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Dissecting the evolvability landscape of the CalB active site toward aromatic substrates
A key event in the directed evolution of enzymes is the systematic use of mutagenesis and selection, a process that can give rise to mutant libraries containing millions of protein variants. To this day, the functional analysis and identification of active variants among such high numbers of mutatio...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821916/ https://www.ncbi.nlm.nih.gov/pubmed/31666622 http://dx.doi.org/10.1038/s41598-019-51940-0 |
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author | Santos, Yossef López de los Chew-Fajardo, Ying Lian Brault, Guillaume Doucet, Nicolas |
author_facet | Santos, Yossef López de los Chew-Fajardo, Ying Lian Brault, Guillaume Doucet, Nicolas |
author_sort | Santos, Yossef López de los |
collection | PubMed |
description | A key event in the directed evolution of enzymes is the systematic use of mutagenesis and selection, a process that can give rise to mutant libraries containing millions of protein variants. To this day, the functional analysis and identification of active variants among such high numbers of mutational possibilities is not a trivial task. Here, we describe a combinatorial semi-rational approach to partly overcome this challenge and help design smaller and smarter mutant libraries. By adapting a liquid medium transesterification assay in organic solvent conditions with a combination of virtual docking, iterative saturation mutagenesis, and residue interaction network (RIN) analysis, we engineered lipase B from P. antarctica (CalB) to improve enzyme recognition and activity against the bulky aromatic substrates and flavoring agents methyl cinnamate and methyl salicylate. Substrate-imprinted docking was used to target active-site positions involved in enzyme-substrate and enzyme-product complexes, in addition to identifying ‘hot spots’ most likely to yield active variants. This iterative semi-rational design strategy allowed selection of CalB variants exhibiting increased activity in just two rounds of site-saturation mutagenesis. Beneficial replacements were observed by screening only 0.308% of the theoretical library size, illustrating how semi-rational approaches with targeted diversity can quickly facilitate the discovery of improved activity variants relevant to a number of biotechnological applications. |
format | Online Article Text |
id | pubmed-6821916 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-68219162019-11-05 Dissecting the evolvability landscape of the CalB active site toward aromatic substrates Santos, Yossef López de los Chew-Fajardo, Ying Lian Brault, Guillaume Doucet, Nicolas Sci Rep Article A key event in the directed evolution of enzymes is the systematic use of mutagenesis and selection, a process that can give rise to mutant libraries containing millions of protein variants. To this day, the functional analysis and identification of active variants among such high numbers of mutational possibilities is not a trivial task. Here, we describe a combinatorial semi-rational approach to partly overcome this challenge and help design smaller and smarter mutant libraries. By adapting a liquid medium transesterification assay in organic solvent conditions with a combination of virtual docking, iterative saturation mutagenesis, and residue interaction network (RIN) analysis, we engineered lipase B from P. antarctica (CalB) to improve enzyme recognition and activity against the bulky aromatic substrates and flavoring agents methyl cinnamate and methyl salicylate. Substrate-imprinted docking was used to target active-site positions involved in enzyme-substrate and enzyme-product complexes, in addition to identifying ‘hot spots’ most likely to yield active variants. This iterative semi-rational design strategy allowed selection of CalB variants exhibiting increased activity in just two rounds of site-saturation mutagenesis. Beneficial replacements were observed by screening only 0.308% of the theoretical library size, illustrating how semi-rational approaches with targeted diversity can quickly facilitate the discovery of improved activity variants relevant to a number of biotechnological applications. Nature Publishing Group UK 2019-10-30 /pmc/articles/PMC6821916/ /pubmed/31666622 http://dx.doi.org/10.1038/s41598-019-51940-0 Text en © The Author(s) 2019 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/. |
spellingShingle | Article Santos, Yossef López de los Chew-Fajardo, Ying Lian Brault, Guillaume Doucet, Nicolas Dissecting the evolvability landscape of the CalB active site toward aromatic substrates |
title | Dissecting the evolvability landscape of the CalB active site toward aromatic substrates |
title_full | Dissecting the evolvability landscape of the CalB active site toward aromatic substrates |
title_fullStr | Dissecting the evolvability landscape of the CalB active site toward aromatic substrates |
title_full_unstemmed | Dissecting the evolvability landscape of the CalB active site toward aromatic substrates |
title_short | Dissecting the evolvability landscape of the CalB active site toward aromatic substrates |
title_sort | dissecting the evolvability landscape of the calb active site toward aromatic substrates |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821916/ https://www.ncbi.nlm.nih.gov/pubmed/31666622 http://dx.doi.org/10.1038/s41598-019-51940-0 |
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