Recombinant protein production in an Escherichia coli reduced genome strain

Abstract

Recently, efforts have been made to improve the properties of E. 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.

Industrial production of recombinant therapeutics in Escherichia coli and its recent advancements

“…Therefore, recent breakthroughs in high-yield production of aglycosylated antibodies in E. coli and in engineering them for increased binding aYnity with Fc receptors indicate that aglycosylated antibodies have become an important family of protein therapeutics produced in E. coli. [45,59,74,83,111] Strain engineering Improved protein yield and quality; simpliWed puriWcation [10,14,92] E. coli N-linked glycosylation N-linked glycosylated protein synthesis [62,77,91,120] Fusion proteins and PEGylation Enhanced pharmacokinetics; novel therapeutic design [41,101,104] E. coli cell-free systems Novel method for protein synthesis at the industrial scale [76,102,131] Strain engineering for improved product yield and quality It is estimated that more than 3% of the enzymatic activities in E. coli are proteolytic at any given time [68]. Recombinant proteins produced in E. coli, both as IBs and soluble proteins, are susceptible to diVerent levels of proteolysis, which is unfavorable for industrial production.…”

Spontaneous Deletion of a 209-Kilobase-Pair Fragment from the Escherichia coli Genome Occurs with Acquisition of Resistance to an Assortment of Infectious Phages

“…The deletion includes the chromosomal section from ompC to wbbL that encodes the rhamnosyl transferase involved in lipopolysaccharide biosynthesis. Strains D198 and JM109 were comparable in their growth characteristics and their abilities to express a recombinant protein.Among the many systems available for heterologous protein expression, the gram-negative bacterium Escherichia coli remains one of the most attractive because of its ability to grow rapidly and at high density on inexpensive substrates, its wellcharacterized genetics, and the availability of a large number of cloning vectors (13,19). However, phage infection of E. coli cultures can lead to serious problems, including a complete loss of the desired bioproduct and the spread of the destructive bacteriophages.…”

Heterologous Production of Curcuminoids

“…However, robust hosts are usually able to inactive or rid themselves of the foreign DNA to minimize the metabolic burden. Several studies proved that genome reductions (removal of transposons, insertion sequence elements, cryptic phages, integrase genes, damaged genes, and genes with unknown function) improve metabolic efficiency and even electroporation efficiency and accurate propagation of foreign DNA that was unstable in other strains (200)(201)(202)(203). These strains should be considered for curcuminoid production.…”