Directed Evolution of Pyruvate Decarboxylase-Negative Saccharomyces cerevisiae, Yielding a C2-Independent, Glucose-Tolerant, and Pyruvate-Hyperproducing Yeast

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 C₂ 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 acetate-independent 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 C₂ independence. Further selection for glucose tolerance in shake flasks resulted in a Pdc^- S. cerevisiae mutant (TAM) that could grow in batch cultures (μ_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 glucose-repressible 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.