Rational engineering of Escherichia coli strains for plasmid biopharmaceutical manufacturing

Abstract

Plasmid DNA (pDNA) has become very attractive as a biopharmaceutical, especially for gene therapy and DNA vaccination. Currently, there are a few products licensed for veterinary applications and numerous plasmids in clinical trials for use in humans. Recent work in both academia and industry demonstrates a need for technological and economical improvement in pDNA manufacturing. Significant progress has been achieved in plasmid design and downstream processing, but there is still a demand for improved production strains. This review focuses on engineering of Escherichia coli strains for plasmid DNA production, understanding the differences between the traditional use of pDNA for recombinant protein production and its role as a biopharmaceutical. We will present recent developments in engineering of E. coli strains, highlight essential genes for improvement of pDNA yield and quality, and analyze the impact of various process strategies on gene expression in pDNA production strains.

Multiple Phenotypic Changes Associated with Large-Scale Horizontal Gene Transfer

“…Foreign proteins may not fold correctly in their new cellular contexts, which could lead to disruption or triggering of stress responses [4], [11]. Recently acquired regions may disrupt flux through cellular systems, leading to the buildup of toxic intermediates [12], [13]. While such costs have been directly observed in laboratory experiments, retrospective studies across genomes add an additional layer of complexity as there exists an inverse correlation between gene retention after HGT and number of protein-protein interactions affected [14].…”

A reduced genome decreases the host carrying capacity for foreign DNA

“…This result indicates the possibility that the removal of genes from MG1655 influenced the replication of foreign DNA in this strain. As the previously reported genes specifically related to the DNA and protein productivity [21] were not comprised in the deletions, there might be some novel functions interfering with replication or expression among those deleted genes. In another word, the regions that were removed most likely had a buffering effect, releasing the stress caused by the replication of exogenous DNA and helping to maintain cellular homeostasis.…”

“…Thus, MDS42 is believed to be an ideal host for plasmid production [20] because plasmids exhibit a greater degree of structural stability in MDS42 compared to MG1655 [12]. However, to date, the increase in the productivity of plasmids inserted into MDS42 has not been well studied [21]. …”

Plasmid DNA production with Escherichia coli GALG20, a pgi-gene knockout strain: Fermentation strategies and impact on downstream processing

“…As earlier reviewed, strain engineering can increase pDNA percentage per cell, reduce genomic DNA and RNA impurities, and decrease process stream or lysate viscosity (Bower and Prather, 2009). Moreover, a carefully designed strain may result in improved plasmid topology and segregational stability, and eventually contribute to increased transfection efficiency of the final plasmid product (Gonç alves et al, 2012). The experiments described in this section were designed to evaluate the impact of strain genotype on the purification and final quality of pDNA.…”

“…absence of deletions and insertion sequences) and the predominance of the supercoiled topology (Prazeres, 2011;Tejeda-Mansir and Montesinos, 2008). These objectives can be met by focusing on the development of: (i) new fermentation strategies (Carnes et al, 2006;Listner et al, 2006), (ii) improved media formulations (O'Kennedy et al, 2000), (iii) optimized plasmid backbones and (iv) rationally engineered E. coli strains (Gonç alves et al, 2012).…”