Role of ROX1, SKN7, and YAP6 Stress Transcription Factors in the Production of Secondary Metabolites in Xanthophyllomyces dendrorhous

Abstract

Xanthophyllomyces dendrorhous is a natural source of astaxanthin and mycosporines. This yeast has been isolated from high and cold mountainous regions around the world, and the production of these secondary metabolites may be a survival strategy against the stress conditions present in its environment. Biosynthesis of astaxanthin is regulated by catabolic repression through the interaction between MIG1 and corepressor CYC8–TUP1. To evaluate the role of the stress-associated transcription factors SKN7, ROX1, and YAP6, we employed an omic and phenotypic approach. Null mutants were constructed and grown in two fermentable carbon sources. The yeast proteome and transcriptome were quantified by iTRAQ and RNA-seq, respectively. The total carotenoid, sterol, and mycosporine contents were determined and compared to the wild-type strain. Each mutant strain showed significant metabolic changes compared to the wild type that were correlated to its phenotype. In a metabolic context, the principal pathways affected were glycolysis/gluconeogenesis, the pentose phosphate (PP) pathway, and the citrate (TCA) cycle. Additionally, fatty acid synthesis was affected. The absence of ROX1 generated a significant decline in carotenoid production. In contrast, a rise in mycosporine and sterol synthesis was shown in the absence of the transcription factors SKN7 and YAP6, respectively.

Unraveling the Molecular Basis of Mycosporine Biosynthesis in Fungi

“…In the case of astaxanthin biosynthesis, it has been observed that mutations in the MIG1 , CYC8, and TUP1 genes lead to an increase in carotenoid production and could be involved in the regulation of catabolic glucose repression [ 29 , 30 , 38 ]. In addition, it has been observed that the products of the CYC8 and TUP1 genes constitute a complex (Cyc8-Tup1) that interacts with different TFs, modifying the expression of other structural genes [ 38 , 39 , 40 ].…”

“…Recognition sequences of TFs involved in the stress response were observed in the IGR1 and IGR2 regions of the MYC cluster, and mycosporine production was measured in deletion mutants of the ROX1 , SKN7 , YAP6 , and OPI1 genes. Mycosporine production was also investigated in deletion mutants of the MIG1 , CYC8 , and TUP1 genes, which, in S. cerevisiae , participate in glucose repression through the Cyc8-Tup1 complex [ 41 ], and in P. rhodozyma , participate in the regulation of the production of secondary metabolites such as astaxanthin [ 29 , 30 ] and mycosporines [ 40 ].…”

“…In S. cerevisiae , Skn7 interacts with the Cyc8–Tup1 complex, recruiting Tup1 for optimal induction of genes for oxidative stress response, osmoregulation, hypoxia response and induction of heat shock proteins in response to oxidative stress [ 41 , 42 , 43 ]. In addition, an RNA-seq comparative analysis of CYC8 and TUP1 gene deletion mutant strains was carried out based on previous results [ 39 , 40 ]. This analysis showed that the expression levels of the messengers of the DDGS and OMT genes were 5.7- and 2.9-fold, and 2- and 1.7-fold higher, respectively, than those in the wild-type parental strain.…”

“…To determine the biological processes that may account for the changes in mycosporine production observed, RNA-seq and proteomic data from previous studies were analyzed [ 39 , 40 ], focusing on mycosporine-related pathways ( Table S2 ). The enzyme 6-phosphogluconate dehydrogenase (Pgd) was more abundant in the Δ cyc8 −/− mutant strain than in the wild-type strain, and the DDGS gene was upregulated ( Figure 7 A).…”

“…Pigments were extracted from cellular pellets by acetone extraction [ 40 ]. In brief, aliquots of cell cultures were collected under the different conditions tested.…”