Recombinant protein production in an Escherichia coli reduced genome strain

Abstract

Recently, efforts have been made to improve the properties of Escherichia coli as a recombinant host by 'genomic surgery'-deleting large segments of the E. coli K12 MG1655 genome without scars. These excised segments included K-islands, which contain a high proportion of transposons, insertion sequences, cryptic phage, damaged, and unknown-function genes. The resulting multiple-deletion strain, designated E. coli MDS40, has a 14% (about 700 genes) smaller genome than the parent strain, E. coli MG1655. The multiple-deletion and parent E. coli strains were cultured in fed-batch fermenters to high cell densities on minimal medium to simulate industrial conditions for evaluating growth and recombinant protein production characteristics. Recombinant protein production and by-product levels were quantified at different controlled growth rates. These results indicate that the multiple-deletion strain's growth behavior and recombinant protein productivity closely matched the parent stain. Thus, the multiple-deletion strain E. coli MDS40 provides a suitable foundation for further genomic reduction.

Figure 1

Growth characteristics for E. coli MDS40 and MG1655 cultured in shake flasks. (A) Cell densities for MDS40 - induced (■) and uninduced (□); E. coli MG1655 - induced (▲) and uninduced (△). (B) Specific CAT activity for E. coli MDS40 - induced (■) and uninduced (□); E. coli MG1655 - induced (▲) and uninduced (△).

Figure 2

Growth characteristics for E. coli MDS40 and MG1655 at controlled growth rates. (A) Cell densities for E. coli MDS40 – induced with 1 mM IPTG (■), 5 mM IPTG (▼), and uninduced (□) at the low growth rate (0.15 hr⁻¹). (B) Cell densities for E. coli MDS40 - induced (●) and uninduced (○) at the intermediate growth rate (0.25 hr⁻¹). Cell densities for E. coli MG1655 - induced (▲) and uninduced (△) at the intermediate growth rate are also shown. (C) Cell densities for E. coli MDS40 – induced with 1 mM IPTG (◆), 5 mM IPTG (▼), and uninduced (◊) at the high growth rate (0.5 hr⁻¹). (D) Specific CAT activity for E. coli MDS40 induced with 1 mM IPTG (■) and 5 mM IPTG (▼) at the low growth rate. (E) Specific CAT activity for E. coli MDS40 (●) and E. coli MG1655 (▲) at the intermediate growth rate. (F) Specific CAT activity for E. coli MDS40 induced with 1 mM IPTG (◆) and 5 mM IPTG (▼) at the high growth rate.

Figure 3

Acetate concentrations in the fermentation broth. (A) E. coli MDS40 – induced with 1 mM IPTG (■), 5 mM IPTG (▼), and uninduced (□) at the low growth rate (0.15 h⁻¹). (B) E. coli MDS40 induced (●) and uninduced (○) at the intermediate growth rate (0.25 h⁻¹). E. coli MG1655 – induced (▲) and uninduced (△). (C) E. coli MDS40 – induced with 1 mM IPTG (◆), 5 mM IPTG (▼), and uninduced (◊) at the high growth rate (0.5 h⁻¹).

Figure 4

Formate concentrations in the fermentation broth. (A) E. coli MDS40 – induced (■) and uninduced (□) at the low growth rate (0.15 h⁻¹). (B) E. coli MDS40 induced (●) and uninduced (○) at the intermediate growth rate (0.25 h⁻¹). E. coli MG1655 – induced (▲) and uninduced (△). (C) E. coli MDS40 – induced (◆) and uninduced (◊) at the high growth rate (0.5 h⁻¹).

Figure 5

Growth characteristics for E. coli MDS40 stressed by acetate in shake flasks. (A) Cell densities for cultures with the acetate addition (●) and no acetate addition (■). (B) Specific CAT activity for cultures with the acetate addition (●) and no acetate addition (■). Error bars indicate 95% confidence intervals.