Neutral genetic drift-based engineering of a sucrose-utilizing enzyme toward glycodiversification

Autor: David Daudé, Alizée Vergès, Magali Remaud-Simeon, Stéphane Emond, Isabelle André, Emmanuelle Cambon, Samuel Tranier
Přispěvatelé: Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Inst Pharmacol & Biol Struct, Dept Biophys Struct,CNRS, Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, French National Research Agency (ANR Project GLUCODESIGN) ANR-08-PCVI-002-02, French Ministry of Research, ANR-08-PCVI-0002,GLUCODESIGN,Ingénierie à façon de glycoenzymes par des outils de criblage virtuel innovants pour le design de vaccins entériques(2008), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Institut de pharmacologie et de biologie structurale (IPBS), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)
Jazyk: angličtina
Rok vydání: 2019
Předmět:
Zdroj: 'ACS Catalysis ', vol: 9, pages: 1241-1252 (2019)
ACS Catalysis
ACS Catalysis, American Chemical Society, 2019, 9 (2), pp.1241-1252. ⟨10.1021/acscatal.8b03609⟩
ACS Catalysis, 2019, 9 (2), pp.1241-1252. ⟨10.1021/acscatal.8b03609⟩
ISSN: 2155-5435
DOI: 10.1021/acscatal.8b03609⟩
Popis: Neutral drift (also called purifying selection) is an attractive approach to generate polymorphic variant libraries for enzyme engineering. Here, we have applied this strategy to modify the substrate specificity of a transglucosylase. Our model enzyme, the amylosucrase from Neisseria polysaccharea, is a glucosylation biocatalyst of prime interest because it uses the widespread substrate sucrose as a glucosyl donor and shows broad acceptor promiscuity. A library of 440 functional amylosucrase variants was generated after four rounds of neutral drift at a low mutation rate. The functional variations present in this library were investigated by assaying the ability of these variants to use an alternative glucosyl donor (p-nitrophenyl-α-d-glucopyranoside, pNP-Glc) and to glucosylate a range of acceptors (including methyl-α-l-rhamnopyranoside, which is not naturally recognized by the parental enzyme). The impact of these mutations on the thermal stability of the variants was also assessed. Large variations of acceptor promiscuity were observed, ranging from the complete loss of detectable activity to a 2-fold increase relative to the parental enzyme. Variants showing increased catalytic efficiency toward the alternative pNP-Glc donor were also identified. Specifically, one variant combining four unprecedented amino acid changes was 25-fold more efficient at utilizing pNP-Glc than the parental enzyme and acquired glucosylation activity toward methyl-α-l-rhamnopyranoside. Enzymes with improved thermal stability were also identified. Overall, our work demonstrates that neutral drift is an effective and powerful strategy to engineer transglycosylases with enhanced or even acquired substrate specificities from small-sized functional libraries compatible with accurate low-throughput multi-parameter analyses.
Databáze: OpenAIRE
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