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

Genes encoding cytochromes P450 of the CYP52 family in the n-alkane-assimil ating yeast Yarrowia lipolytica have been cloned and analyzed, Degenerate P CR primers which were designed for the conserved amino acid sequences of cy tochromes P450ALK of Candida species were used for amplification and isolat ion of genes encoding P450ALK from a genomic DNA library of Y, lipolytica C X161-1B, Seven new genes (YlALK2-YlALK8) were isolated, Five of the seven Y lALK genes were induced by n-alkane under the culture conditions used in th is study, whereas their expression was strictly repressed by glycerol but n ot by glucose, similar to the case of YlALK1, reported previously. Disrupti on of YlALK2, YlALK3, YlALK3 or YlALK6 did not change the growth of Y, lipo lytica on medium containing n-alkanes of various chain lengths. A mutant wi th 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 rt-hexadecan e. These results suggest that the presence of multiple P450ALK species is a rather common phenomenon among the n-alkane-assimilating yeasts and that i n the n-alkane assimilation of Y, lipolytica, YlALK1 functions to assimilat e n-decane and longer molecules, whereas YlALK2 is involved in the assimila tion of molecules longer than n-dodecane; other YlALKs are not significantl y involved in the assimilation of C10-C16 n-alkanes, Copyright (C) 2000 Joh n Wiley & Sons, Ltd.