Evolutionary regain of lost gene circuit function

Evolutionary reversibility - the ability to regain a lost function - is an important problem both in evolutionary and synthetic biology, where repairing natural or synthetic systems broken by evolutionary processes may be valuable. Here, we use a synthetic positive-feedback (PF) gene circuit integrated into haploid Saccharomyces cerevisiae cells to test if the population can restore lost PF function. In previous evolution experiments, mutations in a gene eliminated the fitness costs of PF activation. Since PF activation also provides drug resistance, exposing such compromised or broken mutants to both drug and inducer should create selection pressure to regain drug resistance and possibly PF function. Indeed, evolving seven PF mutant strains in the presence of drug revealed three adaptation scenarios through genomic mutations outside of the PF circuit that elevate PF basal expression, possibly by affecting transcription, translation, degradation and other fundamental cell functions. Nonfunctional mutants gained drug resistance without ever developing high expression, while quasi-functional and dysfunctional PF mutants developed high expression which then diminished, although more slowly for dysfunctional mutants where revertant clones arose. These results highlight how intracellular context, such as the growth rate, can affect regulatory network dynamics and evolutionary dynamics, which has important consequences for understanding the evolution of drug resistance and developing future synthetic biology applications.\n\nSignificance StatementNatural or synthetic genetic modules can lose their function over long-term evolution if the function is costly. How populations can evolve to restore broken functions is poorly understood. To test the reversibility of evolutionary breakdown, we use yeast cell populations with a chromosomally integrated synthetic gene circuit. In previous evolution experiments the gene circuit lost its costly function through various mutations. By exposing such mutant populations to conditions where regaining gene circuit function would be beneficial we find adaptation scenarios with or without repairing lost gene circuit function. These results are important for drug resistance or future synthetic biology applications where loss and regain of function play a significant role.