STRUCTURE AND METABOLIC CONTROL OF THE YARROWIA-LIPOLYTICA PEROXISOMAL 3-OXOACYL-COA-THIOLASE GENE

Using a Yarrowia lipolytica genomic library, several overlapping clone s of the peroxisomal 3-oxoacyl-CoA-thiolase gene, POT1, were isolated. The library was prepared in the bacterial expression vector lambdagt1 1, thus allowing an immunological screening of recombinant bacteriopha ges with specific antibodies raised against purified peroxisomal thiol ase. The isolated POT1 clones hybridized to a 1.4 kb RNA species, whic h was induced approximately 30-fold when oleate was the carbon source. A 3634-bp segment of the cloned DNA was sequenced. This segment conta ined, on both strands, three major overlapping open-reading frames of 678, 1122 and 1242 bp. Northern-hybridization analysis showed that onl y the largest of these reading frames was transcribed. It encodes a pr otein of 414 amino acids and molecular mass 43.059 kDa. Its deduced am ino acid sequence has 30 - 60% identity and 50 - 70% sequence similari ty when compared to other known thiolases. According to both the amoun t (68 - 71%) and location of conserved amino acids, the encoded protei n belongs to the peroxisomal rather than the mitochondrial or cytoplas mic class of thiolases. Compared to bacterial and yeast cytosolic thio lases, the POT1 gene product contains a N-terminal extension of 25 ami no acids which clearly differs from typical mitochondrial import signa ls. One of the isolated clones contained, in addition to the POT1 codi ng sequence, 784 bp of the corresponding 5' flanking region. Neverthel ess, it was efficiently expressed in Escherichia coli suggesting the c orrect recognition of this fungal promoter by the prokaryotic transcri ptional and translational machinery. The Y. lipolytica genomic POT1 ge ne was disrupted by replacing 120 bp of its coding sequence with 2.7 k bp of DNA including the Y. lipolytica LEU2 gene. The resulting DELTApo t1 = LEU2 cells were free of immunologically cross-reacting thiolase. Western-blot analysis showed that the product of the non-disrupted gen e had a molecular mass of approximately 42 kDa. This corresponds well to the molecular mass of purified Y. lipolytica peroxisomal thiolase. Disruption of POT1 abolished the ability of Y. lipolytica cells to gro w on solid media with oleate as a carbon source. This inability to gro w in the presence of oleate suggests both the catabolic function of PO T1 and the absence of additional catabolic thiolases in Y. lipolytica. However, the DELTApot1 = LEU2 cells were unaffected in their ability to elongate externally added tridecanoic acid to its higher-chain-leng th homologues. Hence, another, POT1-independent and biosynthetic 3-oxo acyl-CoA thiolase must be responsible for this reaction in Y. lipolyti ca.