## 2 The Biosynthetic Pathway of Astaxanthin in _X. dendrohous_  
The astaxanthin biosynthetic pathway is encoded by six genes (_crtl_, _crtl_, _crtR_, _crtO_, _crtW_, and _crtZ_), which present an evolutionary pattern (eukaryotic and prokaryotic) characterised by lateral gene transfer and gene duplication events. These genes are more conserved in plants and algae than in any other bacterial phyla, where the structural genes evolve slower than the regulatory genes [44]. This biosynthetic pathway (Figure 1) has been widely studied in _X. dendrohous_[45; 46; 47; 48]. Thus, the biosynthesis begins with a C5 isoprene unit to which prenyl transferases sequentially add three other isoprenic units [49], resulting in the formation of C20 geranylgeranyl-pyrophosphate (GGPP). The active forms of the isoprene unit are isopentenyl-pyrophosphate (IPP) and its allylic isomer dimethylallyl-pyrophosphate (DMAPP). In most eukaryotes, IPP derives from the mevalonate pathway [50], while in prokaryotes and in plant plastids, it is synthesised via the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway, which is also known as the non-mevalonate pathway [51]. The _idi_ gene encodes IPP isomerase, which catalyses the isomerisation of IPP to DMAPP [52], and then both molecules are joined together, generating C10-geranyl pyrophosphate (GPP), the precursor of monoterenes [53]. The addition of a second molecule of IPP to GPP by prenyl transferases gives the C15 precursor of sesquiterpenes, famesyl pyrophosphate (FPP), which is converted into GGPP [54] by the further addition of IPP by the GGPP synthase (encoded by the _crtZ_ gene). Next, phytoene synthase (encoded by the _crtYB_ gene) links two molecules of GGPP in a tail-to-tail manner, yielding phytoene [50; 55]. This is the first carotenoid synthesised in the pathway, which is colourless as it has a symmetrical carotenoid skeleton with only three conjugated double bonds. The structural diversity of carotenoids is generated by further modifications, including desaturations, cyclisations, isomerisations, and oxygenations [56]. The phytoene synthase of fungi is a bifunctional enzyme that has both phytoene synthase and lycopene cyclase activities, which gives rise to \(\beta\)-carotene. In this particular enzyme, encoded by the _crtYB_ gene, the phytoene synthase and lycopene cyclase activities are located in the C-terminal and N-terminal functional domains, respectively. Next, phytoene is desaturated by phytoene desaturase (encoded by the _crtl_ gene) [3; 57] by the incorporation of two, three, four, or five double bonds, thus producing the coloured carotenoids: (i) \(\zeta\)-carotene (yellow, synthesised by some plants, cyanobacteria, and algae); (ii) neurosoprene (yellow, accumulates in _Rhodobacter capsulatus_ and _R. shareoides_); (iii) lycopene (red, found in most eubacteria and fungi) or (iv) 3,\(A\)-didehydescope (found in the mold _Neurospora crassa_) [50], respectively. Although there are acyclic carotenoids, the cyclisation of lycopene is a frequent step in the biosynthesis of carotenoids, forming three types of ionnone rings: \(\beta\)-, \(\varepsilon\)-, and \(\gamma\)(gamma)-rings ([PERSON] 1998). The \(\beta\)-ring is the most common, the \(\varepsilon\)-ring is found in plants and in some algae, and the \(\gamma\)-ring is the rarest one. The lycopene cyclase (also encoded by the _crtYB_ gene) [58] sequentially converts the \(\psi\) acyclic ends of lycopene to \(\beta\) rings to form \(\gamma\)-carotene and \(\beta\)-carotene [55]. The synthesis of xanthophylls involves the oxidation of post-phytoene carotenoid molecules, mainly from \(\alpha\)- and \(\beta\)-carotenes, resulting in oxygenated products with hydroxyl-, epoxy-, and oxo-functional groups. The formation of astaxanthin from \(\beta\)-carotene involves the introduction of a hydroxyl and a keto group at C3 and C4, respectively, for each of the \(\beta\)-ionone rings.