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 fi-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 fi-ring-4- dehydrogenase (CBFD) and 4-hydroxy-fi-ring-4-dehydrogenase (HBFD) genes from Adonis aestivalis outperformed fi-carotene hydroxylase (BCH) and fi-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 mu g/g of dry cell weight (DCW), significantly higher than BKT-D. salina (83.58 +/- 2.40 mu g/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.