Abstract
The sterol regulatory element-binding protein (SREBP) pathway is an integral cellular mechanism that regulates lipid homeostasis, in which transcriptional activator SREBPs regulate the expression of various genes. In the carotenogenic yeast Xanthophyllomyces dendrorhous, Sre1 (the yeast SREBP homolog) regulates lipid biosynthesis and carotenogenesis, among other processes. Despite the characterization of several components of the SREBP pathway across various eukaryotes, the specific elements of this pathway in X. dendrorhous remain largely unknown. This study aimed to explore the potential regulatory mechanisms of the SREBP pathway in X. dendrorhous using the strain CBS.cyp61- as a model, which is known to have Sre1 in its active state under standard culture conditions, resulting in a carotenoid-overproducing phenotype. This strain was subjected to random mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (NTG), followed by a screening methodology that focused on identifying mutants with altered Sre1 activation phenotypes. Single-nucleotide polymorphism (SNP) analysis of 20 selected mutants detected 5439 single-nucleotide variants (SNVs), narrowing them down to 1327 SNPs of interest after a series of filters. Classification based on SNP impact identified 116 candidate genes, including 49 genes with high impact and 68 genes with deleterious moderate-impact mutations. BLAST, InterProScan, and gene ontology enrichment analyses highlighted 25 genes as potential participants in regulating Sre1 in X. dendrorhous. The key findings of this study include the identification of genes potentially encoding proteins involved in protein import/export to the nucleus, sterol biosynthesis, the ubiquitin-proteasome system, protein regulatory activities such as deacetylases, a subset of kinases and proteases, as well as transcription factors that could be influential in SREBP regulation. These findings are expected to significantly contribute to the current understanding of the intricate regulation of the transcription factor Sre1 in X. dendrorhous, providing valuable groundwork for future research and potential biotechnological applications.