Itine shuttle really should be reversible, but previous research indicate that carnitine

Itine shuttle needs to be reversible, but earlier research indicate that carnitine shuttle-mediated export of mitochondrial acetyl units to the yeast cytosol doesn’t happen in vivo. This apparent unidirectionality was investigated by constitutively expressing genes encoding carnitine shuttle-related proteins in an engineered S. cerevisiae strain, in which cytosolic acetyl coenzyme A (acetyl-CoA) synthesis may be switched off by omitting lipoic acid from development media. Laboratory evolution of this strain yielded mutants whose development on glucose, in the absence of lipoic acid, was L-carnitine dependent, indicating that in vivo export of mitochondrial acetyl units for the cytosol occurred by means of the carnitine shuttle. The mitochondrial pyruvate dehydrogenase complex was identified as the predominant supply of acetyl-CoA within the evolved strains. Whole-genome sequencing revealed mutations in genes involved in mitochondrial fatty acid synthesis (MCT1), nuclear-mitochondrial communication (RTG2), and encoding a carnitine acetyltransferase (YAT2). Introduction of those mutations into the nonevolved parental strain enabled L-carnitine-dependent development on glucose. This study indicates intramitochondrial acetyl-CoA concentration and constitutive expression of carnitine shuttle genes as crucial elements in enabling in vivo export of mitochondrial acetyl units by way of the carnitine shuttle. IMPORTANCEThis study demonstrates, for the very first time, that Saccharomyces cerevisiae might be engineered to employ the carnitine shuttle for export of acetyl moieties in the mitochondria and, thereby, to act because the sole supply of cytosolic acetyl-CoA. Additional optimization of this ATP-independent mechanism for cytosolic acetyl-CoA provision can contribute to efficient, yeastbased production of industrially relevant compounds derived from this precursor. The strains constructed in this study, whose growth on glucose will depend on a functional carnitine shuttle, provide worthwhile models for further functional evaluation and engineering of this shuttle in yeast along with other eukaryotes.Received 25 March 2016 Accepted 31 March 2016 Published 3 Could 2016 Citation Van Rossum HM, Kozak BU, Niemeijer MS, Dykstra JC, Luttik MAH, Daran J-MG, Van Maris AJA, Pronk JT. 2016. Needs for carnitine shuttle-mediated translocation of mitochondrial acetyl moieties to the yeast cytosol. mBio 7(3):e00520-16. doi:10.1128/mBio.00520-16. Editor Caroline S. Harwood, University of Washington Copyright 2016 van Rossum et al. This can be an open-access post distributed beneath the terms with the Inventive Commons Attribution 4.0 International license. Address correspondence to Jack T. Pronk, J.GIP Protein Gene ID T.DSG3 Protein supplier Pronk@TUDelft.PMID:23310954 nl. This article is a direct contribution from a Fellow from the American Academy of Microbiology. External solicited reviewers: Sylvie Dequin, INRA; Danilo Porro, University of Milano-Bicocca.In eukaryotes, metabolic compartmentation necessitates mechanisms for translocation of metabolites in between cellular compartments. Acetyl coenzyme A (acetyl-CoA) is an essential precursor in cytosolic and mitochondrial biosynthetic pathways and, in addition, is involved in cellular regulation by acting as an acetyl donor for acetylation of nuclear and cytosolic proteins (1). Eukaryotes have evolved several mechanisms for synthesis and intracellular transport of acetyl-CoA within and among cellular compartments (six). 1 of those mechanisms, the carnitine shuttle, plays a crucial role in translocation of acetyl units between cellular.