The impact of DNA methyltransferase inhibitors on carotenoid metabolism in Dunaliella salina.

Epigenetic regulation refers to heritable changes in gene expression that occur without alteration of the DNA sequence, primarily mediated by DNA methylation. In this study, we applied two DNA methyltransferase (DNMT) inhibitors, zebularine (Zeb) and 5-azacytidine (5-Azac), to alter the epigenetic landscape of Dunaliella salina and observe the resulting phenotypic changes. Long-term (40-day) treatment revealed that, on Day 40, Zeb dose-dependently increased the proportion of 9-cis β-carotene from 52.4% in the control to 82.0% at 50 μM, and further to 91.9% at 100 μM. In contrast, 5-Azac (200 μM) promoted the accumulation of α-carotenoids (α-carotene and lutein) while reducing β-carotene content. To explain these long-term metabolic shifts, we performed short-term (4-day) treatments to assess global DNA methylation and transcriptional responses of key carotenogenic genes. Unexpectedly, both DNMT inhibitors were associated with a modest increase in 5-methylcytosine levels in D. salina, contrary to the canonical expectation of demethylation. qRT-PCR revealed that 100 μM Zeb broadly upregulated carotenogenic genes, especially β-carotene isomerase (DsBCI), correlating with 9-cis enrichment. In contrast, 200 μM 5-Azac suppressed phytoene synthase (DsPSY) while upregulating lycopene ε-cyclase (DsLCYE) and β-carotene hydroxylase (DsBCH), redirecting flux toward α-carotenoids. Overall, chemical inhibition of DNA methyltransferases reprogrammed the carotenoid metabolism of D. salina. Zeb treatment markedly elevated 9-cis β-carotene levels, whereas 5-Azac promoted a relative enrichment of α-carotenoids. These findings implicate epigenetic regulation in carotenoid metabolism in D. salina and could provide a basis for future strategies aimed at the biotechnological production of high-value 9-cis β-carotene.