Comparative transcriptome profiling analyses during the lag phase uncover YAP1, PDR1, PDR3, RPN4, and HSF1 as key regulatory genes in genomic adaptation to the lignocellulose derived inhibitor HMF for Saccharomyces cerevisiae

Abstract

Background: The yeast Saccharomyces cerevisiae is able to adapt and in situ detoxify lignocellulose derived inhibitors such as furfural and HMF. The length of lag phase for cell growth in response to the inhibitor challenge has been used to measure tolerance of strain performance. Mechanisms of yeast tolerance at the genome level remain unknown. Using systems biology approach, this study investigated comparative transcriptome profiling, metabolic profiling, cell growth response, and gene regulatory interactions of yeast strains and selective gene deletion mutations in response to HMF challenges during the lag phase of growth.

Results: We identified 365 candidate genes and found at least 3 significant components involving some of these genes that enable yeast adaptation and tolerance to HMF in yeast. First, functional enzyme coding genes such as ARI1, ADH6, ADH7, and OYE3, as well as gene interactions involved in the biotransformation and inhibitor detoxification were the direct driving force to reduce HMF damages in cells. Expressions of these genes were regulated by YAP1 and its closely related regulons. Second, a large number of PDR genes, mainly regulated by PDR1 and PDR3, were induced during the lag phase and the PDR gene family-centered functions, including specific and multiple functions involving cellular transport such as TPO1, TPO4, RSB1, PDR5, PDR15, YOR1, and SNQ2, promoted cellular adaptation and survival in order to cope with the inhibitor stress. Third, expressed genes involving degradation of damaged proteins and protein modifications such as SHP1 and SSA4, regulated by RPN4, HSF1, and other co-regulators, were necessary for yeast cells to survive and adapt the HMF stress. A deletion mutation strain Δrpn4 was unable to recover the growth in the presence of HMF.

Conclusions: Complex gene interactions and regulatory networks as well as co-regulations exist in yeast adaptation and tolerance to the lignocellulose derived inhibitor HMF. Both induced and repressed genes involving diversified functional categories are accountable for adaptation and energy rebalancing in yeast to survive and adapt the HMF stress during the lag phase of growth. Transcription factor genes YAP1, PDR1, PDR3, RPN4, and HSF1 appeared to play key regulatory rules for global adaptation in the yeast S. cerevisiae.

Figure 1

Yeast growth and metabolic profile response to HMF. Comparisons of cell growth and metabolic profiles of Saccharomyces cerevisiae NRRL Y-12632 between an HMF treatment (30 mM) and an untreated condition. A. Cell growth as measured by OD₆₀₀ for HMF treated condition (grey circle) and control (open circle). B. Glucose consumption (filled circle) and ethanol conversion (open circle) for HMF treated condition versus glucose (filled square) and ethanol (open square) for control. C. HMF (grey square) and its conversion product furandimethanol (FDM) (open square) for HMF treated condition versus HMF (grey triangle) and FDM (open triangle) for the control.

Figure 2

Transcriptome response to HMF during the lag phase. Hierarchical clustering of genes showing significant differential expression under HMF stress and displaying a 2-fold change for at least one time point compared with 0 h during the lag phase. Scales of the expression are indicated by an integrated color bar at the bottom.

Figure 3

Functional categories of repressed gene expression. Distribution of functional categories of repressed gene expressions by HMF treatment.

Figure 4

Expression response of important transcription factor genes. Expression patterns of seven selective genes encoding important transcription factors for positively regulating gene expression response to HMF stress.

Figure 5

DNA binding sites in promoter region. DNA binding sites for seven selective transcription factor genes YAP1, YAP5, YAP6, PDR1, PDR3, RPN4, and HSF1 in the promoter regions (from -1000 to -1)analyzed based on YEASTRACT.

Figure 6

Significant positive gene regulatory networks. Regulatory interaction networks between transcription factors (filled green and green arrows) and induced genes for functional reduction enzymes (filled blue and blue arrows), PDR gene family (filled orange and orange arrows), and proteasome function (filled pink and pink arrows). Scales of the expression are indicated by an integrated color bar at the right bottom corner.

Figure 7

Deletion mutants growth response to HMF. Cell growth of deletion mutations and the parental wild type BY4742 (WT) on SC medium without HMF (A) and in the presence of 15 mM HMF (B) as measured by OD₆₀₀ over time. Legend for each mutation is provided by a color code.

Figure 8

The qRT-PCR for PDR gene family. Expression abundance and gene interactions affected by deletion mutation Δpdr1 (A) and Δpdr3 (B) for selected PDR genes in response to HMF challenge compared with their parental wild type strain BY4742. Mean values are presented with error bars of standard deviations. Legend of value specificity is provided.

Figure 9

Pathways affected toward TCA cycle. Pathways involved in metabolisms of serine, alanine, proline, lysine, and arginine toward TCA cycle for ATP and NAD(P)H regeneration are significantly affected by HMF challenge. Bolded letters and arrowed lines indicate the levels of expressions and pathways are statistically significant. Enhanced expressions and pathways are in green and repressed in red. Black letters and arrowed lines indicate normal expressions and pathways.

Figure 10

Yeast response to HMF. A schematic diagram of gene regulatory networks involving selective genes and significant regulatory elements in yeast response to HMF stress.