Molecular Cloning Of Carotenoid Isomerase Gene From Dunaliella Bardawil And Construction Of The Heterologous Expression System

Carotenoids(lycopene,?-carotene,zeaxanthin et.al)are pigments of important practical significance which are commercially used in food colorants,nutrition health products,cosmetics and medicines.Dunaliella bardawil as a member of the Dunaliella can produce large amounts of ?-carotene under high irradiance,nutrient depletion and high salt concentration conditions.The ?-carotene metabolic pathway of D.bardawil is catalyzed and regulated by various enzymes.Carotenoid isomerase(CRTISO)is one of the key enzymes in the carotenoid biosynthetic pathway.Cis-lycopene can isomerize to all-trans lycopene under the catalysis of CRTISO and the product changes to ?-or ?-carotene by cyclase.In this paper,a full-length cDNA encoding CRTISO from D.bardawil(DbCRTISO)was isolated by reverse transcription PCR,degenerate PCR and rapid amplification of cDNA ends(RACE)for the first time.The full-length cDNA was 2226 bp,including a 1488 bp ORF encoding 495 amino acids with a calculated mass of 54.35 kDa.The genomic DNA and promoter of DbCRTISO were cloned by genome walking.The length of genomic DNA sequence of DbCRTISO was 7068 bp,containing 16 exons and 15 introns,and the promoter was 2126 bp.A typical CRTISO conserved domain called carot_isom was found in the DbCRTISO.The DbCRTISO was found the closest relatives to the Chlamydomonas reinhardtii by analysis of homologous alignment and phylogenetic tree.Various regulatory elements such as GATA box,CCAAT box and GT1GMSCAM4 were forecasted in the promoter sequence by using the bioinformatics tools,suggesting that the expression of DbCRTISO was regulated by light,temperature and salt.The discovery of salt regulation element helped study the D.bardawil carotenoids biosynthesis mechanism in the high salinity environment at transcriptional level.To verify the genes function of the carotenogenesis enzymes of D.bardawil,the plasmids pACCRT-GGPS-IB,pACCRT-GGPS-I-PSY,pACCRT-E-DbPDZ and pACCRT-E-DbPDZC were constructed with different combinations and replacement of carotenogenic genes from D.bardawil and Pantoea ananatis to expressed in Escherichia coli DH5?.The DbGGPS and DbPSY of D.bardawil can replace the crt E and crt B of P.ananatis to make the transformants accumulate lycopene.DbPDS,DbZDS and DbCRTISO,however,cannot replace the crt ? of P.ananatis to transform phytoene to lycopene.It is possible that ?-carotene isomerase(Z-ISO)is involved in this process.