Escherichia coli Host Cell Modifications in Continuous Culture Affecting Heterologous Protein Overproduction: A Population Dynamics Study

Abstract

There are many published studies of plasmid segregational instability in Escherichia coli in the literature. However, the formation of plasmid-free segregants can be controlled by the addition of selective chemical agents like antibiotics. This solution has become commonplace in both the laboratory and industry. On the other hand, host cell modifications, which result in low production of plasmid-encoded protein and lead to loss of culture productivity, have not been adequately addressed. Continuous culture of an inducible (ptac) Escherichia coli vector containing strain, RB791(pKN), was characterized by strong dynamic changes in the cell population and product (beta-lactamase) expression. Long-term cultivation resulted in the loss of high-level production of beta-lactamase. Loss of productivity was not due to the formation of plasmid-free cells or structural modifications to the plasmid; instead, continuous operation resulted in a culture dominated by irreversibly altered, low-producing cells. Two distinct classes of lac- mutants which inhibited induction were identified (Y- and I(s)).

Effects of Plasmid Copy Number and Runaway Plasmid Replication on Overproduction and Excretion of β‐Lactamase from Escherichia coli

“…Induction M IPTG does not cause immediate runaway plasmid replication. The mutants therefore have a selective growth advantage over unaltered cells and quickly displace the unaltered cell population after induction during continuous culture (48). As seen in Figure 5, because of the low viability of the unaltered cell population after induction, the lac permease mutantstake over batch cultures if IPTG is added early.…”

Influence of The Expression Vector And of Its Elements On Recombinant hPRL Bacterial Synthesis: The Co-Directional Orientation of Replication And Transcription is Highly Critical

“…The transformed E. coli HB2151 strain (Luck et al 1986), RRI (Sambrook and Russel 2001) and RB791 (Fu et al 1992), containing the p1813-hPRL plasmid, were grown in LB medium (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl) supplemented with kanamycin (50 g/mL) at 37°C, while shaking at 180 rpm. When the optical density (OD) reached 0.4-0.8 A600, the cells were induced with 0.1 mM isopropyl-β-D-thiogalactoside (IPTG, Sigma, São Paulo, Brazil) and cultured for 9 h at 37°C.…”