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Isolation and Analysis of the Cppsy Gene and Promoter from Chlorella protothecoides CS-41.

Li M, Cui Y, Gan Z, Shi C, Shi X - Mar Drugs (2015)

Bottom Line: Analysis revealed several candidate motifs for the promoter, which exhibited light- and methyl jasmonate (MeJA)-responsive characteristics, as well as some typical domains universally discovered in promoter sequences, such as the TATA-box and CAAT-box.Light- and MeJA treatment showed that the Cppsy expression level was significantly enhanced by light and MeJA.These results provide a basis for genetically modifying the carotenoid biosynthesis pathway in C. protothecoides.

View Article: PubMed Central - PubMed

Affiliation: MOST-USDA Joint Research Center for Food Safety, School of Agriculture and Biology, and State Key Lab of Microbial Metabolism, Shanghai Jiao Tong University, Shanghai 200240, China. lmeiya@126.com.

ABSTRACT
Phytoene synthase (PSY) catalyzes the condensation of two molecules of geranylgeranyl pyrophosphate to form phytoene, the first colorless carotene in the carotenoid biosynthesis pathway. So it is regarded as the crucial enzyme for carotenoid production, and has unsurprisingly been involved in genetic engineering studies of carotenoid production. In this study, the psy gene from Chlorella protothecoides CS-41, designated Cppsy, was cloned using rapid amplification of cDNA ends. The full-length DNA was 2488 bp, and the corresponding cDNA was 1143 bp, which encoded 380 amino acids. Computational analysis suggested that this protein belongs to the Isoprenoid_Biosyn_C1 superfamily. It contained the consensus sequence, including three predicted substrate-Mg(2+) binding sites. The Cppsy gene promoter was also cloned and characterized. Analysis revealed several candidate motifs for the promoter, which exhibited light- and methyl jasmonate (MeJA)-responsive characteristics, as well as some typical domains universally discovered in promoter sequences, such as the TATA-box and CAAT-box. Light- and MeJA treatment showed that the Cppsy expression level was significantly enhanced by light and MeJA. These results provide a basis for genetically modifying the carotenoid biosynthesis pathway in C. protothecoides.

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(A) Alignment of the selective PSY-deduced amino acid sequences from different algae produced with the GeneDoc program using Clustal W. The alignment indicates aspartate-rich regions/substrate-Mg2+ binding sites (DXXXD). The three DXXXD motifs are shown by the red boxes. Cppsy, Cvpsy, Mspsy, Olpsy, Dbpsy, Dspsy, Hppsy, Cspsy, Czpsy, Vcpsy, Ntpsy, Atpsy, and Zmpsy are the PSY of Chlorella protothecoides CS-41, Chlorella variabilis, Micromonas sp. RCC299, Ostreococcus lucimarinus, Duanliella bardawil, Duanliella salina, Haematococcus pluvialis, Coccomyxa subellipsodiea C-169, Chromochloris zofingiensis, Volvox carteri f. nagariensis, Nicotiana tabacum, Arabidopsis thaliana, and Zea mays, respectively; (B) Three-dimensional model structure of CpPSY. Comparative modeling was performed using homology-based three-dimensional structural modeling. The three aspartate-rich motifs (DXXXD) are colored in orange (DELVD), yellow (DELYD), and magenta (DEGED); others are shown in green. The N-terminus and C-terminus are also shown; (C) High-performance liquid chromatography trace and UV spectrum of carotenoid pigments in the E. coli heterologous complementation system. Pigments extracted from E. coli cells transformed with pACCRT-E and pUC-psy together (1), pUC-psy only (2), and pACCRT-EB only (3). Absorbance was recorded at 285 nm. The peak indicated by the arrow is phytoene.
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marinedrugs-13-06620-f003: (A) Alignment of the selective PSY-deduced amino acid sequences from different algae produced with the GeneDoc program using Clustal W. The alignment indicates aspartate-rich regions/substrate-Mg2+ binding sites (DXXXD). The three DXXXD motifs are shown by the red boxes. Cppsy, Cvpsy, Mspsy, Olpsy, Dbpsy, Dspsy, Hppsy, Cspsy, Czpsy, Vcpsy, Ntpsy, Atpsy, and Zmpsy are the PSY of Chlorella protothecoides CS-41, Chlorella variabilis, Micromonas sp. RCC299, Ostreococcus lucimarinus, Duanliella bardawil, Duanliella salina, Haematococcus pluvialis, Coccomyxa subellipsodiea C-169, Chromochloris zofingiensis, Volvox carteri f. nagariensis, Nicotiana tabacum, Arabidopsis thaliana, and Zea mays, respectively; (B) Three-dimensional model structure of CpPSY. Comparative modeling was performed using homology-based three-dimensional structural modeling. The three aspartate-rich motifs (DXXXD) are colored in orange (DELVD), yellow (DELYD), and magenta (DEGED); others are shown in green. The N-terminus and C-terminus are also shown; (C) High-performance liquid chromatography trace and UV spectrum of carotenoid pigments in the E. coli heterologous complementation system. Pigments extracted from E. coli cells transformed with pACCRT-E and pUC-psy together (1), pUC-psy only (2), and pACCRT-EB only (3). Absorbance was recorded at 285 nm. The peak indicated by the arrow is phytoene.

Mentions: The deduced amino acid sequence of Cppsy was submitted to NCBI for PSI-BLAST searches and the results showed that Cppsy has high homology with psy genes from other algal species, with 83% identity and 88% positives with psy from Chlorella NC_64A. Cppsy was also highly similar to psy from C. reinhardtii (67% identities, 79% positives), H. pluvialis (63% identities, 77% positives), D. bardawil (68% identities, 80% positives), and D. salina (68% identities, 79% positives), suggesting that Cppsy belongs to the algae psy family. In the algae family, CpPSY belongs to class I of PSY according to Tran’s data [29]. BlastP analysis suggested that this protein has the essential characteristics of PSY. It belongs to the Isoprenoid_Biosyn_C1 superfamily, and contains the consensus sequence, including three predicted substrate-Mg2+ binding sites (aspartate-rich regions) (DXXXD), 130-DELVD-134, 203-DELYD-207, and 256-DEGED-260 (Figure 3A). In other algae and higher plants, there are two (DELVD and DVGED) (Figure 3A); hence, CpPSY has one more DXXXD motif than other PSYs. The abundant 203-DELYD-207 site possibly plays an important role in the function of CpPSY, which should be studied further.


Isolation and Analysis of the Cppsy Gene and Promoter from Chlorella protothecoides CS-41.

Li M, Cui Y, Gan Z, Shi C, Shi X - Mar Drugs (2015)

(A) Alignment of the selective PSY-deduced amino acid sequences from different algae produced with the GeneDoc program using Clustal W. The alignment indicates aspartate-rich regions/substrate-Mg2+ binding sites (DXXXD). The three DXXXD motifs are shown by the red boxes. Cppsy, Cvpsy, Mspsy, Olpsy, Dbpsy, Dspsy, Hppsy, Cspsy, Czpsy, Vcpsy, Ntpsy, Atpsy, and Zmpsy are the PSY of Chlorella protothecoides CS-41, Chlorella variabilis, Micromonas sp. RCC299, Ostreococcus lucimarinus, Duanliella bardawil, Duanliella salina, Haematococcus pluvialis, Coccomyxa subellipsodiea C-169, Chromochloris zofingiensis, Volvox carteri f. nagariensis, Nicotiana tabacum, Arabidopsis thaliana, and Zea mays, respectively; (B) Three-dimensional model structure of CpPSY. Comparative modeling was performed using homology-based three-dimensional structural modeling. The three aspartate-rich motifs (DXXXD) are colored in orange (DELVD), yellow (DELYD), and magenta (DEGED); others are shown in green. The N-terminus and C-terminus are also shown; (C) High-performance liquid chromatography trace and UV spectrum of carotenoid pigments in the E. coli heterologous complementation system. Pigments extracted from E. coli cells transformed with pACCRT-E and pUC-psy together (1), pUC-psy only (2), and pACCRT-EB only (3). Absorbance was recorded at 285 nm. The peak indicated by the arrow is phytoene.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4663545&req=5

marinedrugs-13-06620-f003: (A) Alignment of the selective PSY-deduced amino acid sequences from different algae produced with the GeneDoc program using Clustal W. The alignment indicates aspartate-rich regions/substrate-Mg2+ binding sites (DXXXD). The three DXXXD motifs are shown by the red boxes. Cppsy, Cvpsy, Mspsy, Olpsy, Dbpsy, Dspsy, Hppsy, Cspsy, Czpsy, Vcpsy, Ntpsy, Atpsy, and Zmpsy are the PSY of Chlorella protothecoides CS-41, Chlorella variabilis, Micromonas sp. RCC299, Ostreococcus lucimarinus, Duanliella bardawil, Duanliella salina, Haematococcus pluvialis, Coccomyxa subellipsodiea C-169, Chromochloris zofingiensis, Volvox carteri f. nagariensis, Nicotiana tabacum, Arabidopsis thaliana, and Zea mays, respectively; (B) Three-dimensional model structure of CpPSY. Comparative modeling was performed using homology-based three-dimensional structural modeling. The three aspartate-rich motifs (DXXXD) are colored in orange (DELVD), yellow (DELYD), and magenta (DEGED); others are shown in green. The N-terminus and C-terminus are also shown; (C) High-performance liquid chromatography trace and UV spectrum of carotenoid pigments in the E. coli heterologous complementation system. Pigments extracted from E. coli cells transformed with pACCRT-E and pUC-psy together (1), pUC-psy only (2), and pACCRT-EB only (3). Absorbance was recorded at 285 nm. The peak indicated by the arrow is phytoene.
Mentions: The deduced amino acid sequence of Cppsy was submitted to NCBI for PSI-BLAST searches and the results showed that Cppsy has high homology with psy genes from other algal species, with 83% identity and 88% positives with psy from Chlorella NC_64A. Cppsy was also highly similar to psy from C. reinhardtii (67% identities, 79% positives), H. pluvialis (63% identities, 77% positives), D. bardawil (68% identities, 80% positives), and D. salina (68% identities, 79% positives), suggesting that Cppsy belongs to the algae psy family. In the algae family, CpPSY belongs to class I of PSY according to Tran’s data [29]. BlastP analysis suggested that this protein has the essential characteristics of PSY. It belongs to the Isoprenoid_Biosyn_C1 superfamily, and contains the consensus sequence, including three predicted substrate-Mg2+ binding sites (aspartate-rich regions) (DXXXD), 130-DELVD-134, 203-DELYD-207, and 256-DEGED-260 (Figure 3A). In other algae and higher plants, there are two (DELVD and DVGED) (Figure 3A); hence, CpPSY has one more DXXXD motif than other PSYs. The abundant 203-DELYD-207 site possibly plays an important role in the function of CpPSY, which should be studied further.

Bottom Line: Analysis revealed several candidate motifs for the promoter, which exhibited light- and methyl jasmonate (MeJA)-responsive characteristics, as well as some typical domains universally discovered in promoter sequences, such as the TATA-box and CAAT-box.Light- and MeJA treatment showed that the Cppsy expression level was significantly enhanced by light and MeJA.These results provide a basis for genetically modifying the carotenoid biosynthesis pathway in C. protothecoides.

View Article: PubMed Central - PubMed

Affiliation: MOST-USDA Joint Research Center for Food Safety, School of Agriculture and Biology, and State Key Lab of Microbial Metabolism, Shanghai Jiao Tong University, Shanghai 200240, China. lmeiya@126.com.

ABSTRACT
Phytoene synthase (PSY) catalyzes the condensation of two molecules of geranylgeranyl pyrophosphate to form phytoene, the first colorless carotene in the carotenoid biosynthesis pathway. So it is regarded as the crucial enzyme for carotenoid production, and has unsurprisingly been involved in genetic engineering studies of carotenoid production. In this study, the psy gene from Chlorella protothecoides CS-41, designated Cppsy, was cloned using rapid amplification of cDNA ends. The full-length DNA was 2488 bp, and the corresponding cDNA was 1143 bp, which encoded 380 amino acids. Computational analysis suggested that this protein belongs to the Isoprenoid_Biosyn_C1 superfamily. It contained the consensus sequence, including three predicted substrate-Mg(2+) binding sites. The Cppsy gene promoter was also cloned and characterized. Analysis revealed several candidate motifs for the promoter, which exhibited light- and methyl jasmonate (MeJA)-responsive characteristics, as well as some typical domains universally discovered in promoter sequences, such as the TATA-box and CAAT-box. Light- and MeJA treatment showed that the Cppsy expression level was significantly enhanced by light and MeJA. These results provide a basis for genetically modifying the carotenoid biosynthesis pathway in C. protothecoides.

Show MeSH
Related in: MedlinePlus