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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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Unweighted pair group method with arithmetic mean (UPGMA) tree analysis of the indicated plant, algal and bacterial ZEP amino acid sequences. The GenBank accession numbers for other species are as follows: Nicotiana plumbaginifolia (X95732.1); Lycopersicon esculentum (Z83835.1); Capsicum annuum (Q96375); Daucus carota (ABB52077.1); Arabidopsis thaliana (AAG17703); Ricinus comunis (XP_002523587.1); Vitis vinifera (AAR11195.1); Hordeum vulgare (BAK08085.1); Chlorella variabilis (EFN52633.1); Chamydomonas sp (AAO48941.1); Volvox carteri (XP_002953670.1); Chlamydomonas reinhardtii (XP_001701701.1); Sphaerobacter thermophilus DSM 20745 (ACZ40773.1); Ktedonobacter racemifer DSM 44963 (ZP_06974439.1); Lacinutrix sp. (AEH02389.1); Marinomonas mediterranea MMB-1 (ADZ93016.1). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances correspond to the number of amino acid substitutions per site and were computed using the Poisson correction method.
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marinedrugs-10-01955-f003: Unweighted pair group method with arithmetic mean (UPGMA) tree analysis of the indicated plant, algal and bacterial ZEP amino acid sequences. The GenBank accession numbers for other species are as follows: Nicotiana plumbaginifolia (X95732.1); Lycopersicon esculentum (Z83835.1); Capsicum annuum (Q96375); Daucus carota (ABB52077.1); Arabidopsis thaliana (AAG17703); Ricinus comunis (XP_002523587.1); Vitis vinifera (AAR11195.1); Hordeum vulgare (BAK08085.1); Chlorella variabilis (EFN52633.1); Chamydomonas sp (AAO48941.1); Volvox carteri (XP_002953670.1); Chlamydomonas reinhardtii (XP_001701701.1); Sphaerobacter thermophilus DSM 20745 (ACZ40773.1); Ktedonobacter racemifer DSM 44963 (ZP_06974439.1); Lacinutrix sp. (AEH02389.1); Marinomonas mediterranea MMB-1 (ADZ93016.1). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances correspond to the number of amino acid substitutions per site and were computed using the Poisson correction method.

Mentions: The BlastP search results demonstrated that the cloned CzZEP showed the highest overall homology sequence with other ZEP from green algae, such as Volvox carteri (identity, 66% and similarity, 77%), Chlorella variabilis (identity, 69% and similarity, 79%) and Chlamydomonas reinhardtii (identity, 69% and similarity, 80%). The GC content of the Czzep coding region was 52.4%, which was lower than that of V. carteri (60.5%), C. variabilis (68.1%) or of C. reinhardtii (68.8%). The phylogenetic analysis of ZEP from green algae, plants and bacteria is illustrated in Figure 3. This analysis was performed in MEGA5 software [29] using the unweighted pair group method with arithmetic mean (UPGMA) method. The predicted CzZEP forms a cluster with the ZEP of the green algae studied, which are phylogenetically close to ZEP of plants (between 60% and 63% of identity and between 73% and 76% similarity). CzZEP was distantly related to bacterial zeaxanthin epoxidases (between 30% and 34% of identity and 45%–50% of similarity).


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)

Unweighted pair group method with arithmetic mean (UPGMA) tree analysis of the indicated plant, algal and bacterial ZEP amino acid sequences. The GenBank accession numbers for other species are as follows: Nicotiana plumbaginifolia (X95732.1); Lycopersicon esculentum (Z83835.1); Capsicum annuum (Q96375); Daucus carota (ABB52077.1); Arabidopsis thaliana (AAG17703); Ricinus comunis (XP_002523587.1); Vitis vinifera (AAR11195.1); Hordeum vulgare (BAK08085.1); Chlorella variabilis (EFN52633.1); Chamydomonas sp (AAO48941.1); Volvox carteri (XP_002953670.1); Chlamydomonas reinhardtii (XP_001701701.1); Sphaerobacter thermophilus DSM 20745 (ACZ40773.1); Ktedonobacter racemifer DSM 44963 (ZP_06974439.1); Lacinutrix sp. (AEH02389.1); Marinomonas mediterranea MMB-1 (ADZ93016.1). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances correspond to the number of amino acid substitutions per site and were computed using the Poisson correction method.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

marinedrugs-10-01955-f003: Unweighted pair group method with arithmetic mean (UPGMA) tree analysis of the indicated plant, algal and bacterial ZEP amino acid sequences. The GenBank accession numbers for other species are as follows: Nicotiana plumbaginifolia (X95732.1); Lycopersicon esculentum (Z83835.1); Capsicum annuum (Q96375); Daucus carota (ABB52077.1); Arabidopsis thaliana (AAG17703); Ricinus comunis (XP_002523587.1); Vitis vinifera (AAR11195.1); Hordeum vulgare (BAK08085.1); Chlorella variabilis (EFN52633.1); Chamydomonas sp (AAO48941.1); Volvox carteri (XP_002953670.1); Chlamydomonas reinhardtii (XP_001701701.1); Sphaerobacter thermophilus DSM 20745 (ACZ40773.1); Ktedonobacter racemifer DSM 44963 (ZP_06974439.1); Lacinutrix sp. (AEH02389.1); Marinomonas mediterranea MMB-1 (ADZ93016.1). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances correspond to the number of amino acid substitutions per site and were computed using the Poisson correction method.
Mentions: The BlastP search results demonstrated that the cloned CzZEP showed the highest overall homology sequence with other ZEP from green algae, such as Volvox carteri (identity, 66% and similarity, 77%), Chlorella variabilis (identity, 69% and similarity, 79%) and Chlamydomonas reinhardtii (identity, 69% and similarity, 80%). The GC content of the Czzep coding region was 52.4%, which was lower than that of V. carteri (60.5%), C. variabilis (68.1%) or of C. reinhardtii (68.8%). The phylogenetic analysis of ZEP from green algae, plants and bacteria is illustrated in Figure 3. This analysis was performed in MEGA5 software [29] using the unweighted pair group method with arithmetic mean (UPGMA) method. The predicted CzZEP forms a cluster with the ZEP of the green algae studied, which are phylogenetically close to ZEP of plants (between 60% and 63% of identity and between 73% and 76% similarity). CzZEP was distantly related to bacterial zeaxanthin epoxidases (between 30% and 34% of identity and 45%–50% of similarity).

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