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Gene editing enables rapid engineering of complex antibiotic assembly lines

Author

Listed:
  • Wei Li Thong

    (The University of Manchester)

  • Yingxin Zhang

    (The University of Manchester)

  • Ying Zhuo

    (The University of Manchester)

  • Katherine J. Robins

    (The University of Manchester)

  • Joanna K. Fyans

    (The University of Manchester)

  • Abigail J. Herbert

    (The University of Manchester)

  • Brian J. C. Law

    (The University of Manchester)

  • Jason Micklefield

    (The University of Manchester)

Abstract

Re-engineering biosynthetic assembly lines, including nonribosomal peptide synthetases (NRPS) and related megasynthase enzymes, is a powerful route to new antibiotics and other bioactive natural products that are too complex for chemical synthesis. However, engineering megasynthases is very challenging using current methods. Here, we describe how CRISPR-Cas9 gene editing can be exploited to rapidly engineer one of the most complex megasynthase assembly lines in nature, the 2.0 MDa NRPS enzymes that deliver the lipopeptide antibiotic enduracidin. Gene editing was used to exchange subdomains within the NRPS, altering substrate selectivity, leading to ten new lipopeptide variants in good yields. In contrast, attempts to engineer the same NRPS using a conventional homologous recombination-mediated gene knockout and complementation approach resulted in only traces of new enduracidin variants. In addition to exchanging subdomains within the enduracidin NRPS, subdomains from a range of NRPS enzymes of diverse bacterial origins were also successfully utilized.

Suggested Citation

  • Wei Li Thong & Yingxin Zhang & Ying Zhuo & Katherine J. Robins & Joanna K. Fyans & Abigail J. Herbert & Brian J. C. Law & Jason Micklefield, 2021. "Gene editing enables rapid engineering of complex antibiotic assembly lines," Nature Communications, Nature, vol. 12(1), pages 1-10, December.
    • Handle: RePEc:nat:natcom:v:12:y:2021:i:1:d:10.1038_s41467-021-27139-1
    DOI: 10.1038/s41467-021-27139-1
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    References listed on IDEAS

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    1. Eric D. Brown & Gerard D. Wright, 2016. "Antibacterial drug discovery in the resistance era," Nature, Nature, vol. 529(7586), pages 336-343, January.
    2. Ewen Callaway & David Cyranoski, 2015. "Anti-parasite drugs sweep Nobel prize in medicine 2015," Nature, Nature, vol. 526(7572), pages 174-175, October.
    3. Kei Kudo & Takuya Hashimoto & Junko Hashimoto & Ikuko Kozone & Noritaka Kagaya & Reiko Ueoka & Takehiro Nishimura & Mamoru Komatsu & Hikaru Suenaga & Haruo Ikeda & Kazuo Shin-ya, 2020. "In vitro Cas9-assisted editing of modular polyketide synthase genes to produce desired natural product derivatives," Nature Communications, Nature, vol. 11(1), pages 1-12, December.
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