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Scientific
Publications - Work Done by Microbiology Reader
Philip Groeneveld, Nicola Rolley, Douglas B. Kell, Steven L. Kelly and Diane E. Kelly, Metabolic Control Analysis and engineering of the yeast sterol biosynthetic pathway, 10th Meeting of the International Study Group of Biothermokinetics (BTK), September 7-10 2002, Bordeaux-Arcachon, France, Abstract ABSTRACT Sterols are essential components of eukaryotic membranes and contribute to important physiological characteristics, such as osmotic robustness and hormonal effects on growth (Daum et al., 1999), but most importantly, the sterol pathway has shown to be a highly effective target for antifungal drug development. The incidence of life-threatening fungal infections has been increased, particularly among patients who are immuno-compromised by Human Immunodeficiency Virus infection (HIV causing AIDS) and those receiving immuno-suppressive therapy for organ transplantation or chemotherapy for cancer. Resistance among various fungal pathogens in these patients to antifungal drugs has been a matter of concern. Therefore, development of more effective antifungal agents has become a priority. Our research focuses on the yeast sterol biosynthesis. The type of sterol observed varies between the Kingdoms of Life; in yeast it is ergosterol. Starting from 2-aceto-CoA, ergosterol is synthesized by a sequence of at least 21 enzymes, among them two different cytochrome-P450 heme oxygenases. One P450, lanosterol C-14α demethylase (sterol biosynthetic step 13), encoded by the yeast ERG11-gene, is specifically inhibited by the antifugal compound fluconazole. This particular step (13) is suggested to be important for fungal growth. However, how control on ergosterol biosynthesis in yeast is distributed among this and all other steps in the pathway is still unknown. In the current report, we present the first results of a systematic approach to quantify metabolic control of the ergosterol biosynthesis in the yeast Saccharomyces cerevisiae.
METHOD Mutant system and high-throughput analysis Through modulation of the ERG11 expression in a constructed mutant around the level of a reference strain, we were able to determine the effect of this pathway enzyme on the specific growth rate and sterol concentrations. The ERG11 gene has been put under control of a regulatable promoter, for this purpose a doxycycline-repressible tetO7-CYC1 promoter was used (a kind gift from Professor Hegemann). This promoter is down-regulated in the presence of doxycycline and expresses transcript in the absence of doxycycline at levels comparable to genes controlled by the strong GAL1 promoter (Gari,1997). Thus this mutant allows ideal study of high- and low-level expression of the gene relative to that of the wild type (reference strain YUG37). The effect of doxycycline on the maximum specific growth rate (µmax) of the mutant was determined in aerobic batch cultures by a Bioscreen C instrument; a high-throughput system in which 200 separate cultures each have a working volume of just 100 µl (Bioscreen: http: // www. All microbiology . com/index.shtml).
RESULTS When cultivated without any doxycycline, the µmax of the ERG11-overexpressing mutant was more than 15% higher than the reference yeast strain YUG37 (µmax 0.37 and 0.32 h-1, respectively; see Figure 1A). Upon supplying the culture medium with different amounts of doxycycline, the mutant showed a decrease in the rate of biomass synthesis, i.e., its specific growth rate was impaired (see Figure 1B). The reference strain YUG37 did not show any toxic effects on growth over the concentration-range of doxycycline used (data not shown). In the absence of doxycycline and in the presence of a strong tetO7-CYC promoter (comparable with a GAL1 promoter), a higher level of ERG11 gene-product was expected in the mutant strain. As a consequence, this mutant (ERG-11) should be less sensitive to the antifungal agent fluconazole (which specifically inhibits the ERG11 gene-product and causes growth inhibition) than the reference strain (YUG-37). To test the difference in expression level of ERG11, we cultivated both strains on medium containing fixed amounts of fluconazole without any doxycycline. Indeed the mutant was less sensitive to different amounts of fluconazole than the referent strain (see figure 2A). This supported our hypothesis of a higher ERG11 expression level in the mutant strain. To test the doxycycline-repression of ERG11 in the ERG11-mutant, we supplied the batch cultures of both strains with or without one fixed amount of fluconazole together with or without 200 µM of doxycycline. As expected, as doxycycline was added, the more growth was sensitive to fluconazole in the mutant strain (see figure 2B). These early results indicate that the levels of cytochrome-P450 heme oxygenase (Lanosterol C-14a demethylase, sterol-biosynthetic step 13) in the ERG11 mutant strain could indeed be modulated by doxycycline and that it appears to have significant control on the specific growth rate of S. cerevisiae. Quantification of protein and metabolite levels are underway to determine flux- and concentration-control coefficients. Together with computer simulation of the kinetics of this biochemical network in Gepasi (Mendes & Kell 1998), we will determine the distribution of control and regulation within the sterol biosynthetic pathway in yeast.
REFERENCES Daum, G. et al. (1999). Systematic analysis of yeast strains with possible defects in lipid metabolism. Yeast 15, 601-614 Gari E, Piedrafita L, Aldea M, Herrero E,(1997). A set of vectors with a tetracycline-regulatable promoter system for modulated gene expression in Saccharomyces cerevisiae Yeast.13 (9): 837-848 Mendes, P. & Kell, D. B. (1998). Non-linear optimization of biochemical pathways: applications to metabolic engineering and parameter estimation. Bioinformatics 14, 869-883.
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