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.
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.
Gill
R T
RT
Center for Agricultural Biotechnology, University of Maryland Biotechnology Institute and Department of Chemical Engineering, University of Maryland, College Park, Maryland 20742, USA.
Cha
H J
HJ
Jain
A
A
Rao
G
G
Bentley
W E
WE
D000332:Aerobiosis; D001709:Biotechnology / Q000295:instrumentation / Q000379:methods; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism*; D003470:Culture Media; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D005285:Fermentation; D015964:Gene Expression Regulation, Bacterial; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics*; D007700:Kinetics; D010084:Oxidation-Reduction; D010101:Oxygen Consumption
D003470:Culture Media; D006360:Heat-Shock Proteins; D015500:Chloramphenicol O-Acetyltransferase; D004229:Dithiothreitol