title: In vivo continuous evolution of genes and pathways in yeast.
abstract: Directed evolution remains a powerful, highly generalizable approach for improving the performance of biological systems. However, implementations in eukaryotes rely either on in vitro diversity generation or limited mutational capacities. Here we synthetically optimize the retrotransposon Ty1 to enable in vivo generation of mutant libraries up to 1.6 × 107l-1per round, which is the highest of any in vivo mutational generation approach in yeast. We demonstrate this approach by using in vivo-generated libraries to evolve single enzymes, global transcriptional regulators and multi-gene pathways. When coupled to growth selection, this approach enables in vivo continuous evolution (ICE) of genes and pathways. Through a head-to-head comparison, we find that ICE libraries yield higher-performing variants faster than error-prone PCR-derived libraries. Finally, we demonstrate transferability of ICE to divergent yeasts, including Kluyveromyces lactis and alternative S. cerevisiae strains. Collectively, this work establishes a generic platform for rapid eukaryotic-directed evolution across an array of target cargo.
authors.affiliation: Department of Chemical Engineering, The University of Texas at Austin, 200 East Dean Keeton Street, Stop C0400, Austin, Texas 78712, USA.
authors.forename: Nathan
authors.identifier: 0000-0001-6165-1972
authors.initials: N
authors.lastname: Crook
authors.affiliation: Department of Chemical Engineering, The University of Texas at Austin, 200 East Dean Keeton Street, Stop C0400, Austin, Texas 78712, USA.
authors.forename: Joseph
authors.initials: J
authors.lastname: Abatemarco
authors.affiliation: Department of Chemical Engineering, The University of Texas at Austin, 200 East Dean Keeton Street, Stop C0400, Austin, Texas 78712, USA.
authors.forename: Jie
authors.initials: J
authors.lastname: Sun
authors.affiliation: Department of Chemical Engineering, The University of Texas at Austin, 200 East Dean Keeton Street, Stop C0400, Austin, Texas 78712, USA.
authors.forename: James M
authors.initials: JM
authors.lastname: Wagner
authors.affiliation: Department of Chemical Engineering, The University of Texas at Austin, 200 East Dean Keeton Street, Stop C0400, Austin, Texas 78712, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Schmitz
authors.affiliation: Department of Chemical Engineering, The University of Texas at Austin, 200 East Dean Keeton Street, Stop C0400, Austin, Texas 78712, USA.|Institute for Cellular and Molecular Biology, The University of Texas at Austin, 2500 Speedway Avenue, Austin, Texas 78712, USA.
authors.forename: Hal S
authors.initials: HS
authors.lastname: Alper
mesh_terms: D019020:Directed Molecular Evolution / Q000379:methods*; D015966:Gene Expression Regulation, Fungal; D015723:Gene Library; D005800:Genes, Fungal / Q000235:genetics*; D007716:Kluyveromyces / Q000235:genetics; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D008957:Models, Genetic; D016254:Mutagenesis, Insertional; D009154:Mutation; D018626:Retroelements / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D015003:Yeasts / Q000145:classification / Q000235:genetics* / Q000378:metabolism
chemical_list: D018626:Retroelements