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

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 

机构地区：[1]DOE Great Lakes Bioenergy Research Center,University of Wisconsin-Madison,Madison,WI,USA [2]Department of Chemical and Biological Engineering,University of Wisconsin-Madison,Madison,WI,USA [3]Laboratory of Genetics,Center for Genomic Science Innovation,University of Wisconsin-Madison,Madison,WI,USA [4]Department of Bacteriology,University of Wisconsin-Madison,Madison,WI,USA [5]Department of Chemistry,University of Wisconsin-Madison,Madison,WI,USA [6]Wisconsin Energy Institute,J.F.Crow Institute for the Study of Evolution,University of Wisconsin-Madison,Madison,WI,USA [7]Department of Biomolecular Chemistry,University of Wisconsin-Madison,Madison,WI,USA

出　　处：《Synthetic and Systems Biotechnology》2022年第2期738-749,共12页合成和系统生物技术（英文）

摘　　要：Metabolic engineering strategies have been successfully implemented to improve the production of isobutanol,a next-generation biofuel,in Saccharomyces cerevisiae.Here,we explore how two of these strategies,pathway re-localization and redox cofactor-balancing,affect the performance and physiology of isobutanol producing strains.We 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 enzyme.We then conducted transcriptomic,proteomic and metabolomic analyses to elucidate molecular differences between the engineered strains.Pathway 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 enzymes.Cofactor-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 flux.Functional 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 enzyme.We then demonstrated that this cofactor limitation may be partially recovered by disrupting iron homeostasis with a fra2 mutation,thereby increasing cellular iron levels.The 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 

分 类 号：Q78[生物学—分子生物学]