Harnessing Mass Spectrometry-Based Proteomics for Continuous Directed Evolution

Continuous directed evolution is a powerful Synthetic Biology tool to engineer proteins with desired functions in vivo. Mimicking natural evolution, it involves repeated cycles of high-frequency mutagenesis, selection, and replication within platform cells, where the function of the target gene is tightly linked to the host cell's fitness. However, cells might escape the selection pressure due to the inherent flexibility of their metabolism, which allows for adaptation. Whole-proteome analysis as well as targeted proteomics offer valuable insights into global and specific cellular changes. They can identify modifications in the target protein and its interactors to help understand its evolution and network integration. Using the continuous evolution of the Arabidopsis methionine synthases AtMS1 and AtMS2 as an example, we demonstrate how mass spectrometry-based proteomics can be applied in CDE, propose specific checkpoints for its integration and illustrate its role in informed decision making.