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

Author(s)

Dielle Pierotti Procópio

• Document type: Master's Dissertation
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Engenharia de Alimentos
• Defense date: 2017-03-31

Examining board members

Francisco Maugeri Filho; Gabriela Alves Macedo; Osmar Vaz de Carvalho Netto

• Advisor: Francisco Maugeri Filho; Rosana Goldbeck

Abstract

The yeast Saccharomyces cerevisiae is the most used microorganism to produce ethanol, because it presents an excellent fermentative capacity and is tolerant to the stress generated in industrial fermentation processes. However, this yeast can¿t metabolize complex carbohydrates into ethanol, such as cellulose, the major component of sugarcane bagasse, making it necessary to hydrolyse the cellulose into glucose for subsequent conversion of glucose into ethanol. In this sense, an industrial strain of S. cerevisiae (Pedra-2) was transformed with cellulases from Acremonim strictum, a wild microorganism isolated from the Brazilian Biome, aiming to the production of second generation ethanol through simultaneous saccharification and fermentation (SSF) of cellobiose, cellulose (AVICEL®) and pre-treated sugarcane bagasse (145 °C, 12 minutes, 0.5% (v / v) H2SO4). For the heterologous expression, the vector pRS426 with uracil selection marker (URA +) was used and three vector models were assembled, two of them with isolated expressions of the enzymes endoglucanase and ß-glycosidase, and the third one with simultaneous expression of the two enzymes. Only the yeasts transformed with this vector were subjected to simultaneous saccharification and fermentation tests. The enzyme activities were 0.289 U / mL for endoglucanase, and 0.169 U/mL, for ß-glucosidase. Regarding the fermentations, they were performed using a combination of different substrates: AVICEL® (commercial cellulose) + glucose, cellobiose + glucose, glucose, as well as pretreated sugarcane bagasse (acid 0,5%) + glucose, in semi-anaerobic and anaerobic conditions, at 37 ° C for 120 hours. The semi-anaerobic fermentations did not present good ethanol yields, comparing the fermentations using complex medium with the control, using only glucose. In order to improve the alcoholic fermentation, processes were carried out in anaerobiosis. In such conditions the yeast showed a better specific growth rate in AVICEL + glucose. The highest ethanol content, 14,930 (g / L) occurred at 120 h of fermentation when glucose + cellobiose was used as carbon sources, representing 53% higher than fermentation conducted with glucose alone (control) (AU)

• FAPESP's process: 15/02007-8 - Study of expression of the heterologous cellulases of Acremonium strictum in of Saccharomyces cerevisiae strains aimed at second-generation bioethanol production
• Grantee: Dielle Pierotti Procópio
• Support Opportunities: Scholarships in Brazil - Master