Study of expression of the heterologous cellulases of Acremonium strictum in of Saccharomyces cerevisiae strains aimed at second-generation bioethanol production

Abstract

The yeast Saccharomyces cerevisiae is the microorganism most used to produce ethanol for presenting an excellent fermentative capacity and be tolerant to stress generated in industrial fermentation processes. However, this yeast can not metabolize complex carbohydrates into ethanol, such as cellulose, the major component of sugarcane bagasse making it necessary to hydrolysis of cellulose to glucose for subsequent conversiofn of glucose into ethanol. Thus, this project aims to perform the heterologous expression of cellulases originating in Acremonim strictum, wild microorganism isolated of the Brazilian biome, in S. cerevisiae to produce second-generation ethanol by simultaneous saccharification and fermentation of sugar cane bagasse. Will be studied two strains of Saccharomyces cerevisiae: an industrial strain (Pedra-2) and a laboratory strain (FY23). The vector used for expression of cellulases: Endoglucanase (006 gene) and ²-glucosidase (249 gene) were isolated of A. strictum in previous studies, will be the vector P423-TEF that presenting histidine (HIS3) and auxotrophic marker. After confirming the expression of enzymes, will be fermentation performed using a combination of different substrates: Avicel (commercial paper), cellobiose, glucose, molasses, and sugar cane bagasse in natura and pretreated (burst steam and steam explosion delignification). Fermentations will be held in shakers anaerobically to 30°C for 120 hours. Subsequently the products generated during fermentation will be analyzed by high performance liquid chromatography (HPLC) and biomass generated will be quantified by gravimetry (dry mass). Finally, among the fermentations conducted, that one put forward the best substrate conversion factor in ethanol (YP / S) will be conducted in a bench fermenter. (AU)