*3.2. Ά-Carotene and ΅-Carotene Synthesis by Lycopene Cyclases*

All carotenoids in oxygenic phototrophs are dicyclic carotenoids; Ά-carotene, ΅-carotene and their derivatives, are derived from lycopene (Figures 1 and 2). Exceptionally, myxol glycosides and oscillol diglycosides in cyanobacteria are monocyclic and acyclic carotenoids, respectively. 

Lycopene is cyclized into either Ά-carotene through ·-carotene, or ΅-carotene through ·- carotene or Έ-carotene. Three distinct families of lycopene cyclases have been identified in carotenogenetic organisms [13,62,63]. One large family contains CrtY in some bacteria except cyanobacteria, and CrtL (CrtL-b, Lcy-b) in some cyanobacteria and land plants. Lycopene <sup>Ή</sup>cyclases (CrtL-e, Lcy-e) from land plants and lycopene Ά-monocyclases (CrtYm, CrtLm) from bacteria are also included. Their amino acid sequences exhibit a significant five conserved regions [39,62,64], and have an NAD(P)/ 

FAD-binding motif [65]. Note that Maresca *et al.* [63] divide this family into two CrtY and CrtL families. Three enzymes from Rhodophyta, *Cyanidioschyzon merolae* [38], and Chlorophyceae, *Dunaliella salina* [39] and *Haematococcus pluvialis* [40], are functionally confirmed (Table 2). 

Some cyanobacteria also contain these enzymes (Table 2). *Synechococcus elongatus* PCC 7942 contains a functional CrtL [36]. *Prochlorococcus marinus* MED4 contains two lycopene cyclases (Table 2), which have sequence homology to CrtL. CrtL-b exhibits lycopene Ά-cyclase activity, while CrtL-e is a bifunctional enzyme having both lycopene Ή-cyclase and lycopene Ά-cyclase activities [37]. The combination of these two cyclases allows the production of Ά-carotene, ΅- carotene and Ή-carotene. Both enzymes might have originated from the duplication of a single gene. The characteristics of this CrtL-e are somewhat different from those in land plants [66]. In addition, the Ά-end groups of both 

Ά-carotene and ΅-carotene (left half) might be hydroxylated by CrtR to zeaxanthin through Άcryptoxanthin and 3-hydroxy-΅-carotene, respectively, in *P. marinus*. *Acaryochloris marina* MBIC 11017, which produces ΅-carotene, contains only one *crtL*-like gene from genome sequence [14]. 

The second family of lycopene cyclases, heterodimer (*crtYc* and *crtYd*) or monomer (*crtYc-Yd*), has been found in some bacteria, archaea and fungi [62,67], but not in phototrophs. 

Recently, a new family of functional lycopene cyclase, CruA, has been found in *Chlorobaculum* (previously *Chlorobium*) *tepidum* (green sulfur bacterium), and the main product is ·-carotene in *Escherichia coli*, which produces lycopene [68]. Homologous genes, *cruA* and *cruP*, have been found in the genome of *Synechococcus* sp. PCC 7002, and their main products are ·-carotene, in *E. coli*, which produces lycopene [63]. In addition, their homologous genes are widely distributed in cyanobacteria, such as *Synechocystis* sp. PCC 6803 and *Anabaena* sp. PCC 7120; however, these *cruA*- and *cruP*-like genes from both *Synechocystis* sp. PCC 6803 and *Anabaena* sp. PCC 7120 did not show the lycopene dicyclase or monocyclase activities [14]. *S. elongatus* PCC 6301 and PCC 7942, and *A. marina* MBIC 11017 contain *crtL*-, *cruA*- and *cruP*-like genes; consequently, distributions of functional lycopene cyclases (CrtL-, CruA- and CruPlike) in cyanobacteria are unknown. 

Since *Synechocystis* sp. PCC 6803 and *Anabaena* sp. PCC 7120 lack *crtL*-like genes and contain non-functional *cruA*-like genes, there is a possibility to present a fourth new family of lycopene cyclases in these cyanobacteria. Further studies of distributions of functional lycopene cyclases (CrtL- and CruA-like, or others) in cyanobacteria are needed. 

Distribution of ΅-carotene, that is, CrtL-e, is limited in some algae classes (Table 1). Genes and enzymes of CrtL-e are not found in algae. In some species of land plants, the characteristics of CrtL-e were investigated [66], and were shown to have sequence homology with *crtL-b*. Lycopene is first converted to Έ-carotene by CrtL-e, and then to ΅-carotene by CrtL-b. ·- Carotene produced by CrtL-b is not a suitable substrate for CrtL-e. 

## *3.3. Ά-Carotene Derivatives and Their Synthesis*

## 3.3.1. Cyanobacteria

Some cyanobacteria produce zeaxanthin, and some produce both zeaxanthin and nostoxanthin (Figure 3). First, the C-3 and C-3ȝ hydroxyl groups of zeaxanthin are introduced to Ά-carotene by 

Ά-carotene hydroxylase (CrtR) through Ά-cryptoxanthin. Then, the C-2 and C-2ȝ hydroxyl groups of nostoxanthin are introduced by 2,2<sup>ȝ</sup>-Ά-hydroxylase (CrtG) through caloxanthin (Table 2) [13,41–43,47]. The same enzymes, CrtR and CrtG, can also introduce hydroxyl groups to deoxymyxol and myxol to produce myxol and 2-hydroxymyxol, respectively [13,44,47]; consequently, the same enzymes are used in two pathways. 

Cyanobacteria contain two ketocarotenoids, namely, canthaxanthin and 4-ketomyxol. Two distinct Ά-carotene ketolases, CrtO and CrtW, are known, and only seven enzymes are functionally confirmed in four species of cyanobacteria (Table 2) [13]. CrtO catalyzes Άcarotene to echinenone, and the 

final product is canthaxanthin [22,42,45,50,51]. CrtW can introduce a keto group into Άcarotene, zeaxanthin and myxol to produce canthaxanthin, astaxanthin and 4-ketomyxol, respectively 

(Figure 3) [22,27,42,50,52]; therefore, these ketolases are properly used in two pathways, Άcarotene and myxol, depending on the species [13]. 

The pathway and the enzymes to produce the right half of myxol 2<sup>ȝ</sup>-pentoside are still unknown (Figure 3) [13]. 
