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-Mg2+ 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.
Highlights
Lutein is one of more than 750 known naturally occurring carotenoids, and is synthesized by higher plants, bacteria, fungi, and algae
methyl jasmonate (MeJA) treatment showed that the Cppsy expression level was significantly enhanced by light and MeJA
BLAST analysis showed that the nucleotide sequence of this fragment shared about 74% and 73% identities with that of C. reinhardtii and D. salina, respectively, demonstrating that this fragment sequence is derived from a putative phytoene synthase
Summary
Lutein is one of more than 750 known naturally occurring carotenoids, and is synthesized by higher plants, bacteria, fungi, and algae. Since PSY plays a key role in the first step of carotenogenesis, it has unsurprisingly been chosen for genetic engineering studies of carotenoid production. As an efficient lutein-production alga, C. protothecoides CS-41 has high potential for application in the commercial production of lutein; its lutein biosynthesis pathway has not been well studied. Our research group previously cloned other key enzyme genes in the lutein biosynthesis pathway of this alga, such as the phytoene desaturase (pds) (GenBank accession No FJ968162) [24], zeta-carotene desaturase (zds) (GenBank accession No GU269622) [25], and lycopene-ε-cyclase (lyce) (GenBank accession No FJ752528) genes. The psy gene is essential for determining the complete lutein biosynthesis pathway in this alga. This study provides an important theoretical basis for the genetic modification of lutein biosynthesis in C. protothecoides CS-41, including gene sequences, expression promoter candidates, and possible regulatory environmental factors for gene expression
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