title: Enhanced hexose fermentation by Saccharomyces cerevisiae through integration of stoichiometric modeling and genetic screening.
abstract: In order to determine beneficial gene deletions for ethanol production by the yeast Saccharomyces cerevisiae, we performed an in silico gene deletion experiment based on a genome-scale metabolic model. Genes coding for two oxidative phosphorylation reactions (cytochrome c oxidase and ubiquinol cytochrome c reductase) were identified by the model-based simulation as potential deletion targets for enhancing ethanol production and maintaining acceptable overall growth rate in oxygen-limited conditions. Since the two target enzymes are composed of multiple subunits, we conducted a genetic screening study to evaluate the in silico results and compare the effect of deleting various portions of the respiratory enzyme complexes. Over two-thirds of the knockout mutants identified by the in silico study did exhibit experimental behavior in qualitative agreement with model predictions, but the exceptions illustrate the limitation of using a purely stoichiometric model-based approach. Furthermore, there was a substantial quantitative variation in phenotype among the various respiration-deficient mutants that were screened in this study, and three genes encoding respiratory enzyme subunits were identified as the best knockout targets for improving hexose fermentation in microaerobic conditions. Specifically, deletion of either COX9 or QCR9 resulted in higher ethanol production rates than the parental strain by 37% and 27%, respectively, with slight growth disadvantages. Also, deletion of QCR6 led to improved ethanol production rate by 24% with no growth disadvantage. The beneficial effects of these gene deletions were consistently demonstrated in different strain backgrounds and with four common hexoses. The combination of stoichiometric modeling and genetic screening using a systematic knockout collection was useful for narrowing a large set of gene targets and identifying targets of interest.
authors.affiliation: Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA; Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: Josh
authors.initials: J
authors.lastname: Quarterman
authors.affiliation: Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA; School of Food Science and Biotechnology, Kyungpook National University, Buk-Gu, Daegu 702-701, Republic of Korea.
authors.forename: Soo Rin
authors.initials: SR
authors.lastname: Kim
authors.affiliation: Asia Pacific Center for Theoretical Physics, Pohang, Gyeongbuk 790-784, Republic of Korea; Department of Physics, POSTECH, Pohang, Gyeongbuk 790-784, Republic of Korea.
authors.forename: Pan-Jun
authors.initials: PJ
authors.lastname: Kim
authors.affiliation: Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA; Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA. Electronic address: ysjin@illinois.edu.
authors.forename: Yong-Su
authors.initials: YS
authors.lastname: Jin
mesh_terms: D005285:Fermentation / Q000502:physiology*; D006601:Hexoses / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
chemical_list: D006601:Hexoses
keywords: Constraint-based flux analysis; Cytochrome c oxidase; Genome-scale model; Saccharomyces cerevisiae; Ubiquinol cytochrome c reductase