Regulation of Ancillary Reactions around the Central Carbon Metabolism of

E. coli

The central carbon metabolism of Eshechrichia coli includes the glycolysis, tricarboxylic acid cycle (TCA) and pentose phosphate pathway (PPP) providing 12 essential precursor metabolites and cellular energy (ATP, NADH and NADPH) needed for building the entire biomass of the cell. In addition to the central carbon metabolism, there are several bypass and ancillary pathways such as Entner-Doudroff pathway (ED), gluconeogenesis, methylglyoxal shunt and the glyoxylate shunt that help in maintaining cellular homeostasis. Robustness in the central metabolic pathway is controlled by several transcription factors: crp, fnr, arcA, rpiB, cra, iclR, and pdhR etc. However, very less is known about the transcriptional regulation of the ancillary reactions. Even when all necessary precursors are derived from the central carbon metabolic pathway, ancillary reactions are necessary for the sustainability of the cellular process. For example, glyoxylate shunt is indispensable for growth on acetate as it helps reduce the futile cycling of carbon through the TCA cycle. On the other hand, deregulated activation of ancillary reactions will lead to energy and metabolite level imbalance. Here, we show that such ancillary reactions are also essential to shorten the lag phase for growth and to combat the accumulation of metabolic intermediates which otherwise would disturb cellular homeostasis and growth. We elucidate the transcriptional regulation of ancillary reactions through a novel transcription factor, YebK.
Deletion of the transcription factor, yebK, affects the lag phase up on shifting cells from rich medium to minimal medium with different carbon sources like acetate, lactate and propionate. Lactate is first converted to pyruvate and metabolized through gluconeogenesis whereas acetate is metabolized via the TCA cycle, and glyoxylate pathway. The strains deleted for the transcription factor yebK exhibited antagonistic effect for growth on acetate and lactate. For growth on lactate minimal medium, wild type strains had a significantly long lag phase (~15 hours) whereas yebK deleted strains did not exhibit any lag
phase. A long lag phase was evident in acetate minimal medium with yebK deleted strains when L- glutamine was used as a nitrogen source whereas wild-type strains did not have any lag phase.
Lag phase is a distinct growth phase that helps bacteria to prepare for the new environment. Lag phase is the poorly characterized phase in bacterial growth regime mainly because of low number of cells in that phase. The length of the lag phase determines the efficiency to activate all the metabolic enzymes needed for growth on a particular medium. Longer lag phase is usually indicative of failure to activate key metabolic genes. However, it could also be possible that the failure to rapidly turn off pathways that would lead to flux imbalance in the new medium could lead to longer lag phase. Since there is no evidence to support the notion that YebK regulates the metabolic operons for propionate, acetate or lactate directly we hypothesized that YebK may be regulating such futile ancillary metabolic reactions.
Consistent with that the long lag phase observed in wild type strains growing on lactate could be rescued with the deletion of α-ketoglutarate/proton symporter protein, KgtP. ChIP-seq identified the presence of YebK binding site in the middle of the coding region of otherwise constitutive gene, kgtP. Thus, we hypothesize that YebK might be acting as a feed forward loop to activate the ancillary reaction catalyzed by KgtP. To test if YebK could control ancillary reactions globally, conditional mutants were made that could grow only with the activation of ancillary reactions. For example, deletion of the gene pfkA encoding the dedicated step of EMP pathway would demand the activation of ED or PPP pathway for efficient growth. Deletion of yebK in strains deleted for pfkA exhibited a faster growth on glucose than strains with only pfkA deleted indicating the possibility of regulating the ED pathway. Similarly, deletion of sucA and sucB genes of TCA cycle blocks the conversion of α-ketoglutarate to succinyl-coA. Triple knockout strain (â??sucA/â??sucB/â??yebK) experienced a longer lag phase in glucose minimal medium than the double knockout strain (â??sucA/â??sucB). Despite the longer lag phase the triple knockout strains experienced a higher specific growth rate indicating a delayed activation of ancillary reactions consuming alpha ketoglutarate. These results clearly indicate the importance of regulation of the ancillary reactions depending on the accumulation of the metabolic intermediates.
In this study, we elucidate the transcriptional regulation of the ancillary reactions of E. coli by a novel transcription factor, YebK. YebK acts as a feed forward loop to activate or repress several ancillary reactions around TCA cycle and ED pathways. Knowledge of such regulations is highly important while re-designing the metabolic pathways as they would serve to reduce the lag phase and increase the specific growth rate on the desired carbon source.

Fig 1: Metabolic pathway showing the ancillary reactions around the TCA cycle.

Phosphoenol

Pyruvate

Pyruvate

Acetyl

coA

Oxaloacet ate

Citrate

Malate

Glyoxylate

Isocitrate

Known Ancillary reactions

New Ancillary reactions

Ancillary reactions with genes unknown

Fumarate

Succinate

Propionoyl

coA

Succinyl

coA

Alpha Ketogluta rate

Alpha Ketogluta rate (ext)

Propionate

Genes regulated by YebK

Hydroxy butyrate

SSA