RESUMEN
BACKGROUND: Mitochondrial carriers (MCs) can deeply affect the intracellular flux distribution of metabolic pathways. The manipulation of their expression level, to redirect the flux toward the production of a molecule of interest, is an attractive target for the metabolic engineering of eukaryotic microorganisms. The non-conventional yeast Yarrowia lipolytica is able to use a wide range of substrates. As oleaginous yeast, it directs most of the acetyl-CoA therefrom generated towards the synthesis of lipids, which occurs in the cytoplasm. Among them, the odd-chain fatty acids (OCFAs) are promising microbial-based compounds with several applications in the medical, cosmetic, chemical and agricultural industries. RESULTS: In this study, we have identified the MC involved in the Carnitine/Acetyl-Carnitine shuttle in Y. lipolytica, YlCrc1. The Y. lipolytica Ylcrc1 knock-out strain failed to grow on ethanol, acetate and oleic acid, demonstrating the fundamental role of this MC in the transport of acetyl-CoA from peroxisomes and cytoplasm into mitochondria. A metabolic engineering strategy involving the deletion of YlCRC1, and the recombinant expression of propionyl-CoA transferase from Ralstonia eutropha (RePCT), improved propionate utilization and its conversion into OCFAs. These genetic modifications and a lipogenic medium supplemented with glucose and propionate as the sole carbon sources, led to enhanced accumulation of OCFAs in Y. lipolytica. CONCLUSIONS: The Carnitine/Acetyl-Carnitine shuttle of Y. lipolytica involving YlCrc1, is the sole pathway for transporting peroxisomal or cytosolic acetyl-CoA to mitochondria. Manipulation of this carrier can be a promising target for metabolic engineering approaches involving cytosolic acetyl-CoA, as demonstrated by the effect of YlCRC1 deletion on OCFAs synthesis.
Asunto(s)
Carnitina , Yarrowia , Acetilcoenzima A/metabolismo , Carnitina/metabolismo , Acetilcarnitina/metabolismo , Yarrowia/genética , Yarrowia/metabolismo , Ácidos Grasos/metabolismo , Propionatos/metabolismo , Mitocondrias/metabolismo , Ingeniería MetabólicaRESUMEN
The mitochondrial carriers are a family of transport proteins that, with a few exceptions, are found in the inner membranes of mitochondria. They shuttle metabolites, nucleotides, and cofactors through this membrane and thereby connect and/or regulate cytoplasm and matrix functions. ATP-Mg is transported in exchange for phosphate, but no protein has ever been associated with this activity. We have isolated three human cDNAs that encode proteins of 458, 468, and 489 amino acids with 66-75% similarity and with the characteristic features of the mitochondrial carrier family in their C-terminal domains and three EF-hand Ca(2+)-binding motifs in their N-terminal domains. These proteins have been overexpressed in Escherichia coli and reconstituted into phospholipid vesicles. Their transport properties and their targeting to mitochondria demonstrate that they are isoforms of the ATP-Mg/Pi carrier described in the past in whole mitochondria. The tissue specificity of the three isoforms shows that at least one isoform was present in all of the tissues investigated. Because phosphate recycles via the phosphate carrier in mitochondria, the three isoforms of the ATP-Mg/Pi carrier are most likely responsible for the net uptake or efflux of adenine nucleotides into or from the mitochondria and hence for the variation in the matrix adenine nucleotide content, which has been found to change in many physiopathological situations.
Asunto(s)
Antiportadores/química , Proteínas de Unión al Calcio/fisiología , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/fisiología , Proteínas Mitocondriales/química , Proteínas de Transporte de Fosfato/fisiología , Adenosina Trifosfato/química , Secuencias de Aminoácidos , Calcio/química , Catálisis , Citoplasma/metabolismo , ADN Complementario/metabolismo , Difusión , Escherichia coli/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Cinética , Liposomas/metabolismo , Magnesio/química , Proteínas de Transporte de Membrana/metabolismo , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Fosfatos/química , Fosfolípidos/química , Plásmidos/metabolismo , Potasio/metabolismo , Isoformas de Proteínas , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Factores de Tiempo , Distribución TisularRESUMEN
We describe the identification and functional characterization of two Arabidopsis mitochondrial basic amino acid carriers (BAC), AtmBAC1 and AtmBAC2, which are related to the yeast ornithine (Orn) carrier Ort1p, also known as Arg11p. The arg11 mutant requires arginine (Arg) supplementation because it fails to export sufficient ornithine from the mitochondrion to the cytosol where it is converted to arginine. AtmBAC1 and, to a lesser extent, AtmBAC2 partially replaced the function of Ort1p in yeast arg11. The more efficient putative carrier, AtmBAC1, was expressed in E. coli, purified, and reconstituted into phospholipid vesicles, where it transported the basic l-amino acids arginine, lysine, ornithine and histidine (in order of decreasing affinity). AtmBAC1 recognized l-histidine whereas both yeast Ort1p and the mammalian ortholog ORNT1p do not. Also different from ORNT1p, AtmBAC1 did not transport citrulline. AtmBAC1 appeared to be more stereospecific than the yeast and mammalian ornithine carriers, exhibiting greater preference for the l-forms of arginine, lysine and ornithine. By RT-PCR, both AtmBAC1 and AtmBAC2 transcripts were detected in stems, leaves, flowers, siliques, and seedlings. Expression of AtmBAC1 in seedlings is consistent with its involvement in Arg breakdown in early seedling development, i.e. delivery of Arg to mitochondrial arginase. The Km (0.19 mm) for Arg uptake by AtmBAC1 was close to the value we previously determined for the saturable component of Arg uptake into intact mitochondria from soybean seedling cotyledons.