5-Aminolevulinic Acid Drives Coordinated Astaxanthin and Lipid Accumulation in Green Alga Chromochloris zofingiensis

Chromochloris zofingiensis, a photosynthetic microalga, has attracted considerable attention due to its ability to simultaneously accumulate lipids and astaxanthin. However, the induction of lipid and secondary metabolite biosynthesis by abiotic stress is typically accompanied by growth inhibition, resulting in a trade-off between metabolite accumulation and biomass production. In recent years, phytohormones have emerged as an effective strategy for regulating microalgal metabolism, owing to their high specificity and low effective dosage. In this study, 5-aminolevulinic acid (5-ALA) was applied under nitrogen-deficient conditions, and its effects on growth, photosynthesis, lipid metabolism, and carotenoid biosynthesis were systematically evaluated through integrated physiological, biochemical, and transcriptomic analyses. The results showed that 5-ALA had no significant effect on biomass accumulation or photosynthetic performance. However, at 2 μM, 5-ALA exhibited the strongest promotive effect on lipid and astaxanthin accumulation, with total fatty acids (TFA) and triacylglycerol (TAG) contents increasing by 13.3% and 25.7%, respectively, and total carotenoids and astaxanthin contents increasing by 15.6% and 17.2%, respectively. Under semi-continuous cultivation, TAG and astaxanthin productivities were enhanced by 13.9% and 22.9%, reaching 164 mg L−1 d−1 and 2.15 mg L−1 d−1, respectively. Transcriptomic analysis revealed that 5-ALA induced only limited transcriptional changes but enhanced glycolysis, central carbon metabolism, and nitrogen recycling, thereby increasing the supply of carbon precursors and energy. Notably, no significant transcriptional changes were observed in the carotenoid biosynthesis pathway, indicating that the enhanced accumulation of total carotenoids and astaxanthin was likely driven by increased metabolic flux. In terms of lipid metabolism, the upregulation of pathways involved in the conversion of membrane lipids into TAG, together with the downregulation of TAG degradation pathways and enhanced carbon flux, collectively promoted TAG accumulation. Overall, this study demonstrates that supplementation with 2 μM 5-ALA provides a practical and cost-effective strategy for the efficient co-production of lipids and astaxanthin in C. zofingiensis.