Combined transcriptome and proteome analysis of Escherichia coli during high cell density culture

Abstract

Combined transcriptome and proteome analysis was carried out to understand metabolic and physiological changes of Escherichia coli during the high cell density cultivation (HCDC). The expression of genes of TCA cycle enzymes, NADH dehydrogenase and ATPase, was up-regulated during the exponential fed-batch period and was down-regulated afterward. However, expression of most of the genes involved in glycolysis and pentose phosphate pathway was up-regulated at the stationary phase. The expression of most of amino acid biosynthesis genes was down-regulated as cell density increased, which seems to be the major reason for the reduced specific productivity of recombinant proteins during HCDC. The expression of chaperone genes increased with cell density, suggesting that the high cell density condition itself can be stressful to the cells. Severe competition for oxygen at high cell density seemed to make cells use cytochrome bd, which is less efficient but has a high oxygen affinity than cytochrome bo 3 . Population cell density itself strongly affected the expression of porin protein genes, especially ompF, and hence the permeability of the outer membrane. Expression of phosphate starvation genes was most strongly upregulated toward the end of cultivation. It was also found that E (rpoE) plays a more important role than S (rpoS) at the stationary phase of HCDC. These findings should be invaluable in designing metabolic engineering and fermentation strategies for the production of recombinant proteins and metabolites by HCDC of E. coli.

Recombinant protein production in an Escherichia coli reduced genome strain

“…A short-term goal of this project and the focus of this study was to assess the capability of one multiple deletion strain (E. coli MDS40) under industrial conditions. Since conditions in a fermenter, especially high cell densities, are significantly different from those in shake flasks, both of these conditions were examined (Andersson et al, 1996;Gill et al, 2001;Humphrey, 1998;Lee, 1996;Yoon et al, 2003). Under the conditions assessed E. coli MDS40 and its parent strain E. coli MG1655 had similar performance characteristics.…”

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“…Most of these studies have been carried out in flask cultures, whereby non defined conditions were applied [3]. In accordance with good experimental design it is important to ensure that the changes in expression profiles are due to the perturbing event.…”

A New Player in the Biorefineries Field: Phasin PhaP Enhances Tolerance to Solvents and Boosts Ethanol and 1,3-Propanediol Synthesis in Escherichia coli

“…These findings open the possibility of developing new strategies to optimize strains for the synthesis of a diversity of added-value chemicals, such as biofuels and chemical precursors. It has been reported that in high-density cultures, such as those used in industrial fermentations, there is a significant increase in the levels of several chaperones, such as DnaK and GroEL (57). While the results presented in this study constitute a proof of concept, demonstrating that expression of GroEL and PhaP can enhance solvent tolerance and bioproduct yield in small-scale cultivations, further research will be necessary to analyze the beneficial effects of these proteins in bioreactor cultures, in which cells are exposed to multiple stresses.…”