Phosphoenolpyruvate Carboxykinase as the Sole Anaplerotic Enzyme in Saccharomyces cerevisiae

Abstract

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 C 4 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 the PEPCK either from S. cerevisiae or from Actinobacillus succinogenes did not grow 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 , one-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 the 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 C 4 dicarboxylic acids in S. cerevisiae.

Catabolite repression of phosphoenolpyruvate carboxykinase by a zinc finger protein under biotin- and pyruvate carboxylase-deficient conditions in Pichia pastoris

“…The normal function of PEPCK is to catalyse the conversion of OAA to PEP. However, under certain conditions, PEPCK can catalyse the reverse reaction leading to the synthesis of OAA from PEP (Zelle et al, 2010;Blázquez et al, 1995). There is an excellent correlation between derepression of PEPCK and biotin-and PC-independent growth of DROP.…”

“…5a). It is known that aspartate can restore the growth of PC mutants in glucose-ammonium medium due to its conversion to OAA by transamination with a-ketoglutarate (Zelle et al, 2010;Blázquez et al, 1995). When aspartate was added to Bio 2 medium, growth of the WT strain was restored, indicating that the growth defect is primarily due to the deficiency of PC-mediated OAA synthesis (Fig.…”

“…These results indicate that a PC-independent pathway of OAA synthesis (pyruvate carboxylase bypass) is responsible for the growth of DROP in Bio 2 medium. Since enzymes of the glyoxylate cycle such as isocitrate lyase (ICL) and malate synthase (MS) as well as PEPCK are known to substitute for PC and contribute to OAA synthesis under specific growth conditions (Zelle et al, 2010;Blázquez et al, 1995). We examined their expression in WT and DROP strains.…”

“…Growth inhibition is primarily due to the non-replenishment of the citric acid cycle intermediate oxaloacetate (OAA) by the biotin-dependent enzyme PC. When such media are supplemented with aspartate, growth is restored due to replenishment of OAA via transamination of a-ketoglutarate (Zelle et al, 2010;Blázquez et al, 1995). OAA can be synthesized in a PC-and biotin-independent manner via the glyoxylate cycle when yeast cells are cultured in media containing non-fermentative carbon sources such as acetate.…”

“…However, enzymes of the glyoxylate cycle are repressed under glucose-abundant conditions (Blázquez et al, 1995). In S. cerevisiae, strains carrying mutations in the genes encoding PC and pyruvate kinase, phosphoenolpyruvate carboxykinase (PEPCK), which normally has decarboxylating and gluconeogenic functions, can replace the anaplerotic function of PC in glucose-grown cultures (Zelle et al, 2010). While PC-deficient yeast cells cannot grow in a medium containing glucose and ammonium as the sole sources of carbon and nitrogen respectively, certain mutations termed BPC (bypass of pyruvate carboxylase) enable them to grow under these conditions (Blázquez et al, 1995).…”