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Isolation and characterization of the carotenoid biosynthesis genes of Flavobacterium sp. strain R1534
Gene 185 (1997) 35–41
Isolation and characterization of the carotenoid biosynthesis genes of Flavobacterium sp. strain R1534 Luis Pasamontes *, Denis Hug, Michel Tessier, Hans-Peter Hohmann, Joseph Schierle, Adolphus P.G.M. van Loon F. Hoffmann-La Roche Ltd., Vitamins and Fine Chemicals Division, Biotechnology Section 93/4.22, 4070 Basel, Switzerland Received 28 April 1996; revised 15 July 1996; accepted 24 July 1996; Received by A.M. Campbell
Abstract The Gram-negative bacterium Flavobacterium sp. strain R1534 is a natural producer of zeaxanthin. A 14 kb genomic DNA fragment of this organism has been cloned and a 5.1 kb piece containing the carotenoid biosynthesis genes sequenced. The carotenoid biosynthesis cluster consists of five genes arranged in at least two operons. The five genes are necessary and sufficient for the synthesis of zeaxanthin. The encoded proteins have significant homology to the crtE, crtB, crtY, crtI and crtZ gene products of other carotenogenic organisms. Biochemical assignment of the individual gene products was done by HPLC analysis of the carotenoid accumulation in Escherichia coli host strains transformed with plasmids carrying deletions of the Flavobacterium sp. strain R1534 carotenoid biosynthesis cluster. Keywords: Gram-negative bacterium; Cloning; Geranylgeranyl pyrophosphate synthase; Phytoene synthase; Phytoene desaturase; Lycopene cyclase-b-carotene hydroxylase; Zeaxanthin
1. Introduction Over 560 different carotenoids have been described from carotenogenic organisms found among bacteria, yeasts, fungi and plants (Straub, 1987). Their importance for microorganisms has been attributed to their strong antioxidant characteristics which protect the organisms against photooxidative damage ( Tuveson et al., 1988). Flavobacterium sp. strain R1534 is a nonphotosynthetic, Gram-negative bacterium, and a natural producer of the yellow pigment (3R,3R∞)-zeaxanthin (Leuenberger et al., 1973; Britton et al., 1977). Among * Corresponding author. Tel. +41 61 6887838; Fax +41 61 6881645; e-mail: [email protected] Abbreviations: aa, amino acid(s); bp, base pair(s); BSA, bovine serum albumin; CRTE (crtE), geranylgeranyl pyrophosphate synthase (corresponding gene); CRTB (crtB), phytoene synthase; CRTI (crtI ), phytoene desaturase; CRTD (crtD), hydroxyneurosporene desaturase; CRTY (crtY ), lycopene cyclase; CRTZ (crtZ), b-carotene hydroxylase; FPPS, farnesyl pyrophosphate synthase; GCG, Genetics Computer Group, Madison, WI; GGPP, geranylgeranyl pyrophosphate; HPLC, high-performance liquid chromatography; HPPS, hexaprenyl pyrophosphate synthase; ORF, open reading frame; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate; SSC, 0.15 M NaCl/0.015 M Na · citrate pH 7.6. 3
bacteria Flavobacterium may represent the best source for the development of a fermentative production process for (3R,3R∞)-zeaxanthin. Derivatives of Flavobacterium sp. strain R1534, obtained by classical mutagenesis, have attracted in the past two decades wide interest for the development of a large-scale fermentative production of zeaxanthin, although with little success. Mutant strains producing up to 335 mg zeaxanthin/l have been reported (Ninet and Renaut, 1979) but despite this high titer, fermentative large-scale production is still not attractive. Cloning of the carotenoid biosynthesis genes of this organism may allow replacement of the classical mutagenesis approach by a more rational one, using molecular tools to amplify the copy number of relevant genes, deregulate their expression and eliminate bottlenecks in the carotenoid biosynthesis pathway. Although all the necessary genes required to synthesize zeaxanthin have been described from different organisms (Misawa et al., 1990, 1995; Armstrong et al., 1990), the use of the homologous genes in a production strain should be advantageous compared to the use of heterologous genes. The initial steps in the carotenoid biosynthesis up to the phytoene formation have been found to be common to all the carotenoids described so far. The first commit-
0378-1119/97/$17.00 Copyright © 1997 Elsevier Science B.V. All rights reserved PII S 03 7 8 -1 1 1 9 ( 9 6 ) 0 0 6 24 - 5
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L. Pasamontes et al./Gene 185 (1997) 35–41
ting step towards the carotenoid biosynthesis is catalyzed by the enzyme geranylgeranyl pyrophosphate synthase (CRTE) which condenses farnesyl pyrophosphate, the common C carbon precursor of cholesterol, steroids 15 and carotenoids, with an isopentenyl pyrophosphate moiety. Geranylgeranyl pyrophosphate is then converted by the enzymes prephytoene and phytoene synthase into phytoene, a C carbon unit. In bacteria and lower 40 eucaryotes both enzymatic functions are encoded by a single gene (crtB). After phytoene formation the biosynthetic pathways diverge in different species leading to accumulation of different types of carotenoids. In Flavobacterium sp. strain R1534 as in Erwinia herbicola and Erwinia uredovora, phytoene is converted to lycopene by the enzyme phytoene desaturase (CRTI ). The conversion of lycopene to b-carotene involves four desaturation and two cyclization steps. Again, as in E. herbicola and E. uredovora, all these steps are catalyzed by one enzyme, the lycopene cyclase (CRTY ). This is also the case for the hydroxylation of b-carotene to zeaxanthin, which involves two steps both catalyzed by the same enzyme, the b-carotene hydroxylase (CRTZ) (for review, see Armstrong, 1994; Sandmann, 1994). In this report we describe the cloning and sequencing of the carotenoid biosynthesis gene cluster of this bacterium. The cluster consists of all five genes (crtE, crtB, crtI, crtY and crtZ ) of the biosynthetic pathway necessary to synthesize zeaxanthin. Functional assignment for the cloned carotenoid genes was done in Escherichia coli transformed with deletion mutants of the original plasmid carrying all five genes.
2. Results and discussion
2.1. Amplification of a DNA fragment of the crtB gene of Flavobacterium sp. strain R1534 Alignment of the phytoene synthetases (CRTB) of E. herbicola (Armstrong et al., 1990) and R. capsulatus (Armstrong et al., 1989) revealed quite a number of conserved aa stretches among these two enzymes. Two such conserved peptide sequences L( E/D)GFAMD and Q(L/M ) ( T/S )NIARD were chosen for reverse translation to design the degenerate primer #4 (5∞-CTGGAAGG( T/C )TT(C/T )GCTATGGA-3∞) and the reverse primer #5 (5∞-GTCACGAGCGATGTT(G/A)GTCAGCTG-3∞). PCR amplifications with these primers on genomic DNA of Flavobacterium sp. strain R1534 gave a product of about 190 bp named fragment 45F. Cloning and sequencing of this PCR fragment indicated that we had cloned a DNA piece encoding 60 aa, with significant homology to known phytoene synthases from other species. Based on the sequence of fragment 45F two oligonucleotides were
synthesized, primer #7 (5∞-CCTGGATGACGTGCTGGAATATTCC-3∞) and primer #8 (5∞-CAAGGCCCAGATCGCAGGCG-3∞). PCR amplification with these primers on genomic DNA of Flavobacterium R1534 generated a specific DNA probe of 119 bp named 46F which was then used for the library screening as described in Section 2.2. 2.2. Cloning of the Flavobacterium sp. strain R1534 carotenoid biosynthetic genes Cloning of the carotenoid biosynthesis cluster started using the PCR fragment 46F as a probe to hybridize to a Southern blot carrying chromosomal DNA of Flavobacterium sp. strain R1534 digested with different restriction enzymes. A 2.4 kb XhoI-PstI fragment hybridizing to the probe seemed the most appropriate for isolation. The XhoI-PstI partial library was constructed as outlined in the legend of Fig. 1. The insert of the positive transformant, clone 85, was sequenced and revealed sequences not only homologous to the phytoene synthase (CRTB) but also to the phytoene desaturase (CRTI ) of both Erwinia species herbicola and uredovora. Left hand genomic sequences of clone 85 were obtained by the same approach using probe B to screen a partial library made of ClaI-HindIII fragments of approx. 9.2 kb. Sequencing of the 5∞ and 3∞ of the insert of one positive, clone 51, revealed that only the region close to the HindIII site showed relevant homology to genes of the carotenoid biosynthesis of the Erwinia species mentioned above (e.g. crtB gene and crtE gene). To facilitate further sequencing and construction work, the 4.2 kb BamHI-HindIII fragment of clone 51 was further subcloned into the respective sites of pBluescriptII KS(+), resulting in clone 2. Sequencing of the insert of this clone confirmed the presence of genes homologous to Erwinia sp. crtB and crtE genes. These genes were located within 1.8 kb from the HindIII site. The remaining 2.4 kb of this insert had no homology to known carotenoid biosynthesis genes. Additional genomic sequences downstream from the HindIII site were obtained by screening a SalI-HindIII size restricted library (size of fragments approx. 2.8 kb) with probe A. The insert of one positive, clone 59, revealed in addition to known sequences the missing N terminus of the crtI gene, the complete putative lycopene cyclase gene (crtY ) and part of the C terminus of the crtZ gene. To isolate the missing crtZ gene a library made of partially Sau3AI digested genomic DNA of Flavobacterium sp. strain R1534 was screened with probe D, resulting in several positive clones. One transformant designated, clone 6a, had an insert of 4.9 kb comprising the missing part of the crtZ gene. The four independent clones, namely clone 51, clone 85, clone 59 and clone 6a, covering approx. 14 kb of the Flavobacterium sp. strain R1534 genome are shown in Fig. 1. The determined sequence
L. Pasamontes et al./Gene 185 (1997) 35–41
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Fig. 1. Physical map of the organization of the carotenoid biosynthesis cluster in Flavobacterium sp. strain R1534, deduced from the genomic clones obtained. The location of the probes A–D and 46F, used for the screening, are shown as bars. Restriction enzyme site abbreviations are as follows: Ba, BamHI; B, BglII; Bs, BstYI; Bx, BstXI; C, ClaI; H, HindIII; K, KpnI; M, MluI; N, NotI; P, PstI; S, SalI; Sa, Sau3AI; X, XhoI; *more restriction sites present but not shown. Sequences were analyzed using the GCG sequence analysis software package ( Version 8.0) by Genetics Computer, Inc. (Devereux et al., 1984). The determined sequence from the SalI site (bp position 1) to the MluI site (bp position 5188) has been filed to the GenBank under accession No. U62808. Methods: Isolation of genomic DNA of Flavobacterium sp. strain R1534 (ATCC 21588) was basically done as described by Sambrook et al. (1989). Genomic DNA amplifications: The GeneAmp@ DNA amplification kit (Perkin Elmer) was used for PCR. The amplification was carried out with 15 ng of Flavobacterium sp. strain R1534 genomic DNA as template in a total volume of 100 ml using 10 pmol of each primer #4 and #5. In all cases an aliquot of the reaction was analyzed on a 1.5% agarose gel. PCR products of the expected size were excised from the agarose gel and isolated by centrifugation of the gel slices through siliconized glass wool as described by Heery et al. (1990) or using the GENECLEAN Kit (BIO101 Inc.) essentially according to the manufacturer’s protocol. The fragments were subsequentely cloned into the T-tailed EcoRV site of pBluescriptII KS+ made according to Marchuk et al. (1990). Southern blot analysis: For hybridization experiments Flavobacterium sp. strain R1534 genomic DNA (3 mg) was digested with the appropriate restriction enzymes and electrophoresed on a 0.75% agarose gel. The transfer to Zeta-Probe blotting membranes (Bio-Rad ), was done as described (Southern, 1975). Prehybridization and hybridization were in 7% SDS, 1% BSA (fraction V; Boehringer), 0.5 M Na HPO (pH 7.2) at 65°C. After hybridization the membranes were washed twice for 2 4 5 min in 2×SSC, 1% SDS at room temperature and twice for 15 min in 0.1% SSC, 0.1% SDS at 65°C. Library construction: Prior to the partial library construction, Southern blot analysis with a given probe was done in order to identify a specific restriction fragment of interest. Subsequently 10–20 mg of genomic DNA was digested with the appropriate restriction enzymes and electrophoresed on a 0.75% agarose gel. According to comigrating DNA markers, the region of interest was cut out of the gel, the DNA isolated and cloned into the pBluescriptII KS(−) vector. Transformation of the ligation mixture into E. coli strain XL-1 (Stratagene, La Jolla, CA, USA) cells resulted in partial genomic libraries containing the fragment of interest. DNA probes were labeled with [a-32P]dGTP (Amersham International ) by nick-translation according to Sambrook et al. (1989). Probe A is a BstXI-PstI fragment of 184 bp; probe B is a XhoI-NotI fragment of 397 bp; probe C is a BglII-PstI fragment of 536 bp; probe D is a KpnI-BstYI fragment of 376 bp; probe 46F is a 119 bp PCR fragment (see Section 2.1).
from the SalI site (bp position 1) to the MluI site (bp position 5188) has been filed to the GenBank under the accession No. U62808. 2.3. Putative protein coding regions of the cloned sequence Computer analysis of the aforementioned sequence revealed five putative open reading frames (ORFs) encoding proteins with similarity to known crt enzymes ( Table 1). The translation start sites of the ORFs was determined based on the appropriately located sequences homologous to the Shine-Dalgarno (Shine and Dalgarno, 1974) consensus sequence AGG–6-9N–ATG,
the homology to the N-terminal sequences of the respective enzymes of E. herbicola, E. uredovora, Agrobacterium aurantiacum and Alcaligenes sp. strain PC-1 (Misawa et al., 1995) and the strong third position GC bias observed in all five putative ORFs. 2.4. Genomic organization of the crt gene cluster and comparison to the cartenoid biosynthetic cluster of other Gram-negative bacteria The genomic organization of the ORFs within the crt gene cluster of Flavobacterium sp. strain R1534 is different when compared to the carotenoid biosynthesis clusters of other Gram-negative organisms, such as A.
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L. Pasamontes et al./Gene 185 (1997) 35–41
Table 1 Assignment of the ORF’s of the cloned Flavobacterium sp. strain R1534 sequence Gene
Enzyme
Location
Number of amino acids
M
crtE crtB crtI crtY crtZ
geranylgeranyl synthase phytoene synthase phytoene desaturase lycopene cyclase b-carotene hydroxylase
95 – 982 1890 – 979 3371 – 1887 4516 – 3368 5022 – 4513
295 303 494 382 169
31 331 32 615 54 411 42 368 19 282
aurantiacum, E. herbicola and E. uredovora (Fig. 2). In both Erwinia species the transcription of the crtE, crtB, crtY and crtI genes is oriented in the same direction, whereas crtZ has the opposite direction. In Flavobacterium sp. strain R1534 the transcription of the crtE gene is opposite to that of the other genes (e.g. crtZ, crtY, crtI and crtB). In A. aurantiacum all five genes crtW, crtZ, crtY, crtI and crtB are transcribed in the same direction. No resemblance to a consensus −10 or −35 promoter region of E. coli was detected upstream from the transcriptional start sites of crtE and crtZ, the respective first gene of the putative two transcripts in Flavobacterium sp. strain R1534. The genes transcribed in the same direction (crtZ, crtY, crtI and crtB) are grouped so tightly that the TGA stop codon of the
r
preceding gene overlaps the putative ATG of the following gene suggesting a translational coupling of these ORFs (Oppenheim and Yanofsky, 1980). 2.5. Comparison of individual crt genes of Flavobacterium sp. R1534 to their homologue in other organisms All five ORFs of Flavobacterium sp. strain R1534 encoding proteins having homology to known carotenoid biosynthesis enzymes of other species are clustered in 5.1 kb of the sequence ( Fig. 1). The results of the comparison of the aa sequence of the individual crt enzymes to their homologues in A. aurantiacum, E. uredovora and E. herbicola are shown in Fig. 2. The
Fig. 2. Comparison of the carotenoid gene clusters of Flavobacterium sp. strain R1534, A. aurantiacum (Misawa et al., 1995), E. herbicola (Hundle et al., 1994) and E. uredovora (Misawa et al., 1990). The values shown in the boxes indicate the percentages of identical aa compared to Flavobacterium sp. strain R1534.
L. Pasamontes et al./Gene 185 (1997) 35–41
geranylgeranyl pyrophosphate synthase (CRTE) condenses farnesyl pyrophosphate and isopentenyl pyrophosphate in a 1∞–4 head-to-tail reaction to geranylgeranyl pyrophosphate (GGPP) (Math et al., 1992). Like the crtE gene products of E. herbicola and E. uredovora, the GGPP synthase of Flavobacterium sp. strain R1534 ( Fig. 3) shares two highly conserved domains I and II with other prenyltransferases from bacteria and human, e.g. farnesyl pyrophosphate synthase (FPPS, gene fps) (Carattoli et al., 1991), hexaprenyl pyrophosphate synthase (HPPS, gene hps) (Ashby and Edwards, 1990) and GGPP synthases (Hundle et al., 1994; Math et al., 1992). Each of the three synthases are involved in 1∞–4 head-to-tail condensations of C isopentenyl moieties to isoprenoid sub5 strates of various length. Domains I and II are spaced by a stretch of 80–110 aa (yeast HPPS 128 aa) and consist of the motif EX LX DDX DX RRG and 6 2 2–4 4 GX FQX DDX , where X can be any aa (Fig. 3). These 2 2 2 two conserved regions might play an essential role in binding of the reaction partners and/or could represent (part of ) the catalytic center of the enzymes (for review, see Armstrong et al., 1993). The phytoene synthase (CRTB) catalyzes two enzymatic steps. First it catalyzes a head-to-head condensation of two geranylgeranyl pyrophosphates C to the 20
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C carbon unit prephytoene. Second, it catalyzes the 40 rearrangement of the cyclopropyl ring of prephytoene to phytoene (Sandmann and Misawa, 1991). Like their counterparts in A. aurantiacum, E. uredovora, E. herbicola and R. capsulatus, the CRTB of Flavobacterium also has a sequence extending from aa L to aa D 160 172 corresponding to motif (L/M )GXAXQX( T/S )NIXRD shown to have homology to domain II of CRTE (Armstrong et al., 1993). The motif (GAGX 3 GX AX LX AGX EX DX GG) found in the 3 2 2 6 2 2 N-terminal region of all CRTI enzymes, with the exception of the Aphanocapsa CRTI, and perfectly matching the requirements for a bab-binding fold ( Wierenga et al., 1986) for dinucleotides NAD(P)+ or FAD(P)+ needed as cofactors in the desaturation reaction (Armstrong et al., 1990; Bartley et al., 1990) spans in the phytoene desaturase of Flavobacterium from aa G to aa G . A 8 38 second region of homology, located at the C-terminal end of the crtI gene product which is also found in the two Erwinia strains, in R. capsulatus and in N. crassa is located between aa Y and P . This consensus 461 474 sequence, YX( V/A)G(A/G) (G/S )THPGXGXP, also having a bab-binding fold, was proposed to represent conserved structures or functional features required for the interaction between the substrate and the dehydrogenases (Armstrong et al., 1989).
Fig. 3. Localization of the two domains that are conserved between the 1∞–4 head-to-tail condensing enzymes geranylgeranyl pyrophosphate synthases (crtE gene products), farnesyl pyrophosphate synthases (fps), and hexaprenyl pyrophosphate synthase (hps) from different organisms. Only the localization of the boxes having the consensus sequence E*X LX DDX DX RRG and GX F**QX DDX D***, respectively, are shown. 6 2 2-4 4 2 2 2 Numbering is according to the position of the first aa of the respective motif in the protein sequence. The fps sequences from human and yeast are obtained from Wilkin et al. (1990) and Anderson and Rodwell (1989), respectively. The hps sequence of yeast is from Ashby and Edwards (1990). Note the following differences in motifs I and II of N. crassa: *=S, ***=N, and of M. xanthus: **=Y and ***=G.
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L. Pasamontes et al./Gene 185 (1997) 35–41
Fig. 4. (A) Genomic DNA of Flavobacterium sp. strain R1534 carried by the plasmids plyco, p59-2 and pZea-4 used to transform E. coli TG-1 cells to confirm the assignment of the individual crt genes. Restriction enzyme site abbreviations are as follows: B, BamHI; K, KpnI; S, SalI; Sp, SphI. (B) Summary of the crt genes present in the individual plasmid constructions and the main carotenoid detected by HPLC analysis. Methods: Plasmids for carotenoid production in E. coli. Clone 59-2 was obtained by subcloning the HindIII-BamHI fragment of clone 2 ( Fig. 1) into the HindIII-BamHI sites of clone 59 (Fig. 1). The resulting plasmid p59-2 carries the complete ORFs of the crtE, crtB, crtI and crtY gene and should lead to the production of b-carotene. pLyco was obtained by deleting the KpnI fragment, coding for approx. one half (N terminus) of the crtY gene, from the plasmid p59-2. pZea-4 was constructed by ligation of the AscI-SpeI fragment of p59-2, containing the crtE, crtB, crtI and most of the crtY gene with the AscI-XbaI fragment of clone 6a ( Fig. 1), containing the crtZ gene and sequences to complete the truncated crtY gene mentioned above. pZea-4 has therefore all five ORFs of the carotenoid biosynthesis pathway of Flavobacterium sp. strain R1534. Analysis of carotenoids: E. coli XL-1 or JM109 cells carrying different plasmid constructs were grown for the times indicated in the text, usually 24–60 h, in 200–400 ml of LB medium suplemented with 100 mg/ml ampicillin, in shake flasks at 37°C and 220 rpm. The carotenoids present in the microorganisms were extracted with an adequate volume of acetone using a rotation homogenizer (Polytron, Kinematica AG, Luzern, Switzerland ). The homogenate was filtered through the sintered glass of a suction filter into a round bottom flask. The filtrate was evaporated by means of a rotation evaporator at 50°C using a water-jet vacuum. For the zeaxanthin detection the residue was dissolved in n-hexane/acetone (86:14) before analysis with a normal phase HPLC as described ( Weber, 1988). For the detection of b-carotene and lycopene the evaporated extract was dissolved in nhexane/acetone (99:1) and analyzed by HPLC as described (Hengartner et al., 1992).
The b-carotene hydroxylase (CRTZ) that hydroxylates b-carotene to the xanthophyll zeaxanthin is also found in A. aurantiacum, Alcaligenes strain PC-1, E. herbicola and E. uredovora. Recently the b-carotene hydroxylases of A. aurantiacum and E. uredovora have been proposed to be bifunctional, i.e., the CRTZ enzyme thus converting not only b-ionone rings but also 4-ketob-ionone rings to 3-hydroxy-b-ionone and to 3-hydroxy4-keto-b-ionone rings (Misawa et al., 1995). Whether this is also valid for the crtZ gene product of Flavobacterium sp. strain R1534 has yet to be shown, but the high similarity (83%) to the A. aurantiacum enzyme makes this very likely.
2.6. Carotenoid production in E. coli The biochemical assignment of the products of the different Flavobacterium sp. strain R1534 crt genes was confirmed by analyzing the carotenoid accumulation in E. coli host strains, transformed with plasmids carrying the intact or partially deleted variants of the gene cluster, and thus lacking some of the crt gene products. Similar functional assays in E. coli with the Erwinia crt gene cluster have been described by other authors (Hundle et al., 1994; Misawa et al., 1990; Perry et al., 1986). Three different plasmids, pLyco, p59-2 and pZea-4, were constructed as outlined in the legend of Fig. 4 starting
L. Pasamontes et al./Gene 185 (1997) 35–41
from the three genomic isolates clone 2, clone 59 and clone 6a. As expected the pLyco carrying E. coli cells produced lycopene (0.05% of dry weight), those carrying p59-2 produced b-carotene (all-E,9-Z,13-Z, in total 0.03% of dry weight) and the cells having the pZea-4 construct accumulated mainly (3R,3R∞)-zeaxanthin (0.05% of dry weight) and some traces of the precursor b-carotene (0.0009% of dry weight). This confirms that we have cloned all the necessary genes of Flavobacterium sp. strain R1534 for the synthesis of zeaxanthin or their precursors (phytoene, lycopene and b-carotene). The carotenoid biosynthesis genes of Flavobacterium sp. strain R1534 reported in this paper provide the basis for the development of a homologous recombinant strain of Flavobacterium, suitable for the large-scale fermentation of zeaxanthin and other carotenoids.
References Anderson, D.H. and Rodwell, V.W. (1989) Nucleotide sequence and expression in E. coli of the 3-hydroxy-3-methylglutaryl Coenzyme A lyase gene of Pseudomonas mevalonii. J. Bacteriol. 171, 6468–6472. Armstrong, G.A. (1994) Eubacteria show their true colors: genetics of carotenoid biosynthesis from microbes to plants. J. Bacteriol. 176, 4795–4802. Armstrong, G.A., Alberti, M., Leach, F. and Hearst, J.E. (1989) Nucleotide sequence, organization, and nature of the protein products of the carotenoid biosynthesis gene cluster of Rhodobacter capsulatus. Mol. Gen. Genet. 216, 254–268. Armstrong, G.A., Alberti, M. and Hearst, J.E. (1990) Conserved enzymes mediate the early reactions of carotenoid biosynthesis in nonphotosynthetic and photosynthetic prokaryotes. Proc. Natl. Acad. Sci. USA 87, 9975–9979. Armstrong, G.A., Hundle, B.S. and Hearst, J.E. (1993) Evolutionary conservation and structural similarities of carotenoid biosynthesis gene products from photosynthetic and nonphotosynthetic organisms. Methods Enzymol. 214, 297–311. Ashby, M.N. and Edwards, P.A. (1990) Elucidation of the deficiency in two yeast coenzyme Q mutants. J. Biol. Chem. 265, 13157–13164. Bartley, G.E., Schmidhauser, T.J., Yanofsky, C. and Scolnik, P.A. (1990) Carotenoid desaturases from Rhodobacter capsulatus and Neurospora crassa are structurally and functionally conserved and contain domains homologous to flavoprotein disulfide oxidoreductases. J. Biol. Chem. 265, 16020–16024. Britton, G., Brown, D.J., Goodwin, T.W., Leuenberger, F.J. and Schocher, A.J. (1977) The carotenoids of Flavobacterium strain R-1560. Arch. Microbiol. 113, 33–37. Carattoli, A., Romano, N., Ballario, P., Morelli, G. and Macino, G. (1991) The Neurospora crassa carotenoid biosynthetic gene albino 3 reveals highly conserved regions among prenyltransferases. J. Biol. Chem. 266, 5854–5859. Devereux, J., Haeberli, P. and Smithies, O. (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12, 387–395. Heery, D.M., Gannon, H.F. and Powell, R. (1990) A simple method for subcloning DNA fragments from gel slices. Trends Biochem. Sci. 6, 173. Hengartner, U., Bernhard, K., Meyer, K., Englert, G. and Glinz, E. (1992) Synthesis, isolation, and NMR-spectroscopic characterization of fourteen ( Z )-isomers of lycopene and of some acetylenic didehydro- and tetradehydrolycopenes. Helv. Chim. Acta 75, 1848–1865.
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Hundle, B., Alberti, M., Nievelstein, V., Beyer, P., Kleinig, H., Armstrong, G.A., Burke, D.H. and Hearst, J.E. (1994) Functional assignment of Erwinia herbicola Eho10 carotenoid genes expressed in Escherichia coli. Mol. Gen. Genet. 245, 406–416. Leuenberger, F.J., Schocher, A.J., Britton, G. and Goodwin, T.W. (1973) The occurrence and possible biosynthetic significance of 3-hydroxy-b-zeacarotene in a Flavobacterium species. FEBS Lett. 33, 205–207. Marchuk, D., Drumm, M., Saulino, A. and Collins, F.S. (1990) Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR products. Nucleic Acids Res. 19, 1154. Math, S.K., Hearst, J.E. and Poulter, C.D. (1992) The crtE gene in Erwinia herbicola encodes geranylgeranyl diphosphate synthase. Proc. Natl. Acad. Sci. USA 89, 6761–6764. Misawa, N., Nakagawa, M., Kobayashi, K., Yamano, S., Izawa, Y., Nakamura, K. and Harashima, K. (1990) Elucidation of the Erwinia uredovora carotenoid biosynthetic pathway by functional analysis of gene products expressed in Escherichia coli. J. Bacteriol. 172, 6704–6712. Misawa, N., Satomi, Y., Kondo, K., Yokoyama, A., Kajiwara, S., Saito, T., Ohtani, T. and Miki, W. (1995) Structure and functional analysis of a marine bacterial carotenoid biosynthesis gene cluster and astaxanthin biosynthetic pathway proposed at the gene level. J. Bacteriol. 177, 6575–6584. Ninet, L. and Renaut, J. (1979) Carotenoids. In: Peppler, H.J. and Perlmann, D. ( Eds.), Microbial Technology, 2nd ed., Vol. I. Academic Press, New York, pp. 529–544. Oppenheim, D.S. and Yanofsky, C. (1980) Translational coupling during expression of the tryptophan operon of Escherichia coli. Genetics 95, 785–795. Perry, K.L., Simonitch, T.A., Harrison-Lavoie, K.J. and Liu, S.-T. (1986) Cloning and regulation of Erwinia herbicola pigment genes. J. Bacteriol. 168, 607–612. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Sandmann, G. (1994) Review: Carotenoid biosynthesis in microorganisms and plants. Eur. J. Biochem. 223, 7–24. Sandmann, G. and Misawa, N. (1991) New functional assignment of the carotenogenic genes crtB and crtE with constructs of these genes from Erwinia species. FEMS Lett. 90, 253–258. Shine, J. and Dalgarno, L. (1974) The 3∞-terminal sequence of Escherichia coli 16S ribosomal RNA: complementary to nonsense triplets and ribosome binding sites. Proc. Natl. Acad. Sci. USA 71, 1342–1346. Southern, E.M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503–517. Straub, O. (1987) In: Pfander, H. ( Ed.), Key to Carotenoids. Birkhaeuser, Basel. Tuveson, R.W., Larson, R.A. and Kagan, J. (1988) Role of cloned carotenoid genes expressed in Escherichia coli in protecting against inactivation by near-UV light and specific phototoxic molecules. J. Bacteriol. 170, 4675–4680. Weber, S. (1988) Determination of xanthophylls, lutein and zeaxanthin in complete feeds and xanthophyll blends with HPLC. In: Keller, H.E. (Ed.), Analytical Methods for Vitamins and Carotenoids in Feed, pp. 83–85. ROCHE Publications Index Nr. 2101, F. Hoffmann-La Roche Ltd., Basel. Wierenga, R.K., Terpstra, P. and Hol, W.G.J. (1986) Prediction of the occurrence of the ADP-binding bab-fold in proteins, using amino acid sequence fingerprint. J. Mol. Biol. 187, 101–107. Wilkin, D.J., Kutsunai, S.Y. and Edwards, P.A. (1990) Isolation and sequence of the human farnesyl pyrophosphate synthetase cDNA. Coordinate regulation of the mRNAs for farnesylpyrophosphate synthetase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, and 3-hydroxy-3-methylglutaryl coenzyme A synthase by phorbol ester. J. Biol. Chem. 265, 4607–4614.
Isolation and characterization of the carotenoid biosynthesis genes of Flavobacterium sp. strain R1534 Luis Pasamontes *, Denis Hug, Michel Tessier, Hans-Peter Hohmann, Joseph Schierle, Adolphus P.G.M. van Loon F. Hoffmann-La Roche Ltd., Vitamins and Fine Chemicals Division, Biotechnology Section 93/4.22, 4070 Basel, Switzerland Received 28 April 1996; revised 15 July 1996; accepted 24 July 1996; Received by A.M. Campbell
Abstract The Gram-negative bacterium Flavobacterium sp. strain R1534 is a natural producer of zeaxanthin. A 14 kb genomic DNA fragment of this organism has been cloned and a 5.1 kb piece containing the carotenoid biosynthesis genes sequenced. The carotenoid biosynthesis cluster consists of five genes arranged in at least two operons. The five genes are necessary and sufficient for the synthesis of zeaxanthin. The encoded proteins have significant homology to the crtE, crtB, crtY, crtI and crtZ gene products of other carotenogenic organisms. Biochemical assignment of the individual gene products was done by HPLC analysis of the carotenoid accumulation in Escherichia coli host strains transformed with plasmids carrying deletions of the Flavobacterium sp. strain R1534 carotenoid biosynthesis cluster. Keywords: Gram-negative bacterium; Cloning; Geranylgeranyl pyrophosphate synthase; Phytoene synthase; Phytoene desaturase; Lycopene cyclase-b-carotene hydroxylase; Zeaxanthin
1. Introduction Over 560 different carotenoids have been described from carotenogenic organisms found among bacteria, yeasts, fungi and plants (Straub, 1987). Their importance for microorganisms has been attributed to their strong antioxidant characteristics which protect the organisms against photooxidative damage ( Tuveson et al., 1988). Flavobacterium sp. strain R1534 is a nonphotosynthetic, Gram-negative bacterium, and a natural producer of the yellow pigment (3R,3R∞)-zeaxanthin (Leuenberger et al., 1973; Britton et al., 1977). Among * Corresponding author. Tel. +41 61 6887838; Fax +41 61 6881645; e-mail: [email protected] Abbreviations: aa, amino acid(s); bp, base pair(s); BSA, bovine serum albumin; CRTE (crtE), geranylgeranyl pyrophosphate synthase (corresponding gene); CRTB (crtB), phytoene synthase; CRTI (crtI ), phytoene desaturase; CRTD (crtD), hydroxyneurosporene desaturase; CRTY (crtY ), lycopene cyclase; CRTZ (crtZ), b-carotene hydroxylase; FPPS, farnesyl pyrophosphate synthase; GCG, Genetics Computer Group, Madison, WI; GGPP, geranylgeranyl pyrophosphate; HPLC, high-performance liquid chromatography; HPPS, hexaprenyl pyrophosphate synthase; ORF, open reading frame; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate; SSC, 0.15 M NaCl/0.015 M Na · citrate pH 7.6. 3
bacteria Flavobacterium may represent the best source for the development of a fermentative production process for (3R,3R∞)-zeaxanthin. Derivatives of Flavobacterium sp. strain R1534, obtained by classical mutagenesis, have attracted in the past two decades wide interest for the development of a large-scale fermentative production of zeaxanthin, although with little success. Mutant strains producing up to 335 mg zeaxanthin/l have been reported (Ninet and Renaut, 1979) but despite this high titer, fermentative large-scale production is still not attractive. Cloning of the carotenoid biosynthesis genes of this organism may allow replacement of the classical mutagenesis approach by a more rational one, using molecular tools to amplify the copy number of relevant genes, deregulate their expression and eliminate bottlenecks in the carotenoid biosynthesis pathway. Although all the necessary genes required to synthesize zeaxanthin have been described from different organisms (Misawa et al., 1990, 1995; Armstrong et al., 1990), the use of the homologous genes in a production strain should be advantageous compared to the use of heterologous genes. The initial steps in the carotenoid biosynthesis up to the phytoene formation have been found to be common to all the carotenoids described so far. The first commit-
0378-1119/97/$17.00 Copyright © 1997 Elsevier Science B.V. All rights reserved PII S 03 7 8 -1 1 1 9 ( 9 6 ) 0 0 6 24 - 5
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L. Pasamontes et al./Gene 185 (1997) 35–41
ting step towards the carotenoid biosynthesis is catalyzed by the enzyme geranylgeranyl pyrophosphate synthase (CRTE) which condenses farnesyl pyrophosphate, the common C carbon precursor of cholesterol, steroids 15 and carotenoids, with an isopentenyl pyrophosphate moiety. Geranylgeranyl pyrophosphate is then converted by the enzymes prephytoene and phytoene synthase into phytoene, a C carbon unit. In bacteria and lower 40 eucaryotes both enzymatic functions are encoded by a single gene (crtB). After phytoene formation the biosynthetic pathways diverge in different species leading to accumulation of different types of carotenoids. In Flavobacterium sp. strain R1534 as in Erwinia herbicola and Erwinia uredovora, phytoene is converted to lycopene by the enzyme phytoene desaturase (CRTI ). The conversion of lycopene to b-carotene involves four desaturation and two cyclization steps. Again, as in E. herbicola and E. uredovora, all these steps are catalyzed by one enzyme, the lycopene cyclase (CRTY ). This is also the case for the hydroxylation of b-carotene to zeaxanthin, which involves two steps both catalyzed by the same enzyme, the b-carotene hydroxylase (CRTZ) (for review, see Armstrong, 1994; Sandmann, 1994). In this report we describe the cloning and sequencing of the carotenoid biosynthesis gene cluster of this bacterium. The cluster consists of all five genes (crtE, crtB, crtI, crtY and crtZ ) of the biosynthetic pathway necessary to synthesize zeaxanthin. Functional assignment for the cloned carotenoid genes was done in Escherichia coli transformed with deletion mutants of the original plasmid carrying all five genes.
2. Results and discussion
2.1. Amplification of a DNA fragment of the crtB gene of Flavobacterium sp. strain R1534 Alignment of the phytoene synthetases (CRTB) of E. herbicola (Armstrong et al., 1990) and R. capsulatus (Armstrong et al., 1989) revealed quite a number of conserved aa stretches among these two enzymes. Two such conserved peptide sequences L( E/D)GFAMD and Q(L/M ) ( T/S )NIARD were chosen for reverse translation to design the degenerate primer #4 (5∞-CTGGAAGG( T/C )TT(C/T )GCTATGGA-3∞) and the reverse primer #5 (5∞-GTCACGAGCGATGTT(G/A)GTCAGCTG-3∞). PCR amplifications with these primers on genomic DNA of Flavobacterium sp. strain R1534 gave a product of about 190 bp named fragment 45F. Cloning and sequencing of this PCR fragment indicated that we had cloned a DNA piece encoding 60 aa, with significant homology to known phytoene synthases from other species. Based on the sequence of fragment 45F two oligonucleotides were
synthesized, primer #7 (5∞-CCTGGATGACGTGCTGGAATATTCC-3∞) and primer #8 (5∞-CAAGGCCCAGATCGCAGGCG-3∞). PCR amplification with these primers on genomic DNA of Flavobacterium R1534 generated a specific DNA probe of 119 bp named 46F which was then used for the library screening as described in Section 2.2. 2.2. Cloning of the Flavobacterium sp. strain R1534 carotenoid biosynthetic genes Cloning of the carotenoid biosynthesis cluster started using the PCR fragment 46F as a probe to hybridize to a Southern blot carrying chromosomal DNA of Flavobacterium sp. strain R1534 digested with different restriction enzymes. A 2.4 kb XhoI-PstI fragment hybridizing to the probe seemed the most appropriate for isolation. The XhoI-PstI partial library was constructed as outlined in the legend of Fig. 1. The insert of the positive transformant, clone 85, was sequenced and revealed sequences not only homologous to the phytoene synthase (CRTB) but also to the phytoene desaturase (CRTI ) of both Erwinia species herbicola and uredovora. Left hand genomic sequences of clone 85 were obtained by the same approach using probe B to screen a partial library made of ClaI-HindIII fragments of approx. 9.2 kb. Sequencing of the 5∞ and 3∞ of the insert of one positive, clone 51, revealed that only the region close to the HindIII site showed relevant homology to genes of the carotenoid biosynthesis of the Erwinia species mentioned above (e.g. crtB gene and crtE gene). To facilitate further sequencing and construction work, the 4.2 kb BamHI-HindIII fragment of clone 51 was further subcloned into the respective sites of pBluescriptII KS(+), resulting in clone 2. Sequencing of the insert of this clone confirmed the presence of genes homologous to Erwinia sp. crtB and crtE genes. These genes were located within 1.8 kb from the HindIII site. The remaining 2.4 kb of this insert had no homology to known carotenoid biosynthesis genes. Additional genomic sequences downstream from the HindIII site were obtained by screening a SalI-HindIII size restricted library (size of fragments approx. 2.8 kb) with probe A. The insert of one positive, clone 59, revealed in addition to known sequences the missing N terminus of the crtI gene, the complete putative lycopene cyclase gene (crtY ) and part of the C terminus of the crtZ gene. To isolate the missing crtZ gene a library made of partially Sau3AI digested genomic DNA of Flavobacterium sp. strain R1534 was screened with probe D, resulting in several positive clones. One transformant designated, clone 6a, had an insert of 4.9 kb comprising the missing part of the crtZ gene. The four independent clones, namely clone 51, clone 85, clone 59 and clone 6a, covering approx. 14 kb of the Flavobacterium sp. strain R1534 genome are shown in Fig. 1. The determined sequence
L. Pasamontes et al./Gene 185 (1997) 35–41
37
Fig. 1. Physical map of the organization of the carotenoid biosynthesis cluster in Flavobacterium sp. strain R1534, deduced from the genomic clones obtained. The location of the probes A–D and 46F, used for the screening, are shown as bars. Restriction enzyme site abbreviations are as follows: Ba, BamHI; B, BglII; Bs, BstYI; Bx, BstXI; C, ClaI; H, HindIII; K, KpnI; M, MluI; N, NotI; P, PstI; S, SalI; Sa, Sau3AI; X, XhoI; *more restriction sites present but not shown. Sequences were analyzed using the GCG sequence analysis software package ( Version 8.0) by Genetics Computer, Inc. (Devereux et al., 1984). The determined sequence from the SalI site (bp position 1) to the MluI site (bp position 5188) has been filed to the GenBank under accession No. U62808. Methods: Isolation of genomic DNA of Flavobacterium sp. strain R1534 (ATCC 21588) was basically done as described by Sambrook et al. (1989). Genomic DNA amplifications: The GeneAmp@ DNA amplification kit (Perkin Elmer) was used for PCR. The amplification was carried out with 15 ng of Flavobacterium sp. strain R1534 genomic DNA as template in a total volume of 100 ml using 10 pmol of each primer #4 and #5. In all cases an aliquot of the reaction was analyzed on a 1.5% agarose gel. PCR products of the expected size were excised from the agarose gel and isolated by centrifugation of the gel slices through siliconized glass wool as described by Heery et al. (1990) or using the GENECLEAN Kit (BIO101 Inc.) essentially according to the manufacturer’s protocol. The fragments were subsequentely cloned into the T-tailed EcoRV site of pBluescriptII KS+ made according to Marchuk et al. (1990). Southern blot analysis: For hybridization experiments Flavobacterium sp. strain R1534 genomic DNA (3 mg) was digested with the appropriate restriction enzymes and electrophoresed on a 0.75% agarose gel. The transfer to Zeta-Probe blotting membranes (Bio-Rad ), was done as described (Southern, 1975). Prehybridization and hybridization were in 7% SDS, 1% BSA (fraction V; Boehringer), 0.5 M Na HPO (pH 7.2) at 65°C. After hybridization the membranes were washed twice for 2 4 5 min in 2×SSC, 1% SDS at room temperature and twice for 15 min in 0.1% SSC, 0.1% SDS at 65°C. Library construction: Prior to the partial library construction, Southern blot analysis with a given probe was done in order to identify a specific restriction fragment of interest. Subsequently 10–20 mg of genomic DNA was digested with the appropriate restriction enzymes and electrophoresed on a 0.75% agarose gel. According to comigrating DNA markers, the region of interest was cut out of the gel, the DNA isolated and cloned into the pBluescriptII KS(−) vector. Transformation of the ligation mixture into E. coli strain XL-1 (Stratagene, La Jolla, CA, USA) cells resulted in partial genomic libraries containing the fragment of interest. DNA probes were labeled with [a-32P]dGTP (Amersham International ) by nick-translation according to Sambrook et al. (1989). Probe A is a BstXI-PstI fragment of 184 bp; probe B is a XhoI-NotI fragment of 397 bp; probe C is a BglII-PstI fragment of 536 bp; probe D is a KpnI-BstYI fragment of 376 bp; probe 46F is a 119 bp PCR fragment (see Section 2.1).
from the SalI site (bp position 1) to the MluI site (bp position 5188) has been filed to the GenBank under the accession No. U62808. 2.3. Putative protein coding regions of the cloned sequence Computer analysis of the aforementioned sequence revealed five putative open reading frames (ORFs) encoding proteins with similarity to known crt enzymes ( Table 1). The translation start sites of the ORFs was determined based on the appropriately located sequences homologous to the Shine-Dalgarno (Shine and Dalgarno, 1974) consensus sequence AGG–6-9N–ATG,
the homology to the N-terminal sequences of the respective enzymes of E. herbicola, E. uredovora, Agrobacterium aurantiacum and Alcaligenes sp. strain PC-1 (Misawa et al., 1995) and the strong third position GC bias observed in all five putative ORFs. 2.4. Genomic organization of the crt gene cluster and comparison to the cartenoid biosynthetic cluster of other Gram-negative bacteria The genomic organization of the ORFs within the crt gene cluster of Flavobacterium sp. strain R1534 is different when compared to the carotenoid biosynthesis clusters of other Gram-negative organisms, such as A.
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L. Pasamontes et al./Gene 185 (1997) 35–41
Table 1 Assignment of the ORF’s of the cloned Flavobacterium sp. strain R1534 sequence Gene
Enzyme
Location
Number of amino acids
M
crtE crtB crtI crtY crtZ
geranylgeranyl synthase phytoene synthase phytoene desaturase lycopene cyclase b-carotene hydroxylase
95 – 982 1890 – 979 3371 – 1887 4516 – 3368 5022 – 4513
295 303 494 382 169
31 331 32 615 54 411 42 368 19 282
aurantiacum, E. herbicola and E. uredovora (Fig. 2). In both Erwinia species the transcription of the crtE, crtB, crtY and crtI genes is oriented in the same direction, whereas crtZ has the opposite direction. In Flavobacterium sp. strain R1534 the transcription of the crtE gene is opposite to that of the other genes (e.g. crtZ, crtY, crtI and crtB). In A. aurantiacum all five genes crtW, crtZ, crtY, crtI and crtB are transcribed in the same direction. No resemblance to a consensus −10 or −35 promoter region of E. coli was detected upstream from the transcriptional start sites of crtE and crtZ, the respective first gene of the putative two transcripts in Flavobacterium sp. strain R1534. The genes transcribed in the same direction (crtZ, crtY, crtI and crtB) are grouped so tightly that the TGA stop codon of the
r
preceding gene overlaps the putative ATG of the following gene suggesting a translational coupling of these ORFs (Oppenheim and Yanofsky, 1980). 2.5. Comparison of individual crt genes of Flavobacterium sp. R1534 to their homologue in other organisms All five ORFs of Flavobacterium sp. strain R1534 encoding proteins having homology to known carotenoid biosynthesis enzymes of other species are clustered in 5.1 kb of the sequence ( Fig. 1). The results of the comparison of the aa sequence of the individual crt enzymes to their homologues in A. aurantiacum, E. uredovora and E. herbicola are shown in Fig. 2. The
Fig. 2. Comparison of the carotenoid gene clusters of Flavobacterium sp. strain R1534, A. aurantiacum (Misawa et al., 1995), E. herbicola (Hundle et al., 1994) and E. uredovora (Misawa et al., 1990). The values shown in the boxes indicate the percentages of identical aa compared to Flavobacterium sp. strain R1534.
L. Pasamontes et al./Gene 185 (1997) 35–41
geranylgeranyl pyrophosphate synthase (CRTE) condenses farnesyl pyrophosphate and isopentenyl pyrophosphate in a 1∞–4 head-to-tail reaction to geranylgeranyl pyrophosphate (GGPP) (Math et al., 1992). Like the crtE gene products of E. herbicola and E. uredovora, the GGPP synthase of Flavobacterium sp. strain R1534 ( Fig. 3) shares two highly conserved domains I and II with other prenyltransferases from bacteria and human, e.g. farnesyl pyrophosphate synthase (FPPS, gene fps) (Carattoli et al., 1991), hexaprenyl pyrophosphate synthase (HPPS, gene hps) (Ashby and Edwards, 1990) and GGPP synthases (Hundle et al., 1994; Math et al., 1992). Each of the three synthases are involved in 1∞–4 head-to-tail condensations of C isopentenyl moieties to isoprenoid sub5 strates of various length. Domains I and II are spaced by a stretch of 80–110 aa (yeast HPPS 128 aa) and consist of the motif EX LX DDX DX RRG and 6 2 2–4 4 GX FQX DDX , where X can be any aa (Fig. 3). These 2 2 2 two conserved regions might play an essential role in binding of the reaction partners and/or could represent (part of ) the catalytic center of the enzymes (for review, see Armstrong et al., 1993). The phytoene synthase (CRTB) catalyzes two enzymatic steps. First it catalyzes a head-to-head condensation of two geranylgeranyl pyrophosphates C to the 20
39
C carbon unit prephytoene. Second, it catalyzes the 40 rearrangement of the cyclopropyl ring of prephytoene to phytoene (Sandmann and Misawa, 1991). Like their counterparts in A. aurantiacum, E. uredovora, E. herbicola and R. capsulatus, the CRTB of Flavobacterium also has a sequence extending from aa L to aa D 160 172 corresponding to motif (L/M )GXAXQX( T/S )NIXRD shown to have homology to domain II of CRTE (Armstrong et al., 1993). The motif (GAGX 3 GX AX LX AGX EX DX GG) found in the 3 2 2 6 2 2 N-terminal region of all CRTI enzymes, with the exception of the Aphanocapsa CRTI, and perfectly matching the requirements for a bab-binding fold ( Wierenga et al., 1986) for dinucleotides NAD(P)+ or FAD(P)+ needed as cofactors in the desaturation reaction (Armstrong et al., 1990; Bartley et al., 1990) spans in the phytoene desaturase of Flavobacterium from aa G to aa G . A 8 38 second region of homology, located at the C-terminal end of the crtI gene product which is also found in the two Erwinia strains, in R. capsulatus and in N. crassa is located between aa Y and P . This consensus 461 474 sequence, YX( V/A)G(A/G) (G/S )THPGXGXP, also having a bab-binding fold, was proposed to represent conserved structures or functional features required for the interaction between the substrate and the dehydrogenases (Armstrong et al., 1989).
Fig. 3. Localization of the two domains that are conserved between the 1∞–4 head-to-tail condensing enzymes geranylgeranyl pyrophosphate synthases (crtE gene products), farnesyl pyrophosphate synthases (fps), and hexaprenyl pyrophosphate synthase (hps) from different organisms. Only the localization of the boxes having the consensus sequence E*X LX DDX DX RRG and GX F**QX DDX D***, respectively, are shown. 6 2 2-4 4 2 2 2 Numbering is according to the position of the first aa of the respective motif in the protein sequence. The fps sequences from human and yeast are obtained from Wilkin et al. (1990) and Anderson and Rodwell (1989), respectively. The hps sequence of yeast is from Ashby and Edwards (1990). Note the following differences in motifs I and II of N. crassa: *=S, ***=N, and of M. xanthus: **=Y and ***=G.
40
L. Pasamontes et al./Gene 185 (1997) 35–41
Fig. 4. (A) Genomic DNA of Flavobacterium sp. strain R1534 carried by the plasmids plyco, p59-2 and pZea-4 used to transform E. coli TG-1 cells to confirm the assignment of the individual crt genes. Restriction enzyme site abbreviations are as follows: B, BamHI; K, KpnI; S, SalI; Sp, SphI. (B) Summary of the crt genes present in the individual plasmid constructions and the main carotenoid detected by HPLC analysis. Methods: Plasmids for carotenoid production in E. coli. Clone 59-2 was obtained by subcloning the HindIII-BamHI fragment of clone 2 ( Fig. 1) into the HindIII-BamHI sites of clone 59 (Fig. 1). The resulting plasmid p59-2 carries the complete ORFs of the crtE, crtB, crtI and crtY gene and should lead to the production of b-carotene. pLyco was obtained by deleting the KpnI fragment, coding for approx. one half (N terminus) of the crtY gene, from the plasmid p59-2. pZea-4 was constructed by ligation of the AscI-SpeI fragment of p59-2, containing the crtE, crtB, crtI and most of the crtY gene with the AscI-XbaI fragment of clone 6a ( Fig. 1), containing the crtZ gene and sequences to complete the truncated crtY gene mentioned above. pZea-4 has therefore all five ORFs of the carotenoid biosynthesis pathway of Flavobacterium sp. strain R1534. Analysis of carotenoids: E. coli XL-1 or JM109 cells carrying different plasmid constructs were grown for the times indicated in the text, usually 24–60 h, in 200–400 ml of LB medium suplemented with 100 mg/ml ampicillin, in shake flasks at 37°C and 220 rpm. The carotenoids present in the microorganisms were extracted with an adequate volume of acetone using a rotation homogenizer (Polytron, Kinematica AG, Luzern, Switzerland ). The homogenate was filtered through the sintered glass of a suction filter into a round bottom flask. The filtrate was evaporated by means of a rotation evaporator at 50°C using a water-jet vacuum. For the zeaxanthin detection the residue was dissolved in n-hexane/acetone (86:14) before analysis with a normal phase HPLC as described ( Weber, 1988). For the detection of b-carotene and lycopene the evaporated extract was dissolved in nhexane/acetone (99:1) and analyzed by HPLC as described (Hengartner et al., 1992).
The b-carotene hydroxylase (CRTZ) that hydroxylates b-carotene to the xanthophyll zeaxanthin is also found in A. aurantiacum, Alcaligenes strain PC-1, E. herbicola and E. uredovora. Recently the b-carotene hydroxylases of A. aurantiacum and E. uredovora have been proposed to be bifunctional, i.e., the CRTZ enzyme thus converting not only b-ionone rings but also 4-ketob-ionone rings to 3-hydroxy-b-ionone and to 3-hydroxy4-keto-b-ionone rings (Misawa et al., 1995). Whether this is also valid for the crtZ gene product of Flavobacterium sp. strain R1534 has yet to be shown, but the high similarity (83%) to the A. aurantiacum enzyme makes this very likely.
2.6. Carotenoid production in E. coli The biochemical assignment of the products of the different Flavobacterium sp. strain R1534 crt genes was confirmed by analyzing the carotenoid accumulation in E. coli host strains, transformed with plasmids carrying the intact or partially deleted variants of the gene cluster, and thus lacking some of the crt gene products. Similar functional assays in E. coli with the Erwinia crt gene cluster have been described by other authors (Hundle et al., 1994; Misawa et al., 1990; Perry et al., 1986). Three different plasmids, pLyco, p59-2 and pZea-4, were constructed as outlined in the legend of Fig. 4 starting
L. Pasamontes et al./Gene 185 (1997) 35–41
from the three genomic isolates clone 2, clone 59 and clone 6a. As expected the pLyco carrying E. coli cells produced lycopene (0.05% of dry weight), those carrying p59-2 produced b-carotene (all-E,9-Z,13-Z, in total 0.03% of dry weight) and the cells having the pZea-4 construct accumulated mainly (3R,3R∞)-zeaxanthin (0.05% of dry weight) and some traces of the precursor b-carotene (0.0009% of dry weight). This confirms that we have cloned all the necessary genes of Flavobacterium sp. strain R1534 for the synthesis of zeaxanthin or their precursors (phytoene, lycopene and b-carotene). The carotenoid biosynthesis genes of Flavobacterium sp. strain R1534 reported in this paper provide the basis for the development of a homologous recombinant strain of Flavobacterium, suitable for the large-scale fermentation of zeaxanthin and other carotenoids.
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