Stabilizing plasmid copy number to improve recombinant protein production

Grabherr, Reingard; Nilsson, Erik; Striedner, Gerald; Bayer, Karl

doi:10.1002/bit.10104

Cited by 58 publications

(45 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…2A), potentiating the adverse metabolic side effects. This phenomenon can possibly be attributed to the occurrence of the uncharged tRNAs (23,44) that do interact with the plasmid replication control mechanism and additionally trigger the stringent response (45,46). Consequently, the high levels of ppGpp detected for the plasmid-based system may also contribute to the observed growth cessation (47).…”

Section: Discussionmentioning

confidence: 99%

“…High-level protein production leads to malfunctions in the plasmid replication control mechanism. The resultant increase in the PCN induces a self-amplifying cascade of metabolic load which finally produces a fatal metabolic overburden (23). Both phenomena significantly contribute to instabilities and represent distinct limitations of plasmid-based systems in production processes.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Comparative Transcription Profiling and In-Depth Characterization of Plasmid-Based and Plasmid-Free Escherichia coli Expression Systems under Production Conditions

Mairhofer

Scharl

Marisch

et al. 2013

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

Plasmid-based Escherichia coli BL21(DE3) expression systems are extensively used for the production of recombinant proteins. However, the combination of a high gene dosage with strong promoters exerts extremely stressful conditions on producing cells, resulting in a multitude of protective reactions and malfunctions in the host cell with a strong impact on yield and quality of the product. Here, we provide in-depth characterization of plasmid-based perturbations in recombinant protein production. A plasmid-free T7 system with a single copy of the gene of interest (GOI) integrated into the genome was used as a reference. Transcriptomics in combination with a variety of process analytics were used to characterize and compare a plasmid-free T7-based expression system to a conventional pET-plasmid-based expression system, with both expressing human superoxide dismutase in fed-batch cultivations. The plasmid-free system showed a moderate stress response on the transcriptional level, with only minor effects on cell growth. In contrast to this finding, comprehensive changes on the transcriptome level were observed in the plasmid-based expression system and cell growth was heavily impaired by recombinant gene expression. Additionally, we found that the T7 terminator is not a sufficient termination signal. Overall, this work reveals that the major metabolic burden in plasmid-based systems is caused at the level of transcription as a result of overtranscription of the multicopy product gene and transcriptional read-through of T7 RNA polymerase. We therefore conclude that the presence of high levels of extrinsic mRNAs, competing for the limited number of ribosomes, leads to the significantly reduced translation of intrinsic mRNAs. P lasmid-based expression systems have been used for the production of recombinant proteins for more than 4 decades (1, 2). They can be manipulated quickly and easily, and a variety of replicons for use in Escherichia coli have become available (3), allowing, e.g., different expression levels by using plasmids with different copy numbers (4, 5). Plasmids equipped with additional functions can be used to facilitate, for instance, coexpression of proteins assisting correct folding (6) or of tRNAs supporting transcription of rare codons (7). The dissemination of E. coli systems was further strongly supported by the availability of well-established, easy-to-use protocols from molecular manipulation to cell cultivation up to a large scale (8) and by the FDA-proven status of E. coli as a host for production of proteins for clinical use (9).However, E. coli-based production processes are still far from optimal for exploitation of the cellular system, as the expression of heterologous proteins is performed with excessive strength, leading to a rapid exhaustion of the host cell (10) and, hence, loss of yield. One prominent example is the T7 system, combining highcopy-number plasmids with an orthogonal transcription system in the form of the T7 phage RNA polymerase, an enzyme showing an average elongation ...

show abstract

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Comparative Transcription Profiling and In-Depth Characterization of Plasmid-Based and Plasmid-Free Escherichia coli Expression Systems under Production Conditions

Mairhofer

Scharl

Marisch

et al. 2013

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

show abstract

“…At the same time, excessive consumption of amino acids increases the level of uncharged tRNAs, which in turn interferes with plasmid replication control mechanisms resulting in increasing plasmid copy numbers (Teich et al, 1998;Wrobel and Wegrzyn, 1998;Yavachev and Ivanov, 1988). The plasmid replication control mechanism can be restored by eliminating sequence homologies between ColE1 RNA I/RNA II and tRNAs; however, thusly modified pET systems with a stabilized plasmid copy number remain too strong (Grabherr et al, 2002). Processing strategies developed to improve control of the expression rate, such as the transcription tuning concept (Striedner et al, 2003) or the application of lactose as an inducer with lower affinity for the operator binding site (Gombert and Kilikian, 1998;Lin and Hsu, 1997;Studier, 2005), have been partially successful, but the challenge of designing a comprehensive solution for controlling the production scale remains unmet.…”

Section: Introductionmentioning

confidence: 95%

Plasmid‐free T7‐based Escherichia coli expression systems

Striedner

Pfaffenzeller

Luchner

et al. 2010

Biotech & Bioengineering

Self Cite

View full text Add to dashboard Cite

In order to release host cells from plasmid-mediated increases in metabolic load and high gene dosages, we developed a plasmid-free, T7-based E. coli expression system in which the target gene is site-specifically integrated into the genome of the host. With this system, plasmid-loss, a source of instability for conventional expression systems, was eliminated. At the same time, system leakiness, a challenging problem with recombinant systems, was minimized. The efficiency of the T7 RNA polymerase compensates for low gene dosage and provides high rates of recombinant gene expression without fatal consequences to host metabolism. Relative to conventional pET systems, this system permits improved process stability and increases the host cell's capacity for recombinant gene expression, resulting in higher product yields. The stability of the plasmid-free system was proven in chemostat cultivation for 40 generations in a non-induced and for 10 generations in a fully induced state. For this reason plasmid-free systems benefit the development of continuous production processes with E. coli. However, time and effort of the more complex cloning procedure have to be considered in relation to the advantages of plasmid-free systems in upstream-processing.

show abstract

“…However, only a few reports were published to-date about the effects of plasmid DNA on the metabolism of Escherichia coli [1-3]. On the other hand, an optimization of plasmid vectors and host strains has received an important interest in biotechnology, especially due to findng novel applications of plasmids, like gene therapy and development of DNA vaccines [1,4,5].…”

Section: Introductionmentioning

confidence: 99%