Thursday, April 22, 2010 - 9:00 AM
10-03

Production of a highly improved cellulolytic cocktail by an improved Trichoderma reesei CL847 strain

Antoine Margeot1, Fadhel Ben Chaabane1, Laurent Fourage2, Nicolas Lopes-Ferreira1, Etienne Jourdier1, Gilles Ravot2, and Frédéric Monot1. (1) Biotechnology Department, IFP, 1 & 4 avenue de Bois-Préau, 92852, Rueil-Malmaison, France, (2) Protéus, 70 allée Graham Bell, Nimes, 30000, France

Enzymatic hydrolysis of lignocellulose is often considered as the major economic bottleneck of the bioethanol from lignocellulose production process. It is generally admitted that the most efficient organism for the production of cellulolytic enzymes is the imperfect fungus Trichoderma reesei, mainly thanks to its high secretion capacity. The produced enzymatic cocktail contains three important enzymes: endoglucanases (EG), cellobiohydrolases (CBH) and beta-glucosidase (BGL1). The latter one is very poorly secreted by Trichoderma reesei strains and a complete hydrolysis of cellulose often requires supplementation with extra beta-glucosidase, especially to prevent inhibition of CBHs by cellobiose. In order to optimize the produced cocktail, it was decided to obtain and clone a more efficient beta-glucosidase into T. reesei CL847 strain which is an industrial cellulase production strain. The improved beta-glucosidase was evolved by several rounds of L-shuffling, a specific directed evolution method, starting from 3 parental proteins. Compared with native beta-glucosidase, the most evolved enzyme had a 242-fold increased Kcat for the artificial substrate used for screening. Various T. reesei CL847 clones suitably expressing the evolved b-glucosidase were obtained and strain TR-3002 was selected for cellulase production in lab-scale reactors. The kinetics of growth and cellulase production of the new strain were characterized using fed-batch and chemostat modes under different culture conditions. In addition to determination of cellulase and hemicellulase activities, hydrolysis of a steam exploded wheat straw was performed, and demonstrated that the new enzyme mixture allows a 4 fold decrease in cellulase loading without any loss in hydrolysis performance.  

See more of Applications of Biomass Enzyme Technology
See more of General Submissions

See more of The 32nd Symposium on Biotechnology for Fuels and Chemicals (April 19-22, 2010)