title: Engineering and two-stage evolution of a lignocellulosic hydrolysate-tolerant Saccharomyces cerevisiae strain for anaerobic fermentation of xylose from AFEX pretreated corn stover.
abstract: The inability of the yeast Saccharomyces cerevisiae to ferment xylose effectively under anaerobic conditions is a major barrier to economical production of lignocellulosic biofuels. Although genetic approaches have enabled engineering of S. cerevisiae to convert xylose efficiently into ethanol in defined lab medium, few strains are able to ferment xylose from lignocellulosic hydrolysates in the absence of oxygen. This limited xylose conversion is believed to result from small molecules generated during biomass pretreatment and hydrolysis, which induce cellular stress and impair metabolism. Here, we describe the development of a xylose-fermenting S. cerevisiae strain with tolerance to a range of pretreated and hydrolyzed lignocellulose, including Ammonia Fiber Expansion (AFEX)-pretreated corn stover hydrolysate (ACSH). We genetically engineered a hydrolysate-resistant yeast strain with bacterial xylose isomerase and then applied two separate stages of aerobic and anaerobic directed evolution. The emergent S. cerevisiae strain rapidly converted xylose from lab medium and ACSH to ethanol under strict anaerobic conditions. Metabolomic, genetic and biochemical analyses suggested that a missense mutation in GRE3, which was acquired during the anaerobic evolution, contributed toward improved xylose conversion by reducing intracellular production of xylitol, an inhibitor of xylose isomerase. These results validate our combinatorial approach, which utilized phenotypic strain selection, rational engineering and directed evolution for the generation of a robust S. cerevisiae strain with the ability to ferment xylose anaerobically from ACSH.
authors.affiliation: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Lucas S
authors.initials: LS
authors.lastname: Parreiras
authors.affiliation: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Rebecca J
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.initials: R
authors.lastname: Avanasi Narasimhan
authors.affiliation: DOE Great Lakes Bioenergy Research Center, 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.initials: AJ
authors.lastname: Higbee
authors.affiliation: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Alex
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: 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: Li
authors.initials: L
authors.lastname: Qin
authors.affiliation: Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Laura B
authors.initials: LB
authors.lastname: Willis
authors.affiliation: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Benjamin D
authors.initials: BD
authors.lastname: Bice
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Brandi L
authors.initials: BL
authors.lastname: Bonfert
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Rebeca C
authors.initials: RC
authors.lastname: Pinhancos
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Allison J
authors.initials: AJ
authors.lastname: Balloon
authors.affiliation: DOE Great Lakes Bioenergy Research Center, Michigan State University, East Lansing, Michigan, United States of America; Biomass Conversion Research Laboratory, Department of Chemical Engineering and Materials Science, Michigan State University, Lansing, Michigan, United States of America.
authors.forename: Nirmal
authors.initials: N
authors.lastname: Uppugundla
authors.affiliation: DOE Great Lakes Bioenergy Research Center, Michigan State University, East Lansing, Michigan, United States of America; School of Food and Bioengineering, Qilu University of Technology, Jinan, China.
authors.forename: Tongjun
authors.initials: T
authors.lastname: Liu
authors.affiliation: Advanced Biofuels Process Demonstration Unit, Lawrence Berkeley National Laboratory, Emeryville, California, United States of America.
authors.forename: Chenlin
authors.initials: C
authors.lastname: Li
authors.affiliation: Advanced Biofuels Process Demonstration Unit, Lawrence Berkeley National Laboratory, Emeryville, California, United States of America.
authors.forename: Deepti
authors.initials: D
authors.lastname: Tanjore
authors.affiliation: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Irene M
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: Haibo
authors.initials: H
authors.lastname: Li
authors.affiliation: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Edward L
authors.initials: EL
authors.lastname: Pohlmann
authors.affiliation: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Jose
authors.initials: J
authors.lastname: Serate
authors.affiliation: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Sydnor T
authors.initials: ST
authors.lastname: Withers
authors.affiliation: Deconstruction Division, Joint BioEnergy Institute, Emeryville, California, United States of America.
authors.forename: Blake A
authors.initials: BA
authors.lastname: Simmons
authors.affiliation: DOE Great Lakes Bioenergy Research Center, Michigan State University, East Lansing, Michigan, United States of America; Department of Chemical Engineering & Materials Science, Michigan State University, East Lansing, Michigan, United States of America; Department of Biosystems & Agricultural Engineering, Michigan State University, East Lansing Michigan, United States of America; Division of Sustainable Process Engineering, Luleå University of Technology, Luleå, Sweden.
authors.forename: David B
authors.initials: DB
authors.lastname: Hodge
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Westphall
authors.affiliation: Department of 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, Michigan State University, East Lansing, Michigan, United States of America; Biomass Conversion Research Laboratory, Department of Chemical Engineering and Materials Science, Michigan State University, Lansing, Michigan, United States of America.
authors.forename: Bruce E
authors.initials: BE
authors.lastname: Dale
authors.affiliation: DOE Great Lakes Bioenergy Research Center, Michigan State University, East Lansing, Michigan, United States of America; Biomass Conversion Research Laboratory, Department of Chemical Engineering and Materials Science, Michigan State University, Lansing, Michigan, United States of America.
authors.forename: Venkatesh
authors.initials: V
authors.lastname: Balan
authors.affiliation: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: David H
authors.initials: DH
authors.lastname: Keating
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; Department of Bacteriology, 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
authors.affiliation: DOE Great Lakes Bioenergy Research Center, 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: 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.
authors.forename: Trey K
authors.initials: TK
authors.lastname: Sato
mesh_terms: D000641:Ammonia / Q000378:metabolism; D000693:Anaerobiosis; D056804:Biofuels; D018533:Biomass; D000431:Ethanol / Q000378:metabolism; D005285:Fermentation; D005818:Genetic Engineering; D006868:Hydrolysis; D008031:Lignin / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D014994:Xylose / Q000235:genetics / Q000378:metabolism*; D003313:Zea mays / Q000378:metabolism
chemical_list: D056804:Biofuels; C036909:lignocellulose; D000431:Ethanol; D000641:Ammonia; D008031:Lignin; D014994:Xylose