Continuous, high level production and excretion of a plasmid‐encoded protein by Escherichia coli in a two‐stage chemostat

Abstract

The stable continuous overproduction of a plasmidencoded protein, beta-lactamase, for at least 50 days by Escherichia coli K-12, RB791(pKN), with release into the culture medium has been demonstrated in two-stage chemostats. The second-stage culture was continuously induced with 0.1 mM IPTG. Continuous expression of beta-lactamase could not be sustained with this strain in a single-stage chemostat because of cell death and selection for lac(-1) cells. Beta-lactamase production in the second stage was sensitive to the second-stage dilution rate and the distribution of the limiting substrate (i.e., glucose) between the first and second stages. The fraction of viable, excreting cells and the average copy number in the induced culture was measurably higher under those conditions of dilution rate and substrate distribution which yielded high beta-lactamase levels. The best operating conditions found at 20 degrees C were a first-stage dilution rate of 0.12 h(-1), a second-stage dilution rate of 0.03 h(-1), and equal glucose feed supplied to each stage. Enzymatically active beta-lactamase was produced at a level of 25% of total cellular protein with 90% excretion yielding 300 mg beta-lactamase/L that was 50% pure at an OD(600) < 6.

Engineering HlyA hypersecretion inEscherichia coli based on proteomic and microarray analyses

“…Reports of recombinant proteins in E. coli often describe high levels of export to the periplasm (Champion et al, 2003;Rinas et al, 1993), but there are only a few reports of successful attempts to secrete proteins past the outer membrane (Blight and Holland, 1994;Fu et al, 1993;Togna et al, 1993). Efforts to alleviate bottlenecks in secretion pathways via metabolic engineering do not often produce the desired phenotype, primarily due to the low efficiency and high specificity of most secretion systems and an incomplete understanding of their mechanisms.…”

Glycosylation of a recombinant protein in the Tn5B1-4 insect cell line: Influence of ammonia, time of harvest, temperature, and dissolved oxygen

“…Increasing dissolved oxygen concentration increased the sialic acid content of human follicle-stimulating hormone in CHO cells (Chotigeat et al, 1994). Lower temperatures have been used in E. coli to increase the secretion of recombinant ␤-lactamase into the periplasmic space (Fu et al, 1993). These variables have been shown to affect other systems, but how these factors influence glycosylation in the insect cell/baculovirus system has not been systematically studied.…”

A study of the effect of specific growth rate and acetate on recombinant protein production of Escherichia coli JM107

“…Thus all the factors need to be examined together. A number of researchers have worked on the effect of specific growth rate (dilution rate) in continuous culture on the production of recombinant protein (Fu et al 1993;Nancib et al 1992;Brown et al 1985;Park et al 1990) and their results show that product yields were generally higher at low dilution rates. In contrast, little has been reported on the control of specific growth rate in fed-batch culture.…”