Metabolic engineering of Dunaliella salina enhances astaxanthin production: combined effects of heterologous enzyme co-expression and dual-stress optimization.

Astaxanthin is primarily produced by chemical synthesis or extraction from Haematococcus pluvialis, but both approaches face limitations in cost, scalability, or product quality, necessitating alternative production platforms. To explore a potentially cost-effective alternative platform for astaxanthin production, we engineered Dunaliella salina by combinatorial expression of Haematococcus-derived β-carotene ketolase (BKT) and β-carotene hydroxylase (CRTR-B) isoenzymes. The optimal crtR-B1/bkt2 combination (P4 strain) yielded 44.3 µg/g astaxanthin under normal conditions, while also accumulating significant levels of other high-value carotenoids, including β-carotene (5.48 µg/mg) and lutein (1.54-fold higher than wild-type). Under complete nitrogen deficiency, the astaxanthin production of the P4 mutant strain reached 66.20 µg/g, while β-carotene, lutein, and canthaxanthin levels were 3.43-, 1.56-, and 1.75-fold higher than normal conditions, respectively. Dual-stress treatment, i.e., combination of nitrogen deprivation (0 mg/L NaNO₃) and high light intensity (15,000 lx), enhanced astaxanthin production to 89.56 µg/g in the P4 strain, with concurrent increases in canthaxanthin and β-carotene compared with normal culture conditions. Transcriptomic analysis revealed metabolic reprogramming with upregulation of carotenoid biosynthesis (BKT and LUT5), pyruvate metabolism, and photosynthesis genes, alongside downregulation of oxidative phosphorylation. This study demonstrates the combined optimization of isozyme combination and environmental stress to elevate the synthesis of astaxanthin and other carotenoids in D. salina, providing new research ideas and experimental evidence for the future construction of high-yield engineered algal strains.