Dissecting the evolvability landscape of the CalB active site toward aromatic substrates
International audience Abstract 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 am...
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Online Access: | https://hal.archives-ouvertes.fr/hal-03590248 https://doi.org/10.1038/s41598-019-51940-0 |
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ftccsdartic:oai:HAL:hal-03590248v1 2023-05-15T13:59:03+02:00 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 Institut Armand Frappier (INRS-IAF) Réseau International des Instituts Pasteur (RIIP)-Institut National de la Recherche Scientifique Québec (INRS) PROTEO, The Quebec Network for Research on Protein Function, Engineering, and Applications Réseau International des Instituts Pasteur (RIIP)-Institut National de la Recherche Scientifique Québec (INRS)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Recherche Scientifique Québec (INRS)-Université de Sherbrooke (UdeS)-Université Laval Québec (ULaval)-McGill University = Université McGill Montréal, Canada -University of Ottawa Ottawa -Université du Québec à Trois-Rivières (UQTR)-Université de Montréal (UdeM)-TransBiotech, Lévis-Concordia University Montreal -Université du Québec à Montréal = University of Québec in Montréal (UQAM) 2019-12 https://hal.archives-ouvertes.fr/hal-03590248 https://doi.org/10.1038/s41598-019-51940-0 en eng HAL CCSD Nature Publishing Group info:eu-repo/semantics/altIdentifier/doi/10.1038/s41598-019-51940-0 info:eu-repo/semantics/altIdentifier/pmid/31666622 hal-03590248 https://hal.archives-ouvertes.fr/hal-03590248 doi:10.1038/s41598-019-51940-0 PUBMED: 31666622 PUBMEDCENTRAL: PMC6821916 ISSN: 2045-2322 EISSN: 2045-2322 Scientific Reports https://hal.archives-ouvertes.fr/hal-03590248 Scientific Reports, Nature Publishing Group, 2019, 9 (1), pp.15588. ⟨10.1038/s41598-019-51940-0⟩ MESH: Catalytic Domain MESH: Directed Molecular Evolution MESH: Fungal Proteins MESH: Gene Library MESH: Hydrocarbons Aromatic MESH: Lipase MESH: Models Molecular MESH: Mutagenesis [SDV]Life Sciences [q-bio] info:eu-repo/semantics/article Journal articles 2019 ftccsdartic https://doi.org/10.1038/s41598-019-51940-0 2022-03-05T23:26:27Z International audience Abstract 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. Article in Journal/Newspaper Antarc* Antarctica Archive ouverte HAL (Hyper Article en Ligne, CCSD - Centre pour la Communication Scientifique Directe) Scientific Reports 9 1 |
institution |
Open Polar |
collection |
Archive ouverte HAL (Hyper Article en Ligne, CCSD - Centre pour la Communication Scientifique Directe) |
op_collection_id |
ftccsdartic |
language |
English |
topic |
MESH: Catalytic Domain MESH: Directed Molecular Evolution MESH: Fungal Proteins MESH: Gene Library MESH: Hydrocarbons Aromatic MESH: Lipase MESH: Models Molecular MESH: Mutagenesis [SDV]Life Sciences [q-bio] |
spellingShingle |
MESH: Catalytic Domain MESH: Directed Molecular Evolution MESH: Fungal Proteins MESH: Gene Library MESH: Hydrocarbons Aromatic MESH: Lipase MESH: Models Molecular MESH: Mutagenesis [SDV]Life Sciences [q-bio] 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 |
topic_facet |
MESH: Catalytic Domain MESH: Directed Molecular Evolution MESH: Fungal Proteins MESH: Gene Library MESH: Hydrocarbons Aromatic MESH: Lipase MESH: Models Molecular MESH: Mutagenesis [SDV]Life Sciences [q-bio] |
description |
International audience Abstract 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. |
author2 |
Institut Armand Frappier (INRS-IAF) Réseau International des Instituts Pasteur (RIIP)-Institut National de la Recherche Scientifique Québec (INRS) PROTEO, The Quebec Network for Research on Protein Function, Engineering, and Applications Réseau International des Instituts Pasteur (RIIP)-Institut National de la Recherche Scientifique Québec (INRS)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Recherche Scientifique Québec (INRS)-Université de Sherbrooke (UdeS)-Université Laval Québec (ULaval)-McGill University = Université McGill Montréal, Canada -University of Ottawa Ottawa -Université du Québec à Trois-Rivières (UQTR)-Université de Montréal (UdeM)-TransBiotech, Lévis-Concordia University Montreal -Université du Québec à Montréal = University of Québec in Montréal (UQAM) |
format |
Article in Journal/Newspaper |
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 |
title |
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_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_sort |
dissecting the evolvability landscape of the calb active site toward aromatic substrates |
publisher |
HAL CCSD |
publishDate |
2019 |
url |
https://hal.archives-ouvertes.fr/hal-03590248 https://doi.org/10.1038/s41598-019-51940-0 |
genre |
Antarc* Antarctica |
genre_facet |
Antarc* Antarctica |
op_source |
ISSN: 2045-2322 EISSN: 2045-2322 Scientific Reports https://hal.archives-ouvertes.fr/hal-03590248 Scientific Reports, Nature Publishing Group, 2019, 9 (1), pp.15588. ⟨10.1038/s41598-019-51940-0⟩ |
op_relation |
info:eu-repo/semantics/altIdentifier/doi/10.1038/s41598-019-51940-0 info:eu-repo/semantics/altIdentifier/pmid/31666622 hal-03590248 https://hal.archives-ouvertes.fr/hal-03590248 doi:10.1038/s41598-019-51940-0 PUBMED: 31666622 PUBMEDCENTRAL: PMC6821916 |
op_doi |
https://doi.org/10.1038/s41598-019-51940-0 |
container_title |
Scientific Reports |
container_volume |
9 |
container_issue |
1 |
_version_ |
1766267422945312768 |