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CRISPR-Cas9 Editing of the Synthesis of Biodegradable Polyesters Polyhydroxyalkanaotes (PHA) in Pseudomonas putida KT2440

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Enzyme Engineering

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2397))

Abstract

Genome editing technologies allow us to study the metabolic pathways of cells and the contribution of each associated enzyme to various processes, including polyhydroxyalkanoate (PHA) synthesis. These biodegradable polyesters accumulated by a range of bacteria are thermoplastic, elastomeric, and biodegradable, thus have great applicative potential. However, several challenges are associated with PHA production, mainly the cost and shortcomings in their physical properties. The advances in synthetic biology and metabolic engineering provide us with a tool to improve the production process and allow the synthesis of tailor-made PHAs. CRISPR/Cas9 technology represents a new generation of genome editing tools capable of application in nearly all organisms. However, off-target activity is a crucial issue for CRISPR/Cas9 technology, as it can cause genomic instability and disruption of functions of otherwise normal genes. Here, we provide a detailed protocol for scarless deletion of the genes implicated in PHA metabolism of Pseudomonas putida KT2440 using modified CRISPR/Cas9 systems and methodology.

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Author information

Authors and Affiliations

  1. UCD Earth Institute and School of Biomolecular and Biomedical Science, University College Dublin, Belfield, Dublin, Ireland

    Si Liu, Tanja Narancic, Chris Davis & Kevin E. O’Connor

  2. BiOrbic Bioeconomy Research Centre, University College Dublin, Belfield, Dublin, Ireland

    Si Liu, Tanja Narancic, Chris Davis & Kevin E. O’Connor

  3. School of Biomolecular and Biomedical Sciences and BiOrbic SFI Bioeconomy Research Centre, O’Brien Centre for Science, University College Dublin, Belfield, Dublin, Ireland

    Kevin E. O’Connor

Authors
  1. Si Liu
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  2. Tanja Narancic
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  3. Chris Davis
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  4. Kevin E. O’Connor
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Corresponding author

Correspondence to Kevin E. O’Connor .

Editor information

Editors and Affiliations

  1. Department of Biology and Biotechnology, University of Pavia, Pavia, Italy

    Francesca Magnani

  2. Department of Biology and Biotechnology, University of Pavia, Pavia, Italy

    Chiara Marabelli

  3. Department of Chemistry & Biochemistry, University of Bern, Bern, Switzerland

    Francesca Paradisi

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Liu, S., Narancic, T., Davis, C., O’Connor, K.E. (2022). CRISPR-Cas9 Editing of the Synthesis of Biodegradable Polyesters Polyhydroxyalkanaotes (PHA) in Pseudomonas putida KT2440. In: Magnani, F., Marabelli, C., Paradisi, F. (eds) Enzyme Engineering. Methods in Molecular Biology, vol 2397. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1826-4_17

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  • DOI: https://doi.org/10.1007/978-1-0716-1826-4_17

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1825-7

  • Online ISBN: 978-1-0716-1826-4

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