Abstract
Degradation of trans-unsaturated fatty acids was studied in the yeast Saccharomyces cerevisiae. Propagation of yeast cells on trans-9 elaidic acid medium resulted in transcriptional up-regulation of the SPS19 gene, whose promoter contains an oleate response element. This up-regulation depended on the Pip2p-Oaf1p transcription factor and was accompanied by induction of import-competent peroxisomes. Utilization of trans fatty acids as a single carbon and energy source was evaluated by monitoring the formation of clear zones around cell growth on turbid media containing fatty acids dispersed with Tween 80. For metabolizing odd-numbered trans double bonds, cells required the beta-oxidation auxiliary enzyme Delta(3)-Delta(2)-enoyl-CoA isomerase Eci1p. Metabolism of the corresponding even-numbered double bonds proceeded in the absence of Sps19p (2,4-dienoyl-CoA reductase) and Dci1p (Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase). trans-2,trans-4-Dienoyl-CoAs could enter beta-oxidation directly via Fox2p (2-enoyl-CoA hydratase 2 and d-specific 3-hydroxyacyl-CoA dehydrogenase) without the involvement of Sps19p, whereas trans-2,cis-4-dienoyl-CoAs could not. This reductase-independent metabolism of trans-2,trans-4-dienoyl-CoAs resembled the situation postulated for mammalian mitochondria in which oleic acid is degraded through a di-isomerase-dependent pathway. In this hypothetical process, trans-2,trans-4-dienoyl-CoA metabolites are generated by Delta(3)-Delta(2)-enoyl-CoA isomerase and Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase and are degraded by 2-enoyl-CoA hydratase 1 in the absence of 2,4-dienoyl-CoA reductase. Growth of a yeast fox2sps19Delta mutant in which Fox2p was exchanged with rat peroxisomal multifunctional enzyme type 1 on trans-9,trans-12 linolelaidic acid medium gave credence to this theory. We propose an amendment to the current scheme of the carbon flux through beta-oxidation taking into account the dispensability of beta-oxidation auxiliary enzymes for metabolizing trans double bonds at even-numbered positions.
Highlights
Double bonds in naturally occurring unsaturated fatty acids are mostly in the cis configuration
The results showed that transfer of late log-phase cells to liquid elaidic acid medium for 18 h resulted in a 10-fold increase in reporter-gene activity due to the SPS19 oleic acid-response element (ORE), since -galactosidase activity levels were not elevated in the control strain carrying the plasmid vector
In the present study yeast -oxidation was exploited for studying the degradation of unsaturated fatty acids with trans double bonds
Summary
Strains—Escherichia coli strain DH10B was used for all plasmid amplifications and isolations. Strain BJ1991dci1⌬sps19⌬ (yAG935) was constructed by disrupting the SPS19 locus in the BJ1991dci1⌬ strain after transformation with an sps19⌬::LEU2 fragment generated by digesting plasmid pAG129 (11) with ScaI and StuI. Mutant UTL-7Afox[2] cells expressing rat peroxisomal MFE type 1 (yAG1206) or yeast Fox2p (yAG1207) were generated by transformation with the plasmids pYE352-rMFE (17) or pYE352::ScMFE-2 (18), respectively, emulating published strains. UTL-7Afox2sps19⌬ double mutants expressing either native Fox2p or rat peroxisomal MFE type 1 were generated by transforming the strain yAG1181 with the aforementioned plasmids pYE352::ScMFE-2 or pYE352-rMFE, respectively, to generate strains yAG1204 and yAG1203. Transformations, Media, Growth Conditions, and General Methods—Plasmids and fatty acids used are listed in Tables I and II, respectively. Fluorescence microscopy of living yeast cells expressing green fluorescent protein was done according to published methods (9)
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