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.
Overall design: The wild-type strain CBS 6938, the mutants strains FLAG.Sre1-tagged (CBS.FLAG.SRE1N and CBS.cyp61-.FLAG.SRE1) and the mutant CBS.sre1- of Xanthophyllomyces dendrorhous were grown under standard culture conditions, until the late exponential phase of growth for the extraction of RNA for RNA-seq assays and the performance of ChIP-exo assays. For each assay, three biological replicates of each strain were included. For the RNA-seq assays, RNA samples were shipped to Macrogen (Macrogen Inc., Seoul, South Korea) for library preparation and sequencing. For the ChIP-exo assays, formaldehyde-cross linked cell pellets were prepared and shipped to Peconic LLC (State College, PA, USA) for the library preparation and sequencing. Paired-end reads of the RNA-seq and ChIP-exo assays were mapped to the genome assembly of X. dendrorhous included in the BioProject ID: PRJNA637109.
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