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Applied and Environmental Microbiology, August 2004, p. 4872-4879, Vol. 70, No. 8
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.8.4872-4879.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Cloning and Characterization of Three Fatty Alcohol Oxidase Genes from Candida tropicalis Strain ATCC 20336

L. Dudley Eirich,1* David L. Craft,{dagger} Lisa Steinberg,{ddagger} Afreen Asif,1 William H. Eschenfeldt,2 Lucy Stols,2 Mark I. Donnelly,2 and C. Ron Wilson1

Biotechnology Group, Cognis Corporation, Cincinnati, Ohio 45232,1 Environmental Research Division, Argonne National Laboratory, Argonne, Illinois 604392

Received 13 September 2003/ Accepted 29 April 2004

Candida tropicalis (ATCC 20336) converts fatty acids to long-chain dicarboxylic acids via a pathway that includes among other reactions the oxidation of {omega}-hydroxy fatty acids to {omega}-aldehydes by a fatty alcohol oxidase (FAO). Three FAO genes (one gene designated FAO1 and two putative allelic genes designated FAO2a and FAO2b), have been cloned and sequenced from this strain. A comparison of the DNA sequence homology and derived amino acid sequence homology between these three genes and previously published Candida FAO genes indicates that FAO1 and FAO2 are distinct genes. Both genes were individually cloned and expressed in Escherichia coli. The substrate specificity and Km values for the recombinant FAO1 and FAO2 were significantly different. Particularly striking is the fact that FAO1 oxidizes {omega}-hydroxy fatty acids but not 2-alkanols, whereas FAO2 oxidizes 2-alkanols but not {omega}-hydroxy fatty acids. Analysis of extracts of strain H5343 during growth on fatty acids indicated that only FAO1 was highly induced under these conditions. FAO2 contains one CTG codon, which codes for serine (amino acid 177) in C. tropicalis but codes for leucine in E. coli. An FAO2a construct, with a TCG codon (codes for serine in E. coli) substituted for the CTG codon, was prepared and expressed in E. coli. Neither the substrate specificity nor the Km values for the FAO2a variant with a serine at position 177 were radically different from those of the variant with a leucine at that position.

* Corresponding author. Mailing address: Cognis Corporation, Research and Technology, Building 53, 4900 Este Ave., Cincinnati, OH 45232. Phone: (513) 482-2368. Fax: (513) 482-2862. E-mail: dudley-eirich{at}cinci.rr.com.

{dagger} Present address: Food and Drug Administration, Forensic Chemistry Center, Cincinnati, OH 45237.

{ddagger} Present address: Civil Engineering Department, Penn State University, University Park, PA 16802.

Applied and Environmental Microbiology, August 2004, p. 4872-4879, Vol. 70, No. 8
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.8.4872-4879.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.





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