Differential transcription of CYP52 genes of Yarrowia lipolytica during growth on hydrocarbons

English: A large number of monooxygenases contain a haem protein containing cytochrome P450. These moncoxygenases enzymatically catalyze dioxygen activation at the cytochrome P450 haem protein. The cytochrome-P450-dependent monooxygenases are involved in many steps of the biosynthesis and the degradation of compounds such as steroids, fatty acids, prostaglandins, leukotrienes, and n-alkanes. The first enzymatic step of hydrocarbon assimilation is the terminal hydroxylation of the n-alkane by a cytochrome P450 enzyme system. The enzyme system bound to the endoplasmic reticulum (ER), consists of an NADPH-cytochrome P450 reductase involved in transferring electrons (Govindaraj & Poulos, 1997; Nelson & Strobel, 1988), and a cytochrome P450 acting as hydroxylase in a typical monooxygenase reaction. The cytochrome P450 monooxygenase (CYP52) multigene family involved in the terminal hydroxylation of n-alkanes and fatty acids has been characterized in yeast species such as Candida maltosa, Candida tropicolis and Candidaapicola (Lottermoser et al., 1996; Seghezzi et al., 1992; Zirnmer et al., 1.996). These include eight genes in C. maltosa, seven in C. tropicalis. and two In C. apicola. Yarrowia lipolytica, originally classified as a Candida, uses few sugars (mainly glucose) as a carbon source. This yeast readily assimilates organic acids, various polyalcohols, and 'normal parafins. The first of eight Y. lipolytica cytochrome P450 monooxygenase characterized was YIALK 1 (Iida et al., ] 998). Subsequent characterization of the remaining seven Y. lipolytica CYP52 genes showed YIALKl and YIALK2 to be the major P450 forms involved in assimilation of shorter-chain n-alkanes (C10-C16), while the remaining YIALK genes (Y1ALK3 through YIALK8), did not appear to 'be significantly involved in C10-C16 assimilation (Iida et al., 2000). The induction of the eight Y. lipolytica CYP52 genes on longer-chain n-alkanes (C18-C28) and long-chain fatty acids was investigated using gene-specific RT -PCR reactions as well as Northern hybridizations. A PCR-based approach was used to prepare eight gene-specific Y lipolytica CYP52 probes. The probes designed for this study were the same as those used by Iida et al. (2000). Complications that hampered the study included: (i) the insolubility of the very long-chain n-alkanes and fatty acids in water, which necessitated the use of co-solvents such as pristane and Tween 80; (ii) the poor growth of the selected yeast strain under the conditions used by lida et al. (2000) for induction of the genes; (iii) the large number of genes investigated; (iv) the logistical problems associated with comparing a large number of genes under so many growth conditions, and (v) the difficulty in isolating RNA from Y. lipolytica. Induction of the Y. lipolytica CYP52 genes were studied in the presence of glucose, tetradecane, hexadecane, octadecane, mixtures of docosane and pristane, octacosane and pristane, stearic acid and Tween 80 and behenic acid and Tween 80. Negative controls constituted cultures without any substrate as well as cultures containing only either pristane or Tween 80. The first two Y. lipolytica CYP52 genes investigated, YIALK I and YIALK2, showed preferential induction on all the substrates tested. Genes YIALK3 through Y/ALK6 showed relatively weaker induction on the substrates tested when compared to YIALK 1 and YlALK2. No mRNA transcripts were observed for either Y1ALK7 or Y1ALK8 on any of the substrates tested at any of the induction times. These results were not only repeated for both the RT -PCR and Northern hybridization experiments, but were confirmed for the shorter-chain n-alkanes by Iida et al. (2000). Variations in the levels of Y lipolytica CYP52 gene transcripts were observed when using glucose pre-grown or acid pre-grown yeast cells for the induction, experiments. Phylogenetic analysis of the individual Y. lipolytica CYP52 genes suggested that this multigene family evolved and diverged after branching off from the ancestral P450ALK gene. YlALK 1 and 'YIALK2, which were most prominent in the induction studies, grouped together in the phylogenetic analysis. Comparison of the Y. lipolytica CYP52 and peroxisomal β-oxidation MFE2 gene promoter regions revealed oleic acid-response elements involved in activation by fatty acids. Elucidating the transcriptional activating sequences present in the β-oxidation and CYP52 enzyme systems could lead to an understanding of the regulation of enzymes that contribute to the terminal- and β- oxidation reactions occurring within these n-alkane-assimilating yeasts.