Engineering the β-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 β-carotene content, but its carotenoid synthesis pathway is still unclear, especially the synthesis of lycopene, the upstream product of β-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 β-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-β-carotene, which had not been reported before. A new carotenoid 7,7′,9,9′-tetra-cis-β-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 β-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 β-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.