Carotenoid β-Ring Hydroxylase and Ketolase from Marine Bacteria—Promiscuous Enzymes for Synthesizing Functional Xanthophylls

Marine bacteria belonging to genera Paracoccus and Brevundimonas of the α-Proteobacteria class can produce C40-type dicyclic carotenoids containing two β-end groups (β rings) that are modified with keto and hydroxyl groups. These bacteria produce astaxanthin, adonixanthin, and their derivatives, which are ketolated by carotenoid β-ring 4(4′)-ketolase (4(4′)-oxygenase; CrtW) and hydroxylated by carotenoid β-ring 3(3′)-hydroxylase (CrtZ). In addition, the genus Brevundimonas possesses a gene for carotenoid β-ring 2(2′)-hydroxylase (CrtG). This review focuses on these carotenoid β-ring-modifying enzymes that are promiscuous for carotenoid substrates, and pathway engineering for the production of xanthophylls (oxygen-containing carotenoids) in Escherichia coli, using these enzyme genes. Such pathway engineering researches are performed towards efficient production not only of commercially important xanthophylls such as astaxanthin, but also of xanthophylls minor in nature (e.g., β-ring(s)-2(2′)-hydroxylated carotenoids).


Figure 3.
Pathway engineering for the production of functional xanthophylls using the carotenoid biosynthesis genes, crtW, crtZ, and/or crtG, which were isolated from the marine bacteria, Paracoccus sp. strain N81106 or Brevundimonas sp. strain SD212, in addition to the crtE, crtB, crtI, and crtY genes (and crtX) from P. ananatis.
These crt genes have widely used for complementation analysis of carotenoid biosynthesis genes isolated from other organisms, since they are functionally expressed in E. coli with ease [11,[34][35][36][37]. The P. agglomerans gene cluster contained a gene encoding isopentenyl diphosphate (IPP) isomerase (Idi; type 2) [38] in addition to the six crt genes [39]. These seven carotenogenic (carotenoid-biosynthetic) genes were also found to exist in a carotenoid biosynthesis gene cluster of Paracoccus sp. strain N81106 [10,39]. This cluster included an additional gene, designated CrtW, which was elucidated to code for an enzyme responsible for ketocarotenoid formation, that is, CrtW proved to catalyze the synthesis of canthaxanthin from β-carotene by complementation analysis using recombinant E. coli cells that contains the P. ananatis crtE, crtB, crtI, and crtY genes [33] (Figure 3). The hydropathy and transmembrane prediction analyses indicated that CrtW from Paracoccus sp. N81106 contains four transmembrane domains and two other hydrophobic regions, and its topology model is very similar to those for fatty acid desaturases [40]. It should be noted that it is recalcitrant to purify active CrtW and CrtZ proteins, which both are very likely iron-dependent integral membrane proteins, from the recombinant hosts as well as the native hosts, precluding their close enzymatic characterizations.
Two paralogous genes exhibiting significant homology to crtW were isolated from H. pluvialis, and designated bkt [50] or crtO [51]. These genes were renamed bkt1 from crtO and bkt2 from bkt, since "crtO" has been used for the other type of cyanobacterial β-ring 4(4′)-ketolase genes, as shown later [52]. The BKT1 and BKT2 enzymes are very likely to have catalytic function same to the Paracoccus (or Brevundimonas) CrtWs, considering results from the in vitro study on BKT2 with E. coli [42] and pathway engineering researches in higher plants as well as E. coli as the hosts [16,50,51,53].

Carotenoid 3,3′-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, Brevundimonas 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 Brevundimonas sp. SD212 and the bacterial strain P99-3 converted β-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 β-ring 3(3′)-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 β-carotene 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) β ring of monocyclic carotenoids such as deoxymyxol and deoxymyxol 2′-fucoside at the 3 position (Figure 4). Among them, only the Synechocystis sp. PCC 6803CrtR was able to convert β-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 β-ring 3(3′)-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) β ring of β-carotene as the substrate to form zeaxanthin [45,61].

Pathway Engineering for the Synthesis of Functional Xanthophylls via the Incorporation of crtW, crtZ, and/or crtG Genes
The γ-ray-tolerant bacterium D. radiodurans R1 produces the monocyclic carotenoid including the 2-hydroxy-4-keto-β-ring, deinoxanthin [1]. 2,2′-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-β-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· g −1 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][70][71][72][73]. For example, Yoon et al. [73] produced 22 mg· g −1 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· g −1 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· g −1 dry weight (74% of total carotenoids) [75].

Conclusions
This review has focused on the carotenoid β-ring-modifying enzymes, CrtW, CrtZ and CrtG, derived from the marine bacteria of the α-Proteobacteria class, and pathway engineering for the production of xanthophylls in E. coli, using these enzyme genes. Such pathway engineering researches are performed towards efficient production not only of commercially important xanthophylls such as astaxanthin, but also of xanthophylls minor in nature, which are difficult to synthesize chemically, and expected to have beneficial effects on human health as well as anti-oxidation function.