Increase in the astaxanthin synthase gene (crtS) dose by in vivo DNA fragment assembly in Xanthophyllomyces dendrorhous
© Contreras et al.; licensee BioMed Central Ltd. 2013
Received: 12 July 2013
Accepted: 4 October 2013
Published: 9 October 2013
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 (crtS) 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 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 700 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 (ROS) . Among carotenoids, astaxanthin (3,3′-dihydroxy-β,β-carotene-4-4′-dione) 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 .
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 [17, 24–27], in this work, we adapted the DNA assembler methodology to overexpress the gene encoding astaxanthin synthase (crtS) that catalyzes the formation of astaxanthin from beta-carotene [28, 29] 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 67–385. A 1,260 bp locus called DHS3 [GenBank: JN835289.1] was chosen as one of the potential target site, which is located at 2,110 bp downstream of the X. dendrorhous HIS3 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.
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 (data not shown).
Increase of crtSgene dose
Carotenoids in wild-type and transformant strains in ppm (μg per g of dry yeast)
X. dendrorhousstrain [ppm (%)]
( DHS3/ DHS3)
( DHS3/dhs3:: hph+crtS)
(dhs3:: hph+crtS/dhs3:: hph+crtS)
Cultivation time (h)
22.70 ± 11.16
34.31 ± 15.87
189.32 ± 86.51
14.35 ± 11.71
64.21 ± 1.13
188.37 ± 6.05
12.98 ± 4.93
84.79 ± 28.83
197.85 ± 34.65
4.37 ± 1.25
6.90 ± 0.57
7.4 ± 0.01
3.93 ± 0.08
2.36 ± 0.29
0.72 ± 0.22
1.36 ± 0.31
7.01 ± 0.46
2.84 ± 0.01
3.84 ± 0.05
0.54 ± 0.32
0.88 ± 0.03
4.65 ± 0.38
11.47 ± 1.10
5.79 ± 0.01
7.29 ± 0.04
8.85 ± 0.31
1.06 ± 0.16
0. 46 ± 0.12
2.24 ± 0.90
0.73 ± 0
1.41 ± 0.05
0.39 ± 0.12
0.83 ± 0.21
0.46 ± 0.39
3.99 ± 0.37
28.41 ± 3.53
0.5 ± 0.41
6.65 ± 0.01
15.93 ± 0.01
5.50 ± 0.41
11.08 ± 0.07
21.97 ± 0.62
18.81 ± 1.77
128.04 ± 5.06
13.85 ± 0.41
38.05 ± 0.04
154.13 ± 0.09
12.98 ± 0
65.84 ± 0.81
179.21 ± 0.52
0.51 ± 0.15
0.10 ± 0.04
0.54 ± 0.01
0.07 ± 0.04
0.206 ± 0.01
0.45 ± 0.03
0.47 ± 0.20
0.76 ± 0.52
1.72 ± 0.02
1.85 ± 0.02
0.95 ± 0.24
1.49 ± 0.26
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 [17, 25–27], 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 (5,098 kb in total) 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 (rDNA) [17–20]. There are approximately 60 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 DHS3 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 (Table 1), 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 (5,300 ppm) . 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 P450 enzyme, it may also be necessary to increase the cytochrome P450 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.
The experiments performed in this work were approved by the Facultad de Ciencias – Universidad de Chile, Ethics Committee.
Microorganisms, plasmids, media, and enzymes
Strains and plasmids used and built in this work
Genotype or relevant features
Source or reference
F- φ80d lacZΔM15Δ (lacZY-argF) U169 deoR recA1 endA1 hsdR17(rk- mk+) phoA supE44l- thi-1 gyrA96 relA1
ATCC 24230, wild-type. Diploid strain .
(DHS3/dhs3::hph). Heterozygote transformant derived from UCD 67–385 containing an allele of the DHS3 locus with a deletion and a hygromycin B resistance cassette.
(dhs3::hph/dhs3::hph). Homozygote transformant derived from Xd_1H by DRM  with a deletion and a hygromycin B resistance cassette in both alleles of the DHS3 locus.
(DHS3/dhs3::hph+crtS). Heterozygote transformant derived from UCD 67–385 with a deletion in one DHS3 allele and bearing the hygromycin B resistance cassette and the crtS gene expression cassette.
(dhs3::hph+crtS/dhs3::hph+crtS). Homozygote transformant derived from Xd_1H1S by DRM with a deletion in both DHS3 alleles and bearing the hygromycin B resistance cassette and the crtS gene expression cassette.
pBluescript SK- (pBS)
ColE1 ori; AmpR; cloning vector for blue-white selection
pBS containing a cassette of 1.8 kb bearing the resistance Hygromycin B (hph) gene under EF-1 α promoter and gpd transcription terminator of X. dendrorhous in its EcoRV site.
pBS containing the cDNA encoding the X. dendrorhous crtS gene in its EcoRV site.
pBS containing a 2,367 bp cassette bearing the cDNA encoding the X. dendrorhous crtS gene under EF-1α promoter and the actin transcription terminator from X. dendrorhous in the EcoRV site.
X. dendrorhous strains were grown at 22°C with constant agitation in YM medium (1% glucose, 0.3% yeast extract, 0.3% malt extract and 0.5% peptone) or minimal MMV + 2% glucose medium . Yeast transformant selection was performed on YM 1.5% agar plates supplemented with 15 μg/ml hygromycin B. E. coli strains were grown with constant agitation at 37°C in Luria-Bertani (LB) medium and supplemented with 100 μg/ml ampicillin for plasmid selection and 80 μg/ml X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) for recombinant clone selection .
Enzymes were purchased from Promega (Taq DNA pol, restriction enzymes, M-MLV reverse transcriptase) and Pfu DNA pol was purchased from Agilent Technologies.
All of the nucleotides used in this work were purchased from Alpha DNA (Montreal, Canada) or from Integrated DNA Technologies and are listed in Additional file 1: Table S1. PCR reactions were performed in a final volume of 25 μl containing 2 U of Taq DNA pol, 2.5 μl of 10× Taq buffer, 0.5 μl of 10 mM dNTPs, 1 μl of 50 mM MgCl2, 1 μl of 25 μM of each primer and 10–20 ng of template DNA. Amplification was performed in a DNA thermal cycler 2720 (Applied Biosystems) 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. Samples were kept at 4°C until use. The amplicons were separated by 0.8% agarose gel electrophoresis in TAE buffer containing 0.5 μg/ml ethidium bromide  followed by DNA purification using the Glassmilk method  for sequencing or gene expression cassette construction.
To reduce the error rate in DNA amplification for Overlap Extension-PCR (OE-PCR,  and DNA fragment amplification for X. dendrorhous transformation, Pfu DNA pol was used instead of Taq DNA pol following the manufacturer’s instructions.
Cloning and construction of the crtSgene expression cassette
The crtS gene expression cassette was constructed by Overlap Extension-PCR (OE-PCR)  containing 1,674 bp of the cDNA of the crtS gene [GenBank: DQ002007.1] under the control of 393 bp of the X. dendrorhous EF-1α promoter  and 300 bp of the actin transcription terminator of [GenBank: X89898.1]. The EF-1α promoter region was amplified by PCR with primers 1 and 2 (Additional file 1: Table S1) using pMN-hph as a template (Table 2), and the actin gene terminator was amplified from UCD 67–385 genomic DNA using primers 5 and 6 (Additional file 1: Table S1). The crtS cDNA was amplified from pXd_Ex_crtS (Table 2) with primers 3 and 4 (Additional file 1: Table S1). First, the promoter region was joined to the crtS cDNA and then the hybrid product was joined to the actin gene transcription terminator by OE-PCR. The resulting cassette was cloned into the EcoRV site of plasmid pBS (pBS-PTEF-crtS-Tact, Table 2) and was sequenced on both strands using a DYEnamic ET Terminator Kit (Amersham Bioscience) in an ABI 3100 Avant genetic analyzer. DNA sequences were analyzed with Vector NTI Suite 10 (Informax) and bioinformatics programs available online.
X. dendrorhous transformation was performed by electroporation according to  and . Electrocompetent cells were prepared from an exponential culture with DO600nm = 1.2, cultured in YM medium and electroporated using a BioRad Gene Pulser Xcell with PC and CE modules under the following conditions: 125 mF, 600 Ω, 0.45 kV. The yeast were transformed with 4 μl of a mixture of purified DNA fragments (1 μg of each fragment) that were amplified by PCR with Pfu DNA pol and primers with 5′ complementary ends to allow their assembly by homologous recombination. In each transformation event, in addition to the selection marker and/or the crtS gene expression cassette, “up” and “down” DNA fragments were included targeting the insertion into the integration site. The hygromycin B resistance and crtS gene expression cassettes were amplified from plasmids pMN-Hyg and pBS-PTEF-crtS-Tact, respectively, and the “up” and “down” DNA fragments were amplified from X. dendrorhous wild-type genomic DNA. Transformant selection was performed on YM 1.5% agar plates supplemented with 15 μg/ml hygromycin B. As strain UCD 67–385 is diploid , heterozygous transformants were obtained. To obtain homozygous transformants, the double recombinant method (DRM)  was applied. The transformant strains were confirmed as X. dendrorhous by examining the ITS1, 5.8 rRNA gene and ITS2 DNA sequences [42, 43].
RNA extraction, single strand DNA synthesis and RT-qPCR
To measure the relative crtS gene expression, total RNA was extracted from 50 h yeast cultures (late exponential phase of growth) grown at 22°C with constant agitation in YM medium. The cell pellets from 5 ml culture aliquots were frozen with liquid nitrogen and stored at -80°C until use. Total RNA extraction from the cell pellets was performed by mechanical rupture with 0.5 mm glass beads (BioSpec) during 10 min of vortexing, followed by the addition of Tri-Reagent (Ambion). The lysate was incubated for 5 min at room temperature and 200 μl of chloroform per ml of Tri-Reagent used was added, mixed, and centrifuged for 5 min at 4,000 × g. Following this centrifugation, the aqueous phase was recovered. The RNA was precipitated by adding two volumes of isopropanol and incubating at room temperature for 10 min. The RNA was washed with 75% ethanol, suspended in RNase-free H2O and quantified by absorbance determination at 260 nm in a double beam Shimadzu UV-150-20 spectrophotometer.
The synthesis of cDNA was performed according to the M-MLV reverse transcriptase (Invitrogen) manufacturer’s protocol with 5 μg of total RNA in a final volume of 11 μl. The determination of the relative crtS gene expression levels was performed in an Mx3000P quantitative PCR system (Stratagene) using 1 μl of the reverse transcription reaction, 0.25 μM of each primer (Additional file 1: Table S1) and 10 μl of the SensiMix SYBR Green I (Quantace) kit reagent in a final volume of 20 μl. The obtained Ct values were normalized to the respective value of the actin gene [GenBank: X89898.1]  and expressed using the 2-ΔCT method [45, 46].
Carotenoid extraction and RP-HPLC
Carotenoids were extracted from cell pellets from 24, 50 and 96-hours-old yeast cultures (early exponential, late exponential and late stationary phase of growth, respectively) grown at 22°C with constant agitation in YM medium using the acetone extraction method . Total carotenoids were quantified by absorbance at 465 nm using an absorption coefficient of A1% = 2,100. The analyses were performed at least in triplicate, and pigments were normalized relative to the dry weight of the yeast. Carotenoids were separated by RP-HPLC using a reverse phase RP-18 LiChroCART 125–4 (Merck) column with acetonitrile:methanol:isopropanol (85:10:5 v/v) as the mobile phase under isocratic conditions with a 1 ml/min flux. The elusion spectra were recovered using a diode array detector, and carotenoids were identified by their spectra and retention time according to standards.
This work was supported by projects: Fondecyt 11121200 to JA and Fondecyt 1100324 to VC.
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