Generating controlled reducing environments in aerobic recombinant Escherichia coli fermentations: Effects on cell growth, oxygen uptake, heat shock protein expression, and in vivo CAT activity

Abstract

The independent control of culture redox potential (CRP) by the regulated addition of a reducing agent, dithiothreitol (DTT) was demonstrated in aerated recombinant Escherichia coli fermentations. Moderate levels of DTT addition resulted in minimal changes to specific oxygen uptake, growth rate, and dissolved oxygen. Excessive levels of DTT addition were toxic to the cells resulting in cessation of growth. Chloramphenicol acetyltransferase (CAT) activity (nmoles/microgram total protein min.) decreased in batch fermentation experiments with respect to increasing levels of DTT addition. To further investigate the mechanisms affecting CAT activity, experiments were performed to assay heat shock protein expression and specific CAT activity (nmoles/microgram CAT min.). Expression of such molecular chaperones as GroEL and DnaK were found to increase after addition of DTT. Additionally, sigma factor 32 (sigma32) and several proteases were seen to increase dramatically during addition of DTT. Specific CAT activity (nmoles/microgram CAT min. ) varied greatly as DTT was added, however, a minimum in activity was found at the highest level of DTT addition in E. coli strains RR1 [pBR329] and JM105 [pROEX-CAT]. In conjunction, cellular stress was found to reach a maximum at the same levels of DTT. Although DTT addition has the potential for directly affecting intracellular protein folding, the effects felt from the increased stress within the cell are likely the dominant effector. That the effects of DTT were measured within the cytoplasm of the cell suggests that the periplasmic redox potential was also altered. The changes in specific CAT activity, molecular chaperones, and other heat shock proteins, in the presence of minimal growth rate and oxygen uptake alterations, suggest that the ex vivo control of redox potential provides a new process for affecting the yield and conformation of heterologous proteins in aerated E. coli fermentations.

Recombinant protein production in an Escherichia coli reduced genome strain

“…The observed maximum specific CAT activity was comparable for E. coli MDS40 and MG1655. The E. coli MG1655 and MDS40 CAT expression profiles were similar to other reports, even with strain, plasmid and media differences (Cha et al, 2000;Gill et al, 1998;Haddadin and Harcum, 2005;Harcum and Bentley, 1999;Richins et al, 1997). Thus, E. coli MDS40 was capable of good growth and CAT expression under shake flask conditions.…”

Extracellular Oxidoreduction Potential Modifies Carbon and Electron Flow in Escherichia coli

“…To our knowledge, however, a more global study of ORP action on metabolism has not been realized, although Gill et al have shown that extracellular ORP could act on the oxidoreduction state of cytoplasmic molecules, due to the low ORP buffer strength of cytoplasm (9). Furthermore, from a biotechnological point of view, if this parameter can modify metabolic fluxes, it might be an additional physicochemical parameter to take into account for the optimization of processes.…”

“…The similarity of variation between LDH-specific activity and lactate production shows that the apparent activity of LDH is independent of extracellular ORP conditions. Concerning the LDH activity, it could be assumed that its activity was not ORP sensitive, based on the knowledge that E. coli cytoplasm is not well ORP buffered, as has been demonstrated already (9). Measurement of in vitro activity of LDH confirmed the assumption (Fig.…”

Genomic analysis of high-cell-density recombinantEscherichia coli fermentation and ?cell conditioning? for improved recombinant protein yield

“…Specific CAT activity (nanomoles per milligram total protein per minute) (DeLisa et al, 1999;Gill et al, 1998) increased roughly 2.5-fold after induction with IPTG (Table I). Three percent and 1% ethanol followed by IPTG addition resulted in 2.2-and 2.6-fold increases in specific CAT activity, respectively.…”