BACKGROUND
xanthophyllomyces dendrorhous is a basidiomycetous yeast with potential in the biotechnology industry as it is able to synthesize carotenoids, particularly astaxanthin.
carotenoids are natural yellow, orange, or red pigments, and more than  <dig> different carotenoid chemical structures have been described to date  <cit> .
animals are unable to synthesize these pigments de novo and can only obtain them in their diet.
these pigments are currently used as food colorants and have received attention for their ability to alleviate chronic diseases due to their antioxidant properties, which can mitigate the damaging effects of oxidative stress induced by reactive oxygen species   <cit> .
among carotenoids, astaxanthin  is notable based on its antioxidant properties, which are greater than those of beta-carotene or even alpha-tocopherol  <cit> , and the application of astaxanthin in the pharmaceutical and cosmetic industries has recently been explored  <cit> .
astaxanthin has also been widely used in the aquaculture industry as a colorant for cultured salmonids to achieve the flesh color that is preferred by consumers.
in addition, astaxanthin is an essential nutritional component for proper fish growth and reproduction, making this compound a significant factor in aquaculture production costs  <cit> .
x. dendrorhous is one of the few microorganisms that produces astaxanthin.
however, the production of this pigment by wild-type strains is too low  to provide a natural source that is economically competitive with chemical synthesis of this pigment.
therefore, many efforts have attempted to improve the astaxanthin production from x. dendrorhous, including optimization of culture conditions such as glucose concentration  <cit> , oxygen levels  <cit> , ph  <cit> , carbon/nitrogen ratio  <cit> , and light intensities  <cit> , in addition to classic random mutagenesis methods  <cit> .
a promising alternative to increase the astaxanthin yield in this yeast is to overexpress the genes involved in carotenoid synthesis  for which several attempts have been performed .
a small increase in the amount of total carotenoids was obtained by overexpressing the gene encoding phytoene synthase-lycopene cyclase  by integrating multiple copies of this gene into the ribosomal dna  <cit> .
however, this increase was mainly due to increased synthesis of beta-carotene and echinenone, while the astaxanthin content was slightly reduced .
in another experiment in which the gene encoding phytoene desaturase  was overexpressed, the overall carotenoid production decreased including a 50% reduction in the astaxanthin fraction  <cit> .
similarly, the overexpression of the gene encoding isopentenyl pyrophosphate isomerase  led to a decrease in the amount of total carotenoids  <cit> .
in another work attempting to promote metabolite flow towards carotenoid biosynthesis, the cdna encoding the geranylgeranyl pyrophosphate synthase gene  was overexpressed, resulting in a strain with slightly higher carotenoid levels, although the astaxanthin levels were not increased  <cit> .
however, the simultaneous insertion of extra copies of the crtyb and crts  genes in an astaxanthin-overproducing strain obtained by random mutagenesis resulted in transformants with an even higher astaxanthin content  <cit> .
therefore, the overexpression of a single gene is not enough to significantly increase the astaxanthin level in x. dendrorhous, and overexpression of different combinations of carotenogenic genes seemed to provide better results.
one of the major limitations in the genetic manipulation of x. dendrorhous is the limited number of molecular tools available to transform and engineer this yeast.
recently, an in vivo method for dna fragment assembly and yeast transformation, dna assembler, was reported  <cit> .
with this technique, numerous dna fragments are assembled in vivo via homologous recombination at their ends following a single transformation event, allowing the incorporation of an entire heterologous biochemical pathway in saccharomyces cerevisiae.
as the x. dendrorhous homologous recombination machinery has previously been successfully exploited for the development of transformation strategies and gene function analysis  <cit> , in this work, we adapted the dna assembler methodology to overexpress the gene encoding astaxanthin synthase  that catalyzes the formation of astaxanthin from beta-carotene  <cit>  in x. dendrorhous.
RESULTS
in vivo assembly and integration of dna fragments into the genome of x. dendrorhous
to evaluate the feasibility of assembling dna fragments in vivo in x. dendrorhous, a dna cassette containing only the gene that confers resistance to hygromycin b  <cit>  was integrated in the genome of the wild-type x. dendrorhous strain ucd 67– <dig>  a  <dig>  bp locus called dhs <dig>  was chosen as one of the potential target site, which is located at  <dig>  bp downstream of the x. dendrorhous his <dig> gene.
this region is transcribed and encodes an uncharacterized gene product, so we expected that its interruption would not drastically affect the physiology of the yeast.
three dna fragments  were prepared as illustrated in figure 2a.
first, each fragment was individually pcr amplified with a set of primers designed to make the 5′ end of the fragment overlap the 3′ end of the preceding dna fragment.
in this way, the hygromycin b resistance cassette overlaps the two flanking dna fragments targeting the dhs <dig> locus.
the overlapping region contained approximately 100 bp of sequence homology between fragments to allow in vivo homologous recombination between them.
a diploid x. dendrorhous wild-type strain  <cit>  was co-transformed with the three dna fragments by electroporation.
assembly of these fragments and their integration into the genome was accomplished, as nine hygromycin b-resistant transformants were obtained.
pcr analyses confirmed that all of them contained the resistance cassette at the expected integration target.
however, as the starting strain ucd 67– <dig> is diploid  <cit> , a wild-type dhs <dig> allele was still detected in the resulting transformants, indicating that they were heterozygous at this locus.
for this reason, one of the transformants was randomly chosen  to obtain the homozygous strain xd_2h  using the double recombinant method   <cit>  .
panel b of figure  <dig> shows the amplicons obtained from genomic pcr confirming the integration of the hygromycin b resistance cassette into the expected locus in strains xd_1h  and xd_2h .
to the naked eye, the color of the heterozygous and homozygous strains is identical to the wild-type strain and both strains are able to grow in minimal medium .
total carotenoids were quantified and no significant differences were detected between the transformants and the wild-type strain .
thus, the interruption of the dhs <dig> locus did not cause auxotrophy or greatly affected the carotenogenesis in this yeast.
to confirm that this transformation methodology is effective for other integration targets, the resistance cassette was successfully integrated into two other genomic loci.
however, the color phenotype of the transformants was different from the parental strain .
increase of crts gene dose
next, we used the above methodology to increase the crts gene dose.
a crts expression cassette was constructed by oe-pcr  and cloned into plasmid pbluescript sk-, resulting in the plasmid pbs-ptef-crts-tact.
this cassette was integrated in the dhs <dig> locus, as its interruption did not cause auxotrophy or greatly affected the carotenogenesis in this yeast.
four dna fragments  were individually pcr amplified with primers allowing  <dig> to 100 bp of overlap between adjacent fragments, and then these fragments were used to co-transform x. dendrorhous by electroporation .
four hygromycin b-resistant colonies were obtained.
all had the crts gene expression cassettes at the expected locus, as confirmed by pcr analyses with a comprehensive set of primers.
one of these four colonies was randomly selected, named strain xd_1h1s  and subjected to drm to result in a homozygous strain, xd_2h2s .
the pcr amplification confirmed the insertion of the hygromycin b resistance and crts expression cassettes at the dhs <dig> locus in the heterozygous and homozygous transformants are shown in figure  <dig>
to assess whether the increase in the crts gene dose effectively leads to an increase in the crts mrna levels, rt-qpcr analysis was performed for the wild-type, heterozygous and homozygous transformants grown under the same conditions.
total rna was extracted after 50 h of culture  of each strain as it was observed that the crts mrna level reaches its maximum level at this stage  <cit> .
the relative crts expression was normalized to the expression of the actin gene  <cit> , and rt-qpcr analysis revealed that the expression of the crts gene was increased approximately three fold in the xd_2h2s strain with respect to the wild-type strain  at this same growth stage .
carotenoid production
to the naked eye, different pigmentation phenotypes among the wild-type, xd_1h1s and xd_2h2s strains could be noted.
to confirm this observation, the carotenoid content and composition of these strains were analyzed.
total carotenoids were extracted from cell pellets from  <dig>   <dig> and 96-h-old yeast cultures  grown at 22°c in ym  medium with constant agitation.
carotenoids were extracted, quantified by absorbance at 465 nm and their composition was analyzed by rp-hplc.
the results are summarized in table  <dig>  although the total carotenoid production is not affected in strains that overexpress crts, it can be observed that after  <dig> and 95 h of culture, the percentage of astaxanthin in relation to the other carotenoids is higher in strains expressing additional crts gene copies.
nd: not detected.
table values correspond to the average result from three independent cultures ± standard deviations.
percentage relative to total carotenoids is indicated in parentheses.
†: hydroxy or/and keto carotenoid, *: monocyclic carotenoid.
discussion
there are a limited number of currently available molecular tools to manipulate and transform x. dendrorhous.
until this report, transformation of x. dendrorhous usually required the construction of a plasmid for transformation  <cit> , which involves several conventional steps of sequential cloning such as dna amplification, endonuclease digestion, in vitro ligation and transformation.
if the number of expression cassettes to incorporate in a host is increased, these processes will be time consuming and will depend on the availability of restriction sites in the cloning vector.
in contrast, the dna assembler method requires only dna fragments with homologous ends.
we find that this methodology works in x. dendrorhous as we transformed this yeast with three dna fragments to integrate a hygromycin b resistance cassette into its genome.
we also increased the number of crts gene copies in this yeast by transforming it with four dna fragments to be assembled  and integrated in the yeast genome.
we conclude that this methodology can be employed to engineer x. dendrorhous to interrupt or increase gene copy number of several combinations of genes in only one transformation event.
in previous studies attempting to overexpress genes in x. dendrorhous, expression cassettes were integrated into ribosomal dna   <cit> .
there are approximately  <dig> rdna copies in x. dendrorhous <cit> , which favors multiple integrations of the transformant dna into the genome.
however, due to the tandem repeats in this region, the resulting strains are typically unstable as the integrated genes may be lost by homologous recombination.
additionally, the use of this region makes it difficult to evaluate the effect of gene dose on the production of carotenoids, as it is difficult to quantify the number of integrated cassette copies.
for these reasons, we excluded the rdna region as a target site for integration and focused on integration and resultant target site disruption in a locus that does not strongly modify the yeast physiology.
from the analyzed targets, the disruption of locus dhs <dig> fulfilled this requirement, so it was chosen to integrate the additional crts gene copies.
in this work, we studied the effect of altering the metabolic flux between beta-carotene and astaxanthin by increasing the crts gene dose.
although the total carotenoid content in strains with one or two additional crts gene copies was not substantially modified , an increased proportion of astaxanthin relative to the total carotenoid content was obtained in the strain with two additional crts gene copies compared to the wild-type strain.
astaxanthin represented 96% versus 68% of the carotenoids in these strains after 96 h of culture.
in addition, reduced proportions of beta-carotene and intermediate xanthophylls formed during the synthesis of astaxanthin from beta-carotene such as echinenone, hydroxyechinenone, canthaxanthin and phoenicoxanthin.
these observations suggest that astaxanthin synthesis is limited by the amount of astaxanthin synthase substrate, beta-carotene.
thus, to achieve a significantly higher level of astaxanthin production, it is also necessary to increase the synthesis of beta-carotene.
in agreement with this assumption, it has been observed that the overexpression of the crtyb gene in x. dendrorhous resulted in increased beta-carotene production  <cit>  and recently, the simultaneous overexpression of the crtyb and crts genes in a x. dendrorhous strain created via random mutagenesis that already overproduced astaxanthin resulted in a higher astaxanthin content compared to the parental strain   <cit> .
the methodology described in this work should be a helpful tool to evaluate the consequences of overexpressing different combinations of genes involved in carotenoid production in x. dendrorhous.
although beta-carotene was almost completely exhausted in the transformant strain that overexpressed crts, phoenicoxanthin, the xanthophyll precursor to astaxanthin, was still present, although its proportion was reduced from 15% to 6% after 96 h of culture in the wild-type and in the xd_2h2s strains, respectively.
as astaxanthin synthase is a cytochrome p <dig> enzyme, it may also be necessary to increase the cytochrome p <dig> reductase  <cit>  gene dose to enhance the activity of this enzyme.
the dna assembler methodology that has been effective and successful in x. dendrorhous in this study should be useful to overexpress several genes simultaneously to favor the synthesis of carotenoid precursors such as geranylgeranyl pyrophosphate or genes involved in the mevalonate pathway  <cit>  in combination with carotenogenic genes.
thus, using different promoters in the construction of the gene expression cassettes, it should be possible to increase and modulate the production of carotenoids in x. dendrorhous.
CONCLUSIONS
the dna assembler method is a successful technique to transform x. dendrorhous.
this technique allowed an increase in the crts gene copy number in the x. dendrorhous genome.
the overexpression of this gene did not significantly change the total carotenoid production, but there was an increase in the astaxanthin fraction of carotenoids.
as dna assembler requires only dna fragments with homologous ends, this technique could be useful to quickly and easily overexpress several genes simultaneously in x. dendrorhous, saving numerous steps involved in conventional cloning methods.

 xanthophyllomyces dendrorhous is a basidiomycetous yeast with potential in the biotechnology industry as it is able to synthesize carotenoids, particularly astaxanthin. carotenoids are natural yellow, orange, or red pigments, and more than different carotenoid chemical structures have been described to date. animals are unable to synthesize these pigments de novo and can only obtain them in their diet. these pigments are currently used as food colorants and have received attention for their ability to alleviate chronic diseases due to their antioxidant properties, which can mitigate the damaging effects of oxidative stress induced by reactive oxygen species. among carotenoids, astaxanthin is notable based on its antioxidant properties, which are greater than those of beta-carotene or even alpha-tocopherol, and the application of astaxanthin in the pharmaceutical and cosmetic industries has recently been explored. astaxanthin has also been widely used in the aquaculture industry as a colorant for cultured salmonids to achieve the flesh color that is preferred by consumers. in addition, astaxanthin is an essential nutritional component for proper fish growth and reproduction, making this compound a significant factor in aquaculture production costs. x. dendrorhous is one of the few microorganisms that produces astaxanthin. however, the production of this pigment by wild-type strains is too low to provide a natural source that is economically competitive with chemical synthesis of this pigment. therefore, many efforts have attempted to improve the astaxanthin production from x. dendrorhous, including optimization of culture conditions such as glucose concentration, oxygen levels, ph, carbon/nitrogen ratio, and light intensities, in addition to classic random mutagenesis methods. a promising alternative to increase the astaxanthin yield in this yeast is to overexpress the genes involved in carotenoid synthesis for which several attempts have been performed. a small increase in the amount of total carotenoids was obtained by overexpressing the gene encoding phytoene synthase-lycopene cyclase by integrating multiple copies of this gene into the ribosomal dna. however, this increase was mainly due to increased synthesis of beta-carotene and echinenone, while the astaxanthin content was slightly reduced. in another experiment in which the gene encoding phytoene desaturase was overexpressed, the overall carotenoid production decreased including a 50% reduction in the astaxanthin fraction. similarly, the overexpression of the gene encoding isopentenyl pyrophosphate isomerase led to a decrease in the amount of total carotenoids. in another work attempting to promote metabolite flow towards carotenoid biosynthesis, the cdna encoding the geranylgeranyl pyrophosphate synthase gene was overexpressed, resulting in a strain with slightly higher carotenoid levels, although the astaxanthin levels were not increased. however, the simultaneous insertion of extra copies of the crtyb and crts genes in an astaxanthin-overproducing strain obtained by random mutagenesis resulted in transformants with an even higher astaxanthin content. therefore, the overexpression of a single gene is not enough to significantly increase the astaxanthin level in x. dendrorhous, and overexpression of different combinations of carotenogenic genes seemed to provide better results. one of the major limitations in the genetic manipulation of x. dendrorhous is the limited number of molecular tools available to transform and engineer this yeast. recently, an in vivo method for dna fragment assembly and yeast transformation, dna assembler, was reported. with this technique, numerous dna fragments are assembled in vivo via homologous recombination at their ends following a single transformation event, allowing the incorporation of an entire heterologous biochemical pathway in saccharomyces cerevisiae. as the x. dendrorhous homologous recombination machinery has previously been successfully exploited for the development of transformation strategies and gene function analysis, in this work, we adapted the dna assembler methodology to overexpress the gene encoding astaxanthin synthase that catalyzes the formation of astaxanthin from beta-carotene in x. dendrorhous.  in vivo assembly and integration of dna fragments into the genome of x. dendrorhous to evaluate the feasibility of assembling dna fragments in vivo in x. dendrorhous, a dna cassette containing only the gene that confers resistance to hygromycin b was integrated in the genome of the wild-type x. dendrorhous strain ucd 67– a  bp locus called dhs was chosen as one of the potential target site, which is located at  bp downstream of the x. dendrorhous his gene. this region is transcribed and encodes an uncharacterized gene product, so we expected that its interruption would not drastically affect the physiology of the yeast. three dna fragments were prepared as illustrated in figure 2a. first, each fragment was individually pcr amplified with a set of primers designed to make the 5′ end of the fragment overlap the 3′ end of the preceding dna fragment. in this way, the hygromycin b resistance cassette overlaps the two flanking dna fragments targeting the dhs locus. the overlapping region contained approximately 100 bp of sequence homology between fragments to allow in vivo homologous recombination between them. a diploid x. dendrorhous wild-type strain was co-transformed with the three dna fragments by electroporation. assembly of these fragments and their integration into the genome was accomplished, as nine hygromycin b-resistant transformants were obtained. pcr analyses confirmed that all of them contained the resistance cassette at the expected integration target. however, as the starting strain ucd 67– is diploid, a wild-type dhs allele was still detected in the resulting transformants, indicating that they were heterozygous at this locus. for this reason, one of the transformants was randomly chosen to obtain the homozygous strain xd_2h using the double recombinant method. panel b of figure  shows the amplicons obtained from genomic pcr confirming the integration of the hygromycin b resistance cassette into the expected locus in strains xd_1h and xd_2h. to the naked eye, the color of the heterozygous and homozygous strains is identical to the wild-type strain and both strains are able to grow in minimal medium. total carotenoids were quantified and no significant differences were detected between the transformants and the wild-type strain. thus, the interruption of the dhs locus did not cause auxotrophy or greatly affected the carotenogenesis in this yeast. to confirm that this transformation methodology is effective for other integration targets, the resistance cassette was successfully integrated into two other genomic loci. however, the color phenotype of the transformants was different from the parental strain. increase of crts gene dose next, we used the above methodology to increase the crts gene dose. a crts expression cassette was constructed by oe-pcr and cloned into plasmid pbluescript sk-, resulting in the plasmid pbs-ptef-crts-tact. this cassette was integrated in the dhs locus, as its interruption did not cause auxotrophy or greatly affected the carotenogenesis in this yeast. four dna fragments were individually pcr amplified with primers allowing to 100 bp of overlap between adjacent fragments, and then these fragments were used to co-transform x. dendrorhous by electroporation. four hygromycin b-resistant colonies were obtained. all had the crts gene expression cassettes at the expected locus, as confirmed by pcr analyses with a comprehensive set of primers. one of these four colonies was randomly selected, named strain xd_1h1s and subjected to drm to result in a homozygous strain, xd_2h2s. the pcr amplification confirmed the insertion of the hygromycin b resistance and crts expression cassettes at the dhs locus in the heterozygous and homozygous transformants are shown in figure  to assess whether the increase in the crts gene dose effectively leads to an increase in the crts mrna levels, rt-qpcr analysis was performed for the wild-type, heterozygous and homozygous transformants grown under the same conditions. total rna was extracted after 50 h of culture of each strain as it was observed that the crts mrna level reaches its maximum level at this stage. the relative crts expression was normalized to the expression of the actin gene, and rt-qpcr analysis revealed that the expression of the crts gene was increased approximately three fold in the xd_2h2s strain with respect to the wild-type strain at this same growth stage. carotenoid production to the naked eye, different pigmentation phenotypes among the wild-type, xd_1h1s and xd_2h2s strains could be noted. to confirm this observation, the carotenoid content and composition of these strains were analyzed. total carotenoids were extracted from cell pellets from and 96-h-old yeast cultures grown at 22°c in ym medium with constant agitation. carotenoids were extracted, quantified by absorbance at 465 nm and their composition was analyzed by rp-hplc. the results are summarized in table  although the total carotenoid production is not affected in strains that overexpress crts, it can be observed that after and 95 h of culture, the percentage of astaxanthin in relation to the other carotenoids is higher in strains expressing additional crts gene copies. nd: not detected. table values correspond to the average result from three independent cultures ± standard deviations. percentage relative to total carotenoids is indicated in parentheses. †: hydroxy or/and keto carotenoid, *: monocyclic carotenoid. discussion there are a limited number of currently available molecular tools to manipulate and transform x. dendrorhous. until this report, transformation of x. dendrorhous usually required the construction of a plasmid for transformation, which involves several conventional steps of sequential cloning such as dna amplification, endonuclease digestion, in vitro ligation and transformation. if the number of expression cassettes to incorporate in a host is increased, these processes will be time consuming and will depend on the availability of restriction sites in the cloning vector. in contrast, the dna assembler method requires only dna fragments with homologous ends. we find that this methodology works in x. dendrorhous as we transformed this yeast with three dna fragments to integrate a hygromycin b resistance cassette into its genome. we also increased the number of crts gene copies in this yeast by transforming it with four dna fragments to be assembled and integrated in the yeast genome. we conclude that this methodology can be employed to engineer x. dendrorhous to interrupt or increase gene copy number of several combinations of genes in only one transformation event. in previous studies attempting to overexpress genes in x. dendrorhous, expression cassettes were integrated into ribosomal dna. there are approximately rdna copies in x. dendrorhous, which favors multiple integrations of the transformant dna into the genome. however, due to the tandem repeats in this region, the resulting strains are typically unstable as the integrated genes may be lost by homologous recombination. additionally, the use of this region makes it difficult to evaluate the effect of gene dose on the production of carotenoids, as it is difficult to quantify the number of integrated cassette copies. for these reasons, we excluded the rdna region as a target site for integration and focused on integration and resultant target site disruption in a locus that does not strongly modify the yeast physiology. from the analyzed targets, the disruption of locus dhs fulfilled this requirement, so it was chosen to integrate the additional crts gene copies. in this work, we studied the effect of altering the metabolic flux between beta-carotene and astaxanthin by increasing the crts gene dose. although the total carotenoid content in strains with one or two additional crts gene copies was not substantially modified, an increased proportion of astaxanthin relative to the total carotenoid content was obtained in the strain with two additional crts gene copies compared to the wild-type strain. astaxanthin represented 96% versus 68% of the carotenoids in these strains after 96 h of culture. in addition, reduced proportions of beta-carotene and intermediate xanthophylls formed during the synthesis of astaxanthin from beta-carotene such as echinenone, hydroxyechinenone, canthaxanthin and phoenicoxanthin. these observations suggest that astaxanthin synthesis is limited by the amount of astaxanthin synthase substrate, beta-carotene. thus, to achieve a significantly higher level of astaxanthin production, it is also necessary to increase the synthesis of beta-carotene. in agreement with this assumption, it has been observed that the overexpression of the crtyb gene in x. dendrorhous resulted in increased beta-carotene production and recently, the simultaneous overexpression of the crtyb and crts genes in a x. dendrorhous strain created via random mutagenesis that already overproduced astaxanthin resulted in a higher astaxanthin content compared to the parental strain. the methodology described in this work should be a helpful tool to evaluate the consequences of overexpressing different combinations of genes involved in carotenoid production in x. dendrorhous. although beta-carotene was almost completely exhausted in the transformant strain that overexpressed crts, phoenicoxanthin, the xanthophyll precursor to astaxanthin, was still present, although its proportion was reduced from 15% to 6% after 96 h of culture in the wild-type and in the xd_2h2s strains, respectively. as astaxanthin synthase is a cytochrome p enzyme, it may also be necessary to increase the cytochrome p reductase gene dose to enhance the activity of this enzyme. the dna assembler methodology that has been effective and successful in x. dendrorhous in this study should be useful to overexpress several genes simultaneously to favor the synthesis of carotenoid precursors such as geranylgeranyl pyrophosphate or genes involved in the mevalonate pathway in combination with carotenogenic genes. thus, using different promoters in the construction of the gene expression cassettes, it should be possible to increase and modulate the production of carotenoids in x. dendrorhous.  the dna assembler method is a successful technique to transform x. dendrorhous. this technique allowed an increase in the crts gene copy number in the x. dendrorhous genome. the overexpression of this gene did not significantly change the total carotenoid production, but there was an increase in the astaxanthin fraction of carotenoids. as dna assembler requires only dna fragments with homologous ends, this technique could be useful to quickly and easily overexpress several genes simultaneously in x. dendrorhous, saving numerous steps involved in conventional cloning methods. 
in this work, the dna assembler methodology that was previously described in saccharomyces cerevisiae was successfully applied to assemble dna fragments in vivo and integrate these fragments into the genome of x. dendrorhous by homologous recombination in only one transformation event. using this method, the gene encoding astaxanthin synthase was overexpressed in x. dendrorhous and a higher level of astaxanthin was produced. this methodology could be used to easily and rapidly overexpress individual genes or combinations of genes simultaneously in x. dendrorhous, eliminating numerous steps involved in conventional cloning methods. xanthophyllomyces dendrorhous is a basidiomycetous yeast that is relevant to biotechnology, as it can synthesize the carotenoid astaxanthin. however, the astaxanthin levels produced by wild-type strains are low. although different approaches for promoting increased astaxanthin production have been attempted, no commercially competitive results have been obtained thus far. however, a major limitation is the few available molecular tools to manipulate x. dendrorhous. a promising alternative to facilitate the production of carotenoids in this yeast involves the use of genetic modification.
 xanthophyllomyces dendrorhous is a basidiomycetous yeast that is relevant to biotechnology, as it can synthesize the carotenoid astaxanthin. however, the astaxanthin levels produced by wild-type strains are low. although different approaches for promoting increased astaxanthin production have been attempted, no commercially competitive results have been obtained thus far. a promising alternative to facilitate the production of carotenoids in this yeast involves the use of genetic modification. however, a major limitation is the few available molecular tools to manipulate x. dendrorhous.  in this work, the dna assembler methodology that was previously described in saccharomyces cerevisiae was successfully applied to assemble dna fragments in vivo and integrate these fragments into the genome of x. dendrorhous by homologous recombination in only one transformation event. using this method, the gene encoding astaxanthin synthase was overexpressed in x. dendrorhous and a higher level of astaxanthin was produced.  this methodology could be used to easily and rapidly overexpress individual genes or combinations of genes simultaneously in x. dendrorhous, eliminating numerous steps involved in conventional cloning methods. xanthophyllomyces dendrorhousastaxanthin synthasedna assembler 