Directed Evolution of Pyruvate Decarboxylase-Negative Saccharomyces cerevisiae , Yielding a C 2 -Independent, Glucose-Tolerant, and Pyruvate-Hyperproducing Yeast

Abstract

ABSTRACT 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 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 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 (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 .