**3.1. Astaxanthin biosynthesis in** *X. dendrorhous*

authorities [1, 2, 5]. In this context, the society nowadays is looking toward "green" solutions; thus, astaxanthin from natural sources, as from microorganisms, seems to be more favorable than its synthetic counterpart due to structure, function, application and security [25, 26].

pyrophosphate.

66 Progress in Carotenoid Research

**Figure 1.** Overview of *X. dendrorhous* carotenogenesis and related pathways. The steps for carotenoid biosynthesis from IPP, generated via the MVA pathway, are shown. Also, an abbreviated ergosterol synthesis pathway based on the route described in *S. cerevisiae* is schematized (in green), highlighting enzymes whose genes have been functionally characterized in *X. dendrorhous (*homologous to those present in *S. cerevisiae)* [19, 66]. MVA: mevalonate; IPP: isopentenyl pyrophosphate; DMAPP: dimethylallyl pyrophosphate; FPP: farnesyl pyrophosphate; GGPP: geranylgeranyl It is currently believed that the main function of carotenoids in *X. dendrorhous* is to protect the yeast against damage caused by oxidative stress. This hypothesis is supported by the fact that strains that do not produce astaxanthin are more sensitive and grow less in the presence of ROS [23]. In addition, it has been reported that the presence of certain oxygen species increases the total carotenoid content [23, 24]. Other evidence points to the fact that *X. dendrorhous* has significantly lower catalase activity than other yeasts such as *Saccharomyces cerevisiae*, so carotenoids could compensate this low activity to help to preserve the viability of continuously growing yeasts [23]. On the other hand, *X. dendrorhous* does not have a cytosolic version of a superoxide dismutase, and carotenoids could also be compensating the lack of this enzyme [23]. *X. dendrorhous* is the only known yeast that produces astaxanthin *de novo* [22] and it has been approved by the FDA (Food and Drug Administration) for the commercial production of astaxanthin; therefore, it is a good candidate to allow the natural production of astaxanthin [27]. Knowledge about the biology of this yeast has increased and several methodologies to manipulate it have been developed, but still several aspects need to be improved for an economically competitive production of astaxanthin from *X. dendrorhous* [27].

The synthesis pathway preserves the basic steps of carotenoid synthesis in this yeast; however, some steps involve characteristic enzymes in fungi and other characteristic enzymes in *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 one decarboxylation, giving rise to isopentenyl pyrophosphate (IPP, C<sup>5</sup> ), which is the building 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 xanthophylls, of which astaxanthin represents 83–87% of total carotenoids [5].
