Engineering Acetyl Coenzyme A Supply: Functional Expression of a Bacterial Pyruvate Dehydrogenase Complex in the Cytosol of <named-content content-type="genus-species">Saccharomyces cerevisiae</named-content>

ABSTRACT The energetic (ATP) cost of biochemical pathways critically determines the maximum yield of metabolites of vital or commercial relevance. Cytosolic acetyl coenzyme A (acetyl-CoA) is a key precursor for biosynthesis in eukaryotes and for many industrially relevant product pathways that have...

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Autores principales:	Barbara U. Kozak, Harmen M. van Rossum, Marijke A. H. Luttik, Michiel Akeroyd, Kirsten R. Benjamin, Liang Wu, Simon de Vries, Jean-Marc Daran, Jack T. Pronk, Antonius J. A. van Maris
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2014
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/946bda757b034a43b8cc702e6477923b
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spelling	oai:doaj.org-article:946bda757b034a43b8cc702e6477923b2021-11-15T15:45:54ZEngineering Acetyl Coenzyme A Supply: Functional Expression of a Bacterial Pyruvate Dehydrogenase Complex in the Cytosol of <named-content content-type="genus-species">Saccharomyces cerevisiae</named-content>10.1128/mBio.01696-142150-7511https://doaj.org/article/946bda757b034a43b8cc702e6477923b2014-10-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01696-14https://doaj.org/toc/2150-7511ABSTRACT The energetic (ATP) cost of biochemical pathways critically determines the maximum yield of metabolites of vital or commercial relevance. Cytosolic acetyl coenzyme A (acetyl-CoA) is a key precursor for biosynthesis in eukaryotes and for many industrially relevant product pathways that have been introduced into Saccharomyces cerevisiae, such as isoprenoids or lipids. In this yeast, synthesis of cytosolic acetyl-CoA via acetyl-CoA synthetase (ACS) involves hydrolysis of ATP to AMP and pyrophosphate. Here, we demonstrate that expression and assembly in the yeast cytosol of an ATP-independent pyruvate dehydrogenase complex (PDH) from Enterococcus faecalis can fully replace the ACS-dependent pathway for cytosolic acetyl-CoA synthesis. In vivo activity of E. faecalis PDH required simultaneous expression of E. faecalis genes encoding its E1α, E1β, E2, and E3 subunits, as well as genes involved in lipoylation of E2, and addition of lipoate to growth media. A strain lacking ACS that expressed these E. faecalis genes grew at near-wild-type rates on glucose synthetic medium supplemented with lipoate, under aerobic and anaerobic conditions. A physiological comparison of the engineered strain and an isogenic Acs+ reference strain showed small differences in biomass yields and metabolic fluxes. Cellular fractionation and gel filtration studies revealed that the E. faecalis PDH subunits were assembled in the yeast cytosol, with a subunit ratio and enzyme activity similar to values reported for PDH purified from E. faecalis. This study indicates that cytosolic expression and assembly of PDH in eukaryotic industrial microorganisms is a promising option for minimizing the energy costs of precursor supply in acetyl-CoA-dependent product pathways. IMPORTANCE Genetically engineered microorganisms are intensively investigated and applied for production of biofuels and chemicals from renewable sugars. To make such processes economically and environmentally sustainable, the energy (ATP) costs for product formation from sugar must be minimized. Here, we focus on an important ATP-requiring process in baker’s yeast (Saccharomyces cerevisiae): synthesis of cytosolic acetyl coenzyme A, a key precursor for many industrially important products, ranging from biofuels to fragrances. We demonstrate that pyruvate dehydrogenase from the bacterium Enterococcus faecalis, a huge enzyme complex with a size similar to that of a ribosome, can be functionally expressed and assembled in the cytosol of baker’s yeast. Moreover, we show that this ATP-independent mechanism for cytosolic acetyl-CoA synthesis can entirely replace the ATP-costly native yeast pathway. This work provides metabolic engineers with a new option to optimize the performance of baker’s yeast as a “cell factory” for sustainable production of fuels and chemicals.Barbara U. KozakHarmen M. van RossumMarijke A. H. LuttikMichiel AkeroydKirsten R. BenjaminLiang WuSimon de VriesJean-Marc DaranJack T. PronkAntonius J. A. van MarisAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 5, Iss 5 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Barbara U. Kozak Harmen M. van Rossum Marijke A. H. Luttik Michiel Akeroyd Kirsten R. Benjamin Liang Wu Simon de Vries Jean-Marc Daran Jack T. Pronk Antonius J. A. van Maris Engineering Acetyl Coenzyme A Supply: Functional Expression of a Bacterial Pyruvate Dehydrogenase Complex in the Cytosol of <named-content content-type="genus-species">Saccharomyces cerevisiae</named-content>
description	ABSTRACT The energetic (ATP) cost of biochemical pathways critically determines the maximum yield of metabolites of vital or commercial relevance. Cytosolic acetyl coenzyme A (acetyl-CoA) is a key precursor for biosynthesis in eukaryotes and for many industrially relevant product pathways that have been introduced into Saccharomyces cerevisiae, such as isoprenoids or lipids. In this yeast, synthesis of cytosolic acetyl-CoA via acetyl-CoA synthetase (ACS) involves hydrolysis of ATP to AMP and pyrophosphate. Here, we demonstrate that expression and assembly in the yeast cytosol of an ATP-independent pyruvate dehydrogenase complex (PDH) from Enterococcus faecalis can fully replace the ACS-dependent pathway for cytosolic acetyl-CoA synthesis. In vivo activity of E. faecalis PDH required simultaneous expression of E. faecalis genes encoding its E1α, E1β, E2, and E3 subunits, as well as genes involved in lipoylation of E2, and addition of lipoate to growth media. A strain lacking ACS that expressed these E. faecalis genes grew at near-wild-type rates on glucose synthetic medium supplemented with lipoate, under aerobic and anaerobic conditions. A physiological comparison of the engineered strain and an isogenic Acs+ reference strain showed small differences in biomass yields and metabolic fluxes. Cellular fractionation and gel filtration studies revealed that the E. faecalis PDH subunits were assembled in the yeast cytosol, with a subunit ratio and enzyme activity similar to values reported for PDH purified from E. faecalis. This study indicates that cytosolic expression and assembly of PDH in eukaryotic industrial microorganisms is a promising option for minimizing the energy costs of precursor supply in acetyl-CoA-dependent product pathways. IMPORTANCE Genetically engineered microorganisms are intensively investigated and applied for production of biofuels and chemicals from renewable sugars. To make such processes economically and environmentally sustainable, the energy (ATP) costs for product formation from sugar must be minimized. Here, we focus on an important ATP-requiring process in baker’s yeast (Saccharomyces cerevisiae): synthesis of cytosolic acetyl coenzyme A, a key precursor for many industrially important products, ranging from biofuels to fragrances. We demonstrate that pyruvate dehydrogenase from the bacterium Enterococcus faecalis, a huge enzyme complex with a size similar to that of a ribosome, can be functionally expressed and assembled in the cytosol of baker’s yeast. Moreover, we show that this ATP-independent mechanism for cytosolic acetyl-CoA synthesis can entirely replace the ATP-costly native yeast pathway. This work provides metabolic engineers with a new option to optimize the performance of baker’s yeast as a “cell factory” for sustainable production of fuels and chemicals.
format	article
author	Barbara U. Kozak Harmen M. van Rossum Marijke A. H. Luttik Michiel Akeroyd Kirsten R. Benjamin Liang Wu Simon de Vries Jean-Marc Daran Jack T. Pronk Antonius J. A. van Maris
author_facet	Barbara U. Kozak Harmen M. van Rossum Marijke A. H. Luttik Michiel Akeroyd Kirsten R. Benjamin Liang Wu Simon de Vries Jean-Marc Daran Jack T. Pronk Antonius J. A. van Maris
author_sort	Barbara U. Kozak
title	Engineering Acetyl Coenzyme A Supply: Functional Expression of a Bacterial Pyruvate Dehydrogenase Complex in the Cytosol of <named-content content-type="genus-species">Saccharomyces cerevisiae</named-content>
title_short	Engineering Acetyl Coenzyme A Supply: Functional Expression of a Bacterial Pyruvate Dehydrogenase Complex in the Cytosol of <named-content content-type="genus-species">Saccharomyces cerevisiae</named-content>
title_full	Engineering Acetyl Coenzyme A Supply: Functional Expression of a Bacterial Pyruvate Dehydrogenase Complex in the Cytosol of <named-content content-type="genus-species">Saccharomyces cerevisiae</named-content>
title_fullStr	Engineering Acetyl Coenzyme A Supply: Functional Expression of a Bacterial Pyruvate Dehydrogenase Complex in the Cytosol of <named-content content-type="genus-species">Saccharomyces cerevisiae</named-content>
title_full_unstemmed	Engineering Acetyl Coenzyme A Supply: Functional Expression of a Bacterial Pyruvate Dehydrogenase Complex in the Cytosol of <named-content content-type="genus-species">Saccharomyces cerevisiae</named-content>
title_sort	engineering acetyl coenzyme a supply: functional expression of a bacterial pyruvate dehydrogenase complex in the cytosol of <named-content content-type="genus-species">saccharomyces cerevisiae</named-content>
publisher	American Society for Microbiology
publishDate	2014
url	https://doaj.org/article/946bda757b034a43b8cc702e6477923b
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Engineering Acetyl Coenzyme A Supply: Functional Expression of a Bacterial Pyruvate Dehydrogenase Complex in the Cytosol of <named-content content-type="genus-species">Saccharomyces cerevisiae</named-content>

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