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Isolation and characterization of a lycopene ε-cyclase gene of Chlorella (Chromochloris) zofingiensis. Regulation of the carotenogenic pathway by nitrogen and light.

Cordero BF, Couso I, Leon R, Rodriguez H, Vargas MA - Mar Drugs (2012)

Bottom Line: A single copy of Czlcy-e was found in C. zofingiensis.High irradiance stress did not increase mRNA levels of neither lycopene β-cyclase gene (lcy-b) nor lycopene ε-cyclase gene (lcy-e) as compared with low irradiance conditions, whereas the transcript levels of psy, pds, chyB and bkt genes were enhanced, nevertheless triggering the synthesis of the secondary carotenoids astaxanthin, canthaxanthin and zeaxanthin and decreasing the levels of the primary carotenoids α-carotene, lutein, violaxanthin and β-carotene.The combined effect of both high light and nitrogen starvation stresses enhanced significantly the accumulation of these carotenoids as well as the transcript levels of bkt gene, as compared with the effect of only high irradiance stress.

View Article: PubMed Central - PubMed

Affiliation: Institute of Plant Biochemistry and Photosynthesis, CIC Cartuja, University of Seville and CSIC, Avda. Americo Vespucio, n° 49, Seville 41092, Spain. baldomero@ibvf.csic.es

ABSTRACT
The isolation and characterization of the lycopene ε-cyclase gene from the green microalga Chlorella (Chromochloris) zofingiensis (Czlcy-e) was performed. This gene is involved in the formation of the carotenoids α-carotene and lutein. Czlcy-e gene encoded a polypeptide of 654 amino acids. A single copy of Czlcy-e was found in C. zofingiensis. Functional analysis by heterologous complementation in Escherichia coli showed the ability of this protein to convert lycopene to δ-carotene. In addition, the regulation of the carotenogenic pathway by light and nitrogen was also studied in C. zofingiensis. High irradiance stress did not increase mRNA levels of neither lycopene β-cyclase gene (lcy-b) nor lycopene ε-cyclase gene (lcy-e) as compared with low irradiance conditions, whereas the transcript levels of psy, pds, chyB and bkt genes were enhanced, nevertheless triggering the synthesis of the secondary carotenoids astaxanthin, canthaxanthin and zeaxanthin and decreasing the levels of the primary carotenoids α-carotene, lutein, violaxanthin and β-carotene. Nitrogen starvation per se enhanced mRNA levels of all genes considered, except lcy-e and pds, but did not trigger the synthesis of astaxanthin, canthaxanthin nor zeaxanthin. The combined effect of both high light and nitrogen starvation stresses enhanced significantly the accumulation of these carotenoids as well as the transcript levels of bkt gene, as compared with the effect of only high irradiance stress.

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HPLC elution profiles of carotenoids extracted from cultures of E. coli carrying plasmids pAC-LYC (A) and pAC-LYC + pQE-Czlcy-e (B). The absorption spectra and retention time of the corresponding carotenoids are also shown. E. coli BL21 (DE3) cells transformed with the indicated plasmids were isolated in the presence of chloramphenicol (A) or chloramphenicol + ampicillin (B). Lycopene and δ-carotene were identified as described in Experimental Section. Peaks identification: (1) lycopene; (2) δ-carotene.
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marinedrugs-10-02069-f005: HPLC elution profiles of carotenoids extracted from cultures of E. coli carrying plasmids pAC-LYC (A) and pAC-LYC + pQE-Czlcy-e (B). The absorption spectra and retention time of the corresponding carotenoids are also shown. E. coli BL21 (DE3) cells transformed with the indicated plasmids were isolated in the presence of chloramphenicol (A) or chloramphenicol + ampicillin (B). Lycopene and δ-carotene were identified as described in Experimental Section. Peaks identification: (1) lycopene; (2) δ-carotene.

Mentions: In order to check the functionality of the recently isolated gene, the full-length ORF of Czlcy-e was amplified and cloned into pQE-80L vector under the control of the β-galactosidase promoter. The resulting plasmid (pQE-Czlcy-e) was introduced in E. coli engineered to accumulate either lycopene or δ-carotene as final products, due to the presence of the plasmids pAC-LYC or pAC-DELTA, respectively. HPLC analysis of carotenoids extracted from E. coli showed that cells containing pAC-LYC produced lycopene (Figure 5A), while cells co-transformed with both pAC-LYC and pQE-Czlcy-e accumulated δ-carotene (Figure 5B). δ-carotene was also the only carotenoid synthesized by E. coli cells containing pAC-DELTA or both pAC-DELTA and pQE-Czlcy-e (data not shown). As negative controls, E. coli co-transformed with either pAC-LYC or pAC-DELTA and empty pQE-80L were used, resulting in the accumulation of lycopene or δ-carotene, respectively (data not shown). These results indicated that CzLCYe could catalyze the formation of one ε-ring at one of the ends of lineal lycopene to yield δ-carotene, but not the conversion of δ-carotene into ε-carotene (with one ε-ring in each end of lycopene), exhibiting, therefore, monocyclase activity.


Isolation and characterization of a lycopene ε-cyclase gene of Chlorella (Chromochloris) zofingiensis. Regulation of the carotenogenic pathway by nitrogen and light.

Cordero BF, Couso I, Leon R, Rodriguez H, Vargas MA - Mar Drugs (2012)

HPLC elution profiles of carotenoids extracted from cultures of E. coli carrying plasmids pAC-LYC (A) and pAC-LYC + pQE-Czlcy-e (B). The absorption spectra and retention time of the corresponding carotenoids are also shown. E. coli BL21 (DE3) cells transformed with the indicated plasmids were isolated in the presence of chloramphenicol (A) or chloramphenicol + ampicillin (B). Lycopene and δ-carotene were identified as described in Experimental Section. Peaks identification: (1) lycopene; (2) δ-carotene.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475274&req=5

marinedrugs-10-02069-f005: HPLC elution profiles of carotenoids extracted from cultures of E. coli carrying plasmids pAC-LYC (A) and pAC-LYC + pQE-Czlcy-e (B). The absorption spectra and retention time of the corresponding carotenoids are also shown. E. coli BL21 (DE3) cells transformed with the indicated plasmids were isolated in the presence of chloramphenicol (A) or chloramphenicol + ampicillin (B). Lycopene and δ-carotene were identified as described in Experimental Section. Peaks identification: (1) lycopene; (2) δ-carotene.
Mentions: In order to check the functionality of the recently isolated gene, the full-length ORF of Czlcy-e was amplified and cloned into pQE-80L vector under the control of the β-galactosidase promoter. The resulting plasmid (pQE-Czlcy-e) was introduced in E. coli engineered to accumulate either lycopene or δ-carotene as final products, due to the presence of the plasmids pAC-LYC or pAC-DELTA, respectively. HPLC analysis of carotenoids extracted from E. coli showed that cells containing pAC-LYC produced lycopene (Figure 5A), while cells co-transformed with both pAC-LYC and pQE-Czlcy-e accumulated δ-carotene (Figure 5B). δ-carotene was also the only carotenoid synthesized by E. coli cells containing pAC-DELTA or both pAC-DELTA and pQE-Czlcy-e (data not shown). As negative controls, E. coli co-transformed with either pAC-LYC or pAC-DELTA and empty pQE-80L were used, resulting in the accumulation of lycopene or δ-carotene, respectively (data not shown). These results indicated that CzLCYe could catalyze the formation of one ε-ring at one of the ends of lineal lycopene to yield δ-carotene, but not the conversion of δ-carotene into ε-carotene (with one ε-ring in each end of lycopene), exhibiting, therefore, monocyclase activity.

Bottom Line: A single copy of Czlcy-e was found in C. zofingiensis.High irradiance stress did not increase mRNA levels of neither lycopene β-cyclase gene (lcy-b) nor lycopene ε-cyclase gene (lcy-e) as compared with low irradiance conditions, whereas the transcript levels of psy, pds, chyB and bkt genes were enhanced, nevertheless triggering the synthesis of the secondary carotenoids astaxanthin, canthaxanthin and zeaxanthin and decreasing the levels of the primary carotenoids α-carotene, lutein, violaxanthin and β-carotene.The combined effect of both high light and nitrogen starvation stresses enhanced significantly the accumulation of these carotenoids as well as the transcript levels of bkt gene, as compared with the effect of only high irradiance stress.

View Article: PubMed Central - PubMed

Affiliation: Institute of Plant Biochemistry and Photosynthesis, CIC Cartuja, University of Seville and CSIC, Avda. Americo Vespucio, n° 49, Seville 41092, Spain. baldomero@ibvf.csic.es

ABSTRACT
The isolation and characterization of the lycopene ε-cyclase gene from the green microalga Chlorella (Chromochloris) zofingiensis (Czlcy-e) was performed. This gene is involved in the formation of the carotenoids α-carotene and lutein. Czlcy-e gene encoded a polypeptide of 654 amino acids. A single copy of Czlcy-e was found in C. zofingiensis. Functional analysis by heterologous complementation in Escherichia coli showed the ability of this protein to convert lycopene to δ-carotene. In addition, the regulation of the carotenogenic pathway by light and nitrogen was also studied in C. zofingiensis. High irradiance stress did not increase mRNA levels of neither lycopene β-cyclase gene (lcy-b) nor lycopene ε-cyclase gene (lcy-e) as compared with low irradiance conditions, whereas the transcript levels of psy, pds, chyB and bkt genes were enhanced, nevertheless triggering the synthesis of the secondary carotenoids astaxanthin, canthaxanthin and zeaxanthin and decreasing the levels of the primary carotenoids α-carotene, lutein, violaxanthin and β-carotene. Nitrogen starvation per se enhanced mRNA levels of all genes considered, except lcy-e and pds, but did not trigger the synthesis of astaxanthin, canthaxanthin nor zeaxanthin. The combined effect of both high light and nitrogen starvation stresses enhanced significantly the accumulation of these carotenoids as well as the transcript levels of bkt gene, as compared with the effect of only high irradiance stress.

Show MeSH
Related in: MedlinePlus