The S. cerevisiae strains used in this study was the haploid, prototrophic strain CEN.PK113-7D (MATa LEU3). Strains were grown at 30 °C in 2-liter chemostats (Applikon), with a working volume of 1.0 liter as described in van den Berg et al.. Cultures were fed with defined synthetic media based on the medium described by Verduyn et al . For nitrogen-limited growth the medium contained the following: (NH4)2SO4, 1.0 g·l-1; K2SO4, 5.3 g·l-1; KH2PO4, 3.0 g·l-1; MgSO4·7H2O, 0.5 g·l-1; vitamins and trace elements. Glucose concentrations in the feed were designed to obtain approximately 17 g·l-1 residual glucose in the culture broth and thus to avoid transcriptional responses as a result of different degrees of glucose repression. Feed concentrations were 59 g·l-1 glucose (reference strain) and 53 g·l-1 glucose (leu3D strain). The dilution rate was set at 0.10 h-1. The pH was measured online and kept constant at 5.0 by the automatic addition of 2 M KOH with the use of an Applikon ADI 1030 biocontroller. Stirrer speed was 800 rpm and the airflow was 0.5 l.min-1. Dissolved oxygen tension was measured online with an Ingold model 34 100 3002 probe, and was above 50% air saturation. A condenser connected to a cryostat set at 2 °C cooled the off-gas, and oxygen and carbon dioxide were measured off-line. Steady-state samples were taken between 10 and 14 volume changes to avoid strain adaptation due to long term cultivation. Dry weight, metabolite-, and gas profiles were constant over at least 3 volume changes prior to sampling for RNA extraction