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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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Related in: MedlinePlus

RT-qPCR expression analysis of crtS in the wild-type strain and crtS-overexpressing transformants. The crtS transcript level was normalized to the actin mRNA and was determined from RNA extracted after 50 h of culture in YM medium from the parental wild-type UCD 67–385 (WT), the heterozygous Xd_1H1S (DHS3/dhs3::hph+crtS) and homozygous Xd_2H2S (dhs3::hph+crtS/dhs3::hph+crtS) strains. Values are the mean ± standard error of at least three independent cultures. (*P ≤ 0.05; Student’s t test).
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Figure 5: RT-qPCR expression analysis of crtS in the wild-type strain and crtS-overexpressing transformants. The crtS transcript level was normalized to the actin mRNA and was determined from RNA extracted after 50 h of culture in YM medium from the parental wild-type UCD 67–385 (WT), the heterozygous Xd_1H1S (DHS3/dhs3::hph+crtS) and homozygous Xd_2H2S (dhs3::hph+crtS/dhs3::hph+crtS) strains. Values are the mean ± standard error of at least three independent cultures. (*P ≤ 0.05; Student’s t test).

Mentions: 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 (late exponential phase of growth) of each strain as it was observed that the crtS mRNA level reaches its maximum level at this stage [32,33]. The relative crtS expression was normalized to the expression of the actin gene [34], 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 (UCD-67-385) at this same growth stage (Figure 5).


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)

RT-qPCR expression analysis of crtS in the wild-type strain and crtS-overexpressing transformants. The crtS transcript level was normalized to the actin mRNA and was determined from RNA extracted after 50 h of culture in YM medium from the parental wild-type UCD 67–385 (WT), the heterozygous Xd_1H1S (DHS3/dhs3::hph+crtS) and homozygous Xd_2H2S (dhs3::hph+crtS/dhs3::hph+crtS) strains. Values are the mean ± standard error of at least three independent cultures. (*P ≤ 0.05; Student’s t test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: RT-qPCR expression analysis of crtS in the wild-type strain and crtS-overexpressing transformants. The crtS transcript level was normalized to the actin mRNA and was determined from RNA extracted after 50 h of culture in YM medium from the parental wild-type UCD 67–385 (WT), the heterozygous Xd_1H1S (DHS3/dhs3::hph+crtS) and homozygous Xd_2H2S (dhs3::hph+crtS/dhs3::hph+crtS) strains. Values are the mean ± standard error of at least three independent cultures. (*P ≤ 0.05; Student’s t test).
Mentions: 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 (late exponential phase of growth) of each strain as it was observed that the crtS mRNA level reaches its maximum level at this stage [32,33]. The relative crtS expression was normalized to the expression of the actin gene [34], 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 (UCD-67-385) at this same growth stage (Figure 5).

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