A Novel, in Vivo Continuous Evolution Strategy in Saccharomyces Cerevisiae to Create Pathway and Protein Variants

Current evolution techniques in yeast usually require in vitro synthesis of a mutant library, transformation of the created library into yeast cells, and selection of desired phenotypes towards a preferred pressure.  Due to the difficulties of library synthesis and low efficiency of yeast transformation, the traditional directed evolution process is time-consuming and labor-intensive.  Here, we developed a novel in vivo continuous evolution process in which mutagenesis occurs simultaneously with cell growth, resulting in a more streamlined evolution cycle. As a proof-of-concept, we applied in vivo continuous evolution to the development of improved variants of model proteins, global transcriptional regulators, and multigene catabolic pathways in S. cerevisiae over several rounds of in vivo optimization. Taken together, these demonstrations show that in vivo continuous evolution can significantly accelerate the pace of pathway development in yeast.