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Recent Patents on DNA & Gene Sequences
ISSN (Print): 1872-2156
ISSN (Online): 2212-3431
DOI: 10.2174/187221510790410822

Modulation of ColE1-Like Plasmid Replication for Recombinant Gene Expression

Author(s): Manel Camps
Pages 58-73 (16)
ColE1-like plasmids constitute the most popular vectors for recombinant protein expression. ColE1 plasmid replication is tightly controlled by an antisense RNA mechanism that is highly dynamic, tuning plasmid metabolic burden to the physiological state of the host. Plasmid homeostasis is upset upon induction of recombinant protein expression because of non-physiological levels of expression and because of the frequently biased amino acid composition of recombinant proteins. Disregulation of plasmid replication is the main cause of collapse of plasmid-based expression systems because of a simultaneous increase in the metabolic burden (due to increased average copy number) and in the probability of generation of plasmid-free cells (due to increased copy number variation). Interference between regulatory elements of co-resident plasmids causes comparable effects on plasmid stability (plasmid incompatibility). Modulating plasmid copy number for recombinant gene expression aims at achieving a high gene dosage while preserving the stability of the expression system. Here I present strategies targeting plasmid replication for optimizing recombinant gene expression. Specifically, I review approaches aimed at modulating the antisense regulatory system (as well as their implications for plasmid incompatibility) and innovative strategies involving modulation of host factors, of R-loop formation, and of the timing of recombinant gene expression.
Mutagenesis, fermentation, R-loops, RNA degradation, supercoiling, D-loops, plasmid instability, ColE1, dimer catastrophe, plasmid incompatibility
Department of Microbiology and Environmental Toxicology, University of California at Santa Cruz, 1156 High Street, Santa Cruz, California 95064, USA.