In Vivo continuous Evolution of Genes and Pathways in Yeast

Directed evolution serves as a critical bridge between sub-optimal and optimal biological components, even in light of rational design approaches.  While efforts have been made to automate and improve this process for bacterial systems, less attention has been placed on fungal systems.  Here, we demonstrate an in vivo mutagenesis approach in yeast by synthetically optimizing the retrotransposon Ty1 to enable libraries of 10⁸ per liter per round.  We demonstrate this approach by utilizing in vivo-generated libraries in the directed evolution of global transcriptional regulators, single enzymes, and multi-gene pathways.  Fundamentally, this library can be used as an input to screen and/or select for any measurable phenotype, and enables continuous mutant generation and selection for growth-associated phenotypes.  In each case described here, we obtain rapid and significant improvements in performance over days instead of weeks, a clear advantage over traditional in vitro approaches, especially in fungal systems.  Furthermore, we demonstrate transferability of this approach to divergent yeasts such as Kluyveromyces lactis and alternative S. cerevisiae strains.  Finally, we demonstrate how this approach can be coupled with microdroplet technologies to enable rapid screening and selection of novel metabolic production phenotypes.