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Design of a microaerobically inducible replicon for high‐yield plasmid DNA production
Author(s) -
Jaén Karim E.,
Velázquez Daniela,
Sigala JuanCarlos,
Lara Alvaro R.
Publication year - 2019
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.27091
Subject(s) - replicon , plasmid , biology , plasmid preparation , microbiology and biotechnology , escherichia coli , gene , recombinant dna , genetics , pbr322
A pUC‐derived replicon inducible by oxygen limitation was designed and tested in fed‐batch cultures of Escherichia coli . It included the addition of a second inducible copy of rnaII , the positive replication control element. The rnaII gene was expressed from P trc and cloned into pUC18 to test the hypothesis that the ratio of the positive control molecule RNAII to the negative control element, RNAI, was the determinant of plasmid copy number per chromosome (PCN). The construct was evaluated in several E. coli strains. Evaluations of the RNAII/RNAI ratio, PCN and plasmid yield normalized to biomass (Y pDNA/X ) were performed and the initial hypothesis was probed. Furthermore, in high cell‐density cultures in shake flasks, an outstanding amount of 126 mg/L of plasmid was produced. The microaerobically inducible plasmid was obtained by cloning the rnaII gene under the control of the oxygen‐responsive Vitreoscilla stercoraria hemoglobin promoter. For this plasmid, but not for pUC18, the RNAII/RNAI ratio, PCN and Y pDNA/X efficiently increased after the shift to the microaerobic regime in fed‐batch cultures in a 1 L bioreactor. The Y pDNA/X of the inducible plasmid reached 12 mg/g at the end of the fed‐batch but the original pUC18 only reached ca. 6 mg/g. The proposed plasmid is a valuable alternative for the operation and scale‐up of plasmid DNA production processes in which mass transfer limitations will not represent an issue.