Isozyme Function of n-Alkane-inducible Cytochromes P450 in Candida maltosa Revealed by Sequential Gene Disruption

Abstract

An n-alkane-assimilating yeast Candida maltosa contains multiple n-alkane-inducible forms of cytochromes P450 (P450alk), which can be assumed 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 of four major isoforms of P450alk (encoded by ALK1, ALK2, ALK3, and ALK5 genes) was investigated by sequential gene disruption. Auxotrophic markers used for the selection of disrupted strains were regenerated repeatedly through either mitotic recombination between heterozygous alleles of the diploid genome or directed deletion of the marker gene, to allow sequential gene disruptions within a single strain. The strain depleted of all four isoforms could 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 complementation, and enzyme assays after heterologous expression of single isoforms revealed (i) that each of the four individual isoforms is alone sufficient to allow growth on long chain n-alkane; (ii) that the ALK1-encoding isoform is the most versatile and efficient P450alk form, considering both its enzymatic activity and its ability to confer growth on n-alkanes of different chain length; and (iii) that the ALK5-encoding isoform exhibits a rather narrow substrate specificity and thus cannot support the utilization of short chain n-alkanes.

Diversity of Alkane Hydroxylase Systems in the Environment

“…While the P. putida GPo1 system acts on alkanes ranging from pentane to dodecane (C 5 -C 12 ) [42,55], most related membrane-bound alkane hydroxylases solely hydroxylate alkanes containing more than 10 carbon atoms [34] (see below). Several cytochrome P450 isozymes involved in alkane assimilation were recently cloned from the yeasts Candida maltosa, Yarrowia lipolytica and Debaryomyces hansenii [29,56,57]. They belong to the CYP52 gene family of the microsomal class II P450s, and receive electrons from NADPH (nicotinamide adenine dinucleotide phosphate) via FAD-and FMN-containing reductases.…”

A Consensus Sequence for Long-chain Fatty-acid Alcohol Oxidases from Candida Identifies a Family of Genes Involved in Lipid ω-Oxidation in Yeast with Homologues in Plants and Bacteria

“…There has been much interest in cloning and manipulating genes of the alkane/fatty acid oxidation pathway in industrial yeast species (1,13,14). Strains giving high yields of dicarboxylic acids from alkane-or fatty acid-containing media have been produced by selective mutagenesis of the oxidation pathways.…”

Insertional Mutagenesis in the n -Alkane-Assimilating Yeast Yarrowia lipolytica : Generation of Tagged Mutations in Genes Involved in Hydrophobic Substrate Utilization

“…This, and studies on cytochrome P450 regulation in Y. lipolytica and C. maltosa, suggested the existence of several chain length-specific alkane uptake or cytochrome P450 monooxygenase systems catalyzing the primary terminal hydroxylation (17). This was proven for C. maltosa (23,37) and more recently for Y. lipolytica (12), by identifying up to eight cytochrome P450 genes (ALK1 to ALK8) in this species, all belonging to the CYP52 gene family. A Y. lipolytica strain with ALK1 deleted grew very poorly on C 10 but almost normally on C 12 or C 16 (11).…”