Evolverator: An engineered in cellulo yeast system to drive rapid continuous evolution of proteins

In vivo continuous directed evolution generates genetic diversity and selects a target phenotype to generate proteins with desired functionality. However, in current systems, the two processes do not operate simultaneously in the same cell, restricting applications such as evolution of eukaryotic protein-ligand binding. Here, we describe Evolverator Saccharomyces cerevisiae cells that combine inducible targeted mutagenesis with engineered gene circuits that link the emergence of a desired function to graded increases in cell proliferation. Poor ligand binding induces targeted mutagenesis and cells with mutations that improve ligand binding overtake the cell population. By combining strain development with mathematical modeling for systems and process design, we evolved ligand specificities of the human estrogen receptor and more effective variants of the bacterial lacI repressor. Previously undescribed mutations affected residues plausibly involved in ligand binding and residues that exert allosteric effects. Evolverator should aid generation of proteins that bind new targets for many applications.