Optimization of Saccharomyces Cerevisiae for Increased Ethanol Tolerance via Adaptive Laboratory Evolution

Abstract

Saccharomyces cerevisiae has been utilised by industries over the years for ethanol production. Needless to say, it is a ‘must-have’ starter in the winemaking industry. However, over-accumulation of ethanol has been recorded to have a toxic effect on Saccharomyces cerevisiae. Consequently, there is a limit to the level of ethanol production achieved by this industrially relevant yeast, which is detrimental to the industrial setting. Therefore, This study aims to engineer Saccharomyces cerevisiae through Adaptive Laboratory Evolution (ALE) to alleviate the menace of limited ethanol tolerance in Saccharomyces cerevisiae. Wild strain Saccharomyces cerevisiae characterisedfrom previous research was obtained and purified. An ethanol tolerance test was carried out to ascertain the initial tolerance level of the wild strain before ALE. ALE was carried out in 12 generations, gradually increasing ethanol concentration with each successive generation.The wild isolate and the adapted isolates from cycle 7a (36 hours), 7b (72 hours), 10a (36 hours) and 10b (72 hours) were subjected to expressional studies using q-PCR. Four (4) genes identified to be involved in ethanol tolerance were targeted: PCK1, LSC1, ADH2, and ACS1. An internal control gene (ACT1) was also adopted. The wild isolate had an initial ethanol tolerance level of 7.0%, which increased gradually during evolution and peaked at 11.5%. At 12% and 12.5%, the optical density of the isolates began to decline.