Pyruvate carboxylase is the sole anaplerotic enzyme in glucose-grown cultures of wild-type Saccharomyces cerevisiae. Pyruvate carboxylase negative (Pyc(-)) S. cerevisiae strains cannot grow on glucose, unless media are supplemented with C4-compounds, such as aspartic acid. In several succinate-producing prokaryotes, phosphoenolpyruvate carboxykinase (PEPCK) fulfills this anaplerotic role. However, the S. cerevisiae PEPCK encoded by PCK1 is repressed by glucose and is considered to have a purely decarboxylating and gluconeogenic function. This study investigates whether and under which conditions PEPCK can replace the anaplerotic function of pyruvate carboxylase in S. cerevisiae. Pyc(-) S. cerevisiae strains constitutively overexpressing either the PEPCK from S. cerevisiae or from Actinobacillus succinogenes did not grown on glucose as the sole carbon source. However, evolutionary engineering yielded mutants able to grow on glucose as the sole carbon source at a maximum specific growth rate of ca. 0.14 h(-1), half that of the (pyruvate-carboxylase positive) reference strain grown under the same conditions. Growth was dependent on high carbon-dioxide concentrations, indicating that the reaction catalyzed by PEPCK operates near thermodynamic equilibrium. Analysis and reverse engineering of two independently evolved strains showed that single point mutations in pyruvate kinase, which competes with PEPCK for phosphoenolpyruvate, were sufficient to enable the use of PEPCK as sole anaplerotic enzyme. The PEPCK reaction produces one ATP per carboxylation event, whereas the original route through pyruvate kinase and pyruvate carboxylase is ATP-neutral. This increased ATP yield may prove crucial for engineering of efficient and low-cost anaerobic production of C4-dicarboxylic acids in S. cerevisiae.
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