Stabilizing plasmid copy number to improve recombinant protein production.
The key objective for recombinant protein production in bacteria is the maximum exploitation of the cell factory's potential, whereby often strong expression vectors are used to increase product yield. If the metabolic load caused by recombinant expression exceeds the host's capacity, the system exhausts itself, resulting in a loss of protein yield. Excessive plasmid replication is observed after inducing recombinant gene expression, which greatly contributes to metabolic overload of the host cell. The transcriptional and translational machineries are extremely overstrained. By abolishing sequence homology between ColE1 RNA I/RNA II and tRNAs, we were able to restore the plasmid's replication control mechanisms and to keep the plasmid copy number constant throughout the culture process, thereby prolonging metabolic activity and productivity of the bacterial expression system. Because the bacterial host cell is not being exploited beyond its tolerable potential with this method, the constancy of the plasmid copy number level throughout the whole period of the bioprocess provides novel strategies for bioprocess optimization.
Grabherr
Reingard
R
Institute of Applied Microbiology, University of Agricultural Science, Vienna, Muthgasse 18, A-1190 Vienna, Austria. r.grabherr@iam.boku.ac.at
Nilsson
Erik
E
Striedner
Gerald
G
Bayer
Karl
K
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D001483:Base Sequence; D019149:Bioreactors; D003470:Culture Media; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids; D012329:RNA, Bacterial / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D018741:Replication Origin
D001426:Bacterial Proteins; D003470:Culture Media; D017931:DNA Primers; D012329:RNA, Bacterial; C042833:ROM protein, Bacteria; D011994:Recombinant Proteins