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Characterization of the Saccharomyces cerevisiae ERG26 gene encoding the C-3 sterol dehydrogenase (C-4 decarboxylase) involved in sterol biosynthesis.

ABSTRACT: All but two genes involved in the ergosterol biosynthetic pathway in Saccharomyces cerevisiae have been cloned, and their corresponding mutants have been described. The remaining genes encode the C-3 sterol dehydrogenase (C-4 decarboxylase) and the 3-keto sterol reductase and in concert with the C-4 sterol methyloxidase (ERG25) catalyze the sequential removal of the two methyl groups at the sterol C-4 position. The protein sequence of the Nocardia sp NAD(P)-dependent cholesterol dehydrogenase responsible for the conversion of cholesterol to its 3-keto derivative shows 30% similarity to a 329-aa Saccharomyces ORF, YGL001c, suggesting a possible role of YGL001c in sterol decarboxylation. The disruption of the YGL001c ORF was made in a diploid strain, and the segregants were plated onto sterol supplemented media under anaerobic growth conditions. Segregants containing the YGL001c disruption were not viable after transfer to fresh, sterol-supplemented media. However, one segregant was able to grow, and genetic analysis indicated that it contained a hem3 mutation. The YGL001c (ERG26) disruption also was viable in a hem 1Delta strain grown in the presence of ergosterol. Introduction of the erg26 mutation into an erg1 (squalene epoxidase) strain also was viable in ergosterol-supplemented media. We demonstrated that erg26 mutants grown on various sterol and heme-supplemented media accumulate nonesterified carboxylic acid sterols such as 4beta, 14alpha-dimethyl-4alpha-carboxy-cholesta-8,24-dien-3be ta-ol and 4beta-methyl-4alpha-carboxy-cholesta-8,24-dien-3beta-o l, the predicted substrates for the C-3 sterol dehydrogenase. Accumulation of these sterol molecules in a heme-competent erg26 strain results in an accumulation of toxic-oxygenated sterol intermediates that prevent growth, even in the presence of exogenously added sterol.

SUBMITTER: Gachotte D

PROVIDER: S-EPMC24900 | biostudies-literature | 1998 Nov

REPOSITORIES: biostudies-literature

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Characterization of the Saccharomyces cerevisiae ERG26 gene encoding the C-3 sterol dehydrogenase (C-4 decarboxylase) involved in sterol biosynthesis.

Gachotte D D Barbuch R R Gaylor J J Nickel E E Bard M M

Proceedings of the National Academy of Sciences of the United States of America 19981101 23

All but two genes involved in the ergosterol biosynthetic pathway in Saccharomyces cerevisiae have been cloned, and their corresponding mutants have been described. The remaining genes encode the C-3 sterol dehydrogenase (C-4 decarboxylase) and the 3-keto sterol reductase and in concert with the C-4 sterol methyloxidase (ERG25) catalyze the sequential removal of the two methyl groups at the sterol C-4 position. The protein sequence of the Nocardia sp NAD(P)-dependent cholesterol dehydrogenase re ...[more]

PMID: 9811880

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Project description:BackgroundThe only hitherto known biological role of yeast Saccharomyces cerevisiae Tum1 protein is in the tRNA thiolation pathway. The mammalian homologue of the yeast TUM1 gene, the thiosulfate sulfurtransferase (a.k.a. rhodanese) Tst, has been proposed as an obesity-resistance and antidiabetic gene. To assess the role of Tum1 in cell metabolism and the putative functional connection between lipid metabolism and tRNA modification, we analysed evolutionary conservation of the rhodanese protein superfamily, investigated the role of Tum1 in lipid metabolism, and examined the phenotype of yeast strains expressing the mouse homologue of Tum1, TST.ResultsWe analysed evolutionary relationships in the rhodanese superfamily and established that its members are widespread in bacteria, archaea and in all major eukaryotic groups. We found that the amount of sterol esters was significantly higher in the deletion strain tum1Δ than in the wild-type strain. Expression of the mouse TST protein in the deletion strain did not rescue this phenotype. Moreover, although Tum1 deficiency in the thiolation pathway was complemented by re-introducing TUM1, it was not complemented by the introduction of the mouse homologue Tst. We further showed that the tRNA thiolation pathway is not involved in the regulation of sterol ester content in S. cerevisiae, as overexpression of the tEUUC, tKUUU and tQUUG tRNAs did not rescue the lipid phenotype in the tum1Δ deletion strain, and, additionally, deletion of the key gene for the tRNA thiolation pathway, UBA4, did not affect sterol ester content.ConclusionsThe rhodanese superfamily of proteins is widespread in all organisms, and yeast TUM1 is a bona fide orthologue of mammalian Tst thiosulfate sulfurtransferase gene. However, the mouse TST protein cannot functionally replace yeast Tum1 protein, neither in its lipid metabolism-related function, nor in the tRNA thiolation pathway. We show here that Tum1 protein is involved in lipid metabolism by decreasing the sterol ester content in yeast cells, and that this function of Tum1 is not exerted through the tRNA thiolation pathway, but through another, currently unknown pathway.

Project description:Biosynthesis of sterols, which are considered essential components of virtually all eukaryotic membranes, requires molecular oxygen. Anaerobic growth of the yeast Saccharomyces cerevisiae therefore strictly depends on sterol supplementation of synthetic growth media. Neocallimastigomycota are a group of strictly anaerobic fungi which, instead of containing sterols, contain the pentacyclic triterpenoid "sterol surrogate" tetrahymanol, which is formed by cyclization of squalene. Here, we demonstrate that expression of the squalene-tetrahymanol cyclase gene TtTHC1 from the ciliate Tetrahymena thermophila enables synthesis of tetrahymanol by S. cerevisiae Moreover, expression of TtTHC1 enabled exponential growth of anaerobic S. cerevisiae cultures in sterol-free synthetic media. After deletion of the ERG1 gene from a TtTHC1-expressing S. cerevisiae strain, native sterol synthesis was abolished and sustained sterol-free growth was demonstrated under anaerobic as well as aerobic conditions. Anaerobic cultures of TtTHC1-expressing S. cerevisiae on sterol-free medium showed lower specific growth rates and biomass yields than ergosterol-supplemented cultures, while their ethanol yield was higher. This study demonstrated that acquisition of a functional squalene-tetrahymanol cyclase gene offers an immediate growth advantage to S. cerevisiae under anaerobic, sterol-limited conditions and provides the basis for a metabolic engineering strategy to eliminate the oxygen requirements associated with sterol synthesis in yeasts.IMPORTANCE The laboratory experiments described in this report simulate a proposed horizontal gene transfer event during the evolution of strictly anaerobic fungi. The demonstration that expression of a single heterologous gene sufficed to eliminate anaerobic sterol requirements in the model eukaryote Saccharomyces cerevisiae therefore contributes to our understanding of how sterol-independent eukaryotes evolved in anoxic environments. This report provides a proof of principle for a metabolic engineering strategy to eliminate sterol requirements in yeast strains that are applied in large-scale anaerobic industrial processes. The sterol-independent yeast strains described in this report provide a valuable platform for further studies on the physiological roles and impacts of sterols and sterol surrogates in eukaryotic cells.

Dataset Information

Characterization of the Saccharomyces cerevisiae ERG26 gene encoding the C-3 sterol dehydrogenase (C-4 decarboxylase) involved in sterol biosynthesis.

Publications

Characterization of the Saccharomyces cerevisiae ERG26 gene encoding the C-3 sterol dehydrogenase (C-4 decarboxylase) involved in sterol biosynthesis.

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