Genomic organization of the structural genes controlling the astaxanthin biosynthesis pathway of Xanthophyllomyces dendrorhous

The cloning and nucleotide sequence of the genes (idi, crtE, crtYB, crtI and crtS) controlling the astaxanthin biosynthesis pathway of the wild-type ATCC 24230 strain of Xanthophyllomyces dendrorhous in their genomic and cDNA version were obtained. The idi, crtE, crtYB, crtI and crtS genes were cloned, as fragments of 10.9, 11.5, 15.8, 5.9 and 4 kb respectively. The nucleotide sequence data analysis indicates that the idi, crtE, crtYB, crtI and crtS genes have 4, 8, 4, 11, and 17 introns and 5, 9, 5, 12 and 18 exons respectively. In addition, a highly efficient site-directed mutagenesis system was developed by transformation by integration, followed by mitotic recombination (the double recombinant method). Heterozygote idi (idi+/idi-::hph), crtE (crtE+/crtE-::hph), crtYB (crtYB+/crtYB-::hph), crtI (crtI+/crtI-::hph) and crtS (crtS+/crtS-::hph) and homozygote mutants crtYB (crtYB-::hph/crtYB-::hph), crtI (crtI-::hph/crtI-::hph) and crtS (crtS-::hph/crtS::hph) were constructed. All the heterozygote mutants have a pale phenotype and produce less carotenoids than the wild-type strain. The genetic analysis of the crtYB, crtI and crtS loci in the wild-type, heterozygote, and homozygote give evidence of the diploid constitution of ATCC 24230 strains. In addition, the cloning of a truncated form of the crtYB that lacks 153 amino acids of the N-terminal region derived from alternatively spliced mRNA was obtained. Their heterologous expression in Escherichia coli carrying the carotenogenic cluster of Erwinia uredovora result in trans-complementation and give evidence of its functionality in this bacterium, maintaining its phytoene synthase activity but not the lycopene cyclase activity. Key terms: astaxanthin biosynthesis, Xanthophyllomyces dendrorhous, Phaffia rhodozyma. * Corresponding Author: Víctor Cifuentes, Casilla 653, Santiago, Chile; Tel: 56-2-9787346; Fax: 56-2-2727363 ; E-mail: vcifuentes@uchile.cl Received: June 26, 2007. In Revised form: April 28, 2008. Accepted: May 5, 2008 INTRODUCTION In nature, carotenoids are produced on a large scale: over 100 million tons per year. In addition, these pigments are synthesized in a wide variety of structures by plants, algae, bacteria and fungi. One of them, astaxanthin, is the principal carotenoid responsible for the orange-red color of marine invertebrates, fish, and birds and is primarily produced by phytoplankton and the red basidiomycetous yeast Xanthophyllomyces dendrorhous (Andrewes et al., 1976; Johnson and Lewis, 1979; Miller et al., 1976; Johnson, 2003). In this yeast, astaxanthin, like other carotenoids, is a terpenoid pigment that is produced by the terpenoid biosynthetic pathway from the basic C5-isoterpene unit isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP), produced by the isopentenyl pyrophosphate isomerase enzyme encoded by the idi gene. In a following step, DMAPP and IPP are condensed to geranyl pyrophosphate, farnesyl, and geranylgeranyl pyrophosphate (GGPP), possibly through the action of farnesyl pyrophosphate synthase and geranylgeranyl pyrophosphate synthase encoded by the fps NIKLITSCHEK ET AL. Biol Res 41, 2008, 93-108 94 and crtE genes, respectively. Two GGPP molecules are condensed to phytoene by the phytoene synthase enzyme encoded by the crtYB gene, representing the first specific step in the carotenoid biosynthesis. Subsequently, via four desaturations by the action of the phytoene desaturase encoded by the crtI gene, lycopene is produced, which is converted to βcarotene by the lycopene cyclase activity encoded by the crtYB gene after two cyclization reactions. Finally β-carotene is oxidized to astaxanthin by the product of the crtS gene. The crtYB and crtI genes were isolated from a cDNA library of the strain CBS 6938 of X . dendrorhous by heterologous complementation in Escherichia coli strains carrying carotenoid biosynthesis genes from the bacteria Erwinia uredovora (Verdoes et al. , 1999a, Verdoes et al., 1999b). In addition, in the wild-type ATCC 24230 strain of X. dendrorhous, two unexpected cDNA of crtYB and crtI genes have been isolated that correspond to alternative spliced variants, which have numerous stop codons in their sequence (Lodato et al., 2003). The ratio of mature to alternative mRNA for the crtI gene decreased as a function of the age of the culture, while the carotenoids content increased (Lodato et al., 2003). Furthermore, the expression at the mRNA level of the carotenoid biosynthesis idi, crtE, crtYB, crtI, and crtS genes from X. dendrorhous was studied by RT-PCR (Lodato et al. 2003; 2004 and 2007). However, little is known about the genomic organization of these genes in this yeast. In this work, we cloned the idi, crtE, crtYB, crtI, and crtS genes controlling the astaxanthin biosynthesis pathway and their respective cDNAs and determined their genetic organization by sequence analysis and electrophoretic karyotyping. The function of the crtYB, crtI, and crtS genes was inferred from site-directed mutagenesis. MATERIALS AND METHODS Strains, media, plasmid and genomic library All strains used in this study are listed in Table I and the plasmids in Table II. The X. dendrorhous wild-type strain ATCC 24230 was used to obtain genomic DNA for the construction of the genome library and site-directed mutagenesis. E. coli strain DH-5α was used for the propagation of plasmids and the genomic libraries of X. dendrorhous. Three genomic libraries of wild-type X. dendrorhous were used in this study. The first one contains partially digested BamHI DNA fragments inserted in the BamHI site of the YIp5 vector. The other two partial genomic libraries contain EcoRV or PstI DNA fragments in the EcoRV or PstI sites of the Bluescript SKplasmid respectively. X. dendrorhous wild-type strain was grown at 22 °C in baffled flasks containing YM medium in an orbital shaker at 100 rpm. The DNA fragment of 3558 bp containing the crtI gene of X. dendrorhous carried by pL22 plasmid was amplified by PCR using primers PHA1 (5’ AAGCTTA AACTGACGTGCC TC 3’) and PHA4 (5’GAATTCAGAAAGCAAGAAC 3’). The E. coli-transformed cells were plated on selective Luria-Bertani agar plates (Sambrook et al., 1989) and incubated at 37°C overnight. These plates contained ampicillin (100 μg/ml) for plasmid selection and 40 μl of a 2% solution of X-Gal (5b r o m o 4 c h l o r o 3 i n d o l y l β D galactopyranoside) for selection of recombinant clones. The X. dendrorhous transformed cells were plated on YM containing 10 μg/ml of hygromicin B. cDNA synthesis and cloning For the reverse transcription (RT) reaction, total RNA was purified from cellular pellets obtained from 40 ml aliquots of culture by a modified protocol of Chomczynski and Sacchi (Chomczynski and Sacchi, 1987; Lodato et al. , 2004). The total RNA concentration was quantified at 260 nm, and the integrity of the RNA was checked in denaturant agarose gel electrophoresis. The RNA sample was then treated with 1U of DNase I (Roche) in 2.5 mM MgCl2 at 25oC for 30 min. The reaction was stopped by the addition of EDTA at 2.5 mM final concentration and heating at 65oC for 15 95 NIKLITSCHEK ET AL. Biol Res 41, 2008, 93-108 min. The RT reaction was performed in 25 μl final volume with 3 μg of total RNA, 75 pmoles oligodT15-18, 0.5 mM of dNTPs and 200 U of M-MLV reverse transcriptase (Promega) (Lodato et al., 2003, 2004). The reaction mixture was incubated at 42°C for 60 min and then heated to 65°C for 10 min. For the amplification of double-stranded cDNA of carotenoid biosynthesis genes, PCR reactions were performed according to the experimental conditions previously described (Lodato et al., 2003, 2004) in a final volume of 25 μl containing 2 U of Pfu polimerase (Stratagene), 2.5 μl of 10 X Pfu buffer, 0.5 μl of 10 mM dNTPs, 1 μl of 50 mM MgCl2, 1 μl of 25 μM of each primer, and 2 μl of RT reaction product, and the final volume was adjusted with nucleasefree water. Amplification was performed in a DNA Thermal Cycler 2400 (PerkinElmer) or 2720 ABI Thermal cycler as follows: initial denaturation at 95°C for 3 min, 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, synthesis at 72°C for 3 min and a final extension step at 72°C for 10 min.


INTRODUCTION
In nature, carotenoids are produced on a large scale: over 100 million tons per year.In addition, these pigments are synthesized in a wide variety of structures by plants, algae, bacteria and fungi.One of them, astaxanthin, is the principal carotenoid responsible for the orange-red color of marine invertebrates, fish, and birds and is primarily produced by phytoplankton and the red basidiomycetous yeast Xanthophyllomyces dendrorhous (Andrewes et al., 1976;Johnson and Lewis, 1979;Miller et al., 1976;Johnson, 2003).
In this yeast, astaxanthin, like other carotenoids, is a terpenoid pigment that is produced by the terpenoid biosynthetic pathway from the basic C5-isoterpene unit isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP), produced by the isopentenyl pyrophosphate isomerase enzyme encoded by the idi gene.In a following step, DMAPP and IPP are condensed to geranyl pyrophosphate, farnesyl, and geranylgeranyl pyrophosphate (GGPP), possibly through the action of farnesyl pyrophosphate synthase and geranylgeranyl pyrophosphate synthase encoded by the fps and crtE genes, respectively.Two GGPP molecules are condensed to phytoene by the phytoene synthase enzyme encoded by the crtYB gene, representing the first specific step in the carotenoid biosynthesis.Subsequently, via four desaturations by the action of the phytoene desaturase encoded by the crtI gene, lycopene is produced, which is converted to βcarotene by the lycopene cyclase activity encoded by the crtYB gene after two cyclization reactions.Finally β-carotene is oxidized to astaxanthin by the product of the crtS gene.
The crtYB and crtI genes were isolated from a cDNA library of the strain CBS 6938 of X. dendrorhous by heterologous complementation in Escherichia coli strains carrying carotenoid biosynthesis genes from the bacteria Erwinia uredovora (Verdoes et al., 1999a, Verdoes et al., 1999b).In addition, in the wild-type ATCC 24230 strain of X. dendrorhous, two unexpected cDNA of crtYB and crtI genes have been isolated that correspond to alternative spliced variants, which have numerous stop codons in their sequence (Lodato et al., 2003).The ratio of mature to alternative mRNA for the crtI gene decreased as a function of the age of the culture, while the carotenoids content increased (Lodato et al., 2003).Furthermore, the expression at the mRNA level of the carotenoid biosynthesis idi, crtE, crtYB, crtI, and crtS genes from X. dendrorhous was studied by RT-PCR (Lodato et al. 2003;2004 and2007).However, little is known about the genomic organization of these genes in this yeast.
In this work, we cloned the idi, crtE, crtYB, crtI, and crtS genes controlling the astaxanthin biosynthesis pathway and their respective cDNAs and determined their genetic organization by sequence analysis and electrophoretic karyotyping.The function of the crtYB, crtI, and crtS genes was inferred from site-directed mutagenesis.

Strains, media, plasmid and genomic library
All strains used in this study are listed in Table I and the plasmids in Table II.
The X. dendrorhous wild-type strain ATCC 24230 was used to obtain genomic DNA for the construction of the genome library and site-directed mutagenesis.E. coli strain DH-5α was used for the propagation of plasmids and the genomic libraries of X. dendrorhous.Three genomic libraries of wild-type X.dendrorhous were used in this study.The first one contains partially digested BamHI DNA fragments inserted in the BamHI site of the YIp5 vector.The other two partial genomic libraries contain EcoRV or PstI DNA fragments in the EcoRV or PstI sites of the Bluescript SK-plasmid respectively.
X. dendrorhous wild-type strain was grown at 22 °C in baffled flasks containing YM medium in an orbital shaker at 100 rpm.
The E. coli-transformed cells were plated on selective Luria-Bertani agar plates (Sambrook et al., 1989) and incubated at 37°C overnight.These plates contained ampicillin (100 μg/ml) for plasmid selection and 40 μl of a 2% solution of X-Gal (5b r o m o -4 -c h l o r o -3 -i n d o l y l -β -Dgalactopyranoside) for selection of recombinant clones.The X. dendrorhous transformed cells were plated on YM containing 10 μg/ml of hygromicin B.

cDNA synthesis and cloning
For the reverse transcription (RT) reaction, total RNA was purified from cellular pellets obtained from 40 ml aliquots of culture by a modified protocol of Chomczynski and Sacchi (Chomczynski and Sacchi, 1987;Lodato et al., 2004).The total RNA concentration was quantified at 260 nm, and the integrity of the RNA was checked in denaturant agarose gel electrophoresis.The RNA sample was then treated with 1U of DNase I (Roche) in 2.5 mM MgCl2 at 25ºC for 30 min.The reaction was stopped by the addition of EDTA at 2.5 mM final concentration and heating at 65ºC for 15 min.The RT reaction was performed in 25 μl final volume with 3 μg of total RNA, 75 pmoles oligodT15-18, 0.5 mM of dNTPs and 200 U of M-MLV reverse transcriptase (Promega) (Lodato et al., 2003(Lodato et al., , 2004)).The reaction mixture was incubated at 42°C for 60 min and then heated to 65°C for 10 min.For the amplification of double-stranded cDNA of carotenoid biosynthesis genes, PCR reactions were performed according to the experimental conditions previously described (Lodato et al., 2003(Lodato et al., , 2004) ) in a final volume of 25 μl containing 2 U of Pfu polimerase (Stratagene), 2.5 μl of 10 X Pfu buffer, 0.5 μl of 10 mM dNTPs, 1 μl of 50 mM MgC l2 , 1 μl of 25 μM of each primer, and 2 μl of RT reaction product, and the final volume was adjusted with nucleasefree water.Amplification was performed in a DNA Thermal Cycler 2400 (Perkin-Elmer) or 2720 ABI Thermal cycler as follows: initial denaturation at 95°C for 3 min, 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, synthesis at 72°C for 3 min and a final extension step at 72°C for 10 min.The amplicons of the five genes were separated by electrophoresis in 1% agarose gels in TAE buffer containing 0.5 μg/ml of ethidium bromide followed by purification of each DNA fragment using glassmilk (Boyle and Lew, 1995), mixed with plasmid pBluescript SK previously digested with EcoRV and treated with T4 DNA ligase for 16 h at 14°C.Individual ligation mixture reactions for each gene were used to electroporate E. coli DH-5α, plated onto LB agar containing 100 μg/ml ampicillin and X-gal.Transformants were selected as white ampicillin-resistant colonies.

DNA sequence
The nucleotide sequences were determined using a DYEnamic ET terminator Kit Amersham Bioscience in an ABI 3100 Avant genetic analyzer.Each DNA fragment was sequenced by both strands, and the ambiguities were resolved by new sequencing.The sequence data was analyzed using the University of Wisconsin Genetics Computer Group package, version 10.0 (Devereux et al., 1984) and the CLUSTAL W program, version 1.8.(Thompsom et al., 1994).

Pulse field gel electrophoresis
Intact chromosomal DNA of X. dendrorhous was prepared in agarose plugs from cells grown to stationary phase in 100 ml of YM medium, washed twice with 50 mM EDTA pH 7.5 and suspended in 1 ml of a solution containing 10 mg Lysing enzyme per ml of 50 mM EDTA, as previously described (Cifuentes et al., 1997).The chromosomal DNA was separated by contour-clamped homogeneous electric fields (CHEF) in a LKB 2015 pulsaphor on a 1% agarose gel at 14°C in 0.1 M TBE buffer or in a Bio Rad DR II equipment on a 0.85% agarose gel at 14°C in 0.5 X TBE respectively (Sambrook et al., 1989).

Southern blot hybridization
Probes were labeled with [ 32 P]α-dCTP by the random primer technique and Southern blot hybridization was performed according to Sambrook et al. (1989).

Transformation of X. dendrorhous
The transformation of the wild-type strain of X. dendrorhous was performed by electroporation according to a previously described method (Wery et al., 1997).For the electrotransformation of 60 μl of electrocompetent cells, 10 to 20 μg of DNA was used.The electrocompetent cells were prepared from 200 ml YM culture grown until D.O.660 nm = 1.2.Cells were harvested at 5000 x g for 5 min, and the pellet was suspended in 25 ml of potassium phosphate buffer (50 mM potassium phosphate pH 7, 25 mM DTT) and incubated for 15 min at 21°C.The next step was at 4°C, using all the materials previously refrigerated.The cells were washed twice with 25 ml of STM buffer (270 mM sucrose, 10 mM Tris HCl pH 7.5, 1 mM MgC l ).Subsequently, the cells were suspended with 500 μl of STM buffer at 4°C and stored at -70°C.The electroporation conditions were 25 μF, 1000 Ω, 800 V using a BioRad Gene Pulser X Cell with PC and CE Modules.The transformant DNA was linearized with restriction endonucleases according to the cassette type used.The transformant, hygromicin B-resistant colonies were selected on YM with 10 μg/ml hygromicin B. The transformants were identified as X. dendrorhous by the presence of dsRNA elements (Castillo and Cifuentes, 1994) and  ITS1, 5.8 rRNA gene and ITS2 DNA sequences (Reyes et al., 2004).

Cloning of the structural genes of carotenoids biosynthesis pathway
The structural genes of the astaxanthin biosynthesis pathway were isolated from the genomic libraries of X. dendrorhous by PCR using specific primers.Three recombinant plasmids carrying the genes idi (pA1), crtE (pI10), and crtI (pC13) in BamHI fragments of 10.9, 11.5, and 15.8 kb respectively were isolated from a genomic library of X. dendrorhous in the YIp5 plasmid.No positive results were obtained for the crtYB and crtS genes in this genomic library, possibly by the existence of an internal restriction site in these genes.The BamHI inserts of each plasmid were transferred to the pBluescript SK vector and renamed as pXD-A1 (idi), pXD-I10 (crtE), and pXD-C13 (crtI) respectively (Fig. 1).
To determine a single restriction DNA fragment of X. dendrorhous containing either the crtYB or crtS genes, a genomic restriction map was carried out by hybridization experiments.For this, the DNA of X. dendrorhous, in duplicate, was digested with different restriction enzymes, separated by electrophoresis in 0.7% agarose gels, and transferred to a nylon membrane.

Localization of carotenoids biosynthesis genes in the electrophoretic karyotype
In order to study the genomic organization of the carotenoids biosynthesis genes crtE, crtYB, crtI, and crtS of X. dendrorhous, assays to determine the location of each gene in the electrophoretic karyotype of the yeast were performed.For this, the intact chromosomal DNA was separated by pulsed field electrophoresis (Cifuentes et al., 1997), using pulses of 90 seconds at 6 V/cm for 24 hours, followed by DNA transfer to nylon membranes and hybridization using radioactive probes of each gene.As is shown in Figure 2, the crtE and crtI genes were located in the chromosomal band I corresponding to a triplet (Cifuentes et al., 1997).The crtYB gene was located in the chromosomal band II, which also corresponds to a triplet.When a pulsed field gel electrophoresis was carried out using pulses of 500 seconds for 48 hours, followed by pulses of 250 seconds for 12 h at 6 V/cm -conditions that separate the chromosomes of the bands I and II -it was observed that the crtYB gene is located in two different chromosomal bands (Fig. 2B).In addition, the crtS gene was located in two chromosomal bands of the electrophoretic karyotype, using pulses of 90 seconds for 24 hours at 12 V/cm (Fig. 2C).These results could contribute to the evidence supporting the diploid condition and genetic polymorphism of the wild-type strain ATCC 24230 of X. dendrorhous as previously described (Hermosilla et al., 2003;Retamales et al., 1998).In fact, electrophoretic karyotype analysis of wildtype strains of X. dendrorhous shows a large chromosomal polymorphism (Cifuentes et al., 1997;Nagy et al., 1997;Adrio et al., 1995) and could explain the fact that both the crtYB and crtS genes were located on two different chromosomal bands suggesting that their respective homologous chromosomes could be a polymorphic product of chromosomal rearrangement.

Sequence analysis of the carotenoids biosynthesis genes
The genes idi, crtE, crtYB, crtI, and crtS and their respective cDNAs were completely sequenced, and the analysis of sequence allowed the establishment of the exon-intron organization (Fig. 3).According to the sequence data, the idi gene was located in a BamHI fragment of 10,960 bp, containing 4 introns and 5 exons with a coding region of 756 bp corresponding to a peptide of 251 amino acids.The crtE gene was isolated in a BamHI fragment of 11,504 bp, containing 8 introns, and their ORF contained 1,131 bp, which corresponds to a peptide of 376 amino acids.The crtI gene was isolated as a BamHI segment of 15,778 bp, containing 11 introns and 12 exons with an ORF of 1,749 bp encoding a peptide of 582 amino acids.The crtYB gene is located in an EcoRV fragment of 5,917 bp; it contains 4 introns and 5 exons, and its ORF is 2,022 bp, and the peptide encoded has 673 amino acids.Finally the crtS gene is located in a PstI fragment of 3,995 bp; it contains 17 introns and 18 exons, and its ORF has 1,674 bp corresponding to a peptide of 577 amino acids.The DNA sequences of the idi, crtE, crtYB, crtI, and crtS genes and the cDNA sequences from the mRNA of the crtE and crtS genes of the wild-type strain ATCC 24230 of X. dendrorhous have been deposited in the GenBank database under accession numbers DQ235686, DQ012943, DQ016503, DQ028748, DQ002006, DQ016502 and DQ002007, respectively.
On the other hand, the genes crtYB and crtI, in addition to the mature mRNA, produce variants of alternative splicing -amRNA crtYB and amRNA crtI, respectively -as previously reported by our group, suggesting that the splicing to mature or alternative messenger could regulate the cellular concentration of both enzymes depending on the physiological or environmental conditions (Lodato et al., 2003).In both genes, the variants of alternative splicing lead to the formation of premature stop codons in the translation of the three reading frames.However, a correct reading frame of both transcripts may be reestablished downstream from the start codon.In the case of the crtI messenger, if the translation could start internally in the alternative messenger, a truncated phytoene desaturase enzyme without 81 amino acids in the N-terminal of the protein could be synthesized from amRNA crtI.However, the N-terminal of the phytoene desaturase of X. dendrorhous has a dinucleotide binding motif that is conserved in the phytoene desaturases (Lodato et al., 2003), indicating that it is unlikely that an enzyme lacking 81 aminoacids in the N-terminal could remain active.In the case of the crtYB gene, the analysis of the nucleotide sequence of the amRNA indicates that downstream from the initial start codon, an open reading frame is re-established that could be translated to a protein that lacks the first 153 aminoacids of the N-terminal, which constitutes part of the lycopene cyclase domain of the enzyme phytoene-β-carotene synthase (Verdoes et al., 1999b).To determine if the truncated CRTYB has the lycopene cyclase or phytoene synthase activity, a heterologous complementary system was elaborated in E. coli.For this, three E. coli strains were constructed that carried three different carotenogenesis gene arrangements from E. uredovora (DS1B, having the wild-type cluster of carotenogenic genes of E. uredovora; DS1B-ΔcrtB bearing a deletion of the Eu-crtB gene; and DS1B-ΔcrtY bearing the cluster with a deletion of Eu-crtY gene, respectively).These were transformed with the pXD-tcrtYB (Table II) vector carrying cDNA of the truncated version of the gene crtYB that specifies a protein of 520 aminoacids.Results show that the truncated gene may weakly complement the function of the phytoene synthase in the E. coli bearing a deletion of the Eu-crtB of E. uredovora, but not lycopene cyclase in the E. coli strain carrying a deletion of the Eu-crtY gene of E. uredovora (Table III).These results suggest that the lack of the first 153 amino acids completely alters the lycopene cyclase domain of this bifunctional enzyme encoded by crtYB gene of X. dendrorhous.However, the phytoene synthase activity is maintained, although the levels of activity are reduced in comparison to the gene Eu-crtB of E. uredovora.If the alternative splicing of the mRNA of the gene crtYB fulfills a regulatory role in the carotenoids biosynthesis pathway of X. dendrorhous, the existence of a gene product with a single activity, phytoene synthase, could change the composition of intermediates and thus exert a regulatory role in the pathway, a mechanism which should be studied to identify all the processes involved in the carotenogenesis.
Additionally, a blast analysis of the deduced amino acid sequence of the astaxanthin synthase enzyme, the product of the crtS gene, shows a high level of similarity with cytochrome P450 hydroxylase.In other organisms that synthesize astaxanthin, such as the microalgae H. pluvialis and the bacteria Agrobacterium auriantiacum (Misawa et al., 1995) or Paracoccus haeundaensis (Lee and Kim, 2006), the step from βcarotene to astaxanthin using several ketocarotenoids as intermediates are controlled by two genes.The gene crtW that codes for the enzyme β-carotene ketolase catalyzes the introduction of the keto groups, while the gene crtZ that codes for the enzyme β-carotene hydroxylase catalyzes the incorporation of the hydroxyl groups in positions C3 and C3'.However, in X. dendrorhous, the step from β-carotene to astaxanthin is controlled by a single gene, crtS, that codes for the enzyme astaxanthin synthase, which is a cytochrome P450 hydroxylase-type enzyme, subfamily 3A (Ojima et al., 2006), which catalyzes all the reactions to convert β-carotene to astaxanthin.However, it has recently been suggested that CRTS would require an additional gene function, which could play a possible auxiliary role (Martín et al., 2008).This explains the inability to obtain mutants blocked in same step between β-carotene and astaxanthin that accumulate echinenone, 3-hydroxyechinenone or phoenicoxanthin.
Additionally, after nitrosoguanidine (NTG) mutagenesis, we isolated the mutant strain atx5, that shows a yellow phenotype as a result of β-carotene accumulation (Cifuentes et al., 1997).To discover if the mutation of atx5 affects the crtS gene, a 5,920 bp fragment containing the complete crtS gene of strain atx5 was sequenced (GenBank accession number EU713462).The sequence analysis indicates there is only a single base change that corresponds to a guanine (G) in position 376, from the ATG of the genome version, to an adenine (A).This change is located in exon 3, 12 nucleotides from the limit between exon 2 and 3.This transition mutation provokes a change from the Gly to Asp in position 60 of the CRTS protein.According to the literature, the location of this change does not affect important sites for the correct functioning of astaxanthin synthase, such as the oxygen-binding site, binding domain to the Heme group, or the ESLR domain involved in the maintenance of the tridimensional structure (Álvarez et al., 2006).However, since G376A (Gly 60 to Asp) is the only change observed in strain atx5 and considering that glycine is an amino acid that given its short hydrophobic lateral chain, makes no real contributions to the hydrophobic interactions and that Aspartic is a negatively charged amino acid, this change could have not affected the activity of astaxanthin synthase.The aforementioned, coupled with six out of six unsuccessful attempts to transform strain atx5 using as donor DNA the complete wild-type crtS including the promoter region, suggests the involvement of at least another gene, complementary to the crtS gene, in the conversion of β-carotene to astaxanthin in X. dendrorhous.

Directed mutagenesis of crtI, crtYB, and crtS in X. dendrorhous
The cDNA of the genes crtYB and crtI have been cloned by heterologous complementation in E. coli.The crtI gene of X. dendrorhous is expressed in E. coli, which synthesizes phytoene due to the plasmid carrying the genes crtE and crtB of  et al., 1999a).Similarly, the cDNA of the gene crtYB of X. dendrorhous is expressed in different carotenogenic strains of E. coli, providing evidence that it codes for a bifunctional protein with phyotene synthase and lycopene cyclase activity (Verdoes et al., 1999b).However, the functionality of these genes in X. dendrorhous has not been directly demonstrated.Therefore, the effect of mutations in these genes in the carotenoids synthesize in X. dendrorhous was determined.For the study of the functionality of the crtI gene, the wild-type strain was transformed with the plasmid pL22ΔRV, harboring a 1.1 kb deletion of the crtI gene, linearized with BamHI and XhoI.A total of 8 colonies with a pale phenotype were obtained, and one of these, named T5, was selected, and the presence of the 1.1.kbdeletion of the crtI gene was confirmed by PCR using specific primers PHA1 and PHA4.In addition, the PCR analysis also showed the presence of a wild-type copy of the crtI gene, indicating that this strain was heterozygous for the crtI locus, it is crtI + /crtI -, with this crtI -allele being a deletion of 1.1 kb EcoRV fragment of the gene (crtI ΔRV allele), which was integrated in the chromosomal crtI locus by a double recombination or double crossover event and gives evidence that the ATCC 24230 wild-type strain is diploid.Subsequently, the heterozygote T5 strain was transformed with the same lineal DNA plasmid pL22ΔRV, obtaining a large number of white (albino) colonies and few red colonies, some of which showed sectors (wild-type red and pale phenotype).It is possible that these colonies arose by a mitotic recombination event between heterozygote chromosomes in the T5 transformant, reverting to the wild-type condition of red phenotype.Similarly, transformation of the heterozygote T5 strain homozygote double transformants for the deletion mutation crtI ΔRV (crtI ΔRV / crtI ΔRV ) of albino phenotype arise.However, the high number of albino transformants could be a result of two events, one by transformation by integration and the other by mitotic recombination between homologous chromosomes of the heterozygote strain, generating albino homozygotes in both cases.Once the homozygotization has occurred, the wildtype revertant or albino double mutant becomes genetically stable.These results suggest that homozygote mutants can be obtained from the wild-type X.dendrorhous strain through a single genetic transformation event followed by mitotic recombination which we have called the double recombinant method (DRM).From the results shown above, the DRM was developed to obtain deletion homozygote mutants of the gene crtI and insertion of a hygromycin B-resistant cassette.For this, cells of the wild-type strain were transformed with the plasmid pXD-crtI::hph previously linearized with BamHI and XhoI restriction enzymes.As a result, 15 heterozygote pale colonies resistant to 10 μg/ml of hygromicin B were obtained.
The genetic analysis of these colonies by PCR using specific primers for the crtI and the hph genes and their DNA sequence, indicates that they all bear the hph gene in the crtI locus in addition to a wt allele of the crtI.Cells of one heterozygote pale hygromicin B-resistant colony, named T-I21H, showing the crtI + /crtI::hph genotype, were grown in 50 ml flasks containing 10 ml of YM plus 10 μg/ml of hygromicin B for 3 days.Then, a 0.3 ml aliquot of the culture was diluted 100 times in a 125 ml flask with 30 ml of YM medium supplemented with 50 μg/ml of hygromycin B. After incubation for five days at 22°C, a transition from a paler culture was observed, with a pigmentation of approximately 10% of the wild-type, resulting in a white culture.Dilutions were plated on solid YM containing 10 μg/ml hygromicin B to give between 200 to 500 colonies per dish.Over 90% of colonies developed were white and hygromicin Bresistant.The genetic analysis by PCR shows that these colonies (represented by T-I21H1H) were homozygote for the mutation by insertion of the hph gene in crtI locus; it contains the allele crtI -::hph on both homologous chromosomes (crtI -::hph/ crtI -::hph).The pigment content analysis by HPLC showed that they only produced phytoene, which accumulates due to the absence of the enzyme phytoene desaturase that prevents its conversion to lycopene (Fig. 5 B and C).Subsequently, the double recombinant method was used to obtain mutants of genes crtYB and crtS, inserting the hygromycin B-resistance cassette in each gene.In an initial step, the wild-type strain ATCC 24230 of X. dendrorhous was transformed, in parallel, with the crtYB::hph cassette from plasmid pXD-crtYB::hph linearized with the enzyme XhoI and with the cassette crtS::hph from the plasmid pXD-crtS::hph linearized with SmaI.Five heterozygote transformants of the gene crtYB (crtYB + /crtYB -: :hph) and 12 heterozygote transformants for the gene crtS (crtS + /crtS -::hph) were obtained.One colony of each transformant type (T-YBH1 and T-SH1, respectively) was selected and grown in conditions for enrichment of mitotic recombination, after which thousands of albino (crtYB -/-, nonpigment producing) or yellow (crtS -/-, βcarotene accumulating) colonies were observed, respectively.Similarly to what was observed with the mutants of the gene crtI, in genes crtYB and crtS, more than 90% of the colonies were homozygous after the application of DRM.
In addition, the wild-type and the heterozygote and homozygote strains affected in the crtI, crtYB, and crtS genes were analyzed for their cellular production and composition of carotenoids by HPLC.In all heterozygote strains, T-I21H (crtI +/-), T-YBH1 (crtYB +/-) and T-SH1 (crtS +/-), the production of total carotenoids is less than in the wild-type.However, its composition was similar (Fig. 5 B, D and  F).On the other hand, both the homozygote T-I21H1H (crtI -/-) and T-YBHH2 (crtYB -/ -) do not produce colored pigments and are albino in phenotype (Fig. 5 C and E) and T-SHH2 (crtS -/-) produces β-carotene and was yellow in phenotype (Fig. 5 G).However, the crtI -/-homozygote produces and accumulates the uncolored carotenoid phytoene (peak 10 in box of Fig. 4) because this mutant does not synthesize the phytoene desaturase enzyme that transforms phytoene to lycopene, accumulating the former carotenoid.However, in the homozygote crtYB -/-strain, its albino phenotype is a product of the inability to synthesize phytoene due to the lack of the enzyme phytoene synthase.In addition, the crtS-/-homozygote strain produces and accumulates β-carotene (peak 9 in Fig. 4).These results provide clear evidence of the functionality of the genes crtYB, crtI, and crtS of X. dendrorhous and confirm the previous data obtained from heterologous complementation (Verdoes et al., 1999a;1999b).In addition, it was also possible to obtain heterozygous transformants for the genes idi (idi + /idi -::hph) and crtE (crtE + / crtE -::hph), however, the respective homozygotes using DRM or by transformation of the heterozygotes were not obtained, suggesting a possible essential role for the products of both genes in X. dendrorhous.
Finally, in X. dendrorhous, the astaxanthin biosynthesis from IPP could be controlled by five structural genes, idi, crtE, crtYB, crtI, and crtS, while other organisms such as plants, algae, and bacteria, require a greater number of genes.The intermediaries from isopentenyl pyrophophate to astaxanthin, in the main biosynthetic pathway of X. dendrorhous, except HDCO, DCD, HDC (Andrewes et al., 1976;An et al., 1999) dendrorhous, as in all fungi and eubacteria, the four desaturations required to convert phytoene into lycopene are synthesized by phytoene desaturase, controlled by the crtI gene (Verdoes et al., 1999a).However, in plants and algae, two gene products are required in this step, a phytoene desaturase that converts phytoene to ζ-carotene and ζcarotene desaturase that transforms ζcarotene into lycopene.In addition, in X. dendrorhous in the phytoene synthesis from GGPP and the β-carotene production from lycopene, two enzymatic reactions are catalyzed by a bi-functional gene product with phytoene synthase and lycopene cyclase activity (Verdoes et al., 1999b), which differs from plants, algae, and bacteria in which two independent gene products are required.The four steps to convert β-carotene to astaxanthin in X. dendrorhous are catalyzed by the crtS gene product, a protein of 557 amino acids in which the sequence analysis by blast suggests a cytochrome P450 in accordance with that previously described (Hoshino et al., 2000;Ojima et al., 2006).This is consistent with the first report that cytochrome P450 was involved with astaxanthin production in X. dendrorhous (An et al., 1989, Johnson 2003).Other organisms require at least two genes, crtW and crtZ, encoding a β-carotene oxygenase and a β-carotene hydroxylase respectively.Knowledge about the regulation of the expression of genes controlling the carotenogenesis in X. dendrorhous is limited.In fact, in previous works, it was determined that the crtI mRNA and crtYB mRNA levels decreased during the stationary phase of X. dendrorhous (Lodato et al., 2003).However, the carotenoid production differences between wild-type and astaxanthin-overproducing strains of X. dendrorhous could not be explained by a difference in the carotenogenic transcript at the stationary phase of growth (Lodato et al., 2007;2004).The knowledge of the genomic organization of the structural genes controlling the carotenogenesis and the homozygous and heterozygous mutants obtained carrying deletions of each gene  provides the genetic tools that will permit the study of the mechanism involved in the genetic regulation of the carotenoids biosynthesis pathway of X. dendrorhous.In addition, these site-specific mutants would allow the study of the gene dose effect and how it participates in the final expression of the structural genes of carotenogenesis and its regulation.VAN OOYEN AJJ (1999a) Cloning and characterization of the astaxanthin biosynthetic gene encoding phytoene desaturase of Xanthophyllomyces dendrorhous.Biotechnol Bioeng 63: 750-755 VERDOES JC, KRUBASIK P, SANDMANN G, VAN OOYEN AJJ (1999b) Isolation and functional characterization of a novel type of carotenoid biosynthetic gene from Xanthophyllomyces dendrorhous.Mol Gen Genet 262: 453-461 WERY J, GUTKER D, RENNIERS AC, VERDOES JC, VAN OOYEN AJ (1997) High copy number integration into the ribosomal DNA of the yeast Phaffia rhodozyma.Gene 184: 89-97

Figure 4 .
Figure 4. Schematic representation of the Double Recombinant Method in X. dendrorhous.The locus of interest is represented in gray.The gDNA that flanks the locus of interest is represented in white.The potentially recombinant gDNA region is represented in black.The hygromycin B resistant cassette of X. dendrorhous is represented as an arrow.GENE: represents any one of the five carotenogenesis genes (idi, crtE, crtYB, crtI and crtS) of the yeast.

TABLE I
Strains of E. coli and X. dendrorhous created and used in this study

TABLE III Heterologous
complementation analysis of truncated crtYB gene in E. coli carrying carotenoids biosynthesis genes of E. uredovora REYES E, BARAHONA S, FISCHMAN O, NIKLITSCHEK M, BAEZA M, CIFUENTES V (2004) Genetic polymorphism of clinical and environmental strains of Pichia anómala.Biol Res 37: 747-757 SAMBROOCK J, FRITSCH E F, MANIATIS T (1989) Molecular cloning.A laboratory manual, 2nd ed.Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press THOMPSOM JD, HIGGINS DG, GIBSON TJ (1994) CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.Nucleic Acids Res 22:4673-4680 VERDOES JC, MISAWA N,