Evaluation of acyl coenzyme A oxidase (Aox) isozyme function in the n-alkane-assimilating yeast Yarrowia lipolytica

We have identified five acyl coenzyme A (CoA) oxidase isozymes (Aox1 throug h Aox5) in the n-alkane-assimilating yeast Yarrowia lipolytica, encoded by the POX1 through POX5 genes. The physiological function of these oxidases h as been investigated by gene disruption. Single, double, triple, and quadru ple disruptants were constructed. Global Aox activity was determined as a f unction of time after induction and of substrate chain length. Single null mutations did not affect growth but affected the chain length preference of acyl-CoA oxidase activity, as evidenced by a chain length specificity for Aod and Aox3. Aox2 was shown to be a long chain acyl-CoA oxidase and Aox3 w as found to be active against short-chain fatty acids, whereas Aox5 was act ive against molecules of all chain lengths. Mutations in Aox3 and Aox5 resu lted in an increase in total Aox activity. The growth of mutant strains was analyzed. In the presence of POX1 only, strains did not grow on fatty acid s, whereas PO; alone elicited partial growth, and the growth of the double POX2-POX3 deleted mutant was normal excepted on plates containing oleic aci d as the carbon source. The amounts of Aox protein detected by Western blot ting paralleled the Aox activity levels, demonstrating the regulation of do s in cells according to the POX1 genotype.