The absence of alcoholic fermentation makes pyruvate decarboxylase-negative (Pdc ؊ ) strains of Saccharomyces cerevisiae an interesting platform for further metabolic engineering of central metabolism. However, Pdc ؊ S. cerevisiae strains have two growth defects: (i) growth on synthetic medium in glucose-limited chemostat cultures requires the addition of small amounts of ethanol or acetate and (ii) even in the presence of a C 2 compound, these strains cannot grow in batch cultures on synthetic medium with glucose. We used two subsequent phenotypic selection strategies to obtain a Pdc ؊ strain without these growth defects. An acetateindependent Pdc ؊ mutant was obtained via (otherwise) glucose-limited chemostat cultivation by progressively lowering the acetate content in the feed. Transcriptome analysis did not reveal the mechanisms behind the C 2 independence. Further selection for glucose tolerance in shake flasks resulted in a Pdc ؊ S. cerevisiae mutant (TAM) that could grow in batch cultures ( max ‫؍‬ 0.20 h ؊1 ) on synthetic medium, with glucose as the sole carbon source. Although the exact molecular mechanisms underlying the glucose-tolerant phenotype were not resolved, transcriptome analysis of the TAM strain revealed increased transcript levels of many glucoserepressible genes relative to the isogenic wild type in nitrogen-limited chemostat cultures with excess glucose. In pH-controlled aerobic batch cultures, the TAM strain produced large amounts of pyruvate. By repeated glucose feeding, a pyruvate concentration of 135 g liter ؊1 was obtained, with a specific pyruvate production rate of 6 to 7 mmol g of biomass ؊1 h ؊1 during the exponential-growth phase and an overall yield of 0.54 g of pyruvate g of glucose ؊1 .