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Efficient heterologous transformation of Chlamydomonas reinhardtii npq2 mutant with the zeaxanthin epoxidase gene isolated and characterized from Chlorella zofingiensis.

Couso I, Cordero BF, Vargas MÁ, Rodríguez H - Mar Drugs (2012)

Bottom Line: The Czzep gene was adequately inserted in the pSI105 vector and expressed in npq2.The positive transformants were able to efficiently convert zeaxanthin into violaxanthin, as well as to restore their maximum quantum efficiency of the PSII (Fv/Fm).These results show that Chlamydomonas can be an efficient tool for heterologous expression and metabolic engineering for biotechnological applications.

View Article: PubMed Central - PubMed

Affiliation: Institute of Plant Biochemistry and Photosynthesis, CIC Cartuja, University of Seville and CSIC, Avda. Américo Vespucio no. 49, 41092-Seville, Spain. inmaculada.couso@ibvf.csic.es

ABSTRACT
In the violaxanthin cycle, the violaxanthin de-epoxidase and zeaxanthin epoxidase catalyze the inter-conversion between violaxanthin and zeaxanthin in both plants and green algae. The zeaxanthin epoxidase gene from the green microalga Chlorella zofingiensis (Czzep) has been isolated. This gene encodes a polypeptide of 596 amino acids. A single copy of Czzep has been found in the C. zofingiensis genome by Southern blot analysis. qPCR analysis has shown that transcript levels of Czzep were increased after zeaxanthin formation under high light conditions. The functionality of Czzep gene by heterologous genetic complementation in the Chlamydomonas mutant npq2, which lacks zeaxanthin epoxidase (ZEP) activity and accumulates zeaxanthin in all conditions, was analyzed. The Czzep gene was adequately inserted in the pSI105 vector and expressed in npq2. The positive transformants were able to efficiently convert zeaxanthin into violaxanthin, as well as to restore their maximum quantum efficiency of the PSII (Fv/Fm). These results show that Chlamydomonas can be an efficient tool for heterologous expression and metabolic engineering for biotechnological applications.

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Zeaxanthin and violaxanthin content (a) and mRNA relative abundance of Czzep (b) in cells of C. reinhardtii npq2 mutant (npq2) and nine selected transformants (npq2-1, npq2-4, npq2-5, npq2-7, npq2-8, npq2-9, npq2-10, npq2-13 and npq2-14). a, Zeaxanthin (grey bar) and violaxanthin (black bar); b, Levels of Czzep transcripts were normalized in respect to the housekeeping control gene (cblp). Error bars indicate the standard deviations of four independent measurements.
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marinedrugs-10-01955-f006: Zeaxanthin and violaxanthin content (a) and mRNA relative abundance of Czzep (b) in cells of C. reinhardtii npq2 mutant (npq2) and nine selected transformants (npq2-1, npq2-4, npq2-5, npq2-7, npq2-8, npq2-9, npq2-10, npq2-13 and npq2-14). a, Zeaxanthin (grey bar) and violaxanthin (black bar); b, Levels of Czzep transcripts were normalized in respect to the housekeeping control gene (cblp). Error bars indicate the standard deviations of four independent measurements.

Mentions: Lutein and β-carotene were the major carotenoids both in npq2 mutant and Czzep transformant cells, the levels of those carotenoids being higher in Czzep transformants (ranging from 1.67 to 2.29 mg g∙DW−1 in the case of lutein and 0.78 to 1.19 mg g∙DW−1 in the case of β-carotene) than in npq2 mutant (0.55 mg g∙DW−1 in the case of lutein and 0.41 mg g∙DW−1 in the case of β-carotene). Thus, the npq2-4 transformant showed a content of lutein and β-carotene six- and four-fold higher, respectively, than the mutant. Most of the transformants (approximately 80% of the selected positives) exhibited a lutein and β-carotene contents of 2.8- to 3.2- and 1.8- to 2.5-fold higher than the npq2 mutant, respectively. The violaxanthin and zeaxanthin contents and the relative mRNA levels of the Czzep gene in the npq2 mutant and nine selected Czzep npq2 transformants are illustrated in Figure 6. Most of the transformants analyzed showed a restored zeaxanthin epoxidase activity, some of them exhibiting a total conversion of zeaxanthin into violaxanthin, such as npq2-4, which seems also to exhibit the highest expression pattern of the Czzep gene. Some transformants, however, did not show a total conversion of zeaxanthin into violaxanthin or a restored zeaxanthin epoxidase activity, although in all of them the Czzep gene was adequately transcribed. Nevertheless, the expression levels of Czzep in transformants were rather heterogeneous coinciding with the differences found in the carotenoid levels. In fact, the highest Czzep mRNA level was found in the npq2-4 transformant, which reached about seven-fold the levels of the transformants npq2-7, npq2-5 and npq2-10. This correlated with the highest level of carotenoids shown by npq2-4 transformant. Npq2-9 and npq2-13 transformants also showed a high level of Czzep mRNA, reaching about five times the level of the lowest yielding transformants. Therefore, the functional characterization of the recently isolated Czzep gene could be achieved by expression of this gene in the transformants and the restoration of zeaxanthin epoxidase activity.


Efficient heterologous transformation of Chlamydomonas reinhardtii npq2 mutant with the zeaxanthin epoxidase gene isolated and characterized from Chlorella zofingiensis.

Couso I, Cordero BF, Vargas MÁ, Rodríguez H - Mar Drugs (2012)

Zeaxanthin and violaxanthin content (a) and mRNA relative abundance of Czzep (b) in cells of C. reinhardtii npq2 mutant (npq2) and nine selected transformants (npq2-1, npq2-4, npq2-5, npq2-7, npq2-8, npq2-9, npq2-10, npq2-13 and npq2-14). a, Zeaxanthin (grey bar) and violaxanthin (black bar); b, Levels of Czzep transcripts were normalized in respect to the housekeeping control gene (cblp). Error bars indicate the standard deviations of four independent measurements.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475266&req=5

marinedrugs-10-01955-f006: Zeaxanthin and violaxanthin content (a) and mRNA relative abundance of Czzep (b) in cells of C. reinhardtii npq2 mutant (npq2) and nine selected transformants (npq2-1, npq2-4, npq2-5, npq2-7, npq2-8, npq2-9, npq2-10, npq2-13 and npq2-14). a, Zeaxanthin (grey bar) and violaxanthin (black bar); b, Levels of Czzep transcripts were normalized in respect to the housekeeping control gene (cblp). Error bars indicate the standard deviations of four independent measurements.
Mentions: Lutein and β-carotene were the major carotenoids both in npq2 mutant and Czzep transformant cells, the levels of those carotenoids being higher in Czzep transformants (ranging from 1.67 to 2.29 mg g∙DW−1 in the case of lutein and 0.78 to 1.19 mg g∙DW−1 in the case of β-carotene) than in npq2 mutant (0.55 mg g∙DW−1 in the case of lutein and 0.41 mg g∙DW−1 in the case of β-carotene). Thus, the npq2-4 transformant showed a content of lutein and β-carotene six- and four-fold higher, respectively, than the mutant. Most of the transformants (approximately 80% of the selected positives) exhibited a lutein and β-carotene contents of 2.8- to 3.2- and 1.8- to 2.5-fold higher than the npq2 mutant, respectively. The violaxanthin and zeaxanthin contents and the relative mRNA levels of the Czzep gene in the npq2 mutant and nine selected Czzep npq2 transformants are illustrated in Figure 6. Most of the transformants analyzed showed a restored zeaxanthin epoxidase activity, some of them exhibiting a total conversion of zeaxanthin into violaxanthin, such as npq2-4, which seems also to exhibit the highest expression pattern of the Czzep gene. Some transformants, however, did not show a total conversion of zeaxanthin into violaxanthin or a restored zeaxanthin epoxidase activity, although in all of them the Czzep gene was adequately transcribed. Nevertheless, the expression levels of Czzep in transformants were rather heterogeneous coinciding with the differences found in the carotenoid levels. In fact, the highest Czzep mRNA level was found in the npq2-4 transformant, which reached about seven-fold the levels of the transformants npq2-7, npq2-5 and npq2-10. This correlated with the highest level of carotenoids shown by npq2-4 transformant. Npq2-9 and npq2-13 transformants also showed a high level of Czzep mRNA, reaching about five times the level of the lowest yielding transformants. Therefore, the functional characterization of the recently isolated Czzep gene could be achieved by expression of this gene in the transformants and the restoration of zeaxanthin epoxidase activity.

Bottom Line: The Czzep gene was adequately inserted in the pSI105 vector and expressed in npq2.The positive transformants were able to efficiently convert zeaxanthin into violaxanthin, as well as to restore their maximum quantum efficiency of the PSII (Fv/Fm).These results show that Chlamydomonas can be an efficient tool for heterologous expression and metabolic engineering for biotechnological applications.

View Article: PubMed Central - PubMed

Affiliation: Institute of Plant Biochemistry and Photosynthesis, CIC Cartuja, University of Seville and CSIC, Avda. Américo Vespucio no. 49, 41092-Seville, Spain. inmaculada.couso@ibvf.csic.es

ABSTRACT
In the violaxanthin cycle, the violaxanthin de-epoxidase and zeaxanthin epoxidase catalyze the inter-conversion between violaxanthin and zeaxanthin in both plants and green algae. The zeaxanthin epoxidase gene from the green microalga Chlorella zofingiensis (Czzep) has been isolated. This gene encodes a polypeptide of 596 amino acids. A single copy of Czzep has been found in the C. zofingiensis genome by Southern blot analysis. qPCR analysis has shown that transcript levels of Czzep were increased after zeaxanthin formation under high light conditions. The functionality of Czzep gene by heterologous genetic complementation in the Chlamydomonas mutant npq2, which lacks zeaxanthin epoxidase (ZEP) activity and accumulates zeaxanthin in all conditions, was analyzed. The Czzep gene was adequately inserted in the pSI105 vector and expressed in npq2. The positive transformants were able to efficiently convert zeaxanthin into violaxanthin, as well as to restore their maximum quantum efficiency of the PSII (Fv/Fm). These results show that Chlamydomonas can be an efficient tool for heterologous expression and metabolic engineering for biotechnological applications.

Show MeSH
Related in: MedlinePlus