Limits...
Increase in the astaxanthin synthase gene (crtS) dose by in vivo DNA fragment assembly in Xanthophyllomyces dendrorhous.

Contreras G, Barahona S, Rojas MC, Baeza M, Cifuentes V, Alcaíno J - BMC Biotechnol. (2013)

Bottom Line: Although different approaches for promoting increased astaxanthin production have been attempted, no commercially competitive results have been obtained thus far.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Ciencias Ecológicas, Facultad de Ciencias, Universidad de Chile, Las Palmeras 3425, Casilla, Santiago 653, Chile. jalcainog@u.uchile.cl.

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

Show MeSH

Related in: MedlinePlus

Assembly and integration of the hygromycin B resistance cassette into the genome of X. dendrorhous by DNA assembler. A) Three DNA fragments, DHS3 “up”, hph expression cassette (composed by EF-1α promoter, hph gene and gpd terminator) and DHS3 “down” were amplified by PCR to direct the integration of the hygromycin B cassette into the DHS3 locus of the X. dendrorhous genome. Then, they were co-transformed, assembled (by recombination at their overlapping ends) and integrated into the X. dendrorhous genome. The arrows represent the primers used and the black crosses represent the in vivo homologous recombination event. By this way, the heterozygous strain was obtained, which was submitted to the double recombinant method (DRM) to obtain the homozygous strain. B) Evaluation of the hygromycin B cassette integration by PCR of strains Xd_1H (1H, one hygromycin B cassette copy), Xd_2H (2H, two hygromycin B cassette copies) and parental UCD 67–385 (WT), and control without DNA (-). A scheme representing the primer sets (in arrows and numbers according to Additional file 1: Table S1) that were used and the DNA target are included under each gel photograph. M: Molecular marker, lambda DNA digested with HindIII. C) Color phenotype of strains Xd_1H (1H), Xd_2H (2H) and parental UCD 67–385 (WT) grown on an MMV agar plate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3852557&req=5

Figure 2: Assembly and integration of the hygromycin B resistance cassette into the genome of X. dendrorhous by DNA assembler. A) Three DNA fragments, DHS3 “up”, hph expression cassette (composed by EF-1α promoter, hph gene and gpd terminator) and DHS3 “down” were amplified by PCR to direct the integration of the hygromycin B cassette into the DHS3 locus of the X. dendrorhous genome. Then, they were co-transformed, assembled (by recombination at their overlapping ends) and integrated into the X. dendrorhous genome. The arrows represent the primers used and the black crosses represent the in vivo homologous recombination event. By this way, the heterozygous strain was obtained, which was submitted to the double recombinant method (DRM) to obtain the homozygous strain. B) Evaluation of the hygromycin B cassette integration by PCR of strains Xd_1H (1H, one hygromycin B cassette copy), Xd_2H (2H, two hygromycin B cassette copies) and parental UCD 67–385 (WT), and control without DNA (-). A scheme representing the primer sets (in arrows and numbers according to Additional file 1: Table S1) that were used and the DNA target are included under each gel photograph. M: Molecular marker, lambda DNA digested with HindIII. C) Color phenotype of strains Xd_1H (1H), Xd_2H (2H) and parental UCD 67–385 (WT) grown on an MMV agar plate.

Mentions: Three DNA fragments (454 bp DHS3 “up” for upstream, 1,817 bp hygromycin B resistance cassette and 460 bp DHS3 “down” for downstream) 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 DHS3 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 [31] 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 67–385 is diploid [31], a wild-type DHS3 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 (named Xd_1H, for one hygromycin-resistance cassette copy) to obtain the homozygous strain Xd_2H (2H for two copies of the hygromycin-resistance cassette) using the double recombinant method (DRM) [30] (Figure 2A). Panel B of Figure 2 shows the amplicons obtained from genomic PCR confirming the integration of the hygromycin B resistance cassette into the expected locus in strains Xd_1H (DHS3/dhs3::hph) and Xd_2H (dhs3::hph/dhs3::hph). 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 (Figure 2C). Total carotenoids were quantified and no significant differences were detected between the transformants and the wild-type strain (data not shown). Thus, the interruption of the DHS3 locus did not cause auxotrophy or greatly affected the carotenogenesis in this yeast.


Increase in the astaxanthin synthase gene (crtS) dose by in vivo DNA fragment assembly in Xanthophyllomyces dendrorhous.

Contreras G, Barahona S, Rojas MC, Baeza M, Cifuentes V, Alcaíno J - BMC Biotechnol. (2013)

Assembly and integration of the hygromycin B resistance cassette into the genome of X. dendrorhous by DNA assembler. A) Three DNA fragments, DHS3 “up”, hph expression cassette (composed by EF-1α promoter, hph gene and gpd terminator) and DHS3 “down” were amplified by PCR to direct the integration of the hygromycin B cassette into the DHS3 locus of the X. dendrorhous genome. Then, they were co-transformed, assembled (by recombination at their overlapping ends) and integrated into the X. dendrorhous genome. The arrows represent the primers used and the black crosses represent the in vivo homologous recombination event. By this way, the heterozygous strain was obtained, which was submitted to the double recombinant method (DRM) to obtain the homozygous strain. B) Evaluation of the hygromycin B cassette integration by PCR of strains Xd_1H (1H, one hygromycin B cassette copy), Xd_2H (2H, two hygromycin B cassette copies) and parental UCD 67–385 (WT), and control without DNA (-). A scheme representing the primer sets (in arrows and numbers according to Additional file 1: Table S1) that were used and the DNA target are included under each gel photograph. M: Molecular marker, lambda DNA digested with HindIII. C) Color phenotype of strains Xd_1H (1H), Xd_2H (2H) and parental UCD 67–385 (WT) grown on an MMV agar plate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3852557&req=5

Figure 2: Assembly and integration of the hygromycin B resistance cassette into the genome of X. dendrorhous by DNA assembler. A) Three DNA fragments, DHS3 “up”, hph expression cassette (composed by EF-1α promoter, hph gene and gpd terminator) and DHS3 “down” were amplified by PCR to direct the integration of the hygromycin B cassette into the DHS3 locus of the X. dendrorhous genome. Then, they were co-transformed, assembled (by recombination at their overlapping ends) and integrated into the X. dendrorhous genome. The arrows represent the primers used and the black crosses represent the in vivo homologous recombination event. By this way, the heterozygous strain was obtained, which was submitted to the double recombinant method (DRM) to obtain the homozygous strain. B) Evaluation of the hygromycin B cassette integration by PCR of strains Xd_1H (1H, one hygromycin B cassette copy), Xd_2H (2H, two hygromycin B cassette copies) and parental UCD 67–385 (WT), and control without DNA (-). A scheme representing the primer sets (in arrows and numbers according to Additional file 1: Table S1) that were used and the DNA target are included under each gel photograph. M: Molecular marker, lambda DNA digested with HindIII. C) Color phenotype of strains Xd_1H (1H), Xd_2H (2H) and parental UCD 67–385 (WT) grown on an MMV agar plate.
Mentions: Three DNA fragments (454 bp DHS3 “up” for upstream, 1,817 bp hygromycin B resistance cassette and 460 bp DHS3 “down” for downstream) 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 DHS3 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 [31] 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 67–385 is diploid [31], a wild-type DHS3 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 (named Xd_1H, for one hygromycin-resistance cassette copy) to obtain the homozygous strain Xd_2H (2H for two copies of the hygromycin-resistance cassette) using the double recombinant method (DRM) [30] (Figure 2A). Panel B of Figure 2 shows the amplicons obtained from genomic PCR confirming the integration of the hygromycin B resistance cassette into the expected locus in strains Xd_1H (DHS3/dhs3::hph) and Xd_2H (dhs3::hph/dhs3::hph). 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 (Figure 2C). Total carotenoids were quantified and no significant differences were detected between the transformants and the wild-type strain (data not shown). Thus, the interruption of the DHS3 locus did not cause auxotrophy or greatly affected the carotenogenesis in this yeast.

Bottom Line: Although different approaches for promoting increased astaxanthin production have been attempted, no commercially competitive results have been obtained thus far.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Ciencias Ecológicas, Facultad de Ciencias, Universidad de Chile, Las Palmeras 3425, Casilla, Santiago 653, Chile. jalcainog@u.uchile.cl.

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

Show MeSH
Related in: MedlinePlus