**5. Carotenoid 3,3ȝ-Hydroxylase**

The *crtZ* genes have been found not only in carotenogenic bacteria belonging to genera *Pantoea*, *Paracoccus* and *Brevundimonas*, but also in those belonging to the *Flavobacteriaceae* family [6,39]. Conversion efficiency to astaxanthin in several CrtZs  was compared with recombinant *E. coli* cells that synthesize the carotenoid substrate canthaxanthin due to the presence of the *P. ananatis crtE*, *crtB*, *crtI* and *crtY* gens, and the *Paracoccus* N81106 *crtW* gene, into which each *crtZ* gene from *P. ananatis*, *Paracoccus* sp. N81106, *Paracoccus* sp. PC1, *Brevundimona*s sp. SD212, and marine bacterium strain P99-3 of the *Flavobacteriacea* family was introduced and expressed there [45]. It was consequently shown that the CrtZ enzymes from *Brevundimona*s sp. SD212 and the bacterial strain P99-3 converted Ά-carotene to astaxanthin with the highest and lowest efficiency, respectively, along with the *Paracoccus* N81106 CrtW [45]. 

On the other hands, no *crtZ* sequences have not been found in cyanobacteria, instead genes encoding a new type of Ά-ring 3(3ȝ)-hydroxylases (named CrtR) that exhibited moderate homology to CrtW have been found there [58,59]. The *crtR* genes were isolated from *Synechocystis* sp. strain PCC 6803, *Anabaena* sp. PCC 7120, *Anabaena variabilis*, and *N. punctiforme* [46,58]. An *in vivo* analysis on *crtR* was performed with recombinant *E. coli* cells that synthesize the carotenoid substrate Άcarotene or canthaxanthin, into which each *crtR* gene from *Synechocystis* sp. PCC 6803, *Anabaena* sp. PCC 7120, and *A. variabilis* was introduced and expressed there [46]. This result along with another result [60] indicated that the CrtR-type enzymes can hydroxylate the (un-substituted) Ά ring of monocyclic carotenoids such as deoxymyxol and deoxymyxol 2ȝ-fucoside at the 3 position (Figure 4). Among them, only the *Synechocystis* sp. PCC 6803CrtR was able to convert Ά-carotene to zeaxanthin [46,58,60]. A thermophilic bacterium *Thermus thermophilus* HB27, which grows at temperatures above 75 °C, was found to possess another new type of Ά-ring 3(3ȝ)- hydroxylase of the cytochrome P450 superfamily, named CYP175A1 [61]. The *in vivo* analysis with the gene strongly suggested that this thermostable P450 accepts only the (un-substituted) Ά ring of Ά-carotene as the substrate to form zeaxanthin [45,61]. 

## **6. Carotenoid 2,2ȝ-Hydroxylase**

Carotenoid 2,2ȝ-hydroxylase (Ά-ring 2(2ȝ)-hydroxylase) was first found in the marine bacterium *Brevundimonas* sp. strain SD212, and named CrtG [11]. An *in vivo* analysis on *crtG* was performed with recombinant *E. coli* cells that synthesize each carotenoid substrate (Ά-carotene, zeaxanthin, canthaxanthin, or astaxanthin), into which the *crtG* gene was introduced and expressed there [11]. The result indicated that the CrtG can hydroxylate the Ά rings substituted with 3-hydroxy and/or 4-keto groups in dicyclic carotenoids at the 2(2ȝ)-positions (Figures 1 and 3) [11]. The *crtG* genes were also isolated from soil bacteria *Brevundimonas vesicularis* DC263 and *B. aurantiaca* ATCC 15266 [62]. The *in vivo* analysis with these genes indicated that the *B. aurantiaca* CrtG enzyme (accession no. DQ497427), which exhibited the highest amino acid identity (98.8%) to that of the *Brevundimonas* SD212 CrtG, accepted the (un-substituted) Ά rings of Ά-carotene in addition to the substituted Ά rings as the substrates [62]. A *crtG* gene sequence, whose encoded amino acid sequence was 41%  identical to the *Brevundimonas* sp. SD212 CrtG, was found in a thermophilic cyanobacterium *Thermosynechococcus elongatus*, which synthesized 2-hydroxylated carotenoids such as caloxanthin ((2*R*,3*R*,3<sup>ȝ</sup>*R*)-Ά,Ά-carotene-2,3,3<sup>ȝ</sup>-triol), nostoxanthin ((2*R*,3*R*,2<sup>ȝ</sup>*R*,3<sup>ȝ</sup>*R*)-Ά,Ά-carotene-2,3,2<sup>ȝ</sup>,3<sup>ȝ</sup>-tetrol) (Figure 3), and 2-hydroxymyxol 2<sup>ȝ</sup>fucoside [63]. 
