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

[摘要] English: A large number of monooxygenases contain a haem protein containing cytochrome P450.These moncoxygenases enzymatically catalyze dioxygen activation at the cytochromeP450 haem protein. The cytochrome-P450-dependent monooxygenases are involved inmany steps of the biosynthesis and the degradation of compounds such as steroids, fattyacids, prostaglandins, leukotrienes, and n-alkanes. The first enzymatic step ofhydrocarbon assimilation is the terminal hydroxylation of the n-alkane by a cytochromeP450 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 ashydroxylase in a typical monooxygenase reaction.The cytochrome P450 monooxygenase (CYP52) multigene family involved in theterminal hydroxylation of n-alkanes and fatty acids has been characterized in yeastspecies such as Candida maltosa, Candida tropicolis and Candidaapicola (Lottermoseret 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, originallyclassified as a Candida, uses few sugars (mainly glucose) as a carbon source. This yeastreadily assimilates organic acids, various polyalcohols, and 'normal parafins. The first ofeight Y. lipolytica cytochrome P450 monooxygenase characterized was YIALK 1 (Iida etal., ] 998). Subsequent characterization of the remaining seven Y. lipolytica CYP52 genesshowed YIALKl and YIALK2 to be the major P450 forms involved in assimilation ofshorter-chain n-alkanes (C10-C16), while the remaining YIALK genes (Y1ALK3 throughYIALK8), 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 wellas 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 asthose used by Iida et al. (2000). Complications that hampered the study included: (i) theinsolubility of the very long-chain n-alkanes and fatty acids in water, which necessitatedthe use of co-solvents such as pristane and Tween 80; (ii) the poor growth of the selectedyeast 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 withcomparing a large number of genes under so many growth conditions, and (v) thedifficulty 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 andpristane, stearic acid and Tween 80 and behenic acid and Tween 80. Negative controlsconstituted cultures without any substrate as well as cultures containing only eitherpristane or Tween 80. The first two Y. lipolytica CYP52 genes investigated, YIALK I andYIALK2, showed preferential induction on all the substrates tested. Genes YIALK3through Y/ALK6 showed relatively weaker induction on the substrates tested whencompared to YIALK 1 and YlALK2. No mRNA transcripts were observed for eitherY1ALK7 or Y1ALK8 on any of the substrates tested at any of the induction times. Theseresults were not only repeated for both the RT -PCR and Northern hybridizationexperiments, 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 usingglucose pre-grown or acid pre-grown yeast cells for the induction, experiments.Phylogenetic analysis of the individual Y. lipolytica CYP52 genes suggested that thismultigene family evolved and diverged after branching off from the ancestral P450ALKgene. YlALK 1 and 'YIALK2, which were most prominent in the induction studies,grouped together in the phylogenetic analysis. Comparison of the Y. lipolytica CYP52 andperoxisomal β-oxidation MFE2 gene promoter regions revealed oleic acid-responseelements involved in activation by fatty acids. Elucidating the transcriptional activatingsequences present in the β-oxidation and CYP52 enzyme systems could lead to anunderstanding of the regulation of enzymes that contribute to the terminal- and β-oxidation reactions occurring within these n-alkane-assimilating yeasts.

[发布机构] University of the Free State