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Sterol Regulatory Element-Binding Protein Sre1 regulates carotenogenesis in the red yeast Xanthophyllomyces dendrorhous.

ABSTRACT: Xanthophyllomyces dendrorhous is a basidiomycete yeast that produces carotenoids, mainly astaxanthin. Astaxanthin is an organic pigment of commercial interest due to its antioxidant and coloring properties. X. dendrorhous has a functional Sterol Regulatory Element-Binding Protein (SREBP) pathway, and the Sre1 protein is the SREBP homolog in this yeast. However, how Sre1 is promoting the biosynthesis of sterols and carotenoids in X. dendrorhous is unknown. In this work, comparative RNA-seq analysis between modified X. dendrorhous strains that have an active Sre1 protein and the wild-type was performed to identify Sre1-dependent genes. In addition, Sre1 direct target genes were identified through chromatin immunoprecipitation combined with lambda exonuclease digestion (ChIP-exo) assays. SRE motifs were detected in the promoter regions of several Sre1 direct target genes and were consistent with the SREs described in other yeast species. Sre1 directly regulates genes related to ergosterol biosynthesis as well as genes related to the mevalonate pathway, which synthesizes the building blocks of isoprenoids, including carotenoids. Two carotenogenic genes, crtE and crtR, were also identified as Sre1 direct target genes. Thus, carotenogenesis in X. dendrorhous is regulated by Sre1 through the regulation of the mevalonate pathway and the regulation of the crtE and crtR genes. As the crtR gene encodes a cytochrome P450 reductase, Sre1 regulates pathways that include cytochrome P450 enzymes, such as the biosynthesis of carotenoids and sterols. These results demonstrate that Sre1 is a sterol master regulator that is conserved in X. dendrorhous.

ORGANISM(S): Phaffia rhodozyma

PROVIDER: GSE152739 | GEO | 2020/09/29

REPOSITORIES: GEO

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Project description:Regulation of iron acquisition genes is critical for microbial survival under both iron-limiting conditions (to acquire essential iron) and iron-replete conditions (to limit iron toxicity). In fungi, iron acquisition genes are repressed under iron-replete conditions by a conserved GATA transcriptional regulator. Here we investigate the role of this transcription factor, Sre1, in the cellular responses of the fungal pathogen Histoplasma capsulatum to iron. We showed that cells in which SRE1 levels were diminished by RNA interference were unable to repress siderophore biosynthesis and utilization genes in the presence of abundant iron, and thus produced siderophores even under iron-replete conditions. Mutation of a GATA-containing consensus site found in the promoters of these genes also resulted in inappropriate gene expression under iron-replete conditions. Microarray analysis comparing control and SRE1-depleted strains under conditions of iron limitation or abundance revealed both iron-responsive genes and Sre1-dependent genes, which comprised distinct but overlapping sets. Iron-responsive genes included putative oxidoreductases, metabolic and mitochondrial enzymes, superoxide dismutase, and nitrosative-stress response genes; Sre1-dependent genes were of diverse function. Genes regulated by iron levels and Sre1 included all of the siderophore biosynthetic genes, a gene involved in reductive iron acquisition, an iron-responsive transcription factor, and two catalases. Based on transcriptional profiling and phenotypic analyses, we conclude that Sre1 plays a critical role in the regulation of both traditional iron-responsive genes and iron-independent pathways such as regulation of cell morphology. These data highlight the evolving realization that the effect of Sre1 orthologs on fungal biology extends beyond the iron regulon. For microarray studies, initial cultures of HcLH120 (Control RNAi-1) or 123 (SRE1 RNAi-2) yeast cells were grown in 5 mL HMM, and then passaged 1:25 into 100 mL HMM. After 2 days of growth, the cultures were pelleted, washed in 100 mL of PBS, resuspended in 100 mL of mRPMI pH 6.5 and diluted to an OD600~2 in 1 L of mRPMI. After 24 hours of growth, 200 mL of culture was harvested for each of the three zero time points. Then the cultures were split into 2 X 400 mL, and 10 uM FeSO4 (final concentration) was added to one set of cultures. At each time point (.5, 1, 4, or 8 hours), 100 mL of culture was harvested for RNA extraction.

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Sterol Regulatory Element-Binding Protein Sre1 regulates carotenogenesis in the red yeast Xanthophyllomyces dendrorhous.

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