Transcription factor reveals interconnected regulation of carbon source utilization and carbon catabolite repression in an oleaginous yeast
Cells must sense and respond to available nutrients to survive. Microbes evolved mechanisms to activate genes necessary to utilize a carbon source specifically when it is present. To efficiently grow in mixed carbohydrate environments, microbes repress genes necessary to utilize carbon sources that require substantial resources to catabolize when a simpler carbon source is present, known as carbon catabolite repression. We investigated nutrient utilization in the basidiomycete, oleaginous yeast Rhodotorula (Rhodosporidium) toruloides. R. toruloides is a saprophytic fungus that utilizes the building blocks of complex polysaccharides found in plant cell walls. A transcription factor homologous to the cellulose degradation regulator CLR-2/ClrB in Ascomycete filamentous fungi, which we named Cbr1, is required for cellobiose utilization in R. toruloides. Although the role of CLR-2/ClrB is limited to regulating expression of genes involved in cellulose and hemicellulose utilization, Cbr1 is also involved in fucose and tricarboxylic acid cycle intermediate utilization. Additionally, Cbr1 inhibits carbon catabolite repression specifically during utilization of glucose-glucose disaccharides, which may have evolved to limit the glucose-mediated repression of genes encoding proteins that cleave disaccharides into glucose. In fungi, it is thought carbon source-specific transcription factors activate genes necessary for carbon source utilization, while carbon catabolite repression is regulated by transcription factors that broadly repress all nonpreferred carbon source utilization genes when a preferred carbon source is present. Our data provide evidence for a noncanonical carbon catabolite repression mechanism and suggest transcriptional mechanisms regulating carbon catabolite repression may be less broadly conserved than previously thought. Overall design: 3'mRNA-seq profiling of Rhodotorula (Rhodosporidium) toruloides wild type and cbr1 deletion strains exposed to fucose as the sole carbon source for 8h, wild type, cbr1 deletion, and bgl1bgl2 double deletion strains exposed to cellobiose as the sole carbon source for 4 hours, and wild type, cbr1 deletion, and tct1 deletion exposed to citrate as a sole carbon source for 24 hours. Reads were counted using a GFF3 file generated by the peaks2utr software v. 1.4.1 in order to improve annotation of 3'UTRs. Wild type BAM files from the above experiments, as well as wild type cells exposed to 1% succinate as the sole carbon source for 24 hours, and wild type cells exposed to Vogel's minimal media with NH4Cl and 2% glucose as the sole nitrogen and carbon sources, respectively, were combined and used as the input for peaks2utr. The GFF3 file generated by peaks2utr is included as a supplementary file. Each experiment was performed in biological triplicate.