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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Main Authors: Kevin V. Solomon, Elisa Ovadia, Fujio Yu, Wataru Mizunashi, Michelle A. O’Malley
Format: Article
Language:English
Published: Elsevier 2016-12-01
Series:Metabolic Engineering Communications
Online Access:http://www.sciencedirect.com/science/article/pii/S2214030116300104
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spelling 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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