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 poten tial (CRP) by the regul ated addition of a reducing agent, dithiothreitol (DTT) was demonstrat ed in aerated recombinant Escherichia coli fermentations. Moderate lev els of DTT addition resulted in minimal changes to specific oxygen upt ake, growth rate, and dissolved oxygen. Excessive levels of DTT additi on were toxic to the cells resulting in cessation of growth. Chloramph enicol acetyltransferase (CATI activity (nmoles/mu g total protein min ,) decreased in batch fermentation experiments with respect to increas ing levels of DTT addition, To further investigate the mechanisms affe cting CAT activity, experiments were performed to assay heat shock pro tein expression and specific CAT activity (nmoles/mu g CAT min.). Expr ession of such molecular chaperones as GroEL and DnaK were found to in crease after addition of DTT. Additionally, sigma factor 32 (sigma(32) ) and several proteases were seen to increase dramatically during addi tion of DTT. Specific CAT activity (nmoles/mu g CAT min.) varied great ly as DTT was added, however, a minimum in activity was found at the h ighest level of DTT addition in E. coli strains RR? [pBR329] and JM105 [pROEX-CAT]. In conjunction, cellular stress was found to reach a max imum at the same levels of DTT. Although DTT addition has the potentia l for directly affecting intracellular protein folding, the effects fe lt from the increased stress within the cell are likely the dominant e ffector. That the effects of DTT were measured within the cytoplasm of the cell suggests that the periplasmic redox potential was also alter ed. The changes in specific CAT activity, molecular chaperones, and ot her heat shock proteins, in the presence of minimal growth rate and ox ygen uptake alterations, suggest that the ex vivo control of redox pot ential provides a new process for affecting the yield and conformation of heterologous proteins in aerated E. coli fermentations. (C) 1998 J ohn Wiley & Sons, inc.