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Gene editing enables rapid engineering of complex antibiotic assembly lines

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 challeng...

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Autores principales: Thong, Wei Li, Zhang, Yingxin, Zhuo, Ying, Robins, Katherine J., Fyans, Joanna K., Herbert, Abigail J., Law, Brian J. C., Micklefield, Jason
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8616955/
https://www.ncbi.nlm.nih.gov/pubmed/34824225
http://dx.doi.org/10.1038/s41467-021-27139-1
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author Thong, Wei Li
Zhang, Yingxin
Zhuo, Ying
Robins, Katherine J.
Fyans, Joanna K.
Herbert, Abigail J.
Law, Brian J. C.
Micklefield, Jason
author_facet Thong, Wei Li
Zhang, Yingxin
Zhuo, Ying
Robins, Katherine J.
Fyans, Joanna K.
Herbert, Abigail J.
Law, Brian J. C.
Micklefield, Jason
author_sort Thong, Wei Li
collection PubMed
description 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.
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spelling pubmed-86169552021-12-10 Gene editing enables rapid engineering of complex antibiotic assembly lines Thong, Wei Li Zhang, Yingxin Zhuo, Ying Robins, Katherine J. Fyans, Joanna K. Herbert, Abigail J. Law, Brian J. C. Micklefield, Jason Nat Commun Article 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. Nature Publishing Group UK 2021-11-25 /pmc/articles/PMC8616955/ /pubmed/34824225 http://dx.doi.org/10.1038/s41467-021-27139-1 Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Thong, Wei Li
Zhang, Yingxin
Zhuo, Ying
Robins, Katherine J.
Fyans, Joanna K.
Herbert, Abigail J.
Law, Brian J. C.
Micklefield, Jason
Gene editing enables rapid engineering of complex antibiotic assembly lines
title Gene editing enables rapid engineering of complex antibiotic assembly lines
title_full Gene editing enables rapid engineering of complex antibiotic assembly lines
title_fullStr Gene editing enables rapid engineering of complex antibiotic assembly lines
title_full_unstemmed Gene editing enables rapid engineering of complex antibiotic assembly lines
title_short Gene editing enables rapid engineering of complex antibiotic assembly lines
title_sort gene editing enables rapid engineering of complex antibiotic assembly lines
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8616955/
https://www.ncbi.nlm.nih.gov/pubmed/34824225
http://dx.doi.org/10.1038/s41467-021-27139-1
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