Isolation and Characterization of Canthaxanthin Biosynthesis Genes from the Photosynthetic Bacterium Bradyrhizobiumsp. Strain ORS278

Isolation of a carotenoid gene cluster.The genescrtB and crtI, encoding, respectively, phytoene synthase and phytoene desaturase, two enzymes involved in the initial steps of carotenoid biosynthesis (Fig. 1), have been isolated and characterized in various microorganisms (1, 9, 11, 14, 15, 21). In all of these cases, these genes were found to be adjacent and oriented in the same direction. Comparison of the deduced amino acid sequences of the CrtI and CrtB proteins from Erwinia uredovora, Erwinia herbicola,Flavobacterium sp. strain ATCC 21588, Rhodobacter sphaeroides, and Agrobacterium aurantiacum revealed well-conserved domains at the C-terminal end of CrtI (LVGAGTHPG) and in the central region of CrtB (QLTNIARD). These motifs were chosen for designing the degenerated primers CrtIf (5′-GTNGGNGCRGGCACNCAYCC-3′) and CrtBr (5′-TCGCGRGCRATRTTSGTSARRTG-3′). PCR amplification was performed with a Perkin-Elmer model 2400 thermocycler in a 50-μl (total volume) reaction mixture containing 100 ng of strain ORS278 genomic DNA, each deoxynucleotide triphosphate (200 μM), primers (0.8 μM each), MgCl₂ (1.5 mM), 1.25 U of Taq DNA polymerase (Promega, Charbonières, France), and the buffer supplied with the enzyme. A touchdown PCR (3) was done as follows: initial denaturation at 94°C for 5 min followed by 20 cycles consisting of a 30-s denaturation at 94°C, 30 s at an annealing temperature of 60 to 50°C, and a 1-min primer extension at 72°C, followed by 15 cycles consisting of a 30-s denaturation at 94°C, 30 s at an annealing temperature at 50°C, and a 1-min primer extension at 72°C. After the final elongation step at 72°C for 7 min, the amplified 620-bp fragment obtained (probe A) was purified by a Wizard procedure and was ligated into a pGEM-T vector (Promega). The ABI Prism BigDye Terminator Cycle Sequence Kit (Applied Biosystems, Foster City, Calif.) was used to sequence the cloned PCR product with the universal oligonucleotides M13 forward and M13 reverse. Sequencing reactions were analyzed on an Applied Biosystems model 310 DNA sequencer. The sequence of the amplified 620-bp fragment was highly similar to known CrtB sequences at the amino acid level.

Two specific primers, CrtIBfow.ORS278 (5′-ATTCGCAGCGGCTCGAAGAG-3′) and CrtIBrev.ORS278 (5′-GATCGCCGACATCATCACGC-3′), based on the sequence of the amplified DNA fragment, were designed for PCR screening of a library of the ORS278 strain constructed with the SuperCos I cosmid vector kit (Stratagene, La Jolla, Calif.), as instructed by the manufacturer. Four positive clones were isolated and confirmed by Southern blot analysis by using the 620-bp fragment as a probe. Clone pSTM73, containing an insert of approximately 35 kb, was used to characterize this crt gene cluster.

Structure of the canthaxanthin crt gene cluster.A 6.5-kb region in the inserted DNA fragment of the pSTM73 cosmid, showing a positive hybridization signal to probe A, was sequenced and analyzed as shown in Fig. 2. This nucleotide sequence had five open reading frames (ORFs) encoding proteins with similarity to known Crt enzymes (Fig.3). Based on sequence similarity (45% amino acid identity with CrtY of E. herbicola), one of these ORFs was assigned to a crtYgene which encodes lycopene cyclase, a key enzyme that converts lycopene into the cyclic carotenoid β-carotene. Another ORF was similar in sequence to a crtW gene encoding a β-carotene ketolase that synthesizes canthaxanthin from β-carotene via echinenone (15). This indicated that we had isolated acrt gene cluster involved in canthaxanthin biosynthesis. Four of the five ORFs, identified as crtY, crtI,crtB, and crtW, were found to be clustered in this order in the same orientation, whereas the ORF crtEpreceded these four but was oriented in the opposite direction (Fig.2). The crtY, crtI, and crtB genes are closely linked physically; i.e., the stop codons of crtY andcrtI overlap the start codon of the following ORF, suggesting that these genes are translationally coupled (18). Note that the crtY, crtI, andcrtB genes always occurred in this order and were oriented in the same direction in the other cyclic carotenoid biosynthesis clusters described previously (see Fig. 3).

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Fig. 2.

Organization of the canthaxanthin biosynthesis gene cluster of Bradyrhizobium sp. strain ORS278 and locations of various subcloned fragments. The restriction fragments are inserted into pUC18 (pSTM108, pSTM107, and pSTM51) or pUC19 (pSTM462), thecrt genes are transcribed from the lac promoter of the vector. In the plasmid pSTM78, the insert was obtained by Long PCR using the primers Crt.canta.f (5′-GCAACCGGTACCCGAGTTAATTCGCTCGGAATG-3′) and Crt.canta.r (5′-ATGGTGAAGCTTATGCGGCAGCGGGTTTAGTC-3′) and was cloned into pGEM-T (Promega). In pSTM78, the crtY, crtI,crtB, and crtW genes are under lacpromoter control.

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Fig. 3.

Comparison of the organization of the cyclic carotenoid gene clusters of Bradyrhizobium sp. strain ORS278, A. aurantiacum (15), Flavobacterium sp. strain R1534 (21), E. uredovora (14), andE. herbicola (9). Arrows represent the orientations of ORFs. The percentage values below the genes indicate the percentages of amino acid identity compared toBradyrhizobium sp. strain ORS278.

Phylogenetic trees were constructed with available CrtI and CrtB sequences. The CrtB proteins (Fig. 4) and the CrtI proteins (data not shown) formed two distinct clusters which do not correlate with the taxonomical position of the strains, but rather with the nature of the carotenoid (cyclic or noncyclic) synthesized under the control of crtI and crtB. The fact that α- and γ-Proteobacteria andFlavobacteria group in the same cluster (Fig. 4) suggests that lateral gene transfer has occurred between these phylogenetically unrelated bacteria. Moreover, the fact that strain ORS278 genes are clustered with crtI and crtB genes from nonphotosynthetic strains producing cyclic carotenoids rather than with photosynthetic strains producing photosynthetic carotenoids raises the question of whether an additional copy of the crtI andcrtB genes involved in the biosynthesis of spirilloxanthin does exist. The crtC and crtD genes, which were reported to be involved in the biosynthesis of spirilloxanthin from lycopene (20), have just been isolated in another cosmid which did not overlap the pSTM73 cosmid (E. Giraud and B. Dreyfus, unpublished data). We are currently investigating if another copy of the crtI and crtB genes is physically linked to these crtC and crtD genes, as has been found in photosynthetic bacteria (1, 11).

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Fig. 4.

Phylogenetic tree based on the CrtB sequences and constructed by using the neighbor-joining method (22). Bootstrap values (5), expressed as percentages of 1,000 replications, are given at the branching points. P,Proteobacteria; F, Flavobacteria; a, bicyclic carotenoid; b, acyclic carotenoid; c, monocyclic carotenoid. GenBank accession numbers are as follows: AF218415 , Bradyrhizobiumsp. strain ORS278; D58420 , A. aurantiacum; Y15112 ,Paracoccus marcusii; U62808 , Flavobacterium sp.;M87280 , E. herbicola EHO10; M90698 , E. herbicola 557; D90087 , E. uredovora; AF195122 , R. sphaeroides; X52291, Rhodobacter capsulatus;U87626, Rubrivivax gelatinosus; Z211955,Myxococcus xanthus.

Carotenoid production in Escherichia colitransformants. E. coli transformants carrying the entire crt gene cluster of canthaxanthin from strain ORS278, cloned in pGEMT (pSTM78) or SuperCosI (pSTM73), did not produce any carotenoids (Table 1), suggesting that these genes are not expressed or that their products are not functional in E. coli. Misawa et al. (15) constructed E. coli transformants which accumulate each precursor of the zeaxanthin biosynthesis pathway by introducing various combinations of E. uredovora crt genes. To check the functionality of the different ORFs identified in strain ORS278, we complemented several carotenoid-accumulating E. coli transformants with plasmids carrying various crt genes of strain ORS278 and analyzed carotenoids synthesized by high-pressure liquid chromatography (Table1). The conditions were as follows: 5-μm Hypersil C₁₈column (250 by 4.6 mm; Alltech, Templemars, France), eluent of acetonitrile-methanol-isopropanol (85/10/5, vol/vol/vol), flow rate of 1 ml/min, and detection at 470 nm (450 nm for β-carotene). Peaks were compared and coeluted with standard compounds then identified by their visible spectra and partition coefficients (12).

When plasmid pSTM78 carrying the complete crt cluster of Bradyrhizobium sp. strain ORS278 was introduced into the E. coli transformant that had accumulated geranylgeranyl pyrophosphate (GGPP) as a result of the presence of the crtE gene of E. uredovora, the new transformant obtained was shown to accumulate canthaxanthin. This result indicates that thecrtY, crtI, crtB, andcrtW genes are functional and allow the production of canthaxanthin in E. coli. Nevertheless, the amount of canthaxanthin produced remains lower than in the wild-type strain ORS278. When plasmid pSTM462 carrying the crtE gene ofBradyrhizobium sp. strain ORS278 under the lacpromoter was introduced into an E. coli transformant containing the crtI, crtB, and crtYgenes of E. uredovora, the new transformant accumulated β-carotene, showing the functionality of the crtE gene.

In this study, we cloned and characterized all of the crtgenes of Bradyrhizobium sp. strain ORS278 necessary for canthaxanthin biosynthesis. This is the first report of a cyclic carotenoid biosynthesis gene cluster in a photosynthetic bacterium. It would be interesting to determine the genetic links of this canthaxanthin crt gene cluster to the photosynthetic gene cluster. In Bradyrhizobium sp. strain ORS278, canthaxanthin production is stimulated by light (13), suggesting that the expression of canthaxanthin biosynthesis genes is regulated by photoinduction, as already reported for other pigments in different organisms (2, 7). Production of this pigment could be optimized by identifying the signal transduction system controlling canthaxanthin biosynthesis. However, characterization of the entirecrt gene cluster necessary for canthaxanthin biosynthesis already provides a basis for the construction of a recombinant strain that could overproduce this carotenoid.

Nucleotide sequence accession number.The DNA sequence obtained in this study has been deposited in the GenBank database under accession no. AF218415 .