**7. Pathway Engineering for the Synthesis of Functional Xanthophylls via the Incorporation of** *crtW***,** *crtZ***, and/or** *crtG* **Genes**

Figure 3 shows xanthophylls that were produced in recombinant *E. coli* cells via the incorporation of the marine bacterial *crtW*, *crtZ*, and/or *crtG* genes along with the *Pantoea crtE*, *crtB*, *crtI*, and *crtY* genes. The recombinant *E. coli* strain that expresses the four *Pantoea crt* genes can produce Ά-carotene predominantly (approximately 0.2–1 mg·gƺ1 dry cell weight). The coexpression of the *crtW*, *crtZ*, and/or *crtG* genes in the Ά-carotene-synthesizing *E. coli* cells confer the ability to produce not only commercially important xanthophylls such as astaxanthin but also xanthophylls minor in nature (e.g., Ά-ring(s)-2(2ȝ)-hydroxylated carotenoids), which are difficult to synthesize chemically. Particularly, the chemical synthesis of 2(2ȝ)- hydroxycarotenoids are likely to be recalcitrant, due to high-density around the 1,2- positions of the Ά ring in these xanthophylls. We showed that the coexpression of the *Brevundimonas* sp. SD212 *crtW* gene and the *P. ananatis crtZ* gene in the Άcarotene-synthesizing *E. coli* due to the presence of the four *crt* genes of *P. ananatis* resulted in predominant production of astaxanthin [44,46]. The *Paracoccus* sp. N81106 *crtW* gene was evolved by random mutagenesis to have improved activity [40]. It is also demonstrated that the coexpression of the *crtW* gene and the *crtG* gene from *Brevundimonas* sp. SD212 or from *B. aurantiaca* ATCC 15266 in the Ά-carotenesynthesizing *E. coli* resulted in dominant production of 2,2ȝ-dihydroxycanthaxanthin and 2-hydroxycanthaxanthin, while the substrate canthaxanthin accumulated [11,62]. The coexpression of the *crtZ* gene and the *crtG* gene in the Ά-carotenesynthesizing *E. coli* resulted in predominant production of nostoxanthin along with small amounts of caloxanthin [11,62]. The coexpression of all the three genes (*crtW*, *crtZ*, and *crtG*) in the Ά-carotene-synthesizing *E. coli* resulted in dominant production of 2,2ȝ-dihydroxyastaxanthin and 2-hydroxyastaxanthin [11]. When the *P. ananatis crtX* gene was coexpressed in addition to appropriate combinations of the above *crt* genes in *E. coli*, resultant *E. coli* cells were able to synthesize carotenoid-glycosides such as caloxanthin Ά-D-glucoside [64] and astaxanthin Ά-D-diglucoside [65], as shown in Figure 3. 

The ·-ray-tolerant bacterium *D. radiodurans* R1 produces the monocyclic carotenoid including the 2-hydroxy-4-keto-Ά-ring, deinoxanthin [1]. 2,2<sup>ȝ</sup>-Dihydroxycanthaxanthin was shown to have strong inhibitory effect against lipid peroxidation in a rat brain homogenate [11]. Such minor ketocarotenoids, which include the 2-hydroxy-4-keto-Ά-ring, may have beneficial effects on human health as  well as anti-oxidation function, while few works are present examining their biological functions. 

When carotenoid biosynthesis genes starting from the utilization of FPP are introduced in *E. coli*, as above-mentioned, amounts of carotenoids produced with the recombinant *E. coli* cells are far from the practical use, which was difficult to exceed 1 mg·gƺ1 dry weight. In order to overcome this problem, many pathway engineering researches in *E. coli* have been performed for increasing intracellular concentration of FPP (e.g., recently reviewed [66,67]). For example, the coexpression of the *idi* (type 1) gene from *H. pluvialis*, *Xanthophyllomyces dendrorhous* (renamed from *Phaffia rhodozyma*), or *Saccharomyces cerevisiae*, as well as the *idi* (type 2) from *Streptomyces* sp. strain CL190, was shown to be effective to increase FPP content [68,69]. The introduction of heterologous mevalonate pathway genes in *E. coli* along with an *idi* (type 2) gene has been described to efficiently improve the productivity of carotenoids or sesquiterpenes that are synthesized from FPP [69–73]. For example, Yoon *et al.* [73] produced 22 mg·gƺ1 dry cell weight of lycopene in 72 h using such mevalonate-pathway-engineered *E. coli* cells. On the other hand, production of lycopene reached high levels (near to 20 mg·gƺ1 dry cell weight) in 24-h batch flask culture in pathway-engineered *E. coli*, which reflected results of multi-dimensional gene target search or gene-knockout analysis [74]. These finding should be applied to efficient production of the above-mentioned functional xanthophylls with *E. coli*  cells. 

Pathway engineering researches in higher plants have also been performed for efficient production of astaxanthin, which utilized the marine bacterial *crtW* genes from *Paracoccus* sp. N81106 or *Brevundimonas* sp. SD212, or the *H. pluvialis bkt1* or *bkt2* genes, as reviewed [16,39,53]. For example, the *Brevundimonas* sp. SD212 *crtW* and *crtZ* genes, whose nucleotide sequence is modified to codon usage of higher plants, were successfully overexpressed in the chloroplasts of tobacco plants (*Nicotiana tabacum*), and astaxanthin level produce there reached 5.44 mg·gƺ1 dry weight (74% of total carotenoids) [75]. 
