Homologous and heterologous expression of phytoene synthase gene in marine microalgae Dunaliella salina and its potential as aquaculture feed

Dunaliella salina, a unicellular marine microalga, has emerged as a promising host for genetic manipulation to enhance the production of commercially valuable products, including carotenoids, xanthophylls, vitamins, fatty acids, and enzymes. Carotenoids, in particular, are in high demand in the food, medical, and cosmetic industries. In this study, the focus was on manipulating the phytoene synthase (PSY) gene in Dunaliella salina, which is a key enzyme in the carotenoid biosynthesis pathway. Both homologous (PSY gene from Dunaliella salina) and heterologous (PSY gene from Haematococcus pluvialis) expression of PSY were performed. The PSY genes were cloned into the plant expression vector pMDC45 and then introduced into Dunaliella salina through nuclear transformation using electroporation. Positive clones resistant to hygromycin were identified, and transgenic lines carrying the transformed PSY genes were screened using PCR. Six transgenic lines of PSY-transformed Dunaliella salina were selected for further analysis. Real-time PCR was used to measure the expression levels of the PSY gene and other genes involved in the carotenoid pathway, such as phytoene desaturase (PDS), zeta-carotene desaturase (ZDS), lycopene beta-cyclase (LCYb), and beta-carotene hydroxylase (CHYb). Compared to the wild-type strain, the homologous expression line (DS-2) showed an up to 2.9-fold increase in PSY gene expression. Moreover, the expression of other carotenoid-related genes also increased in both the homologous and heterologous transgenic lines. The enhanced expression of the PSY gene resulted in a significant increase in carotenoid production. The DS-2 transgenic line exhibited a 5.4-fold increase in beta-carotene content, while the HP-4 transgenic line showed a 2.4-fold increase. Additionally, the DS-4 and HP-3 transgenic lines had a 7.6-fold increase in lutein content. Furthermore, the modified D. salina, with increased carotenoid production, was used as a feed for Artemia (brine shrimp). The results indicated that Artemia fed with genetically manipulated D. salina showed improved growth and survival rates. These Artemia also exhibited increased uptake of carotenoids, which are beneficial for their reproductive capacity. This study demonstrates the potential of manipulating the PSY gene in Dunaliella salina to enhance carotenoid production. It also highlights the application of genetically modified D. salina as a valuable feed source to improve the reproductive capacity and overall health of aquatic organisms, such as Artemia.