Genomic analysis of high-cell-density recombinant Escherichia coli fermentation and "cell conditioning" for improved recombinant protein yield.
The Escherichia coli stress gene transcription profile and response to recombinant protein overexpression were substantially altered at high cell density when compared with low cell density. Reverse trascription-polymerase chain reaction RT-PCR-amplified mRNA from low (4 g[DCW]/L) and high-cell-density (43.5 g [DCW]/L) conditions were hybridized with a DNA microarray of Kohara clones encompassing 16% of the E. coli genome, and differentially displayed genes were identified. Transcript-specific RNA dot blots indicated that molecular chaperones (groEL, ibpA, degP), proteases (degP, ftsH), the lysis gene mltB, and DNA damage/bacteriophage-associated gene transcript levels (ftsH, recA, alpA, uvrB) increased 10- to 43-fold at high cell density. In addition, overexpression of recombinant green fluorescent protein (GFP(uv))/chloramphenicol acetyltransferase (CAT) fusion protein did not change the rates of cell growth or cell lysis. The stress gene transcription profile at high cell density was used to evaluate "cell conditioning" strategies to alter the levels of chaperones, proteases, and other intracellular proteins prior to recombinant protein overexpression. Interestingly, the addition of 1 g/L dithiothreitol (DTT) 20 min prior to induction of a GFP(uv)/CAT fusion protein resulted in a twofold increase in CAT activity when compared with the unconditioned controls. In addition, RNA dot blots of five stress genes confirmed that cell conditioning strategies significantly altered the dynamic stress gene response to foreign protein overexpression.
Gill
R T
RT
Department of Chemical Engineering, University of Maryland, College Park, Maryland 20742, USA.
DeLisa
M P
MP
Valdes
J J
JJ
Bentley
W E
WE
D001426:Bacterial Proteins / Q000235:genetics; D001709:Biotechnology / Q000379:methods; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D015342:DNA Probes; D004229:Dithiothreitol; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development*; D005285:Fermentation; D005798:Genes, Bacterial; D005818:Genetic Engineering / Q000379:methods; D049452:Green Fluorescent Proteins; D007700:Kinetics; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D015342:DNA Probes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D015500:Chloramphenicol O-Acetyltransferase; D004229:Dithiothreitol