Genome-wide Fitness Profiles Reveal a Requirement for Autophagy During Yeast Fermentation

Abstract

The ability of cells to respond to environmental changes and adapt their metabolism enables cell survival under stressful conditions. The budding yeast Saccharomyces cerevisiae (S. cerevisiae) is particularly well adapted to the harsh conditions of anaerobic wine fermentation. However, S. cerevisiae gene function has not been previously systematically interrogated under conditions of industrial fermentation. We performed a genome-wide study of essential and nonessential S. cerevisiae gene requirements during grape juice fermentation to identify deletion strains that are either depleted or enriched within the viable fermentative population. Genes that function in autophagy and ubiquitin-proteasome degradation are required for optimal survival during fermentation, whereas genes that function in ribosome assembly and peroxisome biogenesis impair fitness during fermentation. We also uncover fermentation phenotypes for 139 uncharacterized genes with no previously known cellular function. We demonstrate that autophagy is induced early in wine fermentation in a nitrogen-replete environment, suggesting that autophagy may be triggered by other forms of stress that arise during fermentation. These results provide insights into the complex fermentation process and suggest possible means for improvement of industrial fermentation strains.

Keywords: S. cerevisiae; autophagy; environmental stress; fermentation; fitness profiling.

Figure 1

Hierarchical cluster analysis of (A) 300 homozygous and (B) 481 heterozygous diploid deletion strains with a fitness defect at 2, 4, 6, 8, 10, and 14 days of fermentation. Green, depletion; red, enrichment; black, no change; gray, no data. Numbers below the color bar represent the normalized log2 value of the microarray signal vs. day 1. Highlighted genes are in the functionally enriched categories of (A) autoproteolytic processing and modification by ubiquitination, deubiquitination, or (B) proteasome degradation. Genes that are listed twice on the clustergram have both UP and DOWN barcode tags that met the cutoff criteria.

Figure 1

Hierarchical cluster analysis of (A) 300 homozygous and (B) 481 heterozygous diploid deletion strains with a fitness defect at 2, 4, 6, 8, 10, and 14 days of fermentation. Green, depletion; red, enrichment; black, no change; gray, no data. Numbers below the color bar represent the normalized log2 value of the microarray signal vs. day 1. Highlighted genes are in the functionally enriched categories of (A) autoproteolytic processing and modification by ubiquitination, deubiquitination, or (B) proteasome degradation. Genes that are listed twice on the clustergram have both UP and DOWN barcode tags that met the cutoff criteria.

Figure 2

Core and starvation-induced autophagy genes contribute to cellular fitness during fermentation. The schematic is derived from Nakatogawa et al. (2009). Genes are color coded as follows: green, reduced fitness; black, no change in fitness; gray, no data obtained; purple, no homolog in S. cerevisiae.

Figure 3

The β-ring of the proteasome core particle enhances cellular fitness during fermentation. The proteasome core particle α-ring (α1-7) and β-ring subunits (β1-7) are shown, along with the Irc25/Poc4 α-ring assembly chaperone. The proteasome regulatory particle (base and lid subunits) is also shown. Genes are color coded as follows: green, reduced fitness; red, increased fitness; black, no change in fitness; gray, no data obtained.

Figure 4

Hierarchical cluster analysis of (A) 303 homozygous and (B) 466 heterozygous diploid deletion strains with a fitness advantage at 2, 4, 6, 8, 10, and 14 days of fermentation. Green, depletion; red, enrichment; black, no change; gray, no data. Numbers below the color bar represent the normalized log2 value of the microarray signal vs. day 1. Highlighted genes are in the functionally enriched categories of (A) peroxisome or (B) ribosomal proteins.

Figure 4

Hierarchical cluster analysis of (A) 303 homozygous and (B) 466 heterozygous diploid deletion strains with a fitness advantage at 2, 4, 6, 8, 10, and 14 days of fermentation. Green, depletion; red, enrichment; black, no change; gray, no data. Numbers below the color bar represent the normalized log2 value of the microarray signal vs. day 1. Highlighted genes are in the functionally enriched categories of (A) peroxisome or (B) ribosomal proteins.

Figure 5

Disruption of atg3 causes reduced CO₂ and ethanol production during fermentation. EC1118 (red diamonds), wild-type (S288C, light blue circles), and atg3Δ (green squares) homozygous diploid strains were inoculated into synthetic grape juice supplemented with amino acids, and anaerobic fermentation was carried out for 16 days. Weight loss (A), ethanol (B), glucose (C), and fructose (D) measurements were taken every 2 days. Each data point on the graph represents the average of two EC1118, four wild-type, and four atg3Δ fermentations. For all graphs, error bars represent the standard deviation for each data point. (E) Growth chamber analysis of wild-type (S288C) vs. atg3Δ strain growth in YPD at 25°. Cells were inoculated into a multiwell plate at an OD₆₀₀ of 0.1 and grown for 50 h. Each data point is an average of three technical replicates. For each data point, SD < ±0.04.

Figure 6

API processing occurs via autophagy during fermentation. Western blot analysis of cell lysates from wild-type diploid (S288C) and atg19Δ and atg1Δ homozygous diploids at days 1, 2, and 4 of fermentation. Blots were probed with anti-API antibody. Arrows point to the precursor form (pAPI) and the mature form (mAPI) of API. API, aminopeptidase I.

Figure 7

Autophagic bodies accumulate in the vacuole during fermentation. Electron microscopy of pep4Δ/pep4Δ homozygous diploid log phase cells (A) and after two days of fermentation (B). Arrows point to autophagic bodies in the vacuole.

Figure 8

Fermentation kinetics of the homozygous yeast deletion pool (S288C) compared with the EC1118 wine yeast strain in synthetic grape juice. Both strains [EC1118 (red diamonds) and diploid homozygous deletion set (S288C, light blue circles)] were fermented in triplicate. The average values are presented with error bars representing SD. (A) Cell growth curve measured by OD₆₀₀, (B) glucose depletion, (C) ethanol production, and (D) yeast assimilable nitrogen [YAN, milligrams of nitrogen (N) per liter] measured from total ammonium sulfate and amino acids. Orange arrow in (A) marks when autophagic bodies are detected by electron microscopy. YAN, yeast assimilable nitrogen.