**4.1. Regulation of carotenogenesis in** *X. dendrorhous*

first stage of the pathway) pathway [38–40]. Previous studies have shown that in *H. pluvialis*, the intermediate IPP most probably derives from the MEP pathway as it lacks three key enzymes of the mevalonate pathway involved in the formation of IPP from acetoacetyl-CoA [41]. To date, the enzymes required for the conversion of photosynthesis derived products i.e., pyruvate and glyceraldehyde-3-phosphate into isopentenyl pyrophosphate through the DOXP pathway inside *H. pluvialis* chloroplasts has been extensively studied [41], being this,

The carotenogenic pathway described for *H. pluvialis* is presented in **Figure 2**. As mentioned before, the astaxanthin synthesis precursor IPP derives from the DOXP (or MEP) pathway. As in *X dendrorhous*, the first step is the isomerization of IPP to DMAPP. It has been long assumed that this conversion was catalyzed exclusively by isopentenyl pyrophosphate isomerase (IPI, encoded by *ipi* genes in *H. pluvialis*) [39, 42]. However, recent transcriptomic studies suggest that neither of the two *ipi* genes of *H. pluvialis* (*ipi1* and *ipi2*) are upregulated during cellular accumulation of astaxanthin [41]. On the contrary, suggestions have been made that another enzyme of similar activity, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) is more likely to be responsible for catalyzing the interconversion between IPP and DMAPP [41]. However, the contribution of these three enzymes to this step of astaxanthin biosynthesis is still unclear. As in *X. dendrorhous*, the isoprenoid chain is elongated by the addition of a molecule of DMAPP and subsequent additions of three molecules of IPP, being these steps catalyzed by the geranylgeranyl pyrophosphate synthase (GGPS) enzyme giving rise to GGPP [43]. The first committed step of carotenoid synthesis is the formation of phytoene from two molecules of GGPP which are condensed in a head-to-tail manner by the enzyme phytoene synthase (PSY) [43]. It must be noted that the same step in *X. dendrorhous* is catalyzed by the bifunctional enzyme PBS [28]. Then, phytoene is desaturated four times. In *H. pluvialis*, these steps involve two phytoene desaturases (PDS) and a ζ-carotene desaturase (ZDS), and two plastid terminal oxidases (PTOX1, PTOX2) acting as co-factors [44, 45], giving as final product the red colored carotene lycopene [43]. These steps constitute other difference with the synthesis of carotenoids in *X. dendrorhous*, where a single enzyme catalyzes the four desaturations necessary for the synthesis of lycopene from phytoene [34]. Both termini of lycopene are cyclized by lycopene cyclases (LCY-e and LCY-b). In most organisms, cyclization of an extreme of lycopene results in the production of α-carotene (precursor of lutein) and β-carotene (precursor of astaxanthin, among others). In *H. pluvialis,* the carbon flux is directed mainly through the production of β-carotene [41]. The final oxygenation steps that lead to astaxanthin from β-carotene are catalyzed by two different enzymes: a β-carotene ketolase (BKT) and a β-carotene hydroxylase (CrtR-b) [46–48]. This is another difference with the astaxanthin synthesis in *X. dendrorhous*, in which the astaxanthin synthase yields the

the most likely source of IPP in *H. pluvialis* cells.

70 Progress in Carotenoid Research

hydroxylation and ketolation of the β-carotene β-ionone rings [29].

**4. Potential mechanisms that regulate the synthesis of astaxanthin**

The astaxanthin synthesis pathway has been extensively studied, and most of genes and enzymes involved are currently known. More recent studies have focused on elucidating the possible regulation mechanisms of carotenogenesis. In the case of yeast *X. dendrorhous*, special An important function of astaxanthin in *X. dendrorhous* is the inactivation of singlet and oxygen radicals, which is consistent with the fact that astaxanthin production increases in the presence of these reactive oxygen species [5, 23]. In addition, it has been observed that high light intensity inhibits the growth of the yeast and the content of carotenoids. However, at low light intensities, light has a positive regulatory effect on the synthesis of carotenoids [5].

It is known that *X. dendrorhous* is able to grow using various carbon sources, among them: glucose, sucrose, maltose, xylose, starch, succinate, glycerol and ethanol. Several studies have shown that there is a relationship between the carbon source used by the yeast and the synthesis of carotenoids. This effect is observed in both: in the amount of total pigments and in their composition [16, 17].

As in other yeasts, *X. dendrorhous* is capable of carrying out two types of metabolisms depending on the carbon source that is present in the culture medium: i) a fermentative and ii) an aerobic metabolism. In previous studies, it has been shown that astaxanthin production decreases during fermentative metabolism (in presence of fermentable carbon sources as glucose or fructose) and it increases during aerobic metabolism (with non-fermentable carbon sources as succinate or ethanol) [16, 17, 49]. Also, it has been observed that carotenoid content is significantly higher when *X. dendrorhous* is cultivated in complete medium (YM) supplemented with different non-fermentable carbon sources (xylose, succinate, sodium acetate, glycerol and ethanol), compared with the carotenoid content when the yeast is cultured in presence of glucose [17]. In cultures supplemented with glucose as the sole carbon source, carotenogenesis is induced only after the culture medium runs out of glucose. While in cultures using succinate as the sole carbon source, it was observed that the production of carotenoids coincides with the growth of the yeast, increasing steadily until reaching the stationary phase of growth. This shows that the production of carotenoids starts earlier and it is higher when a non-fermentable carbon source is used in cultures [17].

On the other hand, studies of carotenogenic gene transcripts (*crtI*, *crtYB* and *crtS*), show that their levels reach their maximum value at the late exponential phase of growth coinciding with the induction of carotenogenesis, the exhaustion of glucose in the medium and with the beginning of the consumption of ethanol produced as result of sugar fermentation [17, 49]. It has also been observed that the addition of glucose to the culture medium decreases the transcript levels of genes *crtYB*, *crtI* and *crtS*, which correlates with a complete inhibition of pigment synthesis. On the other hand, the addition of ethanol to the culture medium of the yeast causes an induction of the expression of the *crtYB* and *crtS* genes, and promotes the synthesis of carotenoids [16]. Furthermore, the promoter region of the *crtS* gene contains four potential CreA binding motifs [50], which is a negative regulator involved in glucose repression in *Aspergillus nidulans* [51]. According to this background, it is clear that glucose causes suppression of carotenogenesis in *X. dendrorhous.*

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 metabolism.
