Dissecting the evolvability landscape of the CalB active site toward aromatic substrates

• Published in: Scientific Reports
• Main Authors: Santos, Yossef López de Los, Chew-Fajardo, Ying Lian, Brault, Guillaume, Doucet, Nicolas
• Other Authors: Armand-Frappier Santé Biotechnologie Research Centre (INRS-AFSB), Institut National de la Recherche Scientifique Québec (INRS)-Réseau International des Instituts Pasteur (RIIP), PROTEO, The Quebec Network for Research on Protein Function, Engineering, and Applications, Institut National de la Recherche Scientifique Québec (INRS)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Recherche Scientifique Québec (INRS)-Réseau International des Instituts Pasteur (RIIP)-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
• Language: English
• Published: HAL CCSD 2019
• Subjects: 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]; Antarc*; Antarctica
• Online Access: https://hal.science/hal-03590248; https://doi.org/10.1038/s41598-019-51940-0

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.