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Journal of Bacteriology, September 1999, p. 5140-5148, Vol. 181, No. 17
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Evaluation of Acyl Coenzyme A Oxidase (Aox) Isozyme Function in the n-Alkane-Assimilating Yeast Yarrowia lipolytica

Huijie J. Wang,1 Marie-Thérèse Le Dall,1 Yves Waché,2 Céline Laroche,2 Jean-Marc Belin,2 Claude Gaillardin,1 and Jean-Marc Nicaud1,*

Laboratoire de Génétique des Microorganismes, INRA-CNRS, 78850 Thiverval-Grignon,1 and Laboratoire de Biotechnologie, ENSBANA, 21000 Dijon,2 France

Received 1 April 1999/Accepted 21 June 1999

We have identified five acyl coenzyme A (CoA) oxidase isozymes (Aox1 through Aox5) in the n-alkane-assimilating yeast Yarrowia lipolytica, encoded by the POX1 through POX5 genes. The physiological function of these oxidases has been investigated by gene disruption. Single, double, triple, and quadruple disruptants were constructed. Global Aox activity was determined as a function 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 Aox2 and Aox3. Aox2 was shown to be a long-chain acyl-CoA oxidase and Aox3 was found to be active against short-chain fatty acids, whereas Aox5 was active against molecules of all chain lengths. Mutations in Aox4 and Aox5 resulted 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 acids, whereas POX4 alone elicited partial growth, and the growth of the double POX2-POX3-deleted mutant was normal excepted on plates containing oleic acid as the carbon source. The amounts of Aox protein detected by Western blotting paralleled the Aox activity levels, demonstrating the regulation of Aox in cells according to the POX genotype.

* Corresponding author. Mailing address: Laboratoire de Génétique Moléculaire et Cellulaire, INRA-CNRS, 78850 Thiverval-Grignon, France. Phone: 33-01-30-81-54-50. Fax: 33-01-30-81-54-57. E-mail: nicaud{at}platon.grignon.inra.fr.

Journal of Bacteriology, September 1999, p. 5140-5148, Vol. 181, No. 17
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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