Engineering the ß-Carotene Metabolic Pathway of Microalgae Dunaliella To Confirm Its Carotenoid Synthesis Pattern in Comparison To Bacteria and Plants.

Dunaliella salina is the most salt-tolerant eukaryote and has the highest ß-carotene content, but its carotenoid synthesis pathway is still unclear, especially the synthesis of lycopene, the upstream product of ß-carotene. In this study, DsGGPS, DsPSY, DsPDS, DsZISO, DsZDS, DsCRTISO, and DsLYCB genes were cloned from D. salina and expressed in Escherichia coli. A series of carotenoid engineering E. coli strains from phytoene to ß-carotene were obtained. ZISO was first identified from Chlorophyta, while CRTISO was first isolated from algae. It was found that DsZISO and DsCRTISO were essential for isomerization of carotenoids in photosynthetic organisms and could not be replaced by photoisomerization, unlike some plants. DsZDS was found to have weak beta cyclization abilities, and DsLYCB was able to catalyze 7,7',9,9'-tetra-cis-lycopene to generate 7,7',9,9'-tetra-cis-ß-carotene, which had not been reported before. A new carotenoid 7,7',9,9'-tetra-cis-ß-carotene, the beta cyclization product of prolycopene, was discovered. Compared with the bacterial-derived carotenoid synthesis pathway, there is higher specificity and greater efficiency of the carotenoid synthesis pathway in algae. This research experimentally confirmed that the conversion of phytoene to lycopene in D. salina was similar to that of plants and different from bacteria and provided a new possibility for the metabolic engineering of ß-carotene. IMPORTANCE The synthesis mode of all trans-lycopene in bacteria and plants is clear, but there are still doubts in microalgae. Dunaliella is the organism with the highest ß-carotene content, and plant-type and bacterial-type enzyme genes have been found in its carotenoid metabolism pathway. In this study, the entire plant-type enzyme gene was completely cloned into Escherichia coli, and high-efficiency expression was obtained, which proved that carotenoid synthesis of algae is similar to that of plants. In bacteria, CRT can directly catalyze 4-step continuous dehydrogenation to produce all trans-lycopene. In plants, four enzymes (PDS, ZISO, ZDS, and CRTISO) are involved in this process. Although a carotenoid synthetase similar to that of bacteria has been found in algae, it does not play a major role. This research reveals the evolutionary relationship of carotenoid metabolism in bacteria, algae, and plants and is of methodologically innovative significance for molecular evolution research.

Identification of ractopamine glucuronides and determination of bioactive ractopamine residues and its metabolites in food animal urine by ELISA, LC-MS/MS and GC-MS.

Ractopamine glucuronides have been identified in cattle urine sampled by LC-MS/MS. An ELISA method, which was capable of specifically determining (1R, 3R)-ractopamine stereoisomer and its glucuronide metabolites, had more than 100% recovery with an acceptable coefficient of variation in the inter- and intra-assay variation tests for RR-ractopamine. The concentration levels of parent ractopamine and ractopamine glucuronide metabolites as the main components of total ractopamine in cattle and sheep urine showed similar depletion trends, in which the concentration curves increased and reached a climax during the feeding period, and then dropped quickly when entering the withdrawal period. Data from the three methods had very good pair-wise correlations. In the cattle urine samples, the correlation coefficient (R(2)) for parent ractopamine between the ELISA and the LC-MS/MS or GC-MS results were 0.93 or 0.92; R(2) values for parent ractopamine and total ractopamine data measured by LC-MS/MS and GC-MS were 0.9651 and 0.9677, respectively. All R(2) values for data gained from sheep urine samples were >0.95. The study indicated that the close levels of RR-ractopamine stereoisomer in cattle and sheep urine samples may imply the presence of a similar depletion pattern in other livestock, and thus would facilitate an accurate detection and management of ractopamine usage in food safety.

Two new lignans from the stems of Schisandra bicolor.

Two new lignans, named zuihonins E (1) and F (2), were isolated from the stems of Schisandra bicolor Cheng var. tuberculata Law. The structures of the new lignans were elucidated on the basis of extensive spectroscopic analysis, including 1D, 2D NMR, and MS experiments, and their absolute stereochemistry was determined by circular dichroism spectrum. Compounds 1 and 2 did not inhibit the growth of hepatoma carcinoma cell (HepG2), lung carcinoma cell (A549), and human breast carcinoma (MCF-7) cell lines.

The metabolomics of carotenoids in engineered cell factory.

Carotenoids such as beta-carotene, lycopene, and antheraxanthin have plenty of scientific and commercial value. The comprehensive investigation of carotenoids drives people to improve and develop all kinds of analytical techniques to approach or even achieve "versatile" analysis. The metabolic engineering efforts in plants and algae have progressed rapidly, aiming to enable the use of plants and algae as "cell factories" for producing specific or novel carotenoids, such as beta-carotene (provitamin A) in Gold rice, while the emerging technologies of metabolomics support it by providing comprehensive analysis of carotenoids biochemical characterizations. This review describes metabolomics as a high-throughput platform to study carotenoids, including the engineering methods in the plants or algae, the bioinformatics for metabolomics, and the metabolomics of carotenoids in engineered cell factory. Modern systems biology tools, together with the development of genomics and metabolomics databases, will dramatically facilitate the advancement of our knowledge in gene-to-metabolite networks in plants. Metabolomics accompanying genomics, transcriptomics, and proteomics as well as bioinformatics facilitate metabolic engineering efforts towards designing superior biocatalysts in cell factories. Ongoing advances in biological techniques coupled with crucial metabolic networks will further promote plants and algae as attractive platforms for the production of numerous high-value compounds such as carotenoids.

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.

Influence of daily collection and culture medium recycling on the growth and beta-carotene yield of Dunaliella salina.

The halophilic green alga Dunaliella salina has the potential to be cultivated for beta-carotene-rich biomass, however, open-air systems need to be further improved in order to become more competitive and more economical, rather than leave the major beta-carotene consuming market derived from artificially synthesis. A set of daily collection ratios was designed and scaled up with the aim to harvest cell biomass and beta-carotene from D. salina at logarithmic phase; the yields were comparable to the normal culture without daily removal of culture. Daily collection of 1/7.5 volume of algal culture was found to be appropriate to keep the balance between the cell biomass and beta-carotene accumulation. Light intensity as one of the important factors would affect both cell growth and beta-carotene content synchronously. Further, the method of recycling 1/7.5 volume of culture after removal of algae cells was developed in order to decrease input cost for the effective production of beta-carotene, and both the resulting yields of the cell biomass and beta-carotene gained an advantage over those from the normal D. salina culture.

Toxicity of carbon tetrachloride to Dunaliella salina, an environmentally tolerant alga.

Carbon tetrachloride (CCl(4)), a water disinfection by-product, at low environmentally relevant concentrations exerts adverse effects on mammals. The unicellular microalga Dunaliella salina possessing a remarkable degree of environmental adaptation was selected as test organism to investigate low-level exposure to CCl(4). With incubation with less than 0.13% CCl(4), algae responses were similar to control as evidenced by cell growth and levels of beta-carotene, a marker of adaptation. The maximal concentration of CCl(4) that D. salina could tolerate was 0.2%. Algae incubated with more than 0.32% CCl(4) showed decreased growth and reduced beta-carotene levels, which were nondetected after a few days. However, after 98 d, D. salina seemed to revive as evidenced by growth and returned to the biomass similar to control in another 25 d. Randomly amplified polymorphic DNA (RAPD) method was used to compare the genomic DNA difference between control and recovered cells. Polymorphic and repeatable RAPD bands indicated that chronic effects of CCl(4) to D. salina led generation of altered genomic DNA, which may enable the microalga to adapt to survival in an apparently toxic substance environment.

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.

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

Phytoene synthase (Psy) catalyzing the dimerization of two molecules of geranylgeranyl pyrophosphate (GGPP) to phytoene may be rate limiting for the synthesis of beta-carotene in Dunaliella salina. To elucidate the carotenogenic pathway in D. salina, the complete Psy gene was first isolated by genome walking technology and suppression PCR. Subsequently, RT-PCR was performed to obtain the Psy cDNA of 1260 bp, which codes 420 amino acids. The Psy gene of total length of 2982 bp was found to consist of five exons and four introns when compared with the cDNA sequence. The D. salina Psy amino acid has a 78-89% similarity with many other higher plants, but a phylogenic analysis shows a closer relationship between the microalgal and cyanobacterial Psy.