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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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Verification of the plasmid pSI105-Tp1-Czzep insertion in the genome of C. reinhardtii by PCR. C. reinhardtii npq2 cells transformed with the plasmid pSI105-Tp1-Czzep were grown in TAP medium with paromomycin (30 µg mL−1), and paromomycin-resistant colonies were tested by PCR. Lane 10 is the 2log DNA ladder (0.1–10 kb, Biolabs). Lanes 1–9 correspond to some analyzed transformants that were found to be positive.
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marinedrugs-10-01955-f005: Verification of the plasmid pSI105-Tp1-Czzep insertion in the genome of C. reinhardtii by PCR. C. reinhardtii npq2 cells transformed with the plasmid pSI105-Tp1-Czzep were grown in TAP medium with paromomycin (30 µg mL−1), and paromomycin-resistant colonies were tested by PCR. Lane 10 is the 2log DNA ladder (0.1–10 kb, Biolabs). Lanes 1–9 correspond to some analyzed transformants that were found to be positive.

Mentions: Since the use of E. coli (DH5α) engineered to produce zeaxanthin did not allow the functional characterization of the Czzep gene, probably due to the lack of cofactors necessary for enzymatic activity, the functionality of this gene was assayed by heterologous genetic complementation in the C. reinhardtii npq2 mutant. For this purpose, the complete coding region of Czzep was amplified by PCR and cloned between the XhoI and EcoRI restriction sites of the Chlamydomonas expression vector pSI105-Tp1, resulting in the plasmid pSI105-Tp1-zep. C. reinhardtii npq2 mutant cells were transformed with this plasmid, and the transformants were a priori selected on the basis of their paromomycin resistance. The colonies obtained were screened for the insertion of Czzep cDNA in their genome by PCR. Figure 5 shows some of the positive transformants analyzed exhibiting a band of approximately 0.1 kb, which corresponds to the Czzep cDNA integrated in their genome. The primers used for PCR analysis to confirm this integration were G-ZEP-2F and G-ZEP-1R (Table 1) with a final amplification of 75 bp. More than 150 colonies resistant to paromomycin were analyzed and 113 of them (≈75%) were found to be positive.


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)

Verification of the plasmid pSI105-Tp1-Czzep insertion in the genome of C. reinhardtii by PCR. C. reinhardtii npq2 cells transformed with the plasmid pSI105-Tp1-Czzep were grown in TAP medium with paromomycin (30 µg mL−1), and paromomycin-resistant colonies were tested by PCR. Lane 10 is the 2log DNA ladder (0.1–10 kb, Biolabs). Lanes 1–9 correspond to some analyzed transformants that were found to be positive.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

marinedrugs-10-01955-f005: Verification of the plasmid pSI105-Tp1-Czzep insertion in the genome of C. reinhardtii by PCR. C. reinhardtii npq2 cells transformed with the plasmid pSI105-Tp1-Czzep were grown in TAP medium with paromomycin (30 µg mL−1), and paromomycin-resistant colonies were tested by PCR. Lane 10 is the 2log DNA ladder (0.1–10 kb, Biolabs). Lanes 1–9 correspond to some analyzed transformants that were found to be positive.
Mentions: Since the use of E. coli (DH5α) engineered to produce zeaxanthin did not allow the functional characterization of the Czzep gene, probably due to the lack of cofactors necessary for enzymatic activity, the functionality of this gene was assayed by heterologous genetic complementation in the C. reinhardtii npq2 mutant. For this purpose, the complete coding region of Czzep was amplified by PCR and cloned between the XhoI and EcoRI restriction sites of the Chlamydomonas expression vector pSI105-Tp1, resulting in the plasmid pSI105-Tp1-zep. C. reinhardtii npq2 mutant cells were transformed with this plasmid, and the transformants were a priori selected on the basis of their paromomycin resistance. The colonies obtained were screened for the insertion of Czzep cDNA in their genome by PCR. Figure 5 shows some of the positive transformants analyzed exhibiting a band of approximately 0.1 kb, which corresponds to the Czzep cDNA integrated in their genome. The primers used for PCR analysis to confirm this integration were G-ZEP-2F and G-ZEP-1R (Table 1) with a final amplification of 75 bp. More than 150 colonies resistant to paromomycin were analyzed and 113 of them (≈75%) were found to be positive.

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