Isolation and characterization of phytoene desaturase cDNA involved in the beta-carotene biosynthetic pathway in Dunaliella salina.

The green alga Dunaliella salina is one of the best and most important biological sources of beta-carotene; however, to date the molecular basis of the beta-carotene biosynthesis process in D. salina is still unresolved. The dehydrogenation of phytoene is the second step in the carotenoids biosynthetic pathway, and the phytoene-related desaturases are the key enzymes in the beta-carotene biosynthetic pathway. A phytoene desaturase (Pds) cDNA with a 1752 bp open reading frame was cloned by RT-PCR and RACE-PCR methods on the basis of a modified switching mechanism at 5' end of the RNA transcript (SMART) technology from D. salina. The predicted protein sequence displays a high identity (up to 65%) with phytoene desaturases of higher plants and cyanobacteria. The highest amino acid sequence identity (91%) is shared with the phytoene desaturase sequence of Dunaliella bardawil, and a dinucleotide-binding motif lies in the N-terminal. The phylogenetic analysis shows that D. salina Pds is closer to higher plants and cyanobacteria than bacterial and fungi. These results together demonstrated the cloned Pds cDNA of D. salina is a Pds-type gene, and it is postulated that in D. salina the first two dehydrogenations, by which phytoene is converted into zeta-carotene, are carried out by this putative phytoene desaturase.

Zhu YH ，Jiang JG ，Yan Y ，Chen XW ... -  《JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY》

Characterization of cDNA of lycopene beta-cyclase responsible for a high level of beta-carotene accumulation in Dunaliella salina.

Lycopene beta-cyclase (Lyc-B) is the key enzyme in the catalysis of linear lycopene to form cyclic beta-carotene, an indispensable part of the photosynthetic apparatus and an important source of vitamin A in human and animal nutrition. Studies showing that the microalga Dunaliella salina can accumulate a high level of beta-carotene are lacking. We hypothesize that D. salina is closely involved with the catalytic mechanism of Lyc-B and the molecular regulation of its gene. In this study, we used RT-PCR and RACE-PCR to isolate a 2475 bp cDNA with a 1824 bp open reading frame, encoding a putative Lyc-B, from D. salina. Homology studies showed that the deduced amino acid sequence had a significant overall similarity with sequences of other green algae and higher plants, and that it shared the highest sequence identity, up to 64%, with Lyc-B of Chlamydomonas reinhardtii. Codon analysis showed that synonymous codon usage in the enzyme has a strong bias towards codons ending with adenosine. Two motifs were found in the Lyc-B sequence, one at the N terminus, for binding the hypothetical cofactor FAD, and the other was a substrate carrier motif in oxygenic organisms shared by an earlier carotenogenesis enzyme, phytoene desaturase, and Lyc-B. A tertiary structure prediction suggested that the catalytic or binding site structure within LycB from D. salina is superior to that of both H. pluvialis and C. reinhardtii. The LycB protein from D. salina was quite removed from that of H. pluvialis and C. reinhardtii in the phylogenetic tree. Taken as a whole, this information provides insight into the regulatatory mechanism of Lyc-B at the molecular level and the high level of beta-carotene accumulation in the microalga D. salina.

Zhu YH ，Jiang JG ，Chen Q 《Biochemistry and Cell Biology》

Cloning and sequence analysis of the phytoene synthase gene from a unicellular chlorophyte, Dunaliella salina.

Yan Y ，Zhu YH ，Jiang JG ，Song DL ... -  《JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY》

Analysis of an essential carotenogenic enzyme: ζ-carotene desaturase from unicellular Alga Dunaliella salina.

Ye ZW ，Jiang JG 《-》

Isolation and characterization of a stress-inducible Dunaliella salina Lcy-beta gene encoding a functional lycopene beta-cyclase.

The halotolerant green alga Dunaliella salina accumulates large amounts of beta-carotene when exposed to various stress conditions. Although several studies concerning accumulation and biotechnological production of beta-carotene have been published, the molecular basis and regulation of the genes involved in carotenoid biosynthesis in D. salina are still poorly known. In this paper, we report the isolation and regulation of the lycopene beta-cyclase (Lcy-beta) gene by abiotic stress. The function of this gene was determined by heterologous genetic complementation in E. coli. Gene expression and physiological analyses revealed that D. salina Lcy-beta steady-state transcript and carotenoid levels were up-regulated in response to all stress conditions tested (salt, light and nutrient depletion). The results presented here suggest that nutrient availability is a key factor influencing carotenogenesis as well as carotenoid biosynthesis-related gene expression in D. salina.

Ramos A ，Coesel S ，Marques A ，Rodrigues M ，Baumgartner A ，Noronha J ，Rauter A ，Brenig B ，Varela J ... -  《APPLIED MICROBIOLOGY AND BIOTECHNOLOGY》