*4.1.1. Catabolic repression: mechanism and components*

In microorganisms of free life, the availability of nutrients is in constant change and is the main factor regulating their growth and development. For yeasts, as for many other microorganisms, glucose is the preferred source of carbon and energy. Therefore, it is not surprising that glucose, in addition to its function as a nutrient, plays an important regulatory role in the metabolism of microorganisms. Thus, a high concentration of glucose in the medium resembles optimal growth conditions to the cellular machinery, causes the induction of various signal transduction pathways, and the activation or inactivation of various proteins. The regulatory role of glucose is more prominent at the transcription level and the general mechanism of catabolic repression involves a parallel decrease in the transcript levels of the target genes and, consequently, of the proteins they encode [52, 54].

Catabolic repression has been widely studied in *S. cerevisiae* and in general, genes that are under regulation by glucose repression encode enzymes that are involved in gluconeogenesis, Krebs cycle, glyoxylate cycle, respiration, mitochondrial development, uptake and metabolization of carbon sources alternative to glucose (such as the genes *GAL*, *SUC* and *MAL*) and high affinity glucose transporters [54, 55]. On the other hand, genes that encode a variety of transcriptional activators are also repressed by glucose. Finally, a large group of genes encoding proteins that are involved in the response to various types of stress highlights, as they have STRE elements (stress response element) in their promoter regions, which are also repressed in the presence of glucose [54].

the co-repressor complex Cyc8–Tup1 is considered as a global transcriptional co-repressor, because it regulates the expression of more than 180 genes, including those regulated by glucose. The proteins Cyc8 and Tup1 belong to evolutionarily highly conserved protein families, and similar repressors have been described in yeasts, worms, flies and mammals [60]. In *S. cerevisiae,* the *CYC8* and/or *TUP1* genes knock-out mutations are not lethal, but have pleiotropic effects causing slow growth, flocculation, sporulation and loss of certain aspects of

**Figure 3.** Catabolite repression mechanism in yeasts. Scheme of the principal components of the general catabolite repression mechanism. At high levels of glucose, Mig1 is dephosphorylated by phosphatase Glc7 and enters the nucleus where it recognizes regulatory sequences in the target gene promoters. Then, Mig1 recruits a co-repressor complex formed by Cyc8 and Tup1 that represses the expression of the target genes at transcriptional level. In absence of glucose, Mig1 is phosphorylated by the kinase complex Snf1, and loses its interaction with Cyc8-Tup1 complex, being exported

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73

In view of the diverse background that suggests that carotenogenesis is regulated by catabolic repression, recent studies have advanced characterizing the catabolic repression mechanism in *X. dendrorhous*, including its components and role in the regulation of carotenogenesis. Several evidences suggest a functional catabolic repression mechanism in *X. dendrorhous* [16, 56, 59]. For example, extracellular α-glucosidase and invertase activities were not detected in *X. dendrorhous* cultures when glucose was used as a carbon source, suggesting

glucose repression, among others [52, 60, 61].

to the cytoplasm [52].

*4.1.2. Catabolic repression and carotenogenesis regulation in X. dendrorhous*

One of the ways in which glucose influences gene expression is by facilitating the action of negative regulators [52], among them, the Mig1 factor (homologous to CreA in *A. nidulans*) is a DNA binding protein that recognizes and binds to specific sequences called "Mig1 boxes" (with the consensus sequence (G/C)(C/T)GGGG)) in the promoters of the target genes. Several studies have identified genes potentially encoding Mig1 in yeasts like *Kluyveromyces lactis*, *Kluyveromyces marxianus*, *Schizosaccharomyces pombe*, *Candida albicans* [52] and recently in *X. dendrorhous* [56].

In general, glucose repression in *S. cerevisiae* (**Figure 3**) mainly depends on the subcellular localization of the Mig1 regulator. At high glucose levels, the repressor Mig1 is dephosphorylated and localizes at the nucleus where it recognizes and binds to "Mig1 boxes" in the promoter region of the target genes. Then, Mig1 recruits a co-repressor complex formed by the Cyc8 and Tup1 proteins that represses the transcription of the target genes. In the absence of glucose, Mig1 is phosphorylated by the Snf1 kinase complex, loses its interaction with the Cyc8–Tup1 complex, and it is exported to the cytoplasm [57–59]. In *S. cerevisiae*,

It is well known that glucose has a global effect on cellular metabolism; generally, when this sugar is present in the culture medium, the expression of genes involved in the metabolism of alternative to glucose carbon sources and secondary metabolism is repressed [52]. This phenomenon is known as "catabolic repression" or "repression by glucose" [53] and could be responsible for the repression of carotenogenic genes in *X. dendrorhous* during fermentative

In microorganisms of free life, the availability of nutrients is in constant change and is the main factor regulating their growth and development. For yeasts, as for many other microorganisms, glucose is the preferred source of carbon and energy. Therefore, it is not surprising that glucose, in addition to its function as a nutrient, plays an important regulatory role in the metabolism of microorganisms. Thus, a high concentration of glucose in the medium resembles optimal growth conditions to the cellular machinery, causes the induction of various signal transduction pathways, and the activation or inactivation of various proteins. The regulatory role of glucose is more prominent at the transcription level and the general mechanism of catabolic repression involves a parallel decrease in the transcript levels of the target

Catabolic repression has been widely studied in *S. cerevisiae* and in general, genes that are under regulation by glucose repression encode enzymes that are involved in gluconeogenesis, Krebs cycle, glyoxylate cycle, respiration, mitochondrial development, uptake and metabolization of carbon sources alternative to glucose (such as the genes *GAL*, *SUC* and *MAL*) and high affinity glucose transporters [54, 55]. On the other hand, genes that encode a variety of transcriptional activators are also repressed by glucose. Finally, a large group of genes encoding proteins that are involved in the response to various types of stress highlights, as they have STRE elements (stress response element) in their promoter regions, which are

One of the ways in which glucose influences gene expression is by facilitating the action of negative regulators [52], among them, the Mig1 factor (homologous to CreA in *A. nidulans*) is a DNA binding protein that recognizes and binds to specific sequences called "Mig1 boxes" (with the consensus sequence (G/C)(C/T)GGGG)) in the promoters of the target genes. Several studies have identified genes potentially encoding Mig1 in yeasts like *Kluyveromyces lactis*, *Kluyveromyces marxianus*, *Schizosaccharomyces pombe*, *Candida albicans* [52] and recently in *X.* 

In general, glucose repression in *S. cerevisiae* (**Figure 3**) mainly depends on the subcellular localization of the Mig1 regulator. At high glucose levels, the repressor Mig1 is dephosphorylated and localizes at the nucleus where it recognizes and binds to "Mig1 boxes" in the promoter region of the target genes. Then, Mig1 recruits a co-repressor complex formed by the Cyc8 and Tup1 proteins that represses the transcription of the target genes. In the absence of glucose, Mig1 is phosphorylated by the Snf1 kinase complex, loses its interaction with the Cyc8–Tup1 complex, and it is exported to the cytoplasm [57–59]. In *S. cerevisiae*,

metabolism.

72 Progress in Carotenoid Research

*4.1.1. Catabolic repression: mechanism and components*

genes and, consequently, of the proteins they encode [52, 54].

also repressed in the presence of glucose [54].

*dendrorhous* [56].

**Figure 3.** Catabolite repression mechanism in yeasts. Scheme of the principal components of the general catabolite repression mechanism. At high levels of glucose, Mig1 is dephosphorylated by phosphatase Glc7 and enters the nucleus where it recognizes regulatory sequences in the target gene promoters. Then, Mig1 recruits a co-repressor complex formed by Cyc8 and Tup1 that represses the expression of the target genes at transcriptional level. In absence of glucose, Mig1 is phosphorylated by the kinase complex Snf1, and loses its interaction with Cyc8-Tup1 complex, being exported to the cytoplasm [52].

the co-repressor complex Cyc8–Tup1 is considered as a global transcriptional co-repressor, because it regulates the expression of more than 180 genes, including those regulated by glucose. The proteins Cyc8 and Tup1 belong to evolutionarily highly conserved protein families, and similar repressors have been described in yeasts, worms, flies and mammals [60]. In *S. cerevisiae,* the *CYC8* and/or *TUP1* genes knock-out mutations are not lethal, but have pleiotropic effects causing slow growth, flocculation, sporulation and loss of certain aspects of glucose repression, among others [52, 60, 61].

#### *4.1.2. Catabolic repression and carotenogenesis regulation in X. dendrorhous*

In view of the diverse background that suggests that carotenogenesis is regulated by catabolic repression, recent studies have advanced characterizing the catabolic repression mechanism in *X. dendrorhous*, including its components and role in the regulation of carotenogenesis. Several evidences suggest a functional catabolic repression mechanism in *X. dendrorhous* [16, 56, 59]. For example, extracellular α-glucosidase and invertase activities were not detected in *X. dendrorhous* cultures when glucose was used as a carbon source, suggesting catabolic repression of these enzymes [62, 63]. This indicates that this regulatory mechanism is operative in *X. dendrorhous*, as genes encoding glycosyl hydrolases are well known targets of catabolic repression.

analyzed promoter regions [56]. To further address the functional consequences of the *mig1<sup>−</sup>* mutation in *X. dendrorhous*, extracellular invertase activity was evaluated in the wild-type and mutant strains. Invertase (encoded by the *INV* gene) catalyzes the utilization of sucrose as carbon source and it is under catabolic repression in presence of glucose [52]. When invertase

*INV* gene which was evidenced by a higher invertase activity in the mutant strain compared

As mentioned before, the Mig1 factor recruits a co-repressor complex formed by the Cyc8 and Tup1 proteins to perform repression at transcriptional level. Thus, in other study, the role of these co-repressors in catabolic repression and carotenogenesis was assessed in *X. dendrorhous* [59]. The *CYC8* and *TUP1* genes were identified in *X. dendrorhous* and similar analyses to those described for *MIG1*, were performed. These analyses showed that the *cyc8<sup>−</sup>*

at the stationary phase of growth that were approximately 90 and 40% higher in the mutant

the sole carbon source [59]. Also, the participation of these genes (*CYC8* and *TUP1*) in the

led to a derepression of the *INV* gene and to a higher invertase activity in presence of glucose, compared to the wild-type strain [59]. Furthermore, it was demonstrated that similarly to the

carotenogenesis, since these mutant strains continuously produced carotenoids, even when

So, many studies support the hypothesis that carotenogenesis in *X. dendrorhous* is regulated

Ergosterol is the main sterol in fungi, fulfilling similar roles as cholesterol in mammalian cells. Since the synthesis of ergosterol and of fatty acids derive from the same precursors as the synthesis of astaxanthin in *X. dendrorhous*, some studies have focused in the interaction between these related pathways. In Ref. [65] was reported that an astaxanthin overproducing strain obtained by random mutagenesis had a decreased production of ergosterol and of fatty acids, which could lead to precursor accumulation favoring the astaxanthin biosynthesis. Moreover, it was also observed higher transcript levels of carotenogenic genes (*crtI*, *crtYB* and *crtS*) in this strain [65]. In the same line, it was shown that the disruption of the C22-sterol desaturase gene (*CYP61*), involved in one of the last steps of ergosterol synthesis, enhanced carotenoid

In *Cryptococcus neoformans* and mammalian cells, the expression of the 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) encoding gene (*HMGR* gene) that is involved in the synthesis of IPP trough the MVA pathway, is regulated by sterol levels [19]. In accordance,

repression of invertase activity was demonstrated, showing that *cyc8<sup>−</sup>*

and *tup1<sup>−</sup>*

*4.1.3. Relation between carotenogenesis and sterol biosynthesis*

glucose was added to the culture medium [59].

by the catabolic repression mechanism.

production in *X. dendrorhous* [19].

mutations increased the specific carotenoid production, reaching production levels

, respectively, compared to the wild-type when glucose was used as

mutations alleviated the glucose mediated repression of

mutant strain of *X. dendrorhous* cultured

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activity was determined in the wild-type and *mig1<sup>−</sup>*

to the wild-type under these conditions [56].

and *tup1<sup>−</sup>*

mutation, the *cyc8<sup>−</sup>*

and *tup1<sup>−</sup>*

*mig1<sup>−</sup>*

strains *cyc8<sup>−</sup>*

in the presence of glucose, it was observed that the *mig1<sup>−</sup>*

Moreover, according to descriptions of other yeasts, one consequence of catabolic repression is the preferred use of glucose over other alternative carbon sources, deferring their use until glucose has been completely consumed. The preferential use of glucose over an alternative carbon source can be evidenced by a characteristic growth rate change of the microbial culture, known as diauxic growth [59, 64]. This aspect has been evaluated in *X. dendrorhous* through experiments in which the yeast was grown in the presence of glucose and a nonpreferred carbon source such as glycerol or sucrose. In both cases, a diauxic-type growth curve was obtained when the yeast was cultivated with both carbon sources simultaneously, indicating the change in the used carbon source and supporting a functional catabolic repression mechanism in *X. dendrorhous* [56, 59]. On the other hand, genes encoding the principal components of the catabolic repression mechanism has been identified and characterized in *X. dendrorhous.* Among them, a gene (*MIG1*) encoding the Mig1 transcriptional factor was identified [56]. The functionality of this gene was assessed by heterologous complementation in *S. cerevisiae*, showing that the protein encoded by the *MIG1* gene is functional and capable of mediating glucose repression [56]. Also, the function of Mig1 in *X. dendrorhous* was determined by evaluating the effect of a *mig1<sup>−</sup>* mutation on carotenoid production, gene expression and extracellular invertase activity (a known glucose repression target). To evaluate whether carotenoid production is affected in the *mig1<sup>−</sup>* mutant strain, samples were taken from cultures of the wild-type and mutant strains grown in presence of glucose at 5 different time-points representatives of different phases of growth. It was observed that the carotenoid content was higher in the *mig1<sup>−</sup>* mutant strain during almost all phases evaluated and the total carotenoid content at the final phase of growth that was evaluated (stationary phase of growth), was approximately 20% higher in the mutant strain compared to the wild-type [56]. The higher carotenoid production in the *X. dendrorhous mig1<sup>−</sup>* mutant strain strongly suggests a role of Mig1 in the regulation of carotenogenesis in this yeast. Also, in a complementary approach it was demonstrated that when glucose was added to a culture that was previously deprived of this sugar, the carotenoid synthesis stopped in the wild-type strain showing no carotenoid synthesis until 24 h later, while in the case of the *mig1<sup>−</sup>* mutant strain, carotenogenesis did not stop, suggesting that the *mig1<sup>−</sup>* mutation alleviate the glucose mediated repression of carotenogenesis [56]. Also, Marcoleta et al. showed that the addition of glucose decreased the transcript levels of the carotenogenic genes in the wild-type strain [16]. Meanwhile, the *mig1<sup>−</sup>* mutation reverts this repression at the transcriptional level [56]. Additionally, by bioinformatic analysis, possible "Mig1 boxes" were identified in the promoter regions of the carotenogenic genes *crtI*, *crtYB* and *crtS*, precisely those in which a repressing effect of glucose at the transcriptional level was also observed [16]. To confirm whether the *X. dendrorhous MIG1* gene product binds to DNA containing Mig1 boxes, Electrophoretic Mobility Shift Assays (EMSAs) were performed using biotin-labeled DNA fragments of the promoter regions of the *crtI*, *crtYB* and *crtS* containing the potential Mig1 boxes. The results indicated that the Mig1 factor of *X. dendrorhous* is capable of binding specifically to the "Mig1 boxes" present in the analyzed promoter regions [56]. To further address the functional consequences of the *mig1<sup>−</sup>* mutation in *X. dendrorhous*, extracellular invertase activity was evaluated in the wild-type and mutant strains. Invertase (encoded by the *INV* gene) catalyzes the utilization of sucrose as carbon source and it is under catabolic repression in presence of glucose [52]. When invertase activity was determined in the wild-type and *mig1<sup>−</sup>* mutant strain of *X. dendrorhous* cultured in the presence of glucose, it was observed that the *mig1<sup>−</sup>* mutation caused derepression of the *INV* gene which was evidenced by a higher invertase activity in the mutant strain compared to the wild-type under these conditions [56].

As mentioned before, the Mig1 factor recruits a co-repressor complex formed by the Cyc8 and Tup1 proteins to perform repression at transcriptional level. Thus, in other study, the role of these co-repressors in catabolic repression and carotenogenesis was assessed in *X. dendrorhous* [59]. The *CYC8* and *TUP1* genes were identified in *X. dendrorhous* and similar analyses to those described for *MIG1*, were performed. These analyses showed that the *cyc8<sup>−</sup>* and *tup1<sup>−</sup>* mutations increased the specific carotenoid production, reaching production levels at the stationary phase of growth that were approximately 90 and 40% higher in the mutant strains *cyc8<sup>−</sup>* and *tup1<sup>−</sup>* , respectively, compared to the wild-type when glucose was used as the sole carbon source [59]. Also, the participation of these genes (*CYC8* and *TUP1*) in the repression of invertase activity was demonstrated, showing that *cyc8<sup>−</sup>* and *tup1<sup>−</sup>* mutations led to a derepression of the *INV* gene and to a higher invertase activity in presence of glucose, compared to the wild-type strain [59]. Furthermore, it was demonstrated that similarly to the *mig1<sup>−</sup>* mutation, the *cyc8<sup>−</sup>* and *tup1<sup>−</sup>* mutations alleviated the glucose mediated repression of carotenogenesis, since these mutant strains continuously produced carotenoids, even when glucose was added to the culture medium [59].

So, many studies support the hypothesis that carotenogenesis in *X. dendrorhous* is regulated by the catabolic repression mechanism.
