*4.1.3. Relation between carotenogenesis and sterol biosynthesis*

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

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*

expression and extracellular invertase activity (a known glucose repression target). To evalu-

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

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].

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

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

 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

mutation on carotenoid production, gene

mutant strain during almost all phases evaluated and the

mutation alleviate the glucose mediated repression

mutant strain, samples were taken

mutant strain strongly suggests

mutant strain, carotenogen-

of catabolic repression.

74 Progress in Carotenoid Research

was determined by evaluating the effect of a *mig1<sup>−</sup>*

content was higher in the *mig1<sup>−</sup>*

esis did not stop, suggesting that the *mig1<sup>−</sup>*

*mig1<sup>−</sup>*

ate whether carotenoid production is affected in the *mig1<sup>−</sup>*

The higher carotenoid production in the *X. dendrorhous mig1<sup>−</sup>*

carotenoid synthesis until 24 h later, while in the case of the *mig1<sup>−</sup>*

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 production in *X. dendrorhous* [19].

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, the transcript level of the *HMGR* gene in the *X. dendrorhous cyp61<sup>−</sup>* mutant strains was higher than in the wild-type strains. This could explain, at least in part, the increased carotenoid content in these mutants, since the synthesis of carotenoid precursors through the MVA pathway could be favored in these strains, showing an interaction between both biosynthetic pathways and a potential role of sterols in the regulation of carotenogenesis in *X. dendrorhous* [19].

protein synthesis while *bkt* gene expression was dependent on *de novo* protein synthesis [48]. It is still necessary to elucidate the different mechanisms of response to these molecules and therefore understand carotenogenic gene regulation in *H. pluvialis* and potentially enhance its

Microbiological Synthesis of Carotenoids: Pathways and Regulation

http://dx.doi.org/10.5772/intechopen.78343

77

*X. dendrorhous* and *H. pluvialis* are the most promising natural sources for the biological production of astaxanthin, which is used in several industrial applications. Almost all the genes and enzymes involved in the carotenogenesis pathways in both microorganisms are known. Currently, efforts have been directed in order to elucidate the regulatory mechanisms acting on carotenogenesis in these microorganisms. Studies show that multiple and complex carotenogenesis regulatory mechanisms are involved acting at transcriptional, post-transcriptional and translational level, and that they could be different in these microorganisms. Regarding *X. dendrorhous* carotenogenesis, there is evidence that suggest that it is regulated by catabolic repression and by sterols levels, while in *H. pluvialis*, carotenogenesis is induced under stress conditions and it is affected by numerous small molecules like plant hormones

capacity as a commercial astaxanthin producer.

**5. Conclusions**

or their analogs.

**Acknowledgements**

**Conflict of interest**

**Author details**

**References**

FONDECYT 1160202 and FONDECYT 1180520.

Authors declare that they have no conflicts of interest.

\*Address all correspondence to: jalcainog@uchile.cl

Universidad de Chile, Santiago, Chile

10.1186/1475-2859-13-12

Pamela Córdova, Marcelo Baeza, Víctor Cifuentes and Jennifer Alcaíno\*

[1] Mata-Gomez LC, Montanez JC, Mendez-Zavala A, Aguilar CN. Biotechnological production of carotenoids by yeasts: An overview. Microbial Cell Factories. 2014;**13**:12. DOI:

Similar to these results, other studies have demonstrated an increased astaxanthin production in *Phaffia rhodozyma* (anamorphic state of *X. dendrorhous*) when ergosterol levels were reduced by fluconazole treatment [65]. In a similar way, it has been described in *X. dendrorhous* that the mutation of the *CYP51* gene that encodes a cytochrome P450 monooxygenase that catalyzes the C14 demethylation of lanosterol during ergosterol biosynthesis, resulted in a reduced ergosterol production together with an increased carotenoid production compared to the wild-type strain [66]. Moreover, as for the *cyp61<sup>−</sup>* mutation, the *cyp51<sup>−</sup>* mutation in *X. dendrorhous* increased the *HMGR* transcript levels. A possible explanation for the increased carotenoid content in the *cyp61<sup>−</sup>* and *cyp51*<sup>−</sup> mutants could be the greater availability of carotenoid precursors when sterol biosynthesis is affected.

All together these results suggest that in *X. dendrorhous*, sterol levels, possible by a negative feedback mechanism, regulate at least the *HMGR* gene expression and in this way; it contributes to the regulation of carotenoid biosynthesis [19, 66].

#### **4.2. Regulation of carotenogenesis in** *H. pluvialis*

It has been extensively accepted that carotenoid synthesis in *H. pluvialis* is induced under stress conditions such as high light, salinity or carbon to nitrogen ratio [48, 67]. Regulation of the carotenogenic pathway in this microalga can be affected by numerous small molecules like plant hormones or similar compounds. In this context, among the hormones associated with stress response mechanisms and induction of astaxanthin synthesis in *H. pluvialis* are abscisic acid (ABA), jasmonic acid (JA), methyl jasmonate (MJ) or growth regulators like gibberellic acid (GA3), salicylic acid (SA) or brassinosteroid-2,4-epibrassinolide (EBR) [26]. It has been shown that all of these compounds affect the expression of numerous genes involved in astaxanthin synthesis, resulting in an up-regulation from 6- to 10-fold. Among them, SA showed the best results enhancing the astaxanthin production [67].

Studies at the mRNA levels of the carotenogenic genes: *ipi, psy, pds, crtO* and *crtR-b,* encoding the key enzymes of astaxanthin synthesis pathway, and its correlation with algal growth and astaxanthin production, suggested complex and multiple regulatory mechanisms that act at the transcriptional, translational and post-translational levels to regulate carotenogenesis in *H. pluvialis* [44]. Small molecules can exert different and multiple effects on several genes involved in the synthesis of astaxanthin. For example, when *H. pluvialis* was submitted to several nutrient stress conditions, it was observed that expression of carotenogenic genes encoding PSY, PDS, LCY, BKT and CrtR-b enzymes, were up-regulated under all the stress conditions studied. However, the extent of the transcript levels of carotenogenic genes varied among the stress conditions. Some of these genes, as *bkt* and *crtR-b*, were induced only transiently in some conditions. Moreover, studies using various inhibitors indicated that general carotenogenesis genes were regulated at transcriptional and translational levels. The induction of the general carotenoid synthesis genes showed to be independent of cytoplasmic protein synthesis while *bkt* gene expression was dependent on *de novo* protein synthesis [48]. It is still necessary to elucidate the different mechanisms of response to these molecules and therefore understand carotenogenic gene regulation in *H. pluvialis* and potentially enhance its capacity as a commercial astaxanthin producer.
