Monitoring GFP-operon fusion protein expression during high cell density cultivation of Escherichia coli using an on-line optical sensor

Synthesis of an operon fusion protein was investigated in batch and fed-bat ch cultures at high cell densities of recombinant Escherichia coli JM105 [p BAD-GFP::CAT]. Glucose-limited growth was achieved without accumulation of inhibitory byproducts allowing high cell densities (110 g L-1 DCW) to be at tained. This was believed to be the highest reported value for dry cell mas s of E. coli strain JM105 expressing two recombinant proteins. Transcriptio n of the two reporter genes, green fluorescent protein (GFP) and chloramphe nicol acetyltransferase (CAT), was under the control of the p(BAD) promoter of the araBAD (arabinose) operon. Each protein was independently translate d via separate ribosome binding sites. CAT served as a model recombinant pr otein product to illustrate the noninvasive quantitative reporting ability of GFP during high cell density fermentations. Expression of GFP was monito red on-line using an intensity-based optical sensor. A linear correlation b etween the on-line GFP intensity and the enzymatic activity of CAT allowed for in vivo real-time quantitative monitoring of a fermentation product und er conditions of high biomass concentration and high productivity. (C) 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 65: 54-64, 1999.