Genetic Evaluation of Physiological Functions of Thiolase Isozymes in the n -Alkane-Assimilating Yeast Candida tropicalis

Abstract

ABSTRACT The n -alkane-assimilating diploid yeast Candida tropicalis possesses three thiolase isozymes encoded by two pairs of alleles: cytosolic and peroxisomal acetoacetyl-coenzyme A (CoA) thiolases, encoded by CT-T1A and CT-T1B , 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-t1aΔ/t1b Δ null mutation abolished the activity of acetoacetyl-CoA thiolase and resulted in mevalonate auxotrophy. The homozygous ct-t3aΔ/t3b Δ null mutation abolished the activity of 3-ketoacyl-CoA thiolase and resulted in growth deficiency on n -alkanes (C 10 to C 13 ). All thiolase activities in this yeast disappeared with the ct-t1aΔ/t1b Δ and ct-t3aΔ/t3b Δ 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-T1A locus in the ct-t1b Δ null mutant. The truncated acetoacetyl-CoA thiolase was 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 by 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 β-oxidation. In addition to its essential contribution to the β-oxidation of longer-chain fatty acids, 3-ketoacyl-CoA thiolase contributed greatly even to the β-oxidation of a C 4 substrate butyrate.