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Alcohol Acetyltransferase Eat1 Is Located in Yeast Mitochondria.

ABSTRACT: Eat1 is a recently discovered alcohol acetyltransferase responsible for bulk ethyl acetate production in yeasts such as Wickerhamomyces anomalus and Kluyveromyces lactis These yeasts have the potential to become efficient bio-based ethyl acetate producers. However, some fundamental features of Eat1 are still not understood, which hampers the rational engineering of efficient production strains. The cellular location of Eat1 in yeast is one of these features. To reveal its location, Eat1 was fused with yeast-enhanced green fluorescent protein (yEGFP) to allow intracellular tracking. Despite the current assumption that bulk ethyl acetate production occurs in the yeast cytosol, most of Eat1 localized to the mitochondria of Kluyveromyces lactis CBS 2359 ?ku80 We then compared five bulk ethyl acetate-producing yeasts in iron-limited chemostats with glucose as the carbon source. All yeasts produced ethyl acetate under these conditions. This strongly suggests that the mechanism and location of bulk ethyl acetate synthesis are similar in these yeast strains. Furthermore, an in silico analysis showed that Eat1 proteins from various yeasts were mostly predicted as mitochondrial. Altogether, it is concluded that Eat1-catalyzed ethyl acetate production occurs in yeast mitochondria. This study has added new insights into bulk ethyl acetate synthesis in yeast, which is relevant for developing efficient production strains.IMPORTANCE Ethyl acetate is a common bulk chemical that is currently produced from petrochemical sources. Several Eat1-containing yeast strains naturally produce large amounts of ethyl acetate and are potential cell factories for the production of bio-based ethyl acetate. Rational design of the underlying metabolic pathways may result in improved production strains, but it requires fundamental knowledge on the function of Eat1. A key feature is the location of Eat1 in the yeast cell. The precursors for ethyl acetate synthesis can be produced in multiple cellular compartments through different metabolic pathways. The location of Eat1 determines the relevance of each pathway, which will provide future targets for the metabolic engineering of bulk ethyl acetate production in yeast.

SUBMITTER: Kruis AJ

PROVIDER: S-EPMC6146999 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Alcohol Acetyltransferase Eat1 Is Located in Yeast Mitochondria.

Kruis Aleksander J AJ Mars Astrid E AE Kengen Servé W M SWM Borst Jan Willem JW van der Oost John J Weusthuis Ruud A RA

Applied and environmental microbiology 20180917 19

Eat1 is a recently discovered alcohol acetyltransferase responsible for bulk ethyl acetate production in yeasts such as <i>Wickerhamomyces anomalus</i> and <i>Kluyveromyces lactis</i> These yeasts have the potential to become efficient bio-based ethyl acetate producers. However, some fundamental features of Eat1 are still not understood, which hampers the rational engineering of efficient production strains. The cellular location of Eat1 in yeast is one of these features. To reveal its location, ...[more]

PMID: 30054364

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Project description:BackgroundThe mitochondrial electron transport chain oxidizes matrix space NADH as part of the process of oxidative phosphorylation. Mitochondria contain shuttles for the transport of cytoplasmic NADH reducing equivalents into the mitochondrial matrix. Therefore for a long time it was believed that NAD(H) itself was not transported into mitochondria. However evidence has been obtained for the transport of NAD(H) into and out of plant and mammalian mitochondria. Since Saccharomyces cerevisiae mitochondria can directly oxidize cytoplasmic NADH, it remained questionable if mitochondrial NAD(H) transport occurs in this organism.ResultsNAD(H) was lost more extensively from the matrix space of swollen than normal, condensed isolated yeast mitochondria from Saccharomyces cerevisiae. The loss of NAD(H) in swollen organelles caused a greatly decreased respiratory rate when ethanol or other matrix space NAD-linked substrates were oxidized. Adding NAD back to the medium, even in the presence of a membrane-impermeant NADH dehydrogenase inhibitor, restored the respiratory rate of swollen mitochondria oxidizing ethanol, suggesting that NAD is transported into the matrix space. NAD addition did not restore the decreased respiratory rate of swollen mitochondria oxidizing the combination of malate, glutamate, and pyruvate. Therefore the loss of matrix space metabolites is not entirely specific for NAD(H). However, during NAD(H) loss the mitochondrial levels of most other nucleotides were maintained. Either hypotonic swelling or colloid-osmotic swelling due to opening of the yeast mitochondrial unspecific channel (YMUC) in a mannitol medium resulted in decreased NAD-linked respiration. However, the loss of NAD(H) from the matrix space was not mediated by the YMUC, because YMUC inhibitors did not prevent decreased NAD-linked respiration during swelling and YMUC opening without swelling did not cause decreased NAD-linked respiration.ConclusionLoss of endogenous NAD(H) from isolated yeast mitochondria is greatly stimulated by matrix space expansion. NAD(H) loss greatly limits NAD-linked respiration in swollen mitochondria without decreasing the NAD-linked respiratory rate in normal, condensed organelles. NAD addition can totally restore the decreased respiration in swollen mitochondria. In live yeast cells mitochondrial swelling has been observed prior to mitochondrial degradation and cell death. Therefore mitochondrial swelling may stimulate NAD(H) transport to regulate metabolism during these conditions.

Dataset Information

Alcohol Acetyltransferase Eat1 Is Located in Yeast Mitochondria.

Publications

Alcohol Acetyltransferase Eat1 Is Located in Yeast Mitochondria.

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