Sequencing, disruption, and characterization of the Candida albicans sterol methyltransferase (ERG6) gene: drug susceptibility studies in erg6 mutants.

The rise in the frequency of fungal infections and the increased resistance noted to the widely employed azole antifungals make the development of new antifungals imperative for human health. The sterol biosynthetic pathway has been exploited for the development of several antifungal agents (allylamines, morpholines, azoles), but additional potential sites for antifungal agent development are yet to be fully investigated. The sterol methyltransferase gene (ERG6) catalyzes a biosynthetic step not found in humans and has been shown to result in several compromised phenotypes, most notably markedly increased permeability, when disrupted in Saccharomyces cerevisiae.

A yeast sterol auxotroph (erg25) is rescued by addition of azole antifungals and reduced levels of heme.

Genetic disruption of the Saccharomyces cerevisiae C-4 sterol methyl oxidase ERG25 gene leads to sterol auxotrophy. We have characterized a suppression system that requires two mutations to restore viability to this disrupted strain. One suppressor mutation is erg11, which is blocked in 14alpha-demethylation of lanosterol and is itself an auxotroph.

Cloning and characterization of the Saccharomyces cerevisiae C-22 sterol desaturase gene, encoding a second cytochrome P-450 involved in ergosterol biosynthesis.

The ERG5 gene from Saccharomyces cerevisiae was cloned by complementation of an erg5-1 mutation using a negative selection protocol involving screening for nystatin-sensitive transformants. ERG5 is the putative gene encoding the C-22 sterol desaturase required in ergosterol biosynthesis. The functional gene was localized to a 2.

Cloning and characterization of ERG25, the Saccharomyces cerevisiae gene encoding C-4 sterol methyl oxidase.

We have cloned the Saccharomyces cerevisiae C-4 sterol methyl oxidase ERG25 gene. The sterol methyl oxidase performs the first of three enzymic steps required to remove the two C-4 methyl groups leading to cholesterol (animal), ergosterol (fungal), and stigmasterol (plant) biosynthesis. An ergosterol auxotroph, erg25, which fails to demethylate and concomitantly accumulates 4,4-dimethylzy-mosterol, was isolated after mutagenesis.

Sterol synthesis and viability of erg11 (cytochrome P450 lanosterol demethylase) mutations in Saccharomyces cerevisiae and Candida albicans.

The identification of the precise structural features of yeast sterol molecules required for the essential "sparking" function has been a controversial area of research. Recent cloning and gene disruption studies in Saccharomyces cerevisiae have shown that C-24 methylation (ERG6), C-5 desaturation (ERG3) and delta 8-delta 7 isomerization (ERG2) are not required, while C-14 demethylation (ERG11) and C-14 reduction (ERG24) are each required for aerobic viability. Earlier observations had indicated that C-14 demethylase deficient strains could be restored to aerobic growth by suppressor mutations that caused a deficiency in C-5 desaturase.