Gene editing enables rapid engineering of complex antibiotic assembly lines

Wei Li Thong, Yingxin Zhang, Ying Zhuo, Katherine J. Robins, Joanna K. Fyans, Abigail J. Herbert, Brian J. C. Law and Jason Micklefield ()
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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

Nature Communications, 2021, vol. 12, issue 1, 1-10

Abstract: 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.

Date: 2021
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DOI: 10.1038/s41467-021-27139-1

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