How molecular evolution technologies can provide bespoke industrial enzymes : application to biofuels.

Laurent Fourage 1 Jean-Marie Sonet 2, * Frédéric Monot 3 Gilles Ravot 4 Antoine Margeot 3
* Corresponding author
1 TOTAL S.A.
2 Protéus SA
3 IFPEN - IFP Energies nouvelles
4 SAS PIVERT
Abstract : Enzymatic hydrolysis of lignocellulose is one of the major bottlenecks in the development of biological conversion of lignocellulosic biomass to biofuels. One of the most efficient organisms for the production of cellulolytic enzymes is the fungus Trichoderma reesei, mainly thanks to its high secretion capacity. The conversion of cellulose to glucose involves three types of cellulases working in synergy: endoglucanases (EC 3.2.1.4) randomly cleave b-1,4 glycosidic linkages of cellulose, cellobiohydrolases (EC 3.2.1.91) attack cellulose chain ends to produce cellobiose dimers which are converted into glucose by the b-glucosidases (EC 3.2.1 21). Unexpectedly, the amount of b-glucosidase (BGL1) from T. reesei hyperproducing strains represents a very low percentage of the total secreted proteins. A suboptimal content of this enzyme limits the performance of commercial cellulase preparations as cellobiose represents the main inhibitor of the cellulolysis reaction by cellobiohydrolases. This bottleneck can be alleviated either by overexpressing the b-glucosidase in T. reesei or optimized its specific activity. After giving a brief overview of the main available technologies, this example will be used to illustrate the potential of directed evolution technologies to devolop enzymes tailored to fit industrial needs. We describe the L-ShufflingTM strategy implemented with three parental genes originating from microbial biodiversity leading to identification of an efficient b-glucosidase showing a 242-fold increase in specific activity for the pNPGlc substrate compared to WT (Wild Type) Cel3a beta-glucosidase of T. reesei. After expression of the best improved b-glucosidase in T. reesei and secretion of a new enzymatic cocktail, improvement of the glucosidase activity allows a 4-fold decrease of cellulase loading for the saccharification of an industrial pretreated biomass compared to the parental cocktail.
Document type :
Journal articles
Liste complète des métadonnées

Cited literature [23 references]  Display  Hide  Download
https://hal-ifp.archives-ouvertes.fr/hal-00908958
Contributor : Françoise Bertrand <>
Submitted on : Tuesday, November 26, 2013 - 2:14:23 PM
Last modification on : Thursday, February 7, 2019 - 4:26:50 PM
Document(s) archivé(s) le : Monday, March 3, 2014 - 3:45:55 PM

File

A9R4E81.pdf
Publisher files allowed on an open archive

Identifiers

  • HAL Id : hal-00908958, version 1

Collections

IFP | OGST

Citation

Laurent Fourage, Jean-Marie Sonet, Frédéric Monot, Gilles Ravot, Antoine Margeot. How molecular evolution technologies can provide bespoke industrial enzymes : application to biofuels.. Oil & Gas Science and Technology - Revue d'IFP Energies nouvelles, Institut Français du Pétrole, 2013, 68 (4), pp.681-691. ⟨hal-00908958⟩

Export

BibTeX TEI DC DCterms EndNote

Share

Metrics

Record views

271

Files downloads

259

Contact

support.ccsd.cnrs.fr
hal.support@ccsd.cnrs.fr
Logo CCSD