Growth and antifungal homoazasterol production in Geotrichum flavo-brunneum

Abstract

The growth cycle and production of 15-aza-24-methylene-8,14-cholestadiene3f8-ol (15-azasterol) in Geotrichum flavo-brunneum strain NRRL28804 have been studied. During the growth cycle of this organism, morphological changes were noted which corresponded to changes in the pH of the culture medium. A physiological shift from acid to base production also occurred during the growth cycle. Concomitant When S. cerevisiae is exposed to picomolar concentrations of 15-azasterol, variations in the normal cellular sterol pool can be observed (5). Although no major deviation in cell growth is seen at such low concentrations, the yeast synthesizes ergosta-8,14-3,B-ol (ignosterol) rather than ergosta-5,7,22-trien-3,8-ol (ergosterol), the major sterol normally found in yeasts (6). Inhibition by 15-azasterol has been demonstrated on three enzymes of the sterol pathway in S. cerevisiae: A24-sterol methyltransferase (1), 24-methylene sterol A24 (28) Berkeley, was also used. In most experiments, cells were cultured in liquid medium containing 1% tryptone and 0.5% yeast extract with 2% glucose as the carbon source. In experiments to determine the effect of pH on growth, cells were also grown in liquid medium containing yeast nitrogen base (Difco Laboratories) with 2% glucose as the carbon source. Growth of NRRL28804 was measured by the change in dry weight per volume of culture medium. All experiments were conducted at 28°C, and cells were grown aerobically with reciprocal shaking.Mixed culture analysis. Log-phase NRRL28804 cells were combined with log-phase A364a cells in an equal cellular ratio. To determine the change in relative cell number with time, samples were taken periodically and cells were differentiated and enumerated with an American Optical hemacytometer.Determination of an optimal medium for production of inhibitory factor. Optimal conditions were determined by growing NRRL28804 in various media and extracting both the cells and media for the inhibitory factor. Cells were grown in 100 ml of liquid media containing different carbon sources. After 5 days of growth, the cells were harvested by centrifugation at 500 x g for 10 min. The cell pellets were suspended in 25 ml of chloroform and incubated overnight at 4°C. Chloroform extracts were filtered to remove debris and concentrated to 5 ml on a Brinkman rotating evaporator operated at 30°C. The 100-ml medium samples were extracted with 50 ml of chloroform and processed in the same manner.Inhibition by the various extracts was determined by the standard disk method. Antibiotic paper disks were wetted with 25 pd of each extract and allowed to dry for 4 h at room temperature. A lawn containing approximately 106 cells of A364a in the log phase of growth was spread on the tryptone-yeast-glucose medium described above, and the dried disks were immediately placed on top of the yeast cells.

Physiological response of Saccharomyces cerevisiae to 15-azasterol-mediated growth inhibition

“…G. flavo-brunneum strain NRRL 28804 was purchased from the American Type Culture Collection. Growth conditions for NRRL 28804 have been described previously (13). Yeast cells were grown in a liquid medium which contained 0.5% yeast extract, 1.0% tryptone, and 2.0% dextrose and was buffered with 0.1 M potassium succinate to pH 5.5 (designated YTDS medium).…”

“…diene-3,8-ol (15-azasterol) is a potent antimycotic compound that is produced by Geotrichum flavo-brunneum (7,13). Studies have shown that this compound inhibits sterol biosynthesis in fungi (8,18) and in plants (15), causing the accumulation of A5,14 sterols.…”

Temporal expression of transcription and relative copy number of plasmid pSFB-1 in Scytalidium flavo-brunneum

“…Synthesis of sterols is far too complex for all of the information to be on the plasmid. We have shown that during exponential growth, ergosterol is formed in S. flavobrunneum, as it is in most other fungi (9). It seems likely that 15-azasterol represents a digression from normal sterol synthesis.…”

“…The imperfect filamentous fungus Scytalidium flavobrunneum produces a potent antifungal compound, 15-azasterol (2), and reddish brown pigment at the end of the exponential phase of growth and into the stationary phase (9). The ability to synthesize both the pigment and the azasterol were lost simultaneously, and revertants to production of the substances were never observed (R. J. Rodriguez, unpublished data).…”

Inhibition of Phytosterol Biosynthesis by Azasterols

“…Overall, it is shown here that azasterols rank as lead compounds exhibiting interesting specificity towards steroidogenic enzymes. Furthermore, it is quite striking to observe that functional azasterols occur naturally in a few organisms, such as the case of the telluric fungus Geotrichum flavo-brunneum which produces a natural 15-azasterol (15-aza-24-methylene-D-homocholestadiene-3β-ol (Figure 7) [46]. This compound is a suitable inhibitor of the sterol-14-reductase in yeast and other fungi based on structural and charge analogies between its protonated form at cellular pH and the C-14 carbocationic high energy intermediate of the reaction that converts a -8,14 -sterol into a -8 -sterol [47].…”