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Substrate multiplexed protein engineering facilitates promiscuous biocatalytic synthesis

Enzymes with high activity are readily produced through protein engineering, but intentionally and efficiently engineering enzymes for an expanded substrate scope is a contemporary challenge. One approach to address this challenge is Substrate Multiplexed Screening (SUMS), where enzyme activity is m...

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Autores principales: McDonald, Allwin D., Higgins, Peyton M., Buller, Andrew R.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9448781/
https://www.ncbi.nlm.nih.gov/pubmed/36068220
http://dx.doi.org/10.1038/s41467-022-32789-w
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author McDonald, Allwin D.
Higgins, Peyton M.
Buller, Andrew R.
author_facet McDonald, Allwin D.
Higgins, Peyton M.
Buller, Andrew R.
author_sort McDonald, Allwin D.
collection PubMed
description Enzymes with high activity are readily produced through protein engineering, but intentionally and efficiently engineering enzymes for an expanded substrate scope is a contemporary challenge. One approach to address this challenge is Substrate Multiplexed Screening (SUMS), where enzyme activity is measured on competing substrates. SUMS has long been used to rigorously quantitate native enzyme specificity, primarily for in vivo settings. SUMS has more recently found sporadic use as a protein engineering approach but has not been widely adopted by the field, despite its potential utility. Here, we develop principles of how to design and interpret SUMS assays to guide protein engineering. This rich information enables improving activity with multiple substrates simultaneously, identifies enzyme variants with altered scope, and indicates potential mutational hot-spots as sites for further engineering. These advances leverage common laboratory equipment and represent a highly accessible and customizable method for enzyme engineering.
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spelling pubmed-94487812022-09-08 Substrate multiplexed protein engineering facilitates promiscuous biocatalytic synthesis McDonald, Allwin D. Higgins, Peyton M. Buller, Andrew R. Nat Commun Article Enzymes with high activity are readily produced through protein engineering, but intentionally and efficiently engineering enzymes for an expanded substrate scope is a contemporary challenge. One approach to address this challenge is Substrate Multiplexed Screening (SUMS), where enzyme activity is measured on competing substrates. SUMS has long been used to rigorously quantitate native enzyme specificity, primarily for in vivo settings. SUMS has more recently found sporadic use as a protein engineering approach but has not been widely adopted by the field, despite its potential utility. Here, we develop principles of how to design and interpret SUMS assays to guide protein engineering. This rich information enables improving activity with multiple substrates simultaneously, identifies enzyme variants with altered scope, and indicates potential mutational hot-spots as sites for further engineering. These advances leverage common laboratory equipment and represent a highly accessible and customizable method for enzyme engineering. Nature Publishing Group UK 2022-09-06 /pmc/articles/PMC9448781/ /pubmed/36068220 http://dx.doi.org/10.1038/s41467-022-32789-w Text en © The Author(s) 2022 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
McDonald, Allwin D.
Higgins, Peyton M.
Buller, Andrew R.
Substrate multiplexed protein engineering facilitates promiscuous biocatalytic synthesis
title Substrate multiplexed protein engineering facilitates promiscuous biocatalytic synthesis
title_full Substrate multiplexed protein engineering facilitates promiscuous biocatalytic synthesis
title_fullStr Substrate multiplexed protein engineering facilitates promiscuous biocatalytic synthesis
title_full_unstemmed Substrate multiplexed protein engineering facilitates promiscuous biocatalytic synthesis
title_short Substrate multiplexed protein engineering facilitates promiscuous biocatalytic synthesis
title_sort substrate multiplexed protein engineering facilitates promiscuous biocatalytic synthesis
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9448781/
https://www.ncbi.nlm.nih.gov/pubmed/36068220
http://dx.doi.org/10.1038/s41467-022-32789-w
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