Characterization of lycopene ß-cyclase from Dunaliella bardawil for enhanced ß-carotene production and salt tolerance.

Dunaliella can accumulate more ß-carotene (10 % or even more of the dry weight of cells) than any other species. Lycopene ß-cyclase (LcyB) is the key enzyme in the catalysis of lycopene to ß-carotene. In the present research, we used Escherichia coli BL21 (DE3) as host to construct two different types of engineering bacteria, one expressing the D. bardawil LcyB and the other expressing the orthologue Erwinia uredovora crtY. The catalytic ability of LcyB and CrtY were evaluated by comparing the ß-carotene yields of the two E. coli BL21(DE3) strains, whose salt tolerance was simultaneously compared by cultivated them under different NaCl concentrations (1 %, 2 %, and 4 %). We also interfered with the LcyB gene to investigate the effect of LcyB in D. bardawil. Results displayed that the ß-carotene yield of the LcyB-transformant significantly increased by about 48 % compared with the crtY-transformant. Additionally, LcyB was verified to be able to enhance the salt tolerance of E. coli BL21 (DE3). It is concluded that D. bardawil LcyB not only has better catalytic ability but also is able to confer salt tolerance to cells. Interfering D. bardawil LcyB induced the low expression of LcyB and the changes of growth and carotenoids metabolism in D. bardawil.

Enhancing carotenoid accumulation in Dunaliella bardawil by combined treatments with fulvic acid and optimized culture conditions.

Natural carotenoids from microalgae have received more attention as an alternative source. In this study, fulvic acid (FA), a plant growth regulator, was used to enhance carotenoid accumulation in microalgae Dunaliella bardawil rich in lutein. However, the addition of FA promoted pigment synthesis but also exhibited an inhibitory effect on biomass. Therefore, the optimization of culture conditions was performed to further enhance carotenoid accumulation, including high light stress (10,000 lx) and the two-stage cultivation comprising 1-aminocyclopropane-1-carboxylic acid (ACC) and FA. Under both culture conditions, the growth inhibition caused by FA was alleviated, leading to a further increase in the contents of chlorophylls and carotenoids. HPLC analysis revealed that the production of lutein, α-carotene and ß-carotene increased by 0.44-, 0.37- and 0.54-fold under the treatment of 400 mg/L FA with high light intensity and 0.91-, 1.15-0.29-fold under the two-stage cultivation comprising 11 mM ACC and 500 mg/L FA. Furthermore, algal cells under FA treatment and the two-stage cultivation stained with Bodipy505/515 emitted stronger fluorescence under a laser confocal microscope, suggesting that lipid accumulation was increased. Additionally, the transcription levels of carotenogenic genes were also found to be up-regulated by qRT-PCR. These results indicated an enhancement in both the storage capacity and synthesis of carotenoids in D. bardawil. This study revealed the potential application of plant growth regulators in promoting carotenoid accumulation in D. bardawil which could be further improved by optimizing the culture conditions, providing a reference for efficient carotenoid production in microalgae.

Enhancing astaxanthin accumulation through the expression of the plant-derived astaxanthin biosynthetic pathway in Dunaliella salina.

Dunaliella salina, a microalga that thrives under high-saline conditions, is notable for its high ß-carotene content and the absence of a polysaccharide cell wall. These unique characteristics render it a prime candidate as a cellular platform for astaxanthin production. In this study, our initial tests in an E. coli revealed that ß-ring-4-dehydrogenase (CBFD) and 4-hydroxy-ß-ring-4-dehydrogenase (HBFD) genes from Adonis aestivalis outperformed ß-carotene hydroxylase (BCH) and ß-carotene ketolase (BKT) from Haematococcus pluvialis counterparts by two-fold in terms of astaxanthin biosynthesis efficiency. Subsequently, we utilized electroporation to integrate either the BKT gene or the CBFD and HBFD genes into the genome of D. salina. In comparison to wild-type D. salina, strains transformed with BKT or CBFD and HBFD exhibited inhibited growth, underwent color changes to shades of red and yellow, and saw a nearly 50% decline in cell density. HPLC analysis confirmed astaxanthin synthesis in engineered D. salina strains, with CBFD + HBFD-D. salina yielding 134.88 ± 9.12 µg/g of dry cell weight (DCW), significantly higher than BKT-D. salina (83.58 ± 2.40 µg/g). This represents the largest amount of astaxanthin extracted from transgenic D. salina, as reported to date. These findings have significant implications, opening up new avenues for the development of specialized D. salina-based microcell factories for efficient astaxanthin production.

Dual Roles of Two Malic Enzymes in Lipid Biosynthesis and Salt Stress Response in Dunaliella salina.

Triacylglycerols (TAG) from microalgae can be used as feedstocks for biofuel production to address fuel shortages. Most of the current research has focused on the enzymes involved in TAG biosynthesis. In this study, the effects of malic enzyme (ME), which provides precursor and reducing power for TAG biosynthesis, on biomass and lipid accumulation and its response to salt stress in Dunaliella salina were investigated. The overexpression of DsME1 and DsME2 improved the lipid production, which reached 0.243 and 0.253 g/L and were 30.5 and 36.3% higher than wild type, respectively. The transcript levels of DsME1 and DsME2 increased with increasing salt concentration (0, 1, 2, 3, and 4.5 mol/L NaCl), indicating that DsMEs participated in the salt stress response in D. salina. It was found that cis-acting elements associated with the salt stress response were present on the promoters of two DsMEs. The deletion of the MYB binding site (MBS) on the DsME2 promoter confirmed that MBS drives the expression of DsME2 to participate in osmotic regulation in D. salina. In conclusion, MEs are the critical enzymes that play pivotal roles in lipid accumulation and osmotic regulation.

Roles of Two Phytoene Synthases and Orange Protein in Carotenoid Metabolism of the ß-Carotene-Accumulating Dunaliella salina.

Phytoene synthase (PSY) is a key enzyme in carotenoid metabolism and often regulated by orange protein. However, few studies have focused on the functional differentiation of the two PSYs and their regulation by protein interaction in the ß-carotene-accumulating Dunaliella salina CCAP 19/18. In this study, we confirmed that DsPSY1 from D. salina possessed high PSY catalytic activity, whereas DsPSY2 almost had no activity. Two amino acid residues at positions 144 and 285 responsible for substrate binding were associated with the functional variance between DsPSY1 and DsPSY2. Moreover, orange protein from D. salina (DsOR) could interact with DsPSY1/2. DbPSY from Dunaliella sp. FACHB-847 also had high PSY activity, but DbOR could not interact with DbPSY, which might be one reason why it could not highly accumulate ß-carotene. Overexpression of DsOR, especially the mutant DsORHis, could significantly improve the single-cell carotenoid content and change cell morphology (with larger cell size, bigger plastoglobuli, and fragmented starch granules) of D. salina. Overall, DsPSY1 played a dominant role in carotenoid biosynthesis in D. salina, and DsOR promoted carotenoid accumulation, especially ß-carotene via interacting with DsPSY1/2 and regulating the plastid development. Our study provides a new clue for the regulatory mechanism of carotenoid metabolism in Dunaliella. IMPORTANCE Phytoene synthase (PSY) as the key rate-limiting enzyme in carotenoid metabolism can be regulated by various regulators and factors. We found that DsPSY1 played a dominant role in carotenogenesis in the ß-carotene-accumulating Dunaliella salina, and two amino acid residues critical in the substrate binding were associated with the functional variance between DsPSY1 and DsPSY2. Orange protein from D. salina (DsOR) can promote carotenoid accumulation via interacting with DsPSY1/2 and regulating the plastid development, which provides new insights into the molecular mechanism of massive accumulation of ß-carotene in D. salina.

Effects of Piperonyl Butoxide on the Accumulation of Lipid and the Transcript Levels of DtMFPα in Dunaliella tertiolecta.

As one of the sources of biodiesel, microalgae are expected to solve petroleum shortage. In this study, different concentrations of piperonyl butoxide were added to the culture medium to investigate their effects on the growth, pigment content, lipid accumulation, and content of carotenoids in Dunaliella tertiolecta. The results showed that piperonyl butoxide addition significantly decreased the biomass, chlorophyll content, and total carotenoid content but hugely increased the lipid accumulation. With the treatment of 150 ppm piperonyl butoxide combined with 8000 Lux light intensity, the final lipid accumulation and single-cell lipid content were further increased by 21.79 and 76.42% compared to those of the control, respectively. The lipid accumulation in D. tertiolecta is probably related to the increased expression of DtMFPα in D. tertiolecta under the action of piperonyl butoxide. The phylogenetic trees of D. tertiolecta and other oil-rich plants were constructed by multiple sequence alignment of DtMFPα, demonstrating their evolutionary relationship, and the tertiary structure of DtMFPα was predicted. In conclusion, piperonyl butoxide has a significant effect on lipid accumulation in D. tertiolecta, which provides valuable insights into chemical inducers to enhance biodiesel production in microalgae to solve the problem of diesel shortage.