title: Quantitative Trait Loci (QTL)-Guided Metabolic Engineering of a Complex Trait.
abstract: Engineering complex phenotypes for industrial and synthetic biology applications is difficult and often confounds rational design. Bioethanol production from lignocellulosic feedstocks is a complex trait that requires multiple host systems to utilize, detoxify, and metabolize a mixture of sugars and inhibitors present in plant hydrolysates. Here, we demonstrate an integrated approach to discovering and optimizing host factors that impact fitness of Saccharomyces cerevisiae during fermentation of a Miscanthus x giganteus plant hydrolysate. We first used high-resolution Quantitative Trait Loci (QTL) mapping and systematic bulk Reciprocal Hemizygosity Analysis (bRHA) to discover 17 loci that differentiate hydrolysate tolerance between an industrially related (JAY291) and a laboratory (S288C) strain. We then used this data to identify a subset of favorable allelic loci that were most amenable for strain engineering. Guided by this "genetic blueprint", and using a dual-guide Cas9-based method to efficiently perform multikilobase locus replacements, we engineered an S288C-derived strain with superior hydrolysate tolerance than JAY291. Our methods should be generalizable to engineering any complex trait in S. cerevisiae, as well as other organisms.
authors.affiliation: Energy Biosciences Institute and ‡Department of Bioengineering, University of California , Berkeley, California 94720, United States.|Biological Systems and Engineering Division, and ∥Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory , Berkeley, California 94720, United States.
authors.forename: Matthew J
authors.identifier: 0000-0002-9150-0240
authors.initials: MJ
authors.lastname: Maurer
authors.affiliation: Energy Biosciences Institute and ‡Department of Bioengineering, University of California , Berkeley, California 94720, United States.|Biological Systems and Engineering Division, and ∥Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory , Berkeley, California 94720, United States.
authors.forename: Lawrence
authors.initials: L
authors.lastname: Sutardja
authors.affiliation: Energy Biosciences Institute and ‡Department of Bioengineering, University of California , Berkeley, California 94720, United States.|Biological Systems and Engineering Division, and ∥Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory , Berkeley, California 94720, United States.
authors.forename: Dominic
authors.initials: D
authors.lastname: Pinel
authors.affiliation: Energy Biosciences Institute and ‡Department of Bioengineering, University of California , Berkeley, California 94720, United States.|Biological Systems and Engineering Division, and ∥Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory , Berkeley, California 94720, United States.
authors.forename: Stefan
authors.initials: S
authors.lastname: Bauer
authors.affiliation: Energy Biosciences Institute and ‡Department of Bioengineering, University of California , Berkeley, California 94720, United States.|Biological Systems and Engineering Division, and ∥Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory , Berkeley, California 94720, United States.
authors.forename: Amanda L
authors.initials: AL
authors.lastname: Muehlbauer
authors.affiliation: Energy Biosciences Institute and ‡Department of Bioengineering, University of California , Berkeley, California 94720, United States.|Biological Systems and Engineering Division, and ∥Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory , Berkeley, California 94720, United States.
authors.forename: Tyler D
authors.initials: TD
authors.lastname: Ames
authors.affiliation: Energy Biosciences Institute and ‡Department of Bioengineering, University of California , Berkeley, California 94720, United States.|Biological Systems and Engineering Division, and ∥Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory , Berkeley, California 94720, United States.
authors.forename: Jeffrey M
authors.identifier: 0000-0003-2653-1566
authors.initials: JM
authors.lastname: Skerker
authors.affiliation: Energy Biosciences Institute and ‡Department of Bioengineering, University of California , Berkeley, California 94720, United States.|Biological Systems and Engineering Division, and ∥Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory , Berkeley, California 94720, United States.
authors.forename: Adam P
authors.initials: AP
authors.lastname: Arkin
mesh_terms: D000431:Ethanol / Q000378:metabolism; D005285:Fermentation / Q000235:genetics; D006868:Hydrolysis; D060847:Metabolic Engineering / Q000379:methods; D010641:Phenotype; D010944:Plants / Q000378:metabolism; D040641:Quantitative Trait Loci / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D058615:Synthetic Biology / Q000379:methods
chemical_list: D000431:Ethanol
keywords: CRISPR-Cas9; biofuel; genetic engineering; hydrolysate; quantitative trait loci; strain development