**5. Conclusions**

the transcript level of the *HMGR* gene in the *X. dendrorhous cyp61<sup>−</sup>*

wild-type strain [66]. Moreover, as for the *cyp61<sup>−</sup>*

precursors when sterol biosynthesis is affected.

utes to the regulation of carotenoid biosynthesis [19, 66].

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

and *cyp51*<sup>−</sup>

showed the best results enhancing the astaxanthin production [67].

enoid content in the *cyp61<sup>−</sup>*

76 Progress in Carotenoid Research

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].

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

*drorhous* increased the *HMGR* transcript levels. A possible explanation for the increased carot-

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 contrib-

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

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

mutation, the *cyp51<sup>−</sup>*

mutants could be the greater availability of carotenoid

mutant strains was higher

mutation in *X. den-*

*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 or their analogs.
