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 .

pH affects many processes on cell metabolism, such as enzyme kinetics. To enhance the understanding of the living cells, it is therefore indispensable to have a method to monitor the pH in living cells. To accomplish this, a dynamic intracellular pH measurement method applying low concentration benzoic acid pulse was developed. The method was thoroughly validated and successfully implemented for measuring fast dynamic intracellular pH of Saccharomyces cerevisiae in response to a glucose pulse perturbation performed in the BioSCOPE set-up. Fast drop in intracellular pH followed by partial alkalinization was observed following the pulse. The low concentration benzoic acid pulse which was implemented in the method avoids the undesirable effects that may be introduced by benzoic acid to cell metabolism.