Transformation of fatty acids catalyzed by cytochrome P450 monooxygenase enzymes of Candida tropicalis

• Published in: Applied and Environmental Microbiology 2003-10, Vol.69 (10), p.5992-5999
• Main Authors: Eschenfeldt, W.H, Zhang, Y, Samaha, H, Stols, L, Eirich, L.D, Wilson, C.R, Donnelly, M.I
• Format: Article
• Language: English
• Subjects: a,^w-dicarboxylic acids; alkanes; Amino Acid Sequence; Animals; Baculoviridae - genetics; BASIC BIOLOGICAL SCIENCES; Biological and medical sciences; CANDIDA; Candida tropicalis; Candida tropicalis - enzymology; Candida tropicalis - genetics; Carbon; CARBOXYLIC ACIDS; cell lines; Cells, Cultured; cytochrome P-450; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; cytochrome P450; cytochrome P450 monooxygenase; CYTOCHROMES; enzyme activity; ENZYMES; Fatty acids; Fatty Acids - metabolism; Fundamental and applied biological sciences. Psychology; Fungi; gene expression; GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE; genes; Genetic Vectors; Microbiology; microsomes; Microsomes - enzymology; molecular cloning; myristic acid; Myristic Acid - metabolism; NADPH-Ferrihemoprotein Reductase - genetics; NADPH-Ferrihemoprotein Reductase - metabolism; oleic acid; Oleic Acid - metabolism; Oxidation; Peptides - chemistry; Physiology and Biotechnology; recombinant proteins; saturated fatty acids; Spodoptera; Spodoptera frugiperda; Substrates; TRANSFORMATIONS; transgenic insects; unsaturated fatty acids; unspecific monooxygenase; w-hydroxy acids
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/AEM.69.10.5992-5999.2003

Candida tropicalis ATCC 20336 can grow on fatty acids or alkanes as its sole source of carbon and energy, but strains blocked in beta-oxidation convert these substrates to long-chain alpha,omega-dicarboxylic acids (diacids), compounds of potential commercial value (Picataggio et al., Biotechnology 10:894-898, 1992). The initial step in the formation of these diacids, which is thought to be rate limiting, is omega-hydroxylation by a cytochrome P450 (CYP) monooxygenase. C. tropicalis ATCC 20336 contains a family of CYP genes, and when ATCC 20336 or its derivatives are exposed to oleic acid (C18:1), two cytochrome P450s, CYP52A13 and CYP52A17, are consistently strongly induced (Craft et al., this issue). To determine the relative activity of each of these enzymes and their contribution to diacid formation, both cytochrome P450s were expressed separately in insect cells in conjunction with the C. tropicalis cytochrome P450 reductase (NCP). Microsomes prepared from these cells were analyzed for their ability to oxidize fatty acids. CYP52A13 preferentially oxidized oleic acid and other unsaturated acids to omega-hydroxy acids. CYP52A17 also oxidized oleic acid efficiently but converted shorter, saturated fatty acids such as myristic acid (C14:0) much more effectively. Both enzymes, in particular CYP52A17, also oxidized omega-hydroxy fatty acids, ultimately generating the alpha,omega-diacid. Consideration of these different specificities and selectivities will help determine which enzymes to amplify in strains blocked for beta-oxidation to enhance the production of dicarboxylic acids. The activity spectrum also identified other potential oxidation targets for commercial development.