Quantitative trait analysis of yeast biodiversity yields novel gene tools for metabolic engineering

Abstract

Engineering of metabolic pathways by genetic modification has been restricted largely to enzyme-encoding structural genes. The product yield of such pathways is a quantitative genetic trait. Out of 52 Saccharomyces cerevisiae strains phenotyped in small-scale fermentations, we identified strain CBS6412 as having unusually low glycerol production and higher ethanol yield as compared to an industrial reference strain. We mapped the QTLs underlying this quantitative trait with pooled-segregant whole-genome sequencing using 20 superior segregants selected from a total of 257. Plots of SNP variant frequency against SNP chromosomal position revealed one major and one minor locus. Downscaling of the major locus and reciprocal hemizygosity analysis identified an allele of SSK1, ssk1(E330N…K356N), expressing a truncated and partially mistranslated protein, as causative gene. The diploid CBS6412 parent was homozygous for ssk1(E330N…K356N). This allele affected growth and volumetric productivity less than the gene deletion. Introduction of the ssk1(E330N…K356N) allele in the industrial reference strain resulted in stronger reduction of the glycerol/ethanol ratio compared to SSK1 deletion and also compromised volumetric productivity and osmotolerance less. Our results show that polygenic analysis of yeast biodiversity can provide superior novel gene tools for metabolic engineering.

Life cycle assessment of lignocellulosic bioethanol: Environmental impacts and energy balance

“…Xylose fermentation and the presence of inhibitors are challenges to be overcome for increasing the economic feasibility of the processes. Nowadays there are important advances in the development of recombinant yeasts able to resist the harsh condition of industrial operations [24] and also it has been discovered yeasts with the natural ability to ferment both sugars [25].…”

Genomic saturation mutagenesis and polygenic analysis identify novel yeast genes affecting ethyl acetate production, a non-selectable polygenic trait

“…This has now been performed with selectable or easily identifiable traits, allowing very high numbers of segregants to be used 910, but also with small pools of selected segregants for difficult-to-score commercially important traits 11121314151617181920212223242526. These new methodologies have allowed exploration of the huge Saccharomyces cerevisiae biodiversity to identify genes affecting commercially-important traits.…”

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

“…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).…”