ISOZYME FUNCTION OF N-ALKANE-INDUCIBLE CYTOCHROMES P450 IN CANDIDA-MALTOSA REVEALED BY SEQUENTIAL GENE DISRUPTION

An n-alkane-assimilating yeast Candida maltosa contains multiple n-alk ane-inducible forms of cytochromes P450 (P450alk), which can be assume d to catalyze terminal hydroxylation of n-alkanes in the assimilation pathway. Eight structurally related P450alk genes have been identified , In the present study, the function off four major isoforms of P450al k (encoded by ALK1, ALK2, ALK3, and ALK5 genes) was investigated by se quential gene disruption. Auxotrophic markers used for the selection o f disrupted strains were regenerated repeatedly through either mitotic recombination between heterozygous alleles of the diploid genome or d irected deletion of the marker gene, to allow sequential gene disrupti ons within a single strain, The strain depleted of all four isoforms c ould not utilize n-alkanes for growth, providing direct evidence that P450alk is essential for n-alkane assimilation, Growth properties of a series of intermediate disrupted strains, plasmid-based complementati on, and enzyme assays after heterologous expression of single isoforms revealed (i) that each of the four individual isoforms is alone suffi cient to allow growth on long chain n-alkane; (ii) that the ALK1-encod ing isoform is the mesh; versatile and efficient P450alk form, conside ring both its enzymatic activity and its ability to confer growth on n -alkanes of different chain length; and (iii) that the ALK5-encoding i soform exhibits a rather narrow substrate specificity and thus cannot support the utilization of short chain n-alkanes.