Development of Saccharomyces cerevisiae strains through heterologous expression of cellulases and evolutionary engineering aiming for simultaneous saccharification and fermentation of lignocellulosic biomass

Abstract

In order to reduce the dependence on this energy source and increase energy safety, several countries are searching for sustainable alternatives, such as bioethanol. A promising approach to increase this production is the use of the lignocellulosic biomass found in industrial residues, such as the sugarcane bagasse and straw left after first generation bioethanol. Two strategies occur to produce ethanol - SHF (Separate Hydrolysis and Fermentation) and SSF (Simultaneous Saccharification and Fermentation) - although the first process is the most used, the second (SSF) is more optimized and less costly, embracing hydrolysis of lignocellulose and fermentation of resulted reducing sugars within the same compartment. However S. cerevisiae is unable to break carbohydrate into ethanol, such as cellulose, major component of sugarcane bagasse and straw. Nevertheless, the cellulose expression from other microorganisms in Saccharomyces might be an alternative making possible the simultaneous saccharification and fermentation of lignocellulosic biomass into bioethanol. It is also necessary to address the matter that industrial yeast strains experience conditions (temperature and pH) that are different from those in which cellulases achieve optimal activity and secretion requiring adaptation of the population, such as, increase of the temperature during SSF process. In face this, this project aims to employ the SSF process for bioethanol production by inserting cellulases from Acremonium strictum into S. cerevisiae Brazilian strains (PE-2 and SA-1) and carry out evolution to select those more adapted to the enzyme conditions. Consequently, we expect to obtain S. cerevisiae strains that could express and secrete cellulases in order to improve the hydrolysis of the lignocellulosic biomass and, thus, increase the glucose availability. After evolution of the modified strains, we expect to achieve higher secretion and activity of the enzymes and, consequently, higher levels of ethanol in the medium compared with the unchanged strains. For heterologous expression of cellulases from Acremonium strictum in S. cerevisiae, employing the technique CRISPR / Cas9, which allows the integration of genes of interest into the yeast genome with great precision, and efficiency and flexibility. For the development of lines will be using EMS mutagen to stimulate the occurrence of random mutations occur and then selective pressure in culture media unfavorable in order to select strains capable of withstanding the stress conditions. After evolution and confirmation of expression of enzymes, fermentations will be performed using different substrates, including sugarcane bagasse and straw pre-treated. Fermentations will be performed in shakers in anaerobic conditions obtained by evolutionary engineering. Products generated during fermentation are quantified by high-performance liquid chromatography (HPLC). Among the fermentations carried out, that present the best substrate conversion factor and ethanol (YS/P) will be conducted in fermenters bench in order to simulate the conditions of the plants and thus produce 2nd generation ethanol by simultaneous saccharification and fermentation of lignocellulosic biomass. (AU)