Minimized backbone and novel microaerobic promoters boost plasmid DNA production

• Published in: Process biochemistry (1991) 2021-07, Vol.106, p.130-136
• Main Authors: Velazquez, Daniela, Jaén, Karim E., Sigala, Juan-Carlos, Lara, Alvaro R.
• Format: Article
• Language: English
• Subjects: Deoxyribonucleic acid; DNA; E coli; Hemoglobin; Microaerobic cultures; Microaerobic induction; Microaerobic promoters; Plasmid DNA; Plasmids; Promoters; Vitreoscilla hemoglobin
• ISSN: 1359-5113; 1873-3298
• DOI: 10.1016/j.procbio.2021.04.013

[Display omitted] •Microaerobically inducible plasmid was unstable in engineered E. coli.•Minimal plasmids allowed stable microaerobic pDNA production.•High yield microaerobic pDNA production in shake flasks was attained. A novel microaerobically inducible pUC18-derived plasmid was produced in Escherichia coli chromosomally expressing Vitreoscilla hemoglobin. The plasmid contains a copy of the positive replication control molecule rnaII under the control of the Pvgb promoter. The plasmid was unstable in the engineered strain. Sequencing showed that the plasmid was fragmented during preculture development, probably due to interactions between the host and vector genetic elements. Therefore, the rnaII copy under the control of the microaerobic promoters Pvgb, Pyfid, or Pyfidm was inserted in the minimal plasmid pUC57mini. Such plasmids were successfully produced in the engineered strain. The maximum plasmid DNA yield from biomass was 16.6 ± 0.1 mg g−1 in microaerobic cultures of the engineered strain using the Pyifd promoter. This was 14-fold greater than that using the original pUC57mini and the wild-type strain. Our results show the importance of considering host-vector interactions during the biodesign process and the potential of microaerobic induction for intensifying pDNA manufacturing.