Acetyl-CoA is a key intermediate in cellular metabolism. It is involved as a precursor metabolite; it is used for generation of Gibbs free energy, and for production of many industrially relevant compounds. In the yeast S. cerevisiae metabolism involving acetyl-CoA is compartmentalized and may vary with the nutrient supply of a cell. Where acetyl-CoA is metabolized on non-fermentable carbon sources is not fully understood, but as S. cerevisiae cannot synthesize carnitine and we did not provide this via the medium, the glyoxylate shunt is the sole possible route for transferring acetyl-CoA (in form of tricarboxylic acid cycle intermediates) into the mitochondria. Cit2p, Mls1p, Acs1p and Acs2p, are the main enzymes relevant to acetyl-CoA metabolism—either consuming or producing this molecule outside of mitochondria. Therefore, the aim of this study was to investigate the physiological effects of different mutant strains with combinations of these genes deletion (ACS1, ACS2, CIT2 and MLS1) on different carbon sources in S. cerevisiae, and try to understand intracellular transport of intermediates in various mutations with alternative carbon sources.
Irrespective of the nutrient supply to the cell the cytosolic acetyl-CoA pool is indispensable for synthesis of fatty acids and sterols, and it relies completely on acetyl-CoA synthetase activity on all carbon sources. In view of its essential role in the central carbon metabolism, at least one functional isoenzyme of acetyl-CoA synthetase must be located in the cytosol. SCIYC23 (acs2
Δ) lacking ACS2
can grow on minimal medium containing a non-fermentable carbon source like ethanol or acetate, which clearly indicates that Acs1p is subjected to cytosolic distribution under these conditions. If Acs1p was exclusively localized in the peroxisomes, it could not activate acetate to form acetyl-CoA in the cytosol; and acetyl-CoA produced in the peroxisomes cannot be shuttled without the carnitine transport system. Our findings for the acs2
Δ mutant are consistent with earlier reports by van den Berg and Steensma 
, i.e. that inactivation of ACS2
did not cause inability to grow on media with acetate or ethanol as sole carbon source (in complex medium with yeast extract). Since the media used in our study are devoid of carnitine it further confirms that Acs1p is distributed to the cytosol on a non-fermentable carbon source. The ability of acs2
Δ mutants to grow in aerobic, glucose-limited chemostat cultures has also been reported 
, which further supports the conclusion that Acs1p is subjected to cytosolic distribution under de-repressing conditions. Even though it is reported that Cit2p has both N- and C-terminal signal for mitochondrion and peroxisomes, the potential of N-terminal signal can only be recognized by removing the C-terminal sequence 
. Taking this into account together with that recently reported CIT2
is in peroxisomes regardless of synthetic or complex medium 
, strongly indicates that Cit2p is the peroxisomal isoenzyme of citrate synthase that is involved in the glyoxylate shunt, and it therefore seems likely that Acs1p functions in the peroxisomes in synthesis of acetyl-CoA required for use by Cit2p. Furthermore, according to the results of Kunze et al.
, Mls1p remains in the cytosol in ethanol-grown cells and it therefore seems reasonable that there is a dual distribution of Acs1p for cells growing under non-repressing conditions. Similarly, it was also recently shown that fumarase and aconitase were dually localized in the mitochondria and the cytoplasm 
. Overall, Acs1p is suggested to show a dual distribution pattern between the cytoplasm and the peroxisomes, as shown in .
Schematic representing cytosolic and peroxisomal acetyl-CoA metabolism in S. cerevisiae under growth on C2 compounds.
The scenario in does probably not apply to the acs1
Δ strain. For mutant SCIYC05 (acs1
Δ), the inability to grow on a non-fermentable carbon source was observed. Besides the requirement of 1.8 mmol cytosolic acetyl-CoA per gram cells for lipid synthesis in the cytosol 
, metabolism on C2
carbon sources passes through acetyl-CoA for both biosynthesis and energy metabolism. Therefore, inefficient conversion of C2
compounds (ethanol or acetate) to acetyl-CoA could be explained by low Acs2p activity, which may explain the acs1
Δ strain's inability to grow on non-fermentable carbon sources. However, the double mutant strain lacking both ACS1
was shown to be able to grow on non-fermentable carbon sources, which contradicts this hypothesis. Furthermore, expressing the truncated but not the intact Mls1p in the cytoplasm of SCIYC05 (acs1
Δ) restored the growth capacity. This observation strongly suggests that both Cit2p and Mls1p are located in the peroxisomes of cells lacking ACS1
(), and low Acs2p activity therefore cannot explain the inability of growth of the acs1Δ
Outside the mitochondria, acetyl-CoA synthetases serve as the main producers of acetyl-CoA, while citrate synthase 2 catalyzes one of the acetyl-CoA consuming reactions. Deletion of CIT2
breaks the integrity of the glyoxylate shunt. However, in our study the cit2
Δ strain SCIYC06 (cit2
Δ) was shown to be able to grow on minimal media plates containing ethanol, acetate or glycerol as sole carbon source. As shown in and , the disruption in the glyoxylate shunt resulting from the CIT2
deletion can be bypassed by (i) succinate being imported into the mitochondria followed by decarboxylation of malate to pyruvate via malic enzyme (as shown pathway I in ); or (ii) malate being converted by cytosolic malate dehydrogenase Mdh2p to form oxaloacetate, followed by synthesis of phosphoenolpyruvate (PEP) involving PEP carboxykinase (as shown pathway II in ). Thus, citrate or isocitrate from the mitochondria might bypass the Cit2p reaction in the glyoxylate shunt. This is supported by the fact that the mitochondrial citrate synthase Cit1p could act as a shunt for the missing Cit2p via the citrate transporter Ctp1p 
. Although the discrepancy of growth phenotype on solid and liquid medium is unclear so far, the growth conditions are different, which might lead to the different physiology.
Being an essential enzyme, elimination of Mls1p also breaks the integrity of the glyoxylate shunt. According to the plate assay, the MLS1
deletion strain SCIYC07 (mls1
Δ) was unable to grow on non-fermentable nutrient sources, indicating that Mls1p is specific and absolutely required for growth on C2
compounds as carbon source. Surprisingly, this mutant utilized ethanol and acetate derived from the overflow metabolism during growth on glucose in shake flask cultivation, while the biomass was increasing only slightly. After conversion of C2
carbon sources to acetyl-CoA, this compound can be metabolized by different pathways such as the glyoxylate shunt, or the biosynthesis of fatty acids and sterols. Analysis of the extracellular metabolite profile revealed that glyoxylate accumulated. This observation indeed showed that in absence of MLS1
, the glyoxylate shunt was working partially, which is also consistent with the phenomenon that all metabolites from overflow metabolism are slowly consumed. These results suggested an alternative pathway to the activities of Mls1p: the counter-exchange of succinate and fumarate and subsequent intra-mitochondrial conversion of succinate to fumarate, together with the canalization of fumarate to malate. This is supported by the fact of that the mitochondrial succinate-fumarate carrier Sfc1p is indispensible for acetate utilization on minimal medium 
In contrast to the inability of SCIYC05 (acs1Δ) to grow on non-fermentable carbon sources, SCIYC14 (acs1Δ cit2Δ) was observed to be able to grow under such carbon conditions. Although the membranes of subcellular organelles are permeable to C2 compounds such as ethanol and acetate, metabolism on C2 carbon source has to pass through acetyl-CoA for biosynthesis and energy requirement. In SCIYC14 (acs1Δ cit2Δ), the conversion of a C2 carbon source to acetyl-CoA can be realized only by the sole extra-mitochondrial producer, cytosolic Acs2p. The acetyl-CoA units so produced cannot be transported into other compartments without the help of carnitine. Furthermore, the resulting acetyl-CoA units in the cytosol must be channeled into the synthesis of C4 dicarboxylic acids. Taken together with the deficiency of Cit2p activity, Mls1p which is the only candidate enzyme left can condense acetyl-CoA to form C4 metabolites, may well be localized in the cytoplasm of SCIYC14 (acs1Δ cit2Δ) ().
In this study, we have investigated the physiological profiling of S. cerevisiae mutants affected in acetyl-CoA metabolism. Through analyzing the growth phenotypes and metabolite processing changes in different mutants on non-fermentable carbon sources, we have proposed a model of acetyl-CoA metabolism to speculate how various mutations alter carbon distribution. This study should be of interest to those studying acetyl-CoA production in eukaryotic cells and central carbon metabolism in fungi.