title: Comparative transcription profiling and in-depth characterization of plasmid-based and plasmid-free Escherichia coli expression systems under production conditions.
abstract: 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.
authors.affiliation: Department of Biotechnology, University of Natural Resources and Life Sciences, and Department of Biotechnology, ACIB GmbH, Vienna, Austria.
authors.forename: Juergen
authors.initials: J
authors.lastname: Mairhofer
authors.forename: Theresa
authors.initials: T
authors.lastname: Scharl
authors.forename: Karoline
authors.initials: K
authors.lastname: Marisch
authors.forename: Monika
authors.initials: M
authors.lastname: Cserjan-Puschmann
authors.forename: Gerald
authors.initials: G
authors.lastname: Striedner
mesh_terms: D019149:Bioreactors; D001709:Biotechnology / Q000379:methods*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D046228:Microarray Analysis; D010957:Plasmids / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis*
chemical_list: D011994:Recombinant Proteins; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase