- The Δ5 desaturase mutant (P127) of the microalga Lobosphaera incisa is a promising organism for large-scale production of the valuable LC-PUFA dihomo-γ-linolenic acid (DGLA, 20:3 n-6). We examined the potential of P127 for DGLA production under nitrogen (N) starvation conditions, triggering the deposition of DGLA in triacylglycerols, and developed a strategy for optimization of the DGLA productivity in high-density cultures. Towards this end, the effects of initial biomass concentration (1, 2, and 4 g L−1) and PAR irradiance (170 and 400 µmol m−2 s−1) on DGLA and total fatty acid (TFA) production were studied. The highest DGLA and TFA percentages (10 and 38% of dry weight, respectively) were displayed by the cultures initiated at 1 g L−1 and grown under a moderate irradiance. Higher irradiances and lower starting biomass content facilitated oleic acid accumulation at the expense of DGLA. Maximum volumetric productivities of TFA and DGLA were recorded in the cultures started at 2 g L−1 biomass and grown under 400 μmol PAR m−2 s−1. We show that a sufficiently high starting culture density should be combined with a mild light stress to facilitate the production of biomass enriched in DGLA-containing triacylglycerols. 1 Practical applications The ∆5 desaturase mutant of L. incisa, P127, is a rare green source of DGLA, a LC-PUFA with valuable pharmaceutical and neutraceutical properties. We report on the optimization of DGLA production by the nitrogen-starving indoor cultures of P127. Stresses, such as N starvation and/or high irradiances, promote accumulation of lipids by microalgal cells. On the other hand, excessively severe stress is deteriorative for the target LC-PUFA accumulation. An important outcome of the present work is establishing physiological constrains of DGLA productivity and finding an approach to balance starting cell density vs. irradiance in order to maximize DGLA yields. Findings of the present work lay a foundation of the efficient production of DGLA using upscaled cultures of P127. High productivity of DGLA-enriched biomass by P127 under nitrogen starvation is attainable by combining relatively high starting culture density with a mild light stress. By contrast, high irradiances impair DGLA accumulation in the cultures initiated at the biomass content of ≤ 1 gL−1.