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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...

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Autores principales: Santos, Yossef López de los, Chew-Fajardo, Ying Lian, Brault, Guillaume, Doucet, Nicolas
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2019
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.
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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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