Studies in the post-squalene section of sterol biosynthesis may be hampered by the poor availability of authentic standards. The present study used different yeast strains engineered in 3-ketosteroid reductase (Erg27p) to obtain radioactive and non-radioactive intermediates of sterol biosynthesis hardly or not available commercially. Non-radioactive 3-keto 4-monomethyl sterones were purified from non-saponifiable lipids extracted from cells bearing point-mutated 3-ketosteroid reductase. Two strategies were adopted to prepare the radioactive compounds: (1) incubation of cell homogenates of an ERG27-deletant strain with radioactive lanosterol, (2) incubation of growing cells of a strain expressing point-mutated 3-ketosteroid reductase with radioactive acetate. Chemical reduction of both radioactive and non-radioactive 3-keto sterones gave the physiological 3-β OH sterols, as well as the non-physiological 3-α OH isomers. This combined biological and chemical preparation procedure provided otherwise unavailable or hardly available 4-mono-methyl intermediates of sterol biosynthesis, paving the way for research into their roles in physiological and pathological conditions.

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A comparative study on post-squalene sterol synthesis in intact yeast cells and spheroplasts was carried out with strains from three genera (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris) as well as with engineered S. cerevisiae cells altered in regard to the late ergosterol synthesis pathway. A common outcome of incubation experiments with radioactive acetate was that in intact cells the metabolic pathway flows till its specific end product (ergosterol and its precursor, depending on the enzyme deficiency), whereas in spheroplasts the pathway was stalled some step upstream. For example, in spheroplasts from wt strains, non-cyclic triterpenes squalene and oxidosqualene accumulated as though the metabolic path was kept from producing steroid-shaped molecules different from the end product. Accumulation of non-cyclic triterpenes was observed also in spheroplasts from S. cerevisiae cells lacking 3-ketosteroid reductase activity, an enzyme belonging to the C4-demethylase complex. When production of cyclic triterpenes was compromised by loss or poor functionality of oxidosqualene cyclase (EC 5.4.99.7), the difference between intact cells and spheroplasts was still remarkable, yet limited to the different oxido/dioxidosqualene ratio. The characteristics of spheroplasts as non-proliferating cells may partially explain the observed differences in post-squalene pathway from intact cells. We cannot say if the difference in metabolic pathways in spheroplasts and intact cells is a rule. We think, however, that it is worthwhile to search for an answer, as a wider picture of the points where the metabolic pathways are stalled in spheroplasts could provide original ideas about the metabolic network in yeast.