Effect of Nutrient Starvation on 3-Hydroxypropionic Acid Production By Recombinant Escherichia coli at Stationary Phase | AIChE

Effect of Nutrient Starvation on 3-Hydroxypropionic Acid Production By Recombinant Escherichia coli at Stationary Phase


1. Background

Escherichia coli is able to catabolize the extracellular glucose even under a starvation of essential nutrients. It has been reported that the limiting nutrients such as nitrogen, sulfur, magnesium, or phosphorus distinctively affect the carbon central metabolism and the biochemical productivity of E. coli at the stationary growth phase. In this study, the effects of nutritional starvations on the fermentation and metabolic profiles were characterized for a metabolically engineered E. coli strain producing 3-hydroxypropionic acid (3HP).

2. Materials & methods

E. coli strain MG1655(DE3) was transformed with pETM6 derived vectors. The E. coli cells grown in M9 minimal medium containing glucose as a sole carbon source were inoculated in nitrogen, sulfur, magnesium, or phosphorus free M9 minimal medium, and cultivated under aerobic condition at 37oC. Concentrations of glucose, 3HP and byproducts in the culture broth were determined by an HPLC. Intracellular metabolites were extracted from the cells with chloroform-water-methanol and quantified using LC-MS/MS and GC-MS.

3. Results

3HP-producing strain was constructed by overexpression of maloyl-CoA reductase encoded by mcr gene derived from Chloroflexus aurantiacus in E. coli MG1655(DE3). In order to evaluate the effect of single nutrient starvations on 3HP production during the stationary phase, the 3HP-producing strain was cultured in M9 medium lacking nitrogen, sulfur, magnesium, or phosphorus, respectively. The comparison of the fermentation profiles showed that larger 3HP yields were observed under the sulfur (6.0% C-mol 3HP/ C-mol glucose) and magnesium starvation (5.8%) conditions than the starvations of nitrogen (0.2%) and phosphorus (undetectable). The byproduction of acetate was occurred in the all conditions. The specific glucose consumption rate in magnesium starvation (2.31 mmol/gCDW/h) was faster than that in nitrogen, phosphorus or sulfur starvation (0.33, 1.11 or 0.78 mmol/gCDW/h, respectively).

The metabolic profiling analysis of the intermediates of the central carbon metabolism revealed that the concentration of malonyl-CoA, a precursor for 3HP synthesis, was larger in magnesium or sulfur starvation than that in nitrogen or phosphorus starvation. The results indicated that the accumulation of malonyl-CoA enhanced the 3HP production in sulfur and magnesium starvation. The high ATP concentration in magnesium or sulfur starvation would contribute the malonyl-CoA accumulation since acetyl-CoA carboxylase requires ATP for a conversion of acetyl-CoA to malonyl-CoA. The active glucose uptake in magnesium starvation could be explained by an accumulation of phosphoenolpyruvate, the substrate of phosphotransferase system (PTS) for glucose transport. On the other hand, the significant accumulation of α-ketoglutarate in nitrogen starvation (4.1 mM) suggested that the slow glucose uptake was derived from the allostric inhibition of PTS by α-ketoglutarate (Ki = 1.3 mM). These results suggest that the choice of lacking nutrients affected the fermentation and metabolic profiles in the 3HP-producing strain during the stationary phase.