title: Directed Evolution Reveals Unexpected Epistatic Interactions That Alter Metabolic Regulation and Enable Anaerobic Xylose Use by Saccharomyces cerevisiae.
abstract: The inability of native Saccharomyces cerevisiae to convert xylose from plant biomass into biofuels remains a major challenge for the production of renewable bioenergy. Despite extensive knowledge of the regulatory networks controlling carbon metabolism in yeast, little is known about how to reprogram S. cerevisiae to ferment xylose at rates comparable to glucose. Here we combined genome sequencing, proteomic profiling, and metabolomic analyses to identify and characterize the responsible mutations in a series of evolved strains capable of metabolizing xylose aerobically or anaerobically. We report that rapid xylose conversion by engineered and evolved S. cerevisiae strains depends upon epistatic interactions among genes encoding a xylose reductase (GRE3), a component of MAP Kinase (MAPK) signaling (HOG1), a regulator of Protein Kinase A (PKA) signaling (IRA2), and a scaffolding protein for mitochondrial iron-sulfur (Fe-S) cluster biogenesis (ISU1). Interestingly, the mutation in IRA2 only impacted anaerobic xylose consumption and required the loss of ISU1 function, indicating a previously unknown connection between PKA signaling, Fe-S cluster biogenesis, and anaerobiosis. Proteomic and metabolomic comparisons revealed that the xylose-metabolizing mutant strains exhibit altered metabolic pathways relative to the parental strain when grown in xylose. Further analyses revealed that interacting mutations in HOG1 and ISU1 unexpectedly elevated mitochondrial respiratory proteins and enabled rapid aerobic respiration of xylose and other non-fermentable carbon substrates. Our findings suggest a surprising connection between Fe-S cluster biogenesis and signaling that facilitates aerobic respiration and anaerobic fermentation of xylose, underscoring how much remains unknown about the eukaryotic signaling systems that regulate carbon metabolism.
authors.affiliation: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Trey K
authors.identifier: http://orcid.org/0000-0001-6592-9337
authors.initials: TK
authors.lastname: Sato
authors.affiliation: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Mary
authors.initials: M
authors.lastname: Tremaine
authors.affiliation: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Lucas S
authors.identifier: http://orcid.org/0000-0001-6156-4780
authors.initials: LS
authors.lastname: Parreiras
authors.affiliation: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.|Genome Center of Wisconsin, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Hebert
authors.affiliation: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.|Genome Center of Wisconsin, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.|Laboratory of Genetics, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Kevin S
authors.initials: KS
authors.lastname: Myers
authors.affiliation: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.|Genome Center of Wisconsin, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.|Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Alan J
authors.identifier: http://orcid.org/0000-0001-9379-8362
authors.initials: AJ
authors.lastname: Higbee
authors.affiliation: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.|Genome Center of Wisconsin, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.|Laboratory of Genetics, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.|Microbiology Doctoral Training Program, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Maria
authors.initials: M
authors.lastname: Sardi
authors.affiliation: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Sean J
authors.identifier: http://orcid.org/0000-0002-3820-8400
authors.initials: SJ
authors.lastname: McIlwain
authors.affiliation: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Irene M
authors.identifier: http://orcid.org/0000-0001-5968-9437
authors.initials: IM
authors.lastname: Ong
authors.affiliation: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Rebecca J
authors.identifier: http://orcid.org/0000-0001-6727-3378
authors.initials: RJ
authors.lastname: Breuer
authors.affiliation: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Ragothaman
authors.identifier: http://orcid.org/0000-0001-8673-4504
authors.initials: R
authors.lastname: Avanasi Narasimhan
authors.affiliation: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Mick A
authors.identifier: http://orcid.org/0000-0001-9178-2703
authors.initials: MA
authors.lastname: McGee
authors.affiliation: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Quinn
authors.initials: Q
authors.lastname: Dickinson
authors.affiliation: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Alex
authors.identifier: http://orcid.org/0000-0001-7750-6091
authors.initials: A
authors.lastname: La Reau
authors.affiliation: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Dan
authors.identifier: http://orcid.org/0000-0002-7335-3726
authors.initials: D
authors.lastname: Xie
authors.affiliation: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.|Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Mingyuan
authors.identifier: http://orcid.org/0000-0002-6518-9540
authors.initials: M
authors.lastname: Tian
authors.affiliation: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.|Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Reed
authors.affiliation: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Yaoping
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.|Genome Center of Wisconsin, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.|Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.|Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Joshua J
authors.initials: JJ
authors.lastname: Coon
authors.affiliation: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.|Genome Center of Wisconsin, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.|Laboratory of Genetics, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.|Microbiology Doctoral Training Program, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.|Wisconsin Energy Institute, J.F. Crow Institute for the Study of Evolution, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Chris Todd
authors.initials: CT
authors.lastname: Hittinger
authors.affiliation: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.|Genome Center of Wisconsin, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.|Laboratory of Genetics, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.|Microbiology Doctoral Training Program, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Audrey P
authors.initials: AP
authors.lastname: Gasch
authors.affiliation: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.|Microbiology Doctoral Training Program, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.|Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Robert
authors.initials: R
authors.lastname: Landick
mesh_terms: D000693:Anaerobiosis / Q000235:genetics; D019020:Directed Molecular Evolution; D004843:Epistasis, Genetic; D005285:Fermentation; D005818:Genetic Engineering; D005947:Glucose / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000235:genetics; D053858:Metabolic Networks and Pathways / Q000235:genetics; D024101:Mitochondrial Proteins / Q000235:genetics*; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics*; D009154:Mutation; D040901:Proteomics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D014994:Xylose / Q000235:genetics / Q000378:metabolism*
chemical_list: C121520:ISU1 protein, S cerevisiae; D007506:Iron-Sulfur Proteins; D024101:Mitochondrial Proteins; D029701:Saccharomyces cerevisiae Proteins; D014994:Xylose; C088589:HOG1 protein, S cerevisiae; D020928:Mitogen-Activated Protein Kinases; D005947:Glucose