Evolutionary Engineering of Two Robust Brazilian Industrial Yeast Strains for Thermotolerance and Second-Generation Biofuels

Abstract

To meet increased demand for biofuels, biorefineries need to achieve higher yields, which could be achieved by optimizing current processes by using thermotolerant microorganisms in first- and second-generation (2G) biofuel production. 2G biofuel production is comprised of two basic steps before fermentation of the reducing sugars: lignocellulosic biomass pretreament and enzymatic hydrolysis, which is typically performed between 40–55°C. However, Saccharomyces cerevisiae, the most commonly used microorganism, performs fermentation at temperatures up to 35°C, which reduces the enzymatic activity when this yeast is combined in processes such as simultaneous saccharification and fermentation (SSF) and consolidated bioprocessing (CBP). In this context, the use of thermotolerant yeast isolates in SSF and CBP could promote enzymatic hydrolysis at temperatures closer to the optimal of enzymes, increasing the sugars and biofuel yields. Moreover, increased temperatures will reduce cooling costs and contamination by other microorganisms that compete with S. cerevisiae for the released sugars. Therefore, we used adaptive laboratory evolution to evolve two industrial Brazilian S. cerevisiae strains, PE-2 and SA-1, for thermotolerance. Compared to their parental strains, AMY35 (SA-1) and AMY12 (PE-2) had 63% and 61% higher cell growth at 40°C, respectively, and showed an ethanol yield of ∼0.44 g ethanol/g glucose. In addition, the reduced trehalose content in the evolved isolates compared with the respective parental strains at 40°C suggests that other mechanisms are responsible for the thermotolerance. Results showed that our adaptive evolution approach was sufficient to evolve diploid industrial S. cerevisiae strains and that the selected strains had great potential for high-temperature fermentation.