Characterization of nystatin-resistant mutants of Saccharomyces cerevisiae and preparation of sterol intermediates using the mutants

Analysis of sterols of Saccharomyces cerevisiae mutants N3, N15, N26, and N3H, defective in sterol biosynthesis, was performed. Strains N3, N15, and N26 were isolated from their mother strain, M10, by screening with nystatin (Nagai et al. (1980) Mie Med. J. 30, 215-224), and strain N3H was isolated from N3 as a doubly-mutated strain. The main sterols of N3, N15, N26, and N3H were ergosta-7,22-dienol, ergost-8-enol, cholesta-5,7,24-trienol, and ergosta-7,22,24(28)-trienol, respectively. The former three strains were characterized as defective in delta 5-desaturation, delta 8--delta 7 isomerization, and C-24 transmethylation. Strain N3H was found to be defective in delta 5-desaturation as well as in delta 24(28)-reduction. However, the defect of N26 and N3H was suggested to be leaky, since small amounts of ergosterol and ergosta-7,22-dienol were found in these mutants, respectively. In N15, an accumulation (2% in total sterols) of the compound likely to be hydroxylated sterol was found. By aerobic adaptation of these strains, the accumulation of these strains, the accumulations of ergosta-7,22-dienol (22 mg/g dry cells), ergosta-7,22,24(28)-trienol (24 mg), ergosta-8,24(28)-dienol (18 mg), and cholesta-8,24-dienol (22 mg) reached a maximum in N3, N3H, N15, and N26 after 20, 20, 30, and 30 h, respectively. These strains appear to be useful for making 14C-labeled and non-labeled preparations of the above sterols.     

Sterol biosynthesis by symbiotes: cytochrome P450 sterol C-22 desaturase genes from yeastlike symbiotes of rice planthoppers and anobiid beetles

Rice planthoppers and anobiid beetles harbor intracellular yeastlike symbiotes (YLS), whose sterols are nutritionally advantageous for the host insects that cannot synthesize sterols. YLS of anobiid beetles synthesize ergosterol, whereas YLS of planthoppers produce ergosta-5,7,24(28)-trienol, which is a metabolic intermediate in the ergosterol biosynthetic pathway in yeasts. Since sterol C-22 desaturase (ERG5p, CYP61) metabolizes ergosta-5,7,24(28)-trienol into ergosta-5,7,22,24(28)-tetraenol, which is the penultimate compound in the ergosterol biosynthesis, we examined the gene of this enzyme to determine whether this enzyme works in the planthopper YLS. C-22 desaturase genes (ERG5) of YLS of the planthoppers and beetles had four introns in identical positions; such introns are not found in the reported genes of yeasts. Cytochrome P450 cysteine heme-iron ligand signature motif was well conserved among the putative amino acid sequences. The gene expression of the planthopper YLS were strongly suppressed, and the genes possessed nonsense mutations. The accumulation of ergosta-5,7,24(28)-trienol in the planthopper YLS was attributed to the inability of the planthopper YLS to produce functional ERG5p.     

Azasterol inhibitors in yeast. Inhibition of the 24-methylene sterol delta24(28)-reductase and delta24-sterol methyltransferase of Saccharomyces cerevisiae by 23-azacholesterol.

The effects of 23-azacholesterol on sterol biosynthesis and growth of Saccharomyces cervisiae were examined. In the presence of 0.2, 0.5, and 1 micron 23-azacholesterol, aerobically-growing yeast produced a nearly constant amount of ergosta-5,7,22,24(28)-tetraenol (approx. 36% of total sterol) and slowly accumulated zymosterol with a concommitant decline in ergosterol synthesis. Growth and total sterol content of yeast cultures treated with 0.2-1 micron 23-azacholesterol were similar to that of the control culture. Yeast cultures treated with 5 and 10 micron 23-azacholesterol produced mostly zymosterol (58-61% of total sterol), while ergosta-5,7,22,24(28)-tetraenol production declined to less than 10% of total sterol. The observed changes in the distribution of sterols in treated cultures are consistent with inhibition of 24-methylene sterol 24(28)-sterol reductase (total inhibition at 1 micron 23-azacholesterol) and of 24-sterol methyltransferase (71% inhibition at 10 micron 23-azacholesterol). Yeast cultures treated with 10 micron 23-azacholesterol were found to contain 4,4-dimethylcholesta-8,14,24-trienol and 4alpha-methylcholesta-8,14,24-trienol, which were isolated and characterized for the first time.     

Sterols of the cultured dinoflagellate Pyrocystis lunula

Eighteen components of the sterol fraction of $\text{Pyrocystis lunula}$ have been identified. In addition to 4α-methyl sterols (typical dinoflagellate sterols), regular sterols, both with a saturated and Δ⁵-unsaturated skeleton, were isolated, together with Δ⁴-3-keto steroids including the hitherto unknown 23,24R-dimethyl-4,22E-cholestadien-3-one.     

Identification of a Sterol Δ7 Reductase Gene Involved in Desmosterol Biosynthesis in Mortierella alpina 1S-4

Molecular cloning of the gene encoding sterol Δ7 reductase from the filamentous fungus Mortierella alpina 1S-4, which accumulates cholesta-5,24-dienol (desmosterol) as the main sterol, revealed that the open reading frame of this gene, designated MoΔ7SR, consists of 1,404 bp and codes for 468 amino acids with a molecular weight of 53,965. The predicted amino acid sequence of MoΔ7SR showed highest homology of 51% with that of sterol Δ7 reductase (EC 1.3.1.21) from Xenopus laevis (African clawed frog). Heterologous expression of the MoΔ7SR gene in yeast Saccharomyces cerevisiae revealed that MoΔ7SR converts ergosta-5,7-dienol to ergosta-5-enol (campesterol) by the activity of Δ7 reductase. In addition, with gene silencing of MoΔ7SR gene by RNA interference, the transformant accumulated cholesta-5,7,24-trienol up to 10% of the total sterols with a decrease in desmosterol. Cholesta-5,7,24-trienol is not detected in the control strain. This indicates that MoΔ7SR is involved in desmosterol biosynthesis in M. alpina 1S-4. This study is the first report on characterization of sterol Δ7 reductase from a microorganism.     

The action of the systemic fungicides tridemorph and fenpropimorph on sterol biosynthesis by the soil amoeba Acanthamoeba polyphaga

Tridemorph and fenpropimorph, two systemic fungicides known by their inhibitory effects on sterol biosynthesis in fungi and plants, were administered in vivo to the amoeba Acanthamoeba polyphaga. The compounds did not kill the cells, but modified completely their sterol pattern. Fungicide-exposed cells accumulated cyclopropylsterols indicating a partial blockage of the cyclopropane isomerase as in higher plants and delta 8-sterols indicating an inhibition of the delta 8----delta 7 isomerase as in fungi. Three new sterols, 4 alpha-methylergosta-9(11),24(28)-dienol, ergosta-6,8,22-trienol and poriferasta-6,8,22-trienol were isolated and identified, the former from control cells, the two latter from fungicide-treated cells. These results are in accordance with our previous results on the presence of cycloartenol as sterol precursor and confirm our hypothesis on a phylogenetic relationship of Acanthamoeba polyphaga with photosynthetic phyla.     

The structure of a Burkholderia pseudomallei immunophilin-inhibitor complex reveals new approaches to antimicrobial development

TbSMT [Trypanosoma brucei 24-SMT (sterol C-24-methyltransferase)] synthesizes an unconventional 24-alkyl sterol product set consisting of Δ24(25)-, Δ24(28)- and Δ25(27)-olefins. The C-methylation reaction requires Si(β)-face C-24-methyl addition coupled to reversible migration of positive charge from C-24 to C-25. The hydride shifts responsible for charge migration in formation of multiple ergostane olefin isomers catalysed by TbSMT were examined by incubation of a series of sterol acceptors paired with AdoMet (S-adenosyl-L-methionine). Results obtained with zymosterol compared with the corresponding 24-2H and 27-13C derivatives revealed isotopic-sensitive branching in the hydride transfer reaction on the path to form a 24-methyl-Δ24(25)-olefin product (kinetic isotope effect, kH/kD=1.20), and stereospecific CH3→CH2 elimination at the C28 branch and C27 cis-terminal methyl to form Δ24(28) and Δ25(27) products respectively. Cholesta-5,7,22,24-tetraenol converted into ergosta-5,7,22,24(28)-tetraenol and 24β-hydroxy ergosta-5,7,23-trienol (new compound), whereas ergosta-5,24-dienol converted into 24-dimethyl ergosta-5,25(27)-dienol and cholesta-5,7,24-trienol converted into ergosta-5,7,25(27)trienol, ergosta-5,7,24(28)-trienol, ergosta-5,7,24-trienol and 24 dimethyl ergosta-5,7,25(27)-trienol. We made use of our prior research and molecular modelling of 24-SMT to identify contact amino acids that might affect catalysis. Conserved tyrosine residues at positions 66, 177 and 208 in TbSMT were replaced with phenylalanine residues. The substitutions generated variable loss of activity during the course of the first C-1-transfer reaction, which differs from the corresponding Erg6p mutants that afforded a gain in C-2-transfer activity. The results show that differences exist among 24-SMTs in control of C-1- and C-2-transfer activities by interactions of intermediate and aromatic residues in the activated complex and provide an opportunity for rational drug design of a parasite enzyme not synthesized by the human host.     

Effect of griseofulvin on lipid composition and membrane integrity inMicrosporum gypseum

The effect of griseofulvin on lipid constituents and membrane permeability ofMicrosporum gypseum has been investigated. Mycelia grown in medium containing griseofulvin (IC₅₀ concentration) possessed a lower content of total lipids, phospholipids and sterols. This inhibitory effect was further supported by decreased incorporation of [¹⁴C] acetate in total lipids, total phospholipids and sterols. Decrease in total phospholipids was also reflected to a varying extent in all individual phospholipids. An increase in the unsaturated to saturated fatty acid ratio was observed in mycelia grown in medium containing griseofulvin. Membrane permeability was affected by griseofulvin as shown by increased K⁺-efflux and greater leakage of intracellular [³²P] labelled components from prelabelled cells. Our results suggest that the antifungal activity of griseofulvin is partially due to its secondary effect on lipid constituents ofMicrosporum gypseum.     

Two new sterols from Chlorella ellipsoidea

Eight sterols were observed in Chlorella ellipsoidea and the four major components were identified as ergosterol, 5α-ergost-7-en-3β-ol, 22-trans-ergosta-5,8(9),22-trien-3β-ol and ergosta-5,8(9)-dien-3β-ol. This is the first report of the latter two sterols from green plants.     

Identification of a sterol Delta7 reductase gene involved in desmosterol biosynthesis in Mortierella alpina 1S-4

Molecular cloning of the gene encoding sterol Delta7 reductase from the filamentous fungus Mortierella alpina 1S-4, which accumulates cholesta-5,24-dienol (desmosterol) as the main sterol, revealed that the open reading frame of this gene, designated MoDelta7SR, consists of 1,404 bp and codes for 468 amino acids with a molecular weight of 53,965. The predicted amino acid sequence of MoDelta7SR showed highest homology of 51% with that of sterol Delta7 reductase (EC 1.3.1.21) from Xenopus laevis (African clawed frog). Heterologous expression of the MoDelta7SR gene in yeast Saccharomyces cerevisiae revealed that MoDelta7SR converts ergosta-5,7-dienol to ergosta-5-enol (campesterol) by the activity of Delta7 reductase. In addition, with gene silencing of MoDelta7SR gene by RNA interference, the transformant accumulated cholesta-5,7,24-trienol up to 10% of the total sterols with a decrease in desmosterol. Cholesta-5,7,24-trienol is not detected in the control strain. This indicates that MoDelta7SR is involved in desmosterol biosynthesis in M. alpina 1S-4. This study is the first report on characterization of sterol Delta7 reductase from a microorganism.     

PIGMENTS,FATTY ACIDS,AND STEROLS OF THE TOXIC DINOFLAGELLATE GYMNODINIUM CATENATUM1

Cultures and field samples of the toxic dinoflagellate Gymnodinium catenatum Graham from Tasmania, Australia, were analyzed for pigment, fatty acid, and sterol composition. Gymnodinium catenatum contained the characteristic pigments of photosynthetic dinoflagellates, including chlorophyll a, chlorophyll c₂, and the carotenoids peridinin, dinoxanthin, diadinoxanthin, diatoxanthin, and β,β-carotene. In midlogarithmic and early stationary phase cultures, the chlorophyll a content ranged 50–72 pg · cell^?1, total lipids 956–2084 pg · cell^?1, total fatty acids 426–804 pg · cell^?1, and total sterols 8–20 pg · cell^?1. The major fatty acids (in order of decreasing abundance) were 16:0, 22:6(n-3), and 20:5(n-3) (collectively 65–70% of the total fatty acids), followed by 16:1(n-7), 18:2(n-6), and 14:0. This distribution is characteristic of most dinoflagellates, except for the low abundance (<3%) of the fatty acid 18:5(n-3), considered by some authors to be a marker for dinoflagellates. The three major sterols were 4α-methyl-5α-cholest-7-en-3β-ol, 4α,23,24-trimethyl-5α-cholest-22E-en-3β-ol (the dinoflagellate sterol, dinosterol), and 4α,23,24-trimethyl-5α-cholest-7-en-3β-ol. These three sterols comprised about 75% of the total sterols in both logarithmic and early stationary phase cultures, and they were also found in high proportions (22–25%) in natural dinoflagellate bloom samples. 4-Desmethyl sterols, which are common in most microalgae, were only present in trace amounts in G. catenatum. The chemotaxonomic affinities of G. catenatum and the potential for using specific signature lipids for monitoring toxic dinoflagellate blooms are discussed.     

The effect of temperature on the growth and lipid composition of the extremely halophilic coccus,Sarcina marina

Sarcina marina (NCMB 778) grew over the temperature range 20–45°C but no growth was recorded at 15°C or 50°C. At the optimum growth temperature of 34°C the doubling time was 14.5 h.The major polar lipid components, tentatively identified as the diether analogues of phosphatidyl glycerophosphate (PGP), phosphatidyl glycerol (PG), diglycosyl diglyceride (DGD) and triglycosyl diglyceride (TGD), and the major neutral lipid components, tentatively identified as squalene, dihydrosqualene, tetrahydrosqualene, vitamin MK8, geranyl geraniol and di-O-phytanyl glycerol, are identical to those found in other extremely halophilic rods and cocci.The total lipid content varied with growth conditions from 0.6 – 3.2% of the dry cell weight, polar lipids accounted for between 94.3 and 83.6% of the total lipid, the remainder being neutral lipid.In response to both the transition from exponential to stationary phase and a reduction of 14°C in growth temperature, batch cultures showed: (i) an increase in total lipid content; (ii) a decrease in PG and (iii) an increase in PGP. Specific responses to the temperature decrease were (i) increased total lipid content; (ii) no decrease in neutral lipids in stationary phase; (iii) marked reduction in PG and (iv) raised DGD. (i) and (ii) could be mechanisms for increasing membrane fluidity.In common with all other extreme halophiles investigated the alkyl side chains of S. marina polar lipids were identified as the phytanyl (3R, 7R, 11R, 15-tetramethylhexadecyl) group. Its structure did not appear to vary with temperature so that the normal mechanisms for modifying the structure of lipid alkyl side chains to modulate membrane fluidity in response to temperature changes probably does not occur in this group of microorganisms.     

Differential effects of fenpropimorph and fenhexamid, two sterol biosynthesis inhibitor fungicides, on arbuscular mycorrhizal development and sterol metabolism in carrot roots

Sterols composition of transformed carrot roots incubated in presence of increasing concentrations of fenpropimorph (0.02; 0.2; 2 mg l⁻¹) and fenhexamid (0.02; 0.2; 2; 20 mg l⁻¹), colonized or not by Glomus intraradices was determined. In mycorrhizal roots treated with fenpropimorph, normal Δ⁵-sterols were replaced by unusual compounds such as 9β,19-cyclopropylsterols (24-methylpollinastanol), Δ^8,14-sterols (ergosta-8,14-dienol, stigmasta-8,14-dienol), Δ⁸-sterols (Δ⁸ sitosterol) and Δ⁷-sterols (ergosta-7,22-dienol). After application of fenpropimorph, a drastic reduction of the mycorrhizal root growth, root colonization and extraradical fungal development was observed. Application of fenhexamid did not modify sterol profiles and the total colonization of roots. But the arbuscule frequency of the fungal partner was significantly affected.Comparison of the effects caused by the tested fungicides indicates that the usual phytosterols may be involved in symbiosis development. Indeed, observed modifications of root sterols composition could explain the high fenpropimorph toxicity to the AM symbiosis. However, the absence of sterolic modifications in the roots treated with fenhexamid could account for its more limited impact on mycorrhization.     

Sterol and Squalene Content of a Docosahexaenoic-Acid-Producing Thraustochytrid: Influence of Culture Age, Temperature, and Dissolved Oxygen

Thraustochytrid strain ACEM 6063, rich in omega-3 polyunsaturated fatty acids, was cultured at 15°C and 20°C in high (>40%) and low (<5%) dissolved oxygen (DO), and at 25°C in low-DO media. Samples were taken 4, 2, and 0 days before each culture reached peak biomass (T₋₄, T₋₂, and T_p, respectively). Twenty sterols, 13 of which were identified, were detected. Predominant were cholest-5-en-3β-ol, 24-ethylcholesta-5,22E-dien-3β-ol, 24-methylcholesta-5,22E-dien-3β-ol, and 2 coeluting sterols, one of which was 24-ethylcholesta-5,7,22-trien-3β-ol. These 4 sterols comprised 50% to 90% of total sterols. Cultures grown at high DO had simpler sterol profiles than those grown at low DO. Only the 4 sterols mentioned above were present at more than 3% of total sterols in high-DO cultures. In low-DO cultures, up to 6 additional sterols were present at more than 3% of total sterols. Culture age, temperature, and DO influenced squalene and sterol content. Total sterols (as a proportion of total lipids) decreased with increasing culture age. If organisms such as ACEM 6063 are to be used for commercial production of lipid products for human consumption, both their sterol content and factors influencing sterol production need to be characterized thoroughly. Received January 8, 2001; accepted March 6, 2001.     

Sterols of Xanthoria parietina: Evidence for two sterol ‘pools’ and the identification of a novel C,8 triene,ergosta-5,8,22-trien-3β-ol

Sterols extracted from Xanthoria parietina with organic solvents and released by saponification of the residual lichen tissue were analysed by GC-MS. The main components of the solvent-extractable sterols were two C₂₈ trienes and those of the more tightly bound sterols were ergost-5-en-3β-ol and two C₂₉ compounds. The structures of the C₂₈ compounds were shown to be ergosta-5,7,22-trien-3β-ol, Ia (ergosterol) and the previously unreported ergosta-5,8,22-trien-3β-ol, IIa, for which the name lichesterol is proposed. The main C₂₉ sterol was identified as (24R)-24-ethylcholesta-5,22-dien-3β-ol (poriferasterol).     

SALT‐INDUCED ALTERATIONS IN LIPID COMPOSITION OF DIATOM NITZSCHIA LAEVIS (BACILLARIOPHYCEAE) UNDER HETEROTROPHIC CULTURE CONDITION1

The diatom Nitzschia laevis Hust. is a potential producer of eicosapentaenoic acid (EPA). To elucidate its cellular response to salt stress, the effects of salinity on EPA production, lipid composition, and fatty acid distribution in the lipid pool were investigated. The highest contents of total fatty acids, EPA, and polar lipids were all obtained at NaCl of 20 g · L^?1, under which 71.3% of total EPA existed in polar lipid fractions. In N. laevis, high salt concentration might induce the decrease in neutral lipids (NLs), whereas the production of polar lipids, including phospholipids (PLs) and glycolipids (GLs), was enhanced. The degree of fatty acid unsaturation of both neutral and polar lipid fractions increased sharply when NaCl concentration increased from 10 to 20 g · L^?1 but decreased at NaCl concentration of 30 g · L^?1. The amount of total free sterols was increased with the increase in salt concentration. All these changes in lipid and fatty acids suggested a decrease in membrane permeability and fluidity under high salt concentration, which could help the alga acclimate to the salinity stress.     

Developmental Changes in the Sterol Composition and the Glycerol Content of Cuticular and Internal Lipids of Three Species of Flies

The glycerol concentration and the composition of cuticular and internal sterols in three medically and forensically important fly species, viz., Musca domestica, Sarcophaga carnaria, and Calliphora vicina, were analyzed. The cuticular and internal lipid extracts were separated by HPLC‐LLSD, after which the sterol fraction was characterized by GC/MS in total ion current (TIC) mode. The cuticular lipids of M. domestica larvae contained seven sterols, while in pupae and females, six sterols were identified. Five sterols were found in the cuticular lipids of M. domestica males. The internal lipids of M. domestica larvae and pupae contained six and seven sterols, respectively, while those of male and female flies contained only five sterols. Sitosterol, cholesterol, and campesterol were the dominant sterols in M. domestica, while campestanol, stigmasterol, sitostanol, and fucosterol were identified in low concentrations or in traces. In contrast, cuticular and internal lipids of S. carnaria and C. vicina contained only cholesterol. Glycerol was identified in all stages of M. domestica, S. carnaria, and C. vicina. For all the three examined fly species, the present study clearly showed species‐specific developmental changes in the composition of cuticular and internal sterols as well as in the glycerol concentration.     

Effects of light intensity, CO2 and nitrogen supply on lipid class composition of Dunaliella viridis

Lipid class composition of Dunaliella viridis Teodoresco was analysed using thin layer chromatography coupled with flame ionisation detection (TLC/FID technique). D. viridis was cultured under four different photon fluence rates and in darkness, and under two different conditions of CO₂ supply (atmospheric and 1%) with and without nitrogen sufficiency. Nine lipid classes were identified and quantified. Total lipids per cell and acetone-mobile polar lipids decreased with light, while the percentage of sterols and triglycerides increased with increasing irradiance. Total phospholipids increase was related with growth rate while hydrocarbons, wax esters and sterol esters accumulated in darkness. There were almost no changes in total lipids per cell because of nitrogen limitation; however, nitrogen limitation led to higher changes in lipid class composition under 1% CO₂ than under atmospheric CO₂ levels. The main reserve lipid, triglycerides, accumulated in high amounts under 1% CO₂ and nitrogen limitation, increasing from 1% to 22% of total lipids. The ratio sterols/acetone-mobile polar lipids could be an index of the 'light status' independently of nitrogen limitation, while the ratio triglycerides/total phospholipids could indicate any physiological stress uncoupling C and N metabolism and affecting the growth rate. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of ay-9944 on sterol biosynthesis in Chlorella sorokiniana

When Chlorella sorokiniana was grown in the presence of 4 ppm AY-9944 total sterol production was unaltered in comparison to control cultures. However, inhibition of sterol biosynthesis was shown by the accumulation of a number of sterols which were considered to be intermediates in sterol biosynthesis. The sterols which were found in treated cultures were identified as cyclolaudenol, 4α,14α-dimethyl-9β,19-cyclo-5α-ergost-25-en-3β-ol, 4α,14α-dimethyl -5α-ergosta-8,25-dien-3β-ol, 14α-methyl-9β,19-cyclo-5α-ergost-25-en-3β-ol, 24-methylpollinastanol, 14α-methyl-5α-ergost-8-en-3β-ol, 5α-ergost -8(14)-enol, 5α-ergost-8-enol, 5α-ergosta-8(14),22-dienol, 5α-ergosta-8,22-dienol, 5α-ergosta-8,14-dienol, and 5α-ergosta-7,22-dienol, in addition to the normally occurring sterols which are ergosterol, 5α-ergost-7-enol, and ergosta-5,7-dienol.The occurrence of these sterols in the treated culture indicates that AY-9944 is an effective inhibitor of the Δ⁸ → Δ⁷ isomerase and Δ¹⁴-reductase, and also inhibits introduction of the Δ²²-double bond. The occurrence of 14α-dimethyl-5α-ergosta-8,25-dien-3β-ol and 14α-methyl-9β,19-cyclo-5α-ergost -25-en-3β-ol is reported for the first time in living organisms. The presence of 25-methylene sterols suggests that they, and not 24-methylene derivatives, are intermediates in the biosynthesis of sterols in C. sorokiniana.     

Sterol mutants of Chlamydomonas reinhardtii: Characterisation of three strains deficient in C24(28) reductase

Three mutants of Chlamydomonas reinhardtii (strain arg7cw15) were obtained using the strategy of insertional mutagenesis by random plasmid integration with subsequent selection for resistance against the polyene antibiotic nystatin. Sterols were isolated by precipitation with digitonin, fractionated by both normal and argentation TLC, and then analysed by GLC and GC-MS. All the mutants accumulated ergosta-5,7,22,24(28)-tetraenol, ergosta-5,7,24(28)-trienol, ergosta-7,24(28)-dienol, stigmasta-5,7,22,24(28)-tetraenol, stigmasta-5,7,24(28)-trienol, stigmasta-8,24(28)-dienol and stigmasta-7,24(28)-dienol, while ergosterol and 7-dehydroporiferasterol which are the only major sterol components of the original strain were absent in the mutants. It is concluded that all these mutants are impaired in this C24(28) reductase which catalyses the reduction of the C24(28) tetraenol to the corresponding 24-alkyl sterol. There is strong evidence that the same enzyme acts on both the C₂₈ and C₂₉ sterol series. This view is also supported by Southern blot hybridisation analysis revealing that in all three mutants, plasmid insertion occurred at the same site indicating the disruption of the same gene. Due to the insertional nature of the mutations, the strains can be used for cloning the corresponding gene.