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Design of a microaerobically inducible replicon for high-yield plasmid DNA production.

Abstract

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 Ptrc 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 (YpDNA/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 YpDNA/X efficiently increased after the shift to the microaerobic regime in fed-batch cultures in a 1 L bioreactor. The YpDNA/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.

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  • Authors+Show Affiliations

    ,

    Departamento de Procesos y Tecnología, Universidad Autónoma Metropolitana-Cuajimalpa, Mexico City, Mexico.

    ,

    Departamento de Procesos y Tecnología, Universidad Autónoma Metropolitana-Cuajimalpa, Mexico City, Mexico.

    ,

    Departamento de Procesos y Tecnología, Universidad Autónoma Metropolitana-Cuajimalpa, Mexico City, Mexico.

    Departamento de Procesos y Tecnología, Universidad Autónoma Metropolitana-Cuajimalpa, Mexico City, Mexico.

    Source

    Pub Type(s)

    Journal Article

    Language

    eng

    PubMed ID

    31232477

    Citation

    Jaén, Karim E., et al. "Design of a Microaerobically Inducible Replicon for High-yield Plasmid DNA Production." Biotechnology and Bioengineering, 2019.
    Jaén KE, Velázquez D, Sigala JC, et al. Design of a microaerobically inducible replicon for high-yield plasmid DNA production. Biotechnol Bioeng. 2019.
    Jaén, K. E., Velázquez, D., Sigala, J. C., & Lara, A. R. (2019). Design of a microaerobically inducible replicon for high-yield plasmid DNA production. Biotechnology and Bioengineering, doi:10.1002/bit.27091.
    Jaén KE, et al. Design of a Microaerobically Inducible Replicon for High-yield Plasmid DNA Production. Biotechnol Bioeng. 2019 Jun 24; PubMed PMID: 31232477.
    * Article titles in AMA citation format should be in sentence-case
    TY - JOUR T1 - Design of a microaerobically inducible replicon for high-yield plasmid DNA production. AU - Jaén,Karim E, AU - Velázquez,Daniela, AU - Sigala,Juan-Carlos, AU - Lara,Alvaro R, Y1 - 2019/06/24/ PY - 2019/03/14/received PY - 2019/06/07/revised PY - 2019/06/10/accepted PY - 2019/6/25/pubmed PY - 2019/6/25/medline PY - 2019/6/25/entrez KW - microaerobic induction KW - oxygen limitation KW - pUC replicon KW - plasmid DNA KW - rnaII JF - Biotechnology and bioengineering JO - Biotechnol. Bioeng. N2 - 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 Ptrc 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 (YpDNA/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 YpDNA/X efficiently increased after the shift to the microaerobic regime in fed-batch cultures in a 1 L bioreactor. The YpDNA/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. SN - 1097-0290 UR - https://www.unboundmedicine.com/medline/citation/31232477/Design_of_microaerobically_inducible_replicon_for_high-yield_plasmid_DNA_production L2 - https://doi.org/10.1002/bit.27091 DB - PRIME DP - Unbound Medicine ER -