Temperature-Sensitive Mutants of Saccharomyces cerevisiae Essential Genes for Enhanced Ethanol Biofuel Production

Abstract

Biofuel development can serve as a possible approach to reduce GHG emissions and help mitigate the effects of climate change on the environment and human health. In the development of biofuels involving microorganisms, there have been various trade-offs between growth and ethanol production. While previous studies have proposed different approaches to assess these trade-offs, temperature-sensitive (ts) mutants have not been widely explored in this context. These mutants lack the function of a certain essential gene at nonpermissive temperatures and display full to partial function at semi-permissive temperatures. In this project, we performed screenings on a library collection consisting of 600 temperature-sensitive mutants of Saccharomyces cerevisiae essential genes at permissive (21°C) and nonpermissive temperatures (38°C). The aim is to identify strains that exhibit impaired growth at elevated temperatures, but can still maintain active metabolism to efficiently produce ethanol. This is based on the assumption that high temperatures can disrupt cellular processes and functions linked to growth, but not ethanol fermentation because of the changes in metabolic pathways. Initial screenings were conducted using alcohol dehydrogenase assays, and high-performance liquid chromatography was used to verify the high specific production ratios of 15 ts mutants that were selected. The gene ontology analysis confirmed that many of these selected genes are involved in growth-related processes, such as cell cycle regulation and ribosomal biogenesis. Further work should be conducted to dynamically control these strains using optogenetic techniques to improve the fermentation efficiency. Additional experiments should also investigate whether these ts alleles can produce more ethanol than the wild-type (BY4741) through fed-batch fermentations.