Gene editing enables rapid engineering of complex antibiotic assembly lines
AuthorsThong, Wei Li
Robins, Katherine J.; orcid: 0000-0001-5049-4246
Fyans, Joanna K.
Herbert, Abigail J.
Law, Brian J. C.
Micklefield, Jason; orcid: 0000-0001-8951-4873; email: email@example.com
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AbstractAbstract: 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.
CitationNature Communications, volume 12, issue 1, page 6872
PublisherNature Publishing Group UK
DescriptionFrom Springer Nature via Jisc Publications Router
History: received 2021-09-09, accepted 2021-11-02, registration 2021-11-08, pub-electronic 2021-11-25, online 2021-11-25, collection 2021-12
Publication status: Published
Funder: RCUK | Biotechnology and Biological Sciences Research Council (BBSRC); doi: https://doi.org/10.13039/501100000268; Grant(s): BB/L013754/1, BB/N023536/1