title: Employing a combinatorial expression approach to characterize xylose utilization in Saccharomyces cerevisiae.
abstract: Fermentation of xylose, a major constituent of lignocellulose, will be important for expanding sustainable biofuel production. We sought to better understand the effects of intrinsic (genotypic) and extrinsic (growth conditions) variables on optimal gene expression of the Scheffersomyces stipitis xylose utilization pathway in Saccharomyces cerevisiae by using a set of five promoters to simultaneously regulate each gene. Three-gene (xylose reductase, xylitol dehydrogenase (XDH), and xylulokinase) and eight-gene (expanded with non-oxidative pentose phosphate pathway enzymes and pyruvate kinase) promoter libraries were enriched under aerobic and anaerobic conditions or with a mutant XDH with altered cofactor usage. Through characterization of enriched strains, we observed (1) differences in promoter enrichment for the three-gene library depending on whether the pentose phosphate pathway genes were included during the aerobic enrichment; (2) the importance of selection conditions, where some aerobically-enriched strains underperform in anaerobic conditions compared to anaerobically-enriched strains; (3) improved growth rather than improved fermentation product yields for optimized strains carrying the mutant XDH compared to the wild-type XDH.
authors.affiliation: Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA; Energy Biosciences Institute, Berkeley, CA 94720, USA. Electronic address: latimer@berkeley.edu.
authors.forename: Luke N
authors.initials: LN
authors.lastname: Latimer
authors.affiliation: Energy Biosciences Institute, Berkeley, CA 94720, USA; The UC Berkeley & UCSF Graduate Program in Bioengineering, Berkeley, CA 94720, USA; Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA. Electronic address: michael.lee@berkeley.edu.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Lee
authors.affiliation: Department of Nutritional Sciences and Toxicology, University of California, Berkeley, Berkeley, CA 94720, USA. Electronic address: danielmc@berkeley.edu.
authors.forename: Daniel
authors.initials: D
authors.lastname: Medina-Cleghorn
authors.affiliation: Department of Nutritional Sciences and Toxicology, University of California, Berkeley, Berkeley, CA 94720, USA. Electronic address: rkohnz@berkeley.edu.
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Kohnz
authors.affiliation: Department of Nutritional Sciences and Toxicology, University of California, Berkeley, Berkeley, CA 94720, USA. Electronic address: dnomura@berkeley.edu.
authors.forename: Daniel K
authors.initials: DK
authors.lastname: Nomura
authors.affiliation: Energy Biosciences Institute, Berkeley, CA 94720, USA; Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA. Electronic address: jdueber@berkeley.edu.
authors.forename: John E
authors.initials: JE
authors.lastname: Dueber
mesh_terms: D049109:Cell Proliferation / Q000502:physiology; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D020869:Gene Expression Profiling / Q000379:methods*; D015723:Gene Library; D024861:Genetic Enhancement / Q000379:methods*; D060847:Metabolic Engineering / Q000379:methods; D009097:Multienzyme Complexes / Q000502:physiology*; D011401:Promoter Regions, Genetic / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D014994:Xylose / Q000378:metabolism*
chemical_list: D009097:Multienzyme Complexes; D014994:Xylose
keywords: Cofactor balance; Combinatorial expression engineering; Metabolic engineering; Synthetic biology; Xylose utilization