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Culture of Escherichia coli under dissolved oxygen gradients simulated in a two‐compartment scale‐down system: Metabolic response and production of recombinant protein
Author(s) -
SandovalBasurto Edgar A.,
Gosset Guillermo,
Bolívar Francisco,
Ramírez Octavio T.
Publication year - 2004
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.20383
Subject(s) - escherichia coli , compartment (ship) , recombinant dna , oxygen , biochemistry , chemistry , biology , microbiology and biotechnology , organic chemistry , gene , oceanography , geology
A significant problem of large‐scale cultures, but scarcely studied for recombinant E. coli , is the presence of gradients in dissolved oxygen tension (DOT). In this study, the effect of DOT gradients on the metabolic response of E. coli and production of recombinant pre‐proinsulin, accumulated as inclusion bodies, was determined. DOT gradients were simulated in a two‐compartment scale‐down system consisting of two interconnected stirred‐tank bioreactors, one maintained at anoxic conditions and the other at a DOT of at least 6%. Cells were continuously circulated between both vessels to simulate circulation times (t c ) of 20, 50, 90, and 180 sec. A complete kinetic and stoichiometric characterization was performed in the scale‐down system as well as in control cultures maintained at constant DOT in the range of 0–20%. The performance of E. coli cultured under oscillating DOT was significantly affected, even at a t c of 20 sec corresponding to transient exposures of only 13.3 sec to anaerobic conditions. Specific growth rate decreased linearly with t c to a maximum reduction of 30% at the highest t c tested. The negative effect of DOT gradients was even more pronounced for the overall biomass yield on glucose and the maximum concentration and yield of pre‐proinsulin. In these cases, the losses were 9%, 27%, and 20%, respectively, at t c of 20 sec and 65%, 94%, and 87%, respectively, at t c of 180 sec. Acetic, lactic, formic, and succinic acids accumulated during oscillatory DOT cultures, indicating that deviation of carbon flow to anaerobic metabolism was responsible for the observed losses. The results of this study indicate that even very short exposures to anaerobic conditions, typical of large‐scale operations, can substantially reduce recombinant protein productivity. The information presented here is useful for establishing improved rational scale‐up strategies and understanding the behavior of recombinant E. coli exposed to DOT gradients. © 2004 Wiley Periodicals, Inc.