High yield plasmid DNA production under oxygen limitation using microaerobically induced replication
March 4-8, 2018
With the aim of increasing plasmid DNA (pDNA) production under oxygen limitation, a self-inducible replication system was created. An extra copy of the gene coding for rnaII, which is a positive control molecule for pMB1-derived replicons, was placed under control of the lac or trp promoters and cloned in plasmid pUC18. The modified plasmid pUC18-Ptrc rnaII resulted in a strong overexpression of rnaII which in turn triggered the plasmid copy number in more than the double of that of pUC18. Based on this, a microaerobically-inducible plasmid was created by inserting an extra copy of rnaII under control of the microaerobic promoter from the Vitreoscilla hemoglobin (Pvgb). Such plasmid was tested in fed-batch cultures of the strain W3110 recA- in which dissolved oxygen was depleted for nearly 6 h. Upon oxygen depletion, rnaII was efficiently induced and pDNA titer increased steadily for pUC18- Pvgb rnaII, reaching nearly 400 mg/L. In contrast, only 200 mg/L of the unmodified pUC18 were obtained.
In order to improve cellular performance under oxygen limitations, engineered strains expressing the Vitreoscilla hemoglobin encoded in the chromosome, were created. The vgb gene was inserted in BL21 and W3110 strains and the performance of both strains were compared in biphasic aerobic-oxygen limited cultures. Interesting differences were observed in the kinetic behavior, metabolic fluxes distribution and gene expression levels when the vgb gene was expressed in BL21 or W3110 recA- vgb+, therefore, this strain was used for production of the inducible plasmid. The amount of pUC18 produced by W3110 recA- vgb+ under oxygen limitation doubled that of W3110 recA-. However, when pUC18-Pvgb rnaII was used, the engineered strain produced only 20 mg/L. Moreover, the size of the obtained plasmid was strongly shortened. Plasmid sequencing revealed that an important fraction of the origin of replication was lost. These results demonstrate the feasibility of microaerobically-induced pDNA production, and that the performance of genetic constructions depend on the strain used. Furthermore, unexpected changes in plasmid fidelity can arise when using genetically modified strains.
Alvaro Lara and Karim E. Jaén, "High yield plasmid DNA production under oxygen limitation using microaerobically induced replication" in "Microbial Engineering", Prof. Eli Keshavarz-Moore , University College London, England Dr. Barry Buckland, BiologicB, USA Eds, ECI Symposium Series, (2018). https://dc.engconfintl.org/microbial/37