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Comparative functional genomics identifies an iron-limited bottleneck in a Saccharomyces cerevisiae strain with a cytosolic-localized isobutanol pathway

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Synthetic and Systems Biotechnology 2022年第2期7卷 738-749页

作者： Francesca V.Gambacorta Ellen R.Wagner Tyler B.Jacobson mary tremaine Laura K.Muehlbauer Mick A.McGee Justin J.Baerwald Russell L.Wrobel John F.Wolters Mike Place Joshua J.Dietrich Dan Xie Jose Serate Shabda Gajbhiye Lisa Liu Maikayeng Vang-Smith Joshua J.Coon Yaoping Zhang Audrey P.Gasch Daniel Amador-Noguez Chris Todd Hittinger Trey K.Sato Brian F.Pfleger DOE Great Lakes Bioenergy Research Center University of Wisconsin-MadisonMadisonWIUSA Department of Chemical and Biological Engineering University of Wisconsin-MadisonMadisonWIUSA Laboratory of Genetics Center for Genomic Science InnovationUniversity of Wisconsin-MadisonMadisonWIUSA Department of Bacteriology University of Wisconsin-MadisonMadisonWIUSA Department of Chemistry University of Wisconsin-MadisonMadisonWIUSA Wisconsin Energy Institute J.F.Crow Institute for the Study of EvolutionUniversity of Wisconsin-MadisonMadisonWIUSA Department of Biomolecular Chemistry University of Wisconsin-MadisonMadisonWIUSA

Metabolic engineering strategies have been successfully implemented to improve the production of isobutanol,a next-generation biofuel,in Saccharomyces ***,we explore how two of these strategies,pathway re-localization and redox cofactor-balancing,affect the performance and physiology of isobutanol producing *** equipped yeast with isobutanol cassettes which had either a mitochondrial or cytosolic localized isobutanol pathway and used either a redox-imbalanced(NADPH-dependent)or redox-balanced(NADH-dependent)ketol-acid reductoisomerase *** then conducted transcriptomic,proteomic and metabolomic analyses to elucidate molecular differences between the engineered *** localization had a large effect on isobutanol production with the strain expressing the mitochondrial-localized enzymes producing 3.8-fold more isobutanol than strains expressing the cytosolic ***-balancing did not improve isobutanol titers and instead the strain with the redox-imbalanced pathway produced 1.5-fold more isobutanol than the balanced version,albeit at low overall pathway *** genomic analyses suggested that the poor performances of the cytosolic pathway strains were in part due to a shortage in cytosolic Fe-S clusters,which are required cofactors for the dihydroxyacid dehydratase *** then demonstrated that this cofactor limitation may be partially recovered by disrupting iron homeostasis with a fra2 mutation,thereby increasing cellular iron *** resulting isobutanol titer of the fra2 null strain harboring a cytosolic-localized isobutanol pathway outperformed the strain with the mitochondrial-localized pathway by 1.3-fold,demonstrating that both localizations can support flux to isobutanol.

关键词： Saccharomyces cerevisiae Isobutanol Functional genomics analysis Pathway localization Fe incorporation

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