution
- metabolic innovation
- prototrophy restoration
PubMed: MeSH publication types
- Journal Article
- Research Support, Non-U.S. Gov't