**3.2. Biosynthesis of astaxanthin in the microalgae** *H. pluvialis*

Several algae and microalgae are able to produce astaxanthin, but *H. pluvialis* is the dominant specie for commercial astaxanthin [22]. *H. pluvialis* contains 1.5–3% of (3S, 3′S)-astaxanthin by dry weight, mainly as monoesters [37]. The isomer (3S, 3′S)-astaxanthin is the preferred form for human applications, for this reason, *H. pluvialis* is an attractive natural source of this pigment [5].

For the production of pigments, the microalgae *H. pluvialis* is usually grown in a two-stage batch process [5]. The first stage (green stage) is necessary to obtain enough biomass for an efficient carotenoid production. In this stage, microalgae are grown in presence of sufficient nutrient supply, optimum pH and temperature, and low irradiation. Since, the astaxanthin synthesis is induced under stress conditions, the second stage of growth (the reddening stage), consists in exposing the cells to stress conditions such as high light irradiation (sun light), nutrient deprivation (mainly nitrogen and phosphate deprivation), and high temperature and/or high salt concentration [26].

As in *X. dendrorhous*, the biosynthesis of astaxanthin in *H. pluvialis* also begins with the synthesis of IPP, which is a key intermediate of carotenoid synthesis. In general, and depending on the organism, IPP may be produced by two different pathways, the i) MVA pathway (cytosolic) and a ii) pathway located in the chloroplast known as the MEP (Methylerythritol 4-phosphate) or as the DOXP (due to the formation of 1-deoxy-D-xylulose-5-phosphate in the

geranylgeranyl pyrophosphate.

**Figure 2.** Overview of *H. pluvialis* carotenogenesis. The steps for astaxanthin biosynthesis from IPP, generated via the MEP pathway, in *H. pluvialis* are shown. Enzymes involved in each step are listed at left and the corresponding genes are indicated in parenthesis. GA-3P: glyceraldehyde 3-phosphate; DOXP: 1-deoxy-D-xylulose-5-phosphate; MEP: methylerythritol 4-phosphate; IPP: isopentenyl pyrophosphate; DMAPP: dimethylallyl pyrophosphate; GGPP:

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*X. dendrorhous* [28–30]. Many studies have attempted to improve astaxanthin production in *X. dendrorhous*, contributing to our current knowledge of the genetic control of carotenogenesis in this yeast (**Figure 1**). As in other eukaryotes, the synthesis of carotenoids in *X. dendrorhous* derives from the MVA pathway. MVA is formed by the condensation of three molecules of acetyl-CoA [31], followed by two phosphorylation reactions by two different kinases and

ing block of all isoprenoids [5, 20, 31]. IPP is isomerized to dimethylallyl pyrophosphate (DMAPP) by the IPP isomerase, encoded by the *idi* gene [32]. In the next steps, a molecule of DMAPP is sequentially condensed with three molecules of IPP to generate geranylgeranyl pyrophosphate (GGPP, C20); these steps involve the prenyl transferase enzymes farnesyl pyrophosphate synthase and geranylgeranyl pyrophosphate synthase, encoded by the *FPS* and *crtE* genes, respectively [33]. Subsequently, by means of a bifunctional enzyme phytoeneβ-carotene synthase (PBS) with two activities (lycopene cyclase and phytoene synthase), that in fungi are encoded by the same gene (*crtYB* in the case of *X. dendrorhous*), two molecules of GGPP are condensed giving rise to phytoene (C40), the first carotenoid of the pathway, which is colorless [28]. Then, phytoene undergoes four desaturation reactions catalyzed by a single enzyme phytoene desaturase (PDS, product of the *crtI* gene) forming lycopene (a red pigment) [5, 34]. The latter is converted to β-carotene by the lycopene cyclase activity of the bifunctional PBS enzyme. Finally, β-carotene is oxidized by the incorporation of a hydroxyl group in position 3 and a keto group in position 4 of both β-ionone rings of β-carotene generating astaxanthin as the final product. Unlike other organisms that produce astaxanthin, in *X. dendrorhous* a single enzyme catalyzes these last oxidizing steps from β-carotene to astaxanthin named astaxanthin synthase (CrtS, encoded by the *crtS* gene), which is a cytochrome P450 monooxygenase [29]. Astaxanthin synthase requires a redox partner, a cytochrome P450 reductase encoded by the *crtR* gene in *X. dendrorhous* [30, 35, 36], which provides the necessary electrons for the enzyme catalysis. The main pigments produced by *X. dendrorhous* are

), which is the build-

one decarboxylation, giving rise to isopentenyl pyrophosphate (IPP, C<sup>5</sup>

xanthophylls, of which astaxanthin represents 83–87% of total carotenoids [5].

Several algae and microalgae are able to produce astaxanthin, but *H. pluvialis* is the dominant specie for commercial astaxanthin [22]. *H. pluvialis* contains 1.5–3% of (3S, 3′S)-astaxanthin by dry weight, mainly as monoesters [37]. The isomer (3S, 3′S)-astaxanthin is the preferred form for human applications, for this reason, *H. pluvialis* is an attractive natural source of this

For the production of pigments, the microalgae *H. pluvialis* is usually grown in a two-stage batch process [5]. The first stage (green stage) is necessary to obtain enough biomass for an efficient carotenoid production. In this stage, microalgae are grown in presence of sufficient nutrient supply, optimum pH and temperature, and low irradiation. Since, the astaxanthin synthesis is induced under stress conditions, the second stage of growth (the reddening stage), consists in exposing the cells to stress conditions such as high light irradiation (sun light), nutrient deprivation (mainly nitrogen and phosphate deprivation), and high tempera-

As in *X. dendrorhous*, the biosynthesis of astaxanthin in *H. pluvialis* also begins with the synthesis of IPP, which is a key intermediate of carotenoid synthesis. In general, and depending

**3.2. Biosynthesis of astaxanthin in the microalgae** *H. pluvialis*

pigment [5].

68 Progress in Carotenoid Research

ture and/or high salt concentration [26].

**Figure 2.** Overview of *H. pluvialis* carotenogenesis. The steps for astaxanthin biosynthesis from IPP, generated via the MEP pathway, in *H. pluvialis* are shown. Enzymes involved in each step are listed at left and the corresponding genes are indicated in parenthesis. GA-3P: glyceraldehyde 3-phosphate; DOXP: 1-deoxy-D-xylulose-5-phosphate; MEP: methylerythritol 4-phosphate; IPP: isopentenyl pyrophosphate; DMAPP: dimethylallyl pyrophosphate; GGPP: geranylgeranyl pyrophosphate.

on the organism, IPP may be produced by two different pathways, the i) MVA pathway (cytosolic) and a ii) pathway located in the chloroplast known as the MEP (Methylerythritol 4-phosphate) or as the DOXP (due to the formation of 1-deoxy-D-xylulose-5-phosphate in the 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 most likely source of IPP in *H. pluvialis* cells.

emphasis has been placed on understanding the effect of the carbon source on carotenogenesis and how this pathway is affected by other related pathways, such as the synthesis of sterols. In the case of *H. pluvialis*, special interest has been placed on the effect of small molecules on

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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

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

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

is higher when a non-fermentable carbon source is used in cultures [17].

suppression of carotenogenesis in *X. dendrorhous.*

the synthesis of astaxanthin.

their composition [16, 17].

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

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