The cytochrome P450ALK multigene family of ann-alkane-assimilating yeast,Yarrowia lipolytica: cloning and characterization of genes coding for new CYP52 family members

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Abstract
Genes encoding cytochromes P450 of the CYP52 family in the n-alkane-assimilating yeast Yarrowia lipolytica have been cloned and analyzed. Degenerate PCR primers which were designed for the conserved amino acid sequences of cytochromes P450ALK of Candida species were used for amplification and isolation of genes encoding P450ALK from a genomic DNA library of Y. lipolytica CX161-1B. Seven new genes (YlALK2-YlALK8) were isolated. Five of the seven YlALK genes were induced by n-alkane under the culture conditions used in this study, whereas their expression was strictly repressed by glycerol but not by glucose, similar to the case of YlALK1, reported previously. Disruption of YlALK2, YlALK3, YlALK4 or YlALK6 did not change the growth of Y. lipolytica on medium containing n-alkanes of various chain lengths. A mutant with disruptions in both YlALK1 and YlALK2 did not grow well on n-hexadecane, whereas one with disruptions in both YlALK1 and YlALK3, which has the same phenotype as the YlALK1 singly disrupted mutant, grew well on n-hexadecane. These results suggest that the presence of multiple P450ALK species is a rather common phenomenon among the n-alkane-assimilating yeasts and that in the n-alkane assimilation of Y. lipolytica, YlALK1 functions to assimilate n-decane and longer molecules, whereas YlALK2 is involved in the assimilation of molecules longer than n-dodecane; other YlALKs are not significantly involved in the assimilation of C10-C16 n-alkanes.