Genetic evaluation of physiological functions of thiolase isozymes in the n-alkane-assimilating yeast Candida tropicalis

The n-alkane-assimilating diploid yeast Candida tropicalis possesses three thiolase isozymes encoded by trio pairs of alleles: cytosolic and peroxisomal acetoacetyl-coenzyme A (CoA) thiolases, encoded by CT-T1A and CT-TIE, and peroxisomal 3-ketoacyl-CoA thiolase, encoded by CT-T3A and CT-T3B. The physiological functions of these thiolases have been examined by gene disruption, The homozygous ct-t1a Delta/t1b Delta null mutation abolished the activity of acetoacctyl-CoA thiolase and resulted in mevalonate auxotrophy. The homozygous ct-t3a Delta/t3b Delta null mutation abolished the activity of 3-ketoacyl-CoA thiolase and resulted in growth deficiency on fz-alkanes (C-10 to C-13), All thiolase activities in this yeast disappeared with the ct-t1a Delta/t1b Delta and ct-t3a Delta/t3b Delta null mutations. To further clarify the function of peroxisomal acetoacetyl-CoA thiolases, the site-directed mutation leading acetoacetyl-CoA thiolase without a putative C-terminal peroxisomal targeting signal was introduced on the CT-TIA locus in the ct-t1b Delta null mutant. The truncated acetoacetyl-CoA thiolase vies solely present in cytoplasm, and the absence of acetoacetyl-CoA thiolase in peroxisomes had no effect on growth on all carbon sources employed. Growth on butyrate was not affected by a lack of peroxisomal acetoacetyl-CoA thiolase, while a retardation of growth hy a lack of peroxisomal 3-ketoacyl-CoA thiolase was observed. A defect of both peroxisomal isozymes completely inhibited growth on butyrate. These results demonstrated that cytosolic acetoacetyl-CoA thiolase was indispensable for the mevalonate pathway and that both peroxisomal acetoacetyl-CoA thiolase and 3-ketoacyl-CoA thiolase could participate in peroxisomal beta-oxidation. In addition to its essential contribution to the beta-oxidation of longer-chain fatty acids, 3-ketoacyl-CoA thiolase contributed greatly el en to the beta-oxidation of a C-4 substrate butyrate.