Acute Combination of Nitrogen Deprivation and High Irradiance Induces the Simultaneous Accumulation of Astaxanthin and Lutein in Continuous Cultures of the Microalga Chromochloris zofingiensis

Carotenoids play a central role in photosynthesis and cellular protection, and microalgae represent a sustainable platform for their commercial production. Here, we optimized the accumulation of the high-value carotenoids astaxanthin and lutein in continuous photoautotrophic cultures of <i>Chromochloris zofingiensis</i> by modulating nitrogen supply and light intensity. Reducing nitrate availability strongly promoted astaxanthin accumulation, whereas lutein levels remained largely unaffected. For 4% N in the dry biomass, accumulation of astaxanthin was highest and that of lutein lowest, while the opposite was recorded for 9% N. Average irradiance positively affected lutein accumulation independently of nitrate, whereas that of astaxanthin only increased under nitrogen-limiting conditions. Integrated transcriptomics and carotenoid profiling analysis revealed nitrogen availability as the dominant regulatory factor, with a synergistic interaction with light that enhances their individual effects. Nitrate limitation redirected metabolic flux from lycopene toward β-carotene and its subsequent conversion to astaxanthin via BKT1 overexpression, while high irradiance induced CYP97A1 and CYP97C expression, favoring lutein biosynthesis. Together, these findings demonstrate that targeted control of nitrogen and light enables the continuous and programmable production of <i>C. zofingiensis</i> biomass with a specific astaxanthin-to-lutein ratio, highlighting its potential for industrial carotenoid bioprocesses.