Unravelling the genetic basis of ethanol tolerance and maximal ethanol accumulation capacity in yeast for very-high gravity bioethanol production

Concerns about global warming and climate change due to greenhouse gas emissions and the growing energy demand for transportation, heating and industrial processes has encouraged the search for sustainable renewable energy sources. Today, bioethanol is the most common biofuel mainly derived from starch or sugar cane and shows great potential as a building block for bio-based chemicals such as bioplastics. Saccharomyces cerevisiae is currently the preferred micro-organism for bioethanol production due to its highly efficient ethanol productivity and high tolerance to various fermentation-associated stresses such as high ethanol concentrations. Although much research has been done to understand the genetic basis of high ethanol tolerance in yeast, most of the research was focused on low and moderate ethanol tolerance in laboratory yeast strains. Previous research in our laboratory has been initiated to study high ethanol tolerance and maximal ethanol accumulation capacity in industrial yeast strains by pooled-segregant whole-genome sequence analysis in order to find genetic determinants that contribute to both of these important industrial traits. In the first part of this thesis, we have further explored the genetic basis of high ethanol tolerance in a former Brazilian bioethanol production strain VR1 which was originally initiated by the research of Dr. S. Swinnen. Our results indicate that very high ethanol concentrations generally compromise the uptake of nutrients required to fulfil auxotrophic requirements and that therefore auxotrophic mutations lead to lower ethanol tolerance. We have downscaled the last major QTL on chromosome X and identified VPS70 as a new causative allele that contributes to high ethanol tolerance. Additionally, we have confirmed that APJ1 has a negative effect on ethanol tolerance and that a lower expression or deletion of the APJ1 allele improves ethanol tolerance. Introduction of all superior alleles in the inferior background has succeeded in improving ethanol tolerance of the inferior parent strain (BY) to an even higher level of that in the superior parent (VR1-5B). In the second part of this thesis, we have further described the genetic dissection of maximal ethanol accumulation capacity of a sake strain CBS1585. We have identified two more causative genes PTK2 and SHE4, supporting superior maximal ethanol accumulation capacity, which expands our understanding of the genetic determinants involved in general ethanol tolerance as well as in maximal ethanol accumulation capacity in very high gravity fermentations. Unfortunately, we could not improve maximal ethanol accumulation in the strain V1116 wine strain by overexpression of the most promising target genes identified during pooled-segregant gene expression analysis upon stable integration in the genome. However, further engineering in order to obtain higher expression of the integrated constructs has a clear potential to provide positive results for improving maximal ethanol accumulation capacity in a stable manner. Finally, we have introduced a specific combination of causative alleles in the ER18 strain which could clearly improve this strain for ethanol tolerance of cell proliferation and maximal ethanol accumulation capacity. However, it still remains to be investigated to what extent these causative genes are universal determinants so that they can be used to improve other industrial yeast strains for bioethanol production. Altogether, this work has provided new insights into the genetic basis of high ethanol tolerance and maximal ethanol accumulation capacity of industrial yeast strains.status: publishe