Mitochondrial targeting increases specific activity of a heterologous valine assimilation pathway in Saccharomyces cerevisiae
Bio-based isobutantol is a sustainable ‘drop in’ substitute for petroleum-based fuels. However, well-studied production routes, such as the Ehrlich pathway, have yet to be commercialized despite more than a century of research. The more versatile bacterial valine catabolism may be a competitive alte...
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doaj-21ae04ad50514c598fee54f332d745002020-11-24T23:46:43ZengElsevierMetabolic Engineering Communications2214-03012016-12-0136875Mitochondrial targeting increases specific activity of a heterologous valine assimilation pathway in Saccharomyces cerevisiaeKevin V. Solomon0Elisa Ovadia1Fujio Yu2Wataru Mizunashi3Michelle A. O’Malley4Department of Chemical Engineering, University of California Santa Barbara, Santa Barbara, CA 93106, United StatesDepartment of Chemical Engineering, University of California Santa Barbara, Santa Barbara, CA 93106, United StatesScience and Technology Research Center, Inc., Mitsubishi Rayon Group, Yokohama, Kanagawa 227-8502, JapanScience and Technology Research Center, Inc., Mitsubishi Rayon Group, Yokohama, Kanagawa 227-8502, JapanDepartment of Chemical Engineering, University of California Santa Barbara, Santa Barbara, CA 93106, United States; Corresponding author.Bio-based isobutantol is a sustainable ‘drop in’ substitute for petroleum-based fuels. However, well-studied production routes, such as the Ehrlich pathway, have yet to be commercialized despite more than a century of research. The more versatile bacterial valine catabolism may be a competitive alternate route producing not only an isobutanol precursor but several carboxylic acids with applications as biomonomers, and building blocks for other advanced biofuels. Here, we transfer the first two committed steps of the pathway from pathogenic Pseudomonas aeruginosa PAO1 to yeast to evaluate their activity in a safer model organism. Genes encoding the heteroligomeric branched chain keto-acid dehydrogenase (BCKAD; bkdA1, bkdA2, bkdB, lpdV), and the homooligomeric acyl-CoA dehydrogenase (ACD; acd1) were tagged with fluorescence epitopes and targeted for expression in either the mitochondria or cytoplasm of S. cerevisiae. We verified the localization of our constructs with confocal fluorescence microscopy before measuring the activity of tag-free constructs. Despite reduced heterologous expression of mitochondria-targeted enzymes, their specific activities were significantly improved with total enzyme activities up to 138% greater than those of enzymes expressed in the cytoplasm. In total, our results demonstrate that the choice of protein localization in yeast has significant impact on heterologous activity, and suggests a new path forward for isobutanol production. Keywords: Pseudomonas, Isobutanol, Dehydrogenase, Mitochondria, Saccharomyces cerevisiae, Metabolic engineeringhttp://www.sciencedirect.com/science/article/pii/S2214030116300104 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Kevin V. Solomon Elisa Ovadia Fujio Yu Wataru Mizunashi Michelle A. O’Malley |
spellingShingle |
Kevin V. Solomon Elisa Ovadia Fujio Yu Wataru Mizunashi Michelle A. O’Malley Mitochondrial targeting increases specific activity of a heterologous valine assimilation pathway in Saccharomyces cerevisiae Metabolic Engineering Communications |
author_facet |
Kevin V. Solomon Elisa Ovadia Fujio Yu Wataru Mizunashi Michelle A. O’Malley |
author_sort |
Kevin V. Solomon |
title |
Mitochondrial targeting increases specific activity of a heterologous valine assimilation pathway in Saccharomyces cerevisiae |
title_short |
Mitochondrial targeting increases specific activity of a heterologous valine assimilation pathway in Saccharomyces cerevisiae |
title_full |
Mitochondrial targeting increases specific activity of a heterologous valine assimilation pathway in Saccharomyces cerevisiae |
title_fullStr |
Mitochondrial targeting increases specific activity of a heterologous valine assimilation pathway in Saccharomyces cerevisiae |
title_full_unstemmed |
Mitochondrial targeting increases specific activity of a heterologous valine assimilation pathway in Saccharomyces cerevisiae |
title_sort |
mitochondrial targeting increases specific activity of a heterologous valine assimilation pathway in saccharomyces cerevisiae |
publisher |
Elsevier |
series |
Metabolic Engineering Communications |
issn |
2214-0301 |
publishDate |
2016-12-01 |
description |
Bio-based isobutantol is a sustainable ‘drop in’ substitute for petroleum-based fuels. However, well-studied production routes, such as the Ehrlich pathway, have yet to be commercialized despite more than a century of research. The more versatile bacterial valine catabolism may be a competitive alternate route producing not only an isobutanol precursor but several carboxylic acids with applications as biomonomers, and building blocks for other advanced biofuels. Here, we transfer the first two committed steps of the pathway from pathogenic Pseudomonas aeruginosa PAO1 to yeast to evaluate their activity in a safer model organism. Genes encoding the heteroligomeric branched chain keto-acid dehydrogenase (BCKAD; bkdA1, bkdA2, bkdB, lpdV), and the homooligomeric acyl-CoA dehydrogenase (ACD; acd1) were tagged with fluorescence epitopes and targeted for expression in either the mitochondria or cytoplasm of S. cerevisiae. We verified the localization of our constructs with confocal fluorescence microscopy before measuring the activity of tag-free constructs. Despite reduced heterologous expression of mitochondria-targeted enzymes, their specific activities were significantly improved with total enzyme activities up to 138% greater than those of enzymes expressed in the cytoplasm. In total, our results demonstrate that the choice of protein localization in yeast has significant impact on heterologous activity, and suggests a new path forward for isobutanol production. Keywords: Pseudomonas, Isobutanol, Dehydrogenase, Mitochondria, Saccharomyces cerevisiae, Metabolic engineering |
url |
http://www.sciencedirect.com/science/article/pii/S2214030116300104 |
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