Comparative Polygenic Analysis of Maximal Ethanol Accumulation Capacity and Tolerance to High Ethanol Levels of Cell Proliferation in Yeast

Abstract

The yeast Saccharomyces cerevisiae is able to accumulate ≥17% ethanol (v/v) by fermentation in the absence of cell proliferation. The genetic basis of this unique capacity is unknown. Up to now, all research has focused on tolerance of yeast cell proliferation to high ethanol levels. Comparison of maximal ethanol accumulation capacity and ethanol tolerance of cell proliferation in 68 yeast strains showed a poor correlation, but higher ethanol tolerance of cell proliferation clearly increased the likelihood of superior maximal ethanol accumulation capacity. We have applied pooled-segregant whole-genome sequence analysis to identify the polygenic basis of these two complex traits using segregants from a cross of a haploid derivative of the sake strain CBS1585 and the lab strain BY. From a total of 301 segregants, 22 superior segregants accumulating ≥17% ethanol in small-scale fermentations and 32 superior segregants growing in the presence of 18% ethanol, were separately pooled and sequenced. Plotting SNP variant frequency against chromosomal position revealed eleven and eight Quantitative Trait Loci (QTLs) for the two traits, respectively, and showed that the genetic basis of the two traits is partially different. Fine-mapping and Reciprocal Hemizygosity Analysis identified ADE1, URA3, and KIN3, encoding a protein kinase involved in DNA damage repair, as specific causative genes for maximal ethanol accumulation capacity. These genes, as well as the previously identified MKT1 gene, were not linked in this genetic background to tolerance of cell proliferation to high ethanol levels. The superior KIN3 allele contained two SNPs, which are absent in all yeast strains sequenced up to now. This work provides the first insight in the genetic basis of maximal ethanol accumulation capacity in yeast and reveals for the first time the importance of DNA damage repair in yeast ethanol tolerance.

Auxotrophic Mutations Reduce Tolerance of Saccharomyces cerevisiae to Very High Levels of Ethanol Stress

“…It is likely that low to moderate ethanol concentrations have little or no effect on nutrient uptake across the plasma membrane and that, therefore, auxotrophic requirements, which strongly depend on nutrient uptake, have always remained unnoticed as an important factor in ethanol tolerance. Another QTL mapping experiment in a cross between the sake strain CBS 1585 and the laboratory BY strain identified URA3 as a causative gene for maximal ethanol accumulation capacity, but it had only a very weak contribution to ethanol tolerance for cell proliferation in the presence of 18 to 20% ethanol (45). This result may at first seem surprising, but it is not contradictory to our findings because a different genetic background was used in this study.…”

“…The most widely used molecular markers in yeast are natural variations detected as hybridization differences on high-density oligonucleotide arrays (38) or determined by whole-genome sequencing (39,40). Several polygenic traits have been investigated by using these approaches, which led to the identification of loci, genes, and single nucleotide polymorphisms involved in hightemperature growth (39,40), sporulation efficiency (41), mRNA expression profiles (42), acetic acid production (43), resistance to chemical agents (44), high ethanol tolerance (34), maximal ethanol accumulation (45), low glycerol production (46,47), and thermotolerance (48).…”

Engineering alcohol tolerance in yeast

“…S4A–D). Ethanol titers of 115–120 g/L have been reported previously from S288C (11), the inbred laboratory strain used here that is known for its low ethanol resistance (5, 12, 13). However, these studies were typically conducted using chemically undefined (“rich”) media.…”

Metabolomics-driven approach to solving a CoA imbalance for improved 1-butanol production in Escherichia coli

“…Previously, 15 g/L of 1-butanol was produced within engineered E.coli by deletion of fermentative pathways, thereby requiring the use of the 1-butanol pathway as the sole NADH sink under anaerobic conditions (Shen et al, 2011). While this strategy yielded 1-butanol that is regarded as one of the highest biofuel production to date, Friedlander et al, 2016;Inokuma et al, 2010;Pais et al, 2013;Wernick et al, 2016;York and Ingram, 1996) improved titers are desired. In addition to the deletion of NADH consuming pathways, many other metabolic engineering strategies have been applied in order to achieve such high titers.…”