Identification of novel genes involved in neutral lipid storage by quantitative trait loci analysis of Saccharomyces cerevisiae

Abstract

Background: The accumulation of intracellular fat depots is a polygenic trait. Therefore, the extent of lipid storage in the individuals of a species covers a broad range and is determined by many genetic factors. Quantitative trait loci analysis can be used to identify those genetic differences between two strains of the same species that are responsible for the differences in a given phenotype. We used this method and complementary approaches to identify genes in the yeast Saccharomyces cerevisiae that are involved in neutral lipid storage.

Results: We selected two yeast strains, the laboratory strain BY4741 and the wine yeast AWRI1631, with a more than two-fold difference in neutral lipid content. After crossing, sporulation and germination, we used fluorescence activated cell sorting to isolate a subpopulation of cells with the highest neutral lipid content from the pool of segregants. Whole genome sequencing of this subpopulation and of the unsorted pool of segregants implicated several loci that are involved in lipid accumulation. Three of the identified genes, PIG1, PHO23 and RML2, were investigated in more detail. Deletions of these genes and the exchange of the alleles between the two parental strains confirmed that the encoded proteins contribute to neutral lipid storage in S. cerevisiae and that PIG1, PHO23 and RML2 are the major causative genes. Backcrossing of one of the segregants with the parental strains for seven generations revealed additional regions in the genomes of both strains with potential causative genes for the high lipid accumulation phenotype.

Conclusions: We identified several genes that contribute to the phenotype of lipid accumulation in an allele-specific manner. Surprisingly, no allelic variations of genes with known functions in lipid metabolism were found, indicating that the level of storage lipid accumulation is determined by many cellular processes that are not directly related to lipid metabolism.

Keywords: QTL analysis; baker’s yeast; lipid droplet; lipid metabolism; natural variation; polygenic trait; steryl esters; triacylglycerol.

Fig. 1

TAG and SE content of the two yeast strains AWRI1631 and BY4741. The cultures were harvested during exponential growth (exp.) or in stationary phase after 48 h (stat.) of cultivation in minimal medium. The data are the means from a minimum of six independent experiments and their standard deviations. The p-values, calculated with a two-tailed t-test, were < 0.001 for the comparisons of the two strains in both exponential and stationary phase and for both compounds, TAG and SE.

Fig. 2

Analysis of segregants from crossing BY4741 with AWRI1631. a: Frequency distribution of fluorescence intensity of 2288 F1 segregants. The segregants with lower fluorescence than the BY4741 parental strain are shown in blue, the segregants with higher values than the AWRI1631 parental strain in red, and the segregants with intermediate intensity are depicted in gray. The frequencies of alleles that are beneficial for high NL content are shown for a subset of 43 out of the 60 segregants with the highest FI. b: WGS data of a section of chromosome XII, including the PIG1 peak (see Fig. S1 for the analysis of all chromosomes). The figure shows the median ratios between the frequencies of BY4741 and AWRI1631 parental strain-derived SNVs in the X-QTL analysis. Red points: selected subpopulation with high [NL]. Gray points: non-selected population with average [NL]. Each point shows a median AWRI1631:BY4741 ratio for all SNVs in a window of 10,000 bp. Black points: difference between the populations: higher abundance of the red than of the gray signal indicates that this region is enriched for AWRI1631 sequences in the population with high NL content. The gap in the signal marks a region derived exclusively from the BY4741 parent, i.e. a region with no SNVs calls relative to the BY4741 variant calling reference. Shading denotes the parental origin of the genomic region in the F7 generation of the backcrossing experiment selecting for high NL content (BY4741 – blue; AWRI1631 – red) in the BY lineage (upper ribbon) and in the AWRI lineage (lower ribbon)

Fig. 3

Neutral lipid analysis of deletion mutants. TAG and SE content of the AWRI1631 (panel a) and the BY4741 (panel b) strains, deleted for PIG1, PHO23 or RML2, and combinations thereof. These results confirm that the proteins encoded by these three genes are involved in NL metabolism, with varying influence in the two strain backgrounds. The strains were cultivated in minimal medium for 48 h. The data are the means from a minimum of three independent experiments and their standard deviations. The p-values are the results of a two-tailed t-test comparing the respective mutant with the wild-type.

Fig. 4

Neutral lipid analysis of substitution mutants. TAG and SE content of the AWRI1631 (panel a) and the BY4741 (panel b) mutant strains. The genes PIG1, PHO23 or RML2 and combinations of these genes are replaced with the alleles from the other parent strain. The mutants were cultivated in minimal medium for 48 h. The data are the means from a minimum of three independent experiments and their standard deviations. The p-values are the results of a two-tailed t-test comparing the respective mutant with the wild-type.

Fig. 5

Neutral lipid content of the BY and AWRI lineages after backcrossing. The figure shows the frequency distribution of the fluorescence intensity of Nile Red, which correlates with the NL content, of the F1 generation (upper panel), the F7 generation of the BY lineage (middle panel) and the F7 generation of the AWRI lineage (lower panel). The FI of the segregants was normalized to the value of the parental strain BY4741 and according to the log₂ value of the normalized intensity the segregants were distributed into 100 intervals. Dotted lines denote some important classes: line a (44^th class; log₂(ratio of [FI]) = 0) – segregants with the same FI as in the BY4741 parental strain; line b (54^th class; log₂(ratio of [FI]) = 0.37) – the average class of the F1 generation; line c (55^th class; log₂(ratio of [FI]) = 0.40) – the average class of the F7 generation of the BY lineage; line d (62^nd class; log₂(ratio of [FI]) = 0.66) – segregants with the same FI content as in the AWRI1631 parental strain; line e (66^th class; log₂(ratio of [FI]) = 0.81) – the average class of the F7 generation of the AWRI lineage