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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.

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PCR-based analysis of the crtS expression cassette and hygromycin B resistance cassette integration into the genome of X. dendrorhous. PCR analyses of Xd_1H1S (1S, one additional crtS gene copy), Xd_2H2S (2S, two additional crtS gene copies), and parental UCD 67–385 (WT) strains, and the negative control without DNA (-). A scheme representing the primers sets that were used (in arrows and numbers according to Additional file 1: Table S1) and the DNA target are under each gel photograph. The scheme shading is in accordance with Figure 3. M: Molecular marker, lambda DNA digested with HindIII.
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Figure 4: PCR-based analysis of the crtS expression cassette and hygromycin B resistance cassette integration into the genome of X. dendrorhous. PCR analyses of Xd_1H1S (1S, one additional crtS gene copy), Xd_2H2S (2S, two additional crtS gene copies), and parental UCD 67–385 (WT) strains, and the negative control without DNA (-). A scheme representing the primers sets that were used (in arrows and numbers according to Additional file 1: Table S1) and the DNA target are under each gel photograph. The scheme shading is in accordance with Figure 3. M: Molecular marker, lambda DNA digested with HindIII.

Mentions: Next, we used the above methodology to increase the crtS gene dose. A crtS expression cassette was constructed by OE-PCR (overlap extension- PCR) and cloned into plasmid pBluescript SK-, resulting in the plasmid pBS-PTEF-crtS-Tact. This cassette was integrated in the DHS3 locus, as its interruption did not cause auxotrophy or greatly affected the carotenogenesis in this yeast. Four DNA fragments (the hygromycin B-resistance selection marker, the crtS gene expression cassettes, and the DHS3 locus “up” and “down” targeting fragments) were individually PCR amplified with primers allowing 50 to 100 bp of overlap between adjacent fragments, and then these fragments were used to co-transform X. dendrorhous by electroporation (Figure 3). 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 (DHS3/dhs3::hph+crtS, one additional crtS gene copy) and subjected to DRM to result in a homozygous strain, Xd_2H2S (dhs3::hph+crtS/dhs3::hph+crtS). The PCR amplification confirmed the insertion of the hygromycin B resistance and crtS expression cassettes at the DHS3 locus in the heterozygous and homozygous transformants are shown in Figure 4.


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)

PCR-based analysis of the crtS expression cassette and hygromycin B resistance cassette integration into the genome of X. dendrorhous. PCR analyses of Xd_1H1S (1S, one additional crtS gene copy), Xd_2H2S (2S, two additional crtS gene copies), and parental UCD 67–385 (WT) strains, and the negative control without DNA (-). A scheme representing the primers sets that were used (in arrows and numbers according to Additional file 1: Table S1) and the DNA target are under each gel photograph. The scheme shading is in accordance with Figure 3. M: Molecular marker, lambda DNA digested with HindIII.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: PCR-based analysis of the crtS expression cassette and hygromycin B resistance cassette integration into the genome of X. dendrorhous. PCR analyses of Xd_1H1S (1S, one additional crtS gene copy), Xd_2H2S (2S, two additional crtS gene copies), and parental UCD 67–385 (WT) strains, and the negative control without DNA (-). A scheme representing the primers sets that were used (in arrows and numbers according to Additional file 1: Table S1) and the DNA target are under each gel photograph. The scheme shading is in accordance with Figure 3. M: Molecular marker, lambda DNA digested with HindIII.
Mentions: Next, we used the above methodology to increase the crtS gene dose. A crtS expression cassette was constructed by OE-PCR (overlap extension- PCR) and cloned into plasmid pBluescript SK-, resulting in the plasmid pBS-PTEF-crtS-Tact. This cassette was integrated in the DHS3 locus, as its interruption did not cause auxotrophy or greatly affected the carotenogenesis in this yeast. Four DNA fragments (the hygromycin B-resistance selection marker, the crtS gene expression cassettes, and the DHS3 locus “up” and “down” targeting fragments) were individually PCR amplified with primers allowing 50 to 100 bp of overlap between adjacent fragments, and then these fragments were used to co-transform X. dendrorhous by electroporation (Figure 3). 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 (DHS3/dhs3::hph+crtS, one additional crtS gene copy) and subjected to DRM to result in a homozygous strain, Xd_2H2S (dhs3::hph+crtS/dhs3::hph+crtS). The PCR amplification confirmed the insertion of the hygromycin B resistance and crtS expression cassettes at the DHS3 locus in the heterozygous and homozygous transformants are shown in Figure 4.

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