title: Identification of gene knockdown targets conferring enhanced isobutanol and 1-butanol tolerance to Saccharomyces cerevisiae using a tunable RNAi screening approach.
abstract: Improving yeast tolerance to 1-butanol and isobutanol is a step toward enabling high-titer production. To identify previously unknown genetic targets leading to increased tolerance, we establish a tunable RNA interference (RNAi) screening approach. Specifically, we optimized the efficiency and tunability of RNA interference library screening in yeast, ultimately enabling downregulation efficiencies from 0 to 94 %. Using this system, we identified the Hsp70 family as a key regulator of isobutanol tolerance in a single round of screening, with downregulation of these genes conferring up to 64 % increased growth in 12 g/L isobutanol. For 1-butanol, we find through two rounds of iterative screening that the combined downregulation of alcohol dehydrogenase and enolase improves growth up to 3100 % in 10 g/L 1-butanol. Collectively, this work improves the tunability of RNAi in yeast as demonstrated by the discovery of novel effectors for these complex phenotypes.
authors.affiliation: McKetta Department of Chemical Engineering, The University of Texas at Austin, 200 East Dean Keeton Street Stop C0400, Austin, TX, 78712, USA.
authors.forename: Nathan
authors.initials: N
authors.lastname: Crook
authors.affiliation: McKetta Department of Chemical Engineering, The University of Texas at Austin, 200 East Dean Keeton Street Stop C0400, Austin, TX, 78712, USA.
authors.forename: Jie
authors.initials: J
authors.lastname: Sun
authors.affiliation: McKetta Department of Chemical Engineering, The University of Texas at Austin, 200 East Dean Keeton Street Stop C0400, Austin, TX, 78712, USA.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Morse
authors.affiliation: McKetta Department of Chemical Engineering, The University of Texas at Austin, 200 East Dean Keeton Street Stop C0400, Austin, TX, 78712, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Schmitz
authors.affiliation: McKetta Department of Chemical Engineering, The University of Texas at Austin, 200 East Dean Keeton Street Stop C0400, Austin, TX, 78712, USA. halper@utexas.edu.
authors.forename: Hal S
authors.initials: HS
authors.lastname: Alper
mesh_terms: D020001:1-Butanol / Q000633:toxicity*; D000426:Alcohol Dehydrogenase / Q000235:genetics / Q000378:metabolism; D000440:Butanols / Q000633:toxicity*; D004361:Drug Tolerance; D055785:Gene Knockdown Techniques; D005820:Genetic Testing / Q000379:methods*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D010751:Phosphopyruvate Hydratase / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics / Q000254:growth & development
chemical_list: D000440:Butanols; D018840:HSP70 Heat-Shock Proteins; C040507:isobutyl alcohol; D020001:1-Butanol; D000426:Alcohol Dehydrogenase; D010751:Phosphopyruvate Hydratase
keywords: Alcohol dehydrogenase; Butanol tolerance; Hsp70; RNA interference; Yeast