Enhancement of growth and zeaxanthin accumulation via gene expression and metabolic pathways modulation in Chromochloris zofingiensis mutant CZ-Z12

BACKGROUND: Chromochloris zofingiensis is recognized as a promising resource for the production of carotenoids. However, current knowledge regarding its capacity for zeaxanthin accumulation remains limited, which restricts its potential for commercial application. The development of high-yielding strains and an understanding of the underlying metabolic and regulatory mechanisms are critical to advancing large-scale zeaxanthin production. RESULTS: In this study, a novel zeaxanthin-enriched mutant CZ-Z12 was obtained using ethyl methyl sulfonate (EMS). CZ-Z12 displayed a rapid growth rate when cultivated heterotrophically. On Day 6, zeaxanthin content and total fatty acid (TFA) content reached 0.229% and 28.6% of dry weight (DW), representing a 4.1 and 1.9-fold increase compared to wild-type (WT), respectively. Differentially expressed genes (DEGs) were significantly enriched in pathways of ABC transporters, endogenous hormones signal transduction, nucleotide excision repair, glycolysis, purine and pyrimidine metabolism, glutathione metabolism, fatty acid and carotenoid biosynthesis. Genes encoding growth-related proteins, such as vegetative storage protein 2 (VSP2), deoxycytidine triphosphate pyrophosphatase 1 (DCTPP1), and fructose-bisphosphate aldolase (FBA), were up-regulated by 1.4, 1.7, and 2.5-fold, respectively. The key genes encoding enzymes in zeaxanthin biosynthesis, including glutathione reductase (GR), phytoene desaturase (PDS), and β-carotene hydroxylase (CHYb), were up-regulated by 3.1, 2.4, and 2.6-fold, respectively. CONCLUSION: These findings indicate that CZ-Z12 is an efficient strain for zeaxanthin production and highlight the potential for EMS mutagenesis for metabolic enhancement in heterotrophic C. zofingiensis. By modulating gene expression and key metabolic pathways, this study provides new insights and a practical strategy for improving carotenoid biosynthesis in microalgae, thereby supporting the feasible of large-scale, sustainable zeaxanthin production.