Mining novel cis-regulatory elements from the emergent host Rhodosporidium toruloides using transcriptomic data

Abstract

The demand for robust microbial cell factories that produce valuable biomaterials while resisting stresses imposed by current bioprocesses is rapidly growing. Rhodosporidium toruloides is an emerging host that presents desirable features for bioproduction, since it can grow in a wide range of substrates and tolerate a variety of toxic compounds. To explore R. toruloides suitability for application as a cell factory in biorefineries, we sought to understand the transcriptional responses of this yeast when growing under experimental settings that simulated those used in biofuels-related industries. Thus, we performed RNA sequencing of the oleaginous, carotenogenic yeast in different contexts. The first ones were stress-related: two conditions of high temperature (37 and 42°C) and two ethanol concentrations (2 and 4%), while the other used the inexpensive and abundant sugarcane juice as substrate. Differential expression and functional analysis were implemented using transcriptomic data to select differentially expressed genes and enriched pathways from each set-up. A reproducible bioinformatics workflow was developed for mining new regulatory elements. We then predicted, for the first time in this yeast, binding motifs for several transcription factors, including HAC1, ARG80, RPN4, ADR1, and DAL81. Most putative transcription factors uncovered here were involved in stress responses and found in the yeast genome. Our method for motif discovery provides a new realm of possibilities in studying gene regulatory networks, not only for the emerging host R. toruloides, but for other organisms of biotechnological importance.

Keywords: RNA sequencing; biorefineries; cell factory; industrial stress; motifs; sugarcane juice; transcription factor.

Figure 1

Workflow for the discovery of cis-regulatory elements using transcriptomic data.

Figure 2

Venn diagram of differentially expressed genes (DEGs) for all conditions. The Venn diagram shows how many DEGs are shared between all conditions and how many are exclusive for each condition. Numbers outside of the Venn diagram represent the total of DEGs when compared to each respective control.

Figure 3

Enriched kyoto encyclopedia of genes and genomes (KEGG) pathways for Rhodosporidium toruloides grown on sugarcane juice (SCJ). Bubble map showing the biochemical pathways of R. toruloides noted by KEGG enriched in the SCJ condition, as obtained by the GAGE package. Pathways that have an enrichment value greater than 0 are up-regulated, while those that have a value less than 0 are down-regulated. Blue scale inside the bubbles represents the decreasing value of ps. The different sizes of the bubbles define the approximate number of DEGs in each biochemical pathway.

Figure 4

Enriched kyoto encyclopedia of genes and genomes (KEGG) pathways for R. toruloides grown at 42°C. Bubble map showing the biochemical pathways of R. toruloides noted by KEGG that are enriched in the 42°C condition, as obtained by the GAGE package. Pathways that have an enrichment value greater than 0 are up-regulated, while those that have a value less than 0 are down-regulated. Blue scale inside the bubbles represents the decreasing value of ps. The different sizes of the bubbles define the approximate number of DEGs in each biochemical pathway.

Figure 5

Eukaryotic orthologous groups (KOG) selected for TFBS finding. Percentage of DEGs compared to the total number of genes in the R. toruloides genome for each condition are shown as annotated using KOG. These are the most relevant conditions with the greatest amount of DEGs and were selected for further analysis. The remaining KOG annotations are shown in Supplementary Figure S5.

Figure 6

Examples of putative motifs aligned with transcription factors motifs from the JASPAR database using TOMTOM. (A) Top motif is the binding motif for ARG81 from Saccharomyces cerevisiae. Bottom motif is from SCJ condition. Eukaryotic orthologous groups (KOG) class: Amino acid transport and metabolism. Value of p: 1.08e–03. E-value: 1.51e + 00 (B) Top motif for AGR80 from S. cerevisiae. Bottom motif is from 42°C condition. KOG class: Amino acid transport and metabolism. Value of p: 4.98e–03. E-value: 8.76e–01. (C) Top motif for DAL80 from S. cerevisiae. Bottom motif is from ethanol 2% condition. KOG class: Amino acid transport and metabolism. value of p: 3.38e–02. E-value: 5.95e + 00. (D) Top for RPN4 from S. cerevisiae. Bottom motif is from 42°C condition. KOG class: Chromatin structure and dynamics. value of p: 4.15e–03. E-value: 7.31e–01. (E) Top motif for MAC1 from S. cerevisiae. Bottom motif is from ethanol 2% condition. KOG class: Secondary metabolites biosynthesis transport and catabolism. Value of p: 4.15e–03. E-value: 7.30e–01. (F) Top motif for HCM1 from S. cerevisiae. Bottom motif is from SCJ condition. KOG class: Chromatin structure and dynamics. Value of p: 3.44e–02. E-value: 6.05e + 00.

Figure 7

Graph of putative transcription factors and their putative binding sites. Graph showing interaction between putative transcription factors for R. toruloides with the transcription factors (TF) binding motifs predicted using HOMER. Green nodes represent the sugarcane juice condition, orange nodes represent the 42°C condition, dark green nodes are the 2% ethanol, and blue nodes are the 4% ethanol condition. Written inside nodes are the number of the TFBS and the name of the gene class annotated by eukaryotic orthologous groups (KOG) in which that motif was found. Pink nodes are the putative transcription FTs found using TOMTOM.