Transformation of Fatty Acids Catalyzed by Cytochrome P450 Monooxygenase Enzymes of Candida tropicalis

Abstract

Candida tropicalis ATCC 20336 can grow on fatty acids or alkanes as its sole source of carbon and energy, but strains blocked in ␤-oxidation convert these substrates to long-chain ␣,-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 -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 (C 18:1 ), 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 -hydroxy acids. CYP52A17 also oxidized oleic acid efficiently but converted shorter, saturated fatty acids such as myristic acid (C 14:0 ) much more effectively. Both enzymes, in particular CYP52A17, also oxidized -hydroxy fatty acids, ultimately generating the ␣,-diacid. Consideration of these different specificities and selectivities will help determine which enzymes to amplify in strains blocked for ␤-oxidation to enhance the production of dicarboxylic acids. The activity spectrum also identified other potential oxidation targets for commercial development.

Identification of acyltransferases required for cutin biosynthesis and production of cutin with suberin-like monomers

“…Although each of these genes encode a biosynthetic enzyme involved in cutin biosynthesis, the in vivo substrates may not be fully understood. In this regard, despite evidence of functional activity on free fatty acids of plant P450 fatty acid -oxidases from various sources when expressed in yeast or insect cells (26,33,34), the production of -hydroxy fatty acids or dicarboxylic acids in planta by overexpression of these enzymes (Fig 2B) has not previously been reported.…”

Expression and Characterization of CYP52 Genes Involved in the Biosynthesis of Sophorolipid and Alkane Metabolism from Starmerella bombicola

“…CYP52A17 also transformed oleic acid but converted shorter, saturated fatty acids, such as myristic acid, much more effectively. Both enzymes also oxidized -hydroxyl fatty acids to generate the ␣,-diacid (25). In this study, we showed that CYP52M1 metabolized a number of saturated and unsaturated acids to -1 and -hydroxyl fatty acids (Fig.…”

Ethylthiosulfanylate effect on Candida tropicalis

“…Namely, concentration of nucleic acids in C. tropicalis cells under the influence of ETS decreased 3.6-fold for DNA and 2.56-fold for RNA. Simultaneous inhibition of both types of nucleic acids -RNA and DNA usually occurs when the chemotherapeutics act on the integrity of the cell membranes [16].…”

“…It may be a subject for further in-depth study. Chemotherapy drugs of different nature can cause the delay of ontogenetic development of crops, leading to the increase of the polar fraction of lipids and unsaturated fatty acids in cells [16]. Results of investigations of content of total lipids in C. tropicalis cells under the influence of ETS subfungicidal concentration are shown in Table 3.…”

“…The investigation of influence of ethylthiosulfanilate on permeability of the cytoplasmic membranes of yeast was determined photometrically by the release of low-molecular compounds having a maximum absorption at λ-260 nm [16].…”