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

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

Synthetic and Systems Biotechnology (Jun 2022)

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

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

Affiliations

Francesca V. Gambacorta: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI, USA; Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI, USA
Ellen R. Wagner: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI, USA; Laboratory of Genetics, Center for Genomic Science Innovation, University of Wisconsin-Madison, Madison, WI, USA
Tyler B. Jacobson: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI, USA; Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, USA
Mary Tremaine: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI, USA
Laura K. Muehlbauer: Department of Chemistry, University of Wisconsin-Madison, Madison, WI, USA
Mick A. McGee: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI, USA
Justin J. Baerwald: Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI, USA
Russell L. Wrobel: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI, USA; Laboratory of Genetics, Center for Genomic Science Innovation, University of Wisconsin-Madison, Madison, WI, USA; Wisconsin Energy Institute, J. F. Crow Institute for the Study of Evolution, University of Wisconsin-Madison, Madison, WI, USA
John F. Wolters: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI, USA; Laboratory of Genetics, Center for Genomic Science Innovation, University of Wisconsin-Madison, Madison, WI, USA; Wisconsin Energy Institute, J. F. Crow Institute for the Study of Evolution, University of Wisconsin-Madison, Madison, WI, USA
Mike Place: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI, USA
Joshua J. Dietrich: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI, USA; Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI, USA
Dan Xie: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI, USA
Jose Serate: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI, USA
Shabda Gajbhiye: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI, USA
Lisa Liu: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI, USA; Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, USA
Maikayeng Vang-Smith: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI, USA
Joshua J. Coon: Department of Chemistry, University of Wisconsin-Madison, Madison, WI, USA; Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, WI, USA
Yaoping Zhang: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI, USA
Audrey P. Gasch: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI, USA; Laboratory of Genetics, Center for Genomic Science Innovation, University of Wisconsin-Madison, Madison, WI, USA
Daniel Amador-Noguez: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI, USA; Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, USA
Chris Todd Hittinger: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI, USA; Laboratory of Genetics, Center for Genomic Science Innovation, University of Wisconsin-Madison, Madison, WI, USA; Wisconsin Energy Institute, J. F. Crow Institute for the Study of Evolution, University of Wisconsin-Madison, Madison, WI, USA
Trey K. Sato: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI, USA
Brian F. Pfleger: DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI, USA; Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI, USA; Corresponding author. 3629 Engineering Hall, 1415 Engineering Drive, Madison, WI, 53706, United States.

Journal volume & issue: Vol. 7, no. 2
pp. 738 – 749

Abstract

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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.

Published in Synthetic and Systems Biotechnology

ISSN: 2405-805X (Online)
Publisher: KeAi Communications Co., Ltd.
Country of publisher: China
LCC subjects: Technology: Chemical technology: Biotechnology; Science: Biology (General)
Website: https://www.keaipublishing.com/en/journals/synthetic-and-systems-biotechnology/

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