Appl Microbiol Biotechnol (2005) 67: 119–124 DOI 10.1007/s00253-004-1697-0 APPLIED MICROBIAL AND CELL PHYSIOLOGY Tamay Seker . Kasper Møller . Jens Nielsen Analysis of acyl CoA ester intermediates of the mevalonate pathway in Saccharomyces cerevisiae Received: 12 February 2004 / Revised: 14 June 2004 / Accepted: 21 June 2004 / Published online: 22 September 2004 # Springer-Verlag 2004 Abstract The mevalonate pathway plays an important role in providing the cell with a number of essential precursors for the synthesis of biomass constituents. With respect to their chemical structure, the metabolites of this pathway can be divided into two groups: acyl esters [acetoacetyl CoA, acetyl CoA, hydroxymethylglutaryl (HMG) CoA] and phosphorylated metabolites (isopente- nyl pyrophosphate, dimethylallyl pyrophosphate, geranyl pyrophosphate, farnesyl pyrophosphate). In this study, we developed a method for the precise analysis of the intracellular concentration of acetoacetyl CoA, acetyl CoA and HMG CoA; and we used this method for quantification of these metabolites in Saccharomyces cerevisiae, both during batch growth on glucose and on galactose and in glucose-limited chemostat cultures operated at three different dilution rates. The level of the metabolites changed depending on the growth phase/ specific growth rate and the carbon source, in a way which indicated that the synthesis of acetoacetyl CoA and HMG CoA is subject to glucose repression. In the glucose batch, acetyl CoA accumulated during the growth on glucose and, just after glucose depletion, HMG CoA and aceto- acetyl CoA started to accumulate during the growth on ethanol. In the galactose batch, HMG CoA accumulated during the growth on galactose and a high level was maintained into the ethanol growth phase; and the levels of acetyl CoA and HMG CoA were more than two-fold higher in the galactose batch than in the glucose batch. Introduction The mevalonate pathway plays an important role in providing precursors for isoprenoids. The pathway involves more than 20 distinct reactions initiated with acetyl CoA and proceeding through farnesyl pyrophos- phate (FPP), a branch-point intermediate for terpenoids and sterols (Fig. 1). There are many different types of isoprenoids serving a number of different functions, such as sterols having a role in maintaining membrane fluidity, hormones and bile acids, carotenoids, antioxidants, ubiquinones and hemes involved in electron transport and dolichols involved in protein glycolsylation (Lees et al. 1997; Daum et al. 1998; Michal 1999). There is a widespread industrial interest in isoprenoids as they serve as flavors (e.g., limonene), fragrances (e.g., geraniol, citranellol), and anticancer agents (Burke et al. 1997; Crowell 1999). Since many bioactive compounds diverge from the mevalonate pathway, there is a great interest for deregulating the flux through this pathway via metabolic engineering. For industrial production, yeast is an interesting cell factory as it is well suited for large-scale fermentation and has been approved for the production of compounds used both as food ingredients and as pharmaceuticals (reviewed by Barkovich and Liao 2001). In order to design a yeast platform that can serve as an efficient cell factory for the production of different isoprenoids, it is necessary to ensure an efficient precursor supply; and in this context it is important to understand how the in vivo flux through the pathway is regulated. Robust methods for the analysis of pathway intermediates are required to obtain fundamental insights into the regulation of in vivo fluxes. In this study, we developed a method for the quantitative analysis of the first three intermediates of the mevalonate pathway. Employing this method, we analyzed the levels of the CoA esters during different growth conditions of Saccharomyces cerevisiae. Previously described extraction methods for mevalonate pathway intermediates are designed mostly for plant or animal tissues. Since the metabolites in the pathway have a wide diversity of structural and physical properties, T. Seker . K. Møller . J. Nielsen (*) Center for Microbial Biotechnology, Technical University of Denmark, BioCentrum-DTU, Building 223, 2800 Lyngby, Denmark e-mail: jn@biocentrum.dtu.dk Tel.: +45-45252698 Fax: +45-45884148