The Effect of Limiting Phosphate Using the HMS174 Cell Line

Surprising results from a case study using the HMS174 cell line.
Jul 01, 2010
Volume 23, Issue 7


The Escherichia coli recombinant cell line HMS174, when grown in a 2XYS media and fed with phosphate and glucose, reached a fairly high cell density. If phosphate was not added to the culture along with the glucose, the culture seemed to switch from glycolysis to the glyoxylate bypass metabolism of acetate, mimicking a glucose-limiting event. In this culture without phosphate, the level of acetate remained considerably lower, whereas glucose levels rose, suggesting a metabolic shift to using both acetate and glucose as a carbon source. The overall growth of this culture was approximately half that of the culture containing phosphate, but following induction with IPTG, the amount of recombinant protein produced per gram of wet cell weight was very similar for the two cultures. This suggests that even in a phosphate-limited state, HMS174 cells maintained their ability to produce expected amounts of recombinant protein by switching metabolic pathways and metabolizing acetate through the glyoxylate bypass pathway. Ultimately, the HMS174 recombinant host strain may be able to use both glucose and acetate in a mixed feed strategy to maximize recombinant product production.

High cell density fermentation of Escherichia coli hosted recombinant systems is increasingly being used for the production of protein products for vaccines, diagnostics, and therapeutic treatments. Understanding the metabolic needs of high cell density fermentation can help increase the productivity and efficiency of the fermentation as a whole.

Under aerobic respiration conditions, glucose is used as the main carbon source and is fed in a non-limiting fashion to reach high cell densities. Complications can arise when the culture maintains a high growth rate during the exponential phase of growth with the secretion of acetate into the surrounding media. At high enough concentrations, the acetate can inhibit cell growth or recombinant protein production.1 Acetate also can decouple transmembrane pH gradients, affecting amino acid synthesis, osmotic pressure, and intracellular pH.2 It has been shown that by adding yeast extract to the fermentation, either initially or during the feed, acetate formation and its negative effects on the culture can be lessened.3 Other nutrient additions to the growing culture also may have positive effects on growth and product formation.

In this study, HMS174 competent cells containing a pET29a plasmid with a gene of interest were grown to a medium-high cell density in 2XYS media using a fed-batch fermentation method. Two cultures were fed 40% glucose in 2XYS, with and without the addition of phosphate at 20 g/L. Adding phosphate notably increased the culture's ability to sustain a high growth rate without the formation of inhibiting amounts of acetate. In contrast, the growth rate of the culture that was fed glucose without phosphate was half the rate, and after 3 h of growth, the phosphate concentration was undetectable.

Nevertheless, the cell culture maintained the recombinant protein expression fidelity of the culture that was fed glucose with phosphate. Both cultures produced the same amount of recombinant protein per gram of cell paste. In the absence of phosphate, the HMS174 strain seems to undergo the acetate switch and process acetate through the glyoxylate bypass pathway.4 Using an alternative carbon source in the absence of additional phosphate and the accumulation of glucose in the surrounding media may indicate an inhibition of the phosphotransferase system (PTS) for glucose5 or additional pathways that are dependent on a phosphorylated state. It also is noteworthy that the culture fed glucose with phosphate began metabolizing acetate upon induction of the culture with IPTG. These findings indicate that the HMS174 strain, in the absence or presence of excess phosphate, can metabolize acetate very efficiently and thus minimize the effects of acetate formation that would significantly affect other recombinant cell strains.

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