Overproduction of threonine aldolase circumvents the biosynthetic role of pyruvate decarboxylase in glucose-limited chemostat cultures of Saccharomyces cerevisiae

Abstract

Pyruvate decarboxylase-negative (Pdc(-)) mutants of Saccharomyces cerevisiae require small amounts of ethanol or acetate to sustain aerobic, glucose-limited growth. This nutritional requirement has been proposed to originate from (i) a need for cytosolic acetyl coenzyme A (acetyl-CoA) for lipid and lysine biosynthesis and (ii) an inability to export mitochondrial acetyl-CoA to the cytosol. To test this hypothesis and to eliminate the C(2) requirement of Pdc(-) S. cerevisiae, we attempted to introduce an alternative pathway for the synthesis of cytosolic acetyl-CoA. The addition of L-carnitine to growth media did not restore growth of a Pdc(-) strain on glucose, indicating that the C(2) requirement was not solely due to the inability of S. cerevisiae to synthesize this compound. The S. cerevisiae GLY1 gene encodes threonine aldolase (EC 4.1.2.5), which catalyzes the cleavage of threonine to glycine and acetaldehyde. Overexpression of GLY1 enabled a Pdc(-) strain to grow under conditions of carbon limitation in chemostat cultures on glucose as the sole carbon source, indicating that acetaldehyde formed by threonine aldolase served as a precursor for the synthesis of cytosolic acetyl-CoA. Fractionation studies revealed a cytosolic localization of threonine aldolase. The absence of glycine in these cultures indicates that all glycine produced by threonine aldolase was either dissimilated or assimilated. These results confirm the involvement of pyruvate decarboxylase in cytosolic acetyl-CoA synthesis. The Pdc(-) GLY1 overexpressing strain was still glucose sensitive with respect to growth in batch cultivations. Like any other Pdc(-) strain, it failed to grow on excess glucose in batch cultures and excreted pyruvate when transferred from glucose limitation to glucose excess.

FIG. 1.

Concentrations of glucose, metabolites, and biomass after a switch to an aerobic chemostat culture (dilution rate = 0.10 h⁻¹) of the pdc1Δ pdc5Δ pdc6Δ reference strain RWB893(YEplac181) from growth on synthetic medium containing a mixture of glucose and acetate (0.25 M substrate carbon and 10% acetate on a carbon basis) to growth on a synthetic medium containing glucose (0.25 M substrate carbon) as the sole carbon source. The graph shows the washout profile of a single representative culture. An independent replicate experiment yielded the same results.

FIG. 2.

Metabolic responses of aerobic, glucose-limited chemostat cultures (dilution rate = 0.10 h⁻¹) to a 50 mM glucose pulse. (A) S. cerevisiae CEN.PK 113-7D (prototrophic wild-type strain). (B) GLY1-overproducing Pdc⁻ strain RWB893(YEpGLY1). The graphs show single representative glucose pulse experiments for each strain. Independent replicate experiments yielded essentially the same results.