Importance of balanced intracellular formate levels for pHi and CO2 homeostases during mixed-acid fermentation.

During mixed-acid fermentation, Escherichia coli metabolizes glucose to a mixture of acetate, ethanol, formate, lactate, and succinate, together with equimolar amounts of H2 and CO2. These gases are generated via the disproportionation of formate by formate hydrogenlyase (FHL-1). This reaction provides the cell with its only source of internally generated CO2 for carboxylation reactions. Formate is produced by pyruvate formate-lyase (PflB), and its balanced level within the cell is maintained by the activities of PflB, FHL-1, and the bidirectional formate channel, FocA. A formate imbalance results when strains synthesize a FocAN209 variant that continuously and efficiently exports formic acid. During fermentative growth, a FocAN209-producing strain prematurely enters stationary phase and fails to synthesize FHL-1 due to intracellular formate insufficiency. Here, we show that intracellular pH (pHi) of the mutant was increased relative to the parental strain, correlating with reduced ATP levels. Anaerobic growth could be partially restored by cultivation with bicarbonate; however, pHi was lowered only marginally and ATP levels remained low. Determination of the fermentation-product profile revealed that the continuous loss of formate from the cell could not be compensated by bicarbonate supplementation. A severe limitation in both lactate and succinate production was also only partially rescued by bicarbonate. Increased ethanol production indicated that acetyl-CoA was sacrificed in the mutant to re-oxidize reduced pyridine nucleotides. These findings demonstrate that an imbalance in intracellular formate not only negatively impacts carboxylative metabolism but also remodels the spectrum of fermentation products to the detriment of the cell's energy metabolism and pH homeostasis.IMPORTANCEFormate is central to the fermentative metabolism of Escherichia coli. Balanced formate levels are required for DNA synthesis and for CO2 generation by formate hydrogenlyase (FHL-1). An E. coli mutant synthesizing an FocA variant that irreversibly exports formate has a severe growth defect because it cannot make FHL-1, which severely restricts CO2 and negatively impacts pH homeostasis by lowering ATP levels. We show, however, that impaired growth of the mutant can be only partially restored by supplying bicarbonate. The underlying cellular lack of formate cannot be fully compensated by bicarbonate because efficient pyruvate formate-lyase-dependent pyruvate cleavage remodels fermentation away from acetate and toward ethanol production, which ensures redox balance, but generates less ATP by substrate-level phosphorylation.