The George Bennett Lab: Dr. Sagit Shalel-Levanon

It is well known that metabolic products formed by E. coli change according to the growth conditions; factors such as oxygen availability, pH, and nutrient source must be carefully controlled. In industrial situations, care is taken to control pH and dissolved oxygen, and to maintain a reproducible feedstock. However, with the increased use of genetically modified strains, it is useful to be able to predict culture conditions that would be best suited for a particular strain to produce the desired product. One major factor is the aerobic vs. anaerobic nature of the culture; aerobic culture favors faster growth but anaerobic conditions are needed for the formation of certain desired products (e.g. ethanol, lactic acid etc.).

Escherichia coli possess a large number of sensing/regulating systems for the rapid response to changes in oxygen availability and the presence of other electron acceptors. Those regulation systems channel electrons from donor to terminal acceptors such that the overall potential difference is maximized for any given growth condition. The adaptive responses are coordinated by a group of global regulators, which includes the single-component FNR (fumarate, nitrate reduction) protein, and the two-component Arc (aerobic respiration control) system. With the initial onset of anaerobiosis, ArcA is activated, and if these conditions persist, FNR is subsequently activated leading in turn to the up-regulation of ArcA and the amplification of its effect.

Pathway Regulation Strain Construction

The aim of my work is to quantify the contributions of Arc- and Fnr-dependent regulation in catabolism. The metabolic and gene expression patterns of wild-type E. coli, an arcA mutant, an fnr mutant, and a double arcA-fnr mutant, are studied at steady state at different oxygen concentrations. To study the steady state metabolic activity, the concentrations of selected metabolites (such as succinate, lactate, formate, ethanol, and acetate) are measured using HPLC and GC. The gene expression levels are determined using QRT-PCR. Results of the metabolic fluxes and the gene expression pattern will be used to develop a mathematical model for the E. coli oxygen regulatory system and its cellular responses