Staged fermentation improves docosahexaenoic acid productivity by Aurantiochytrium sp. PKU#Mn16: From bench to pilot...

DHA (docosahexaenoic acid, C22:6) is a vital omega-3 fatty acid with profound roles in human health, yet sustainable production remains constrained by process inefficiencies. Thraustochytrids are recognized as prolific DHA producers; however, fermentation optimization often relies on empirical adjustments rather than mechanistic insights. Here, we systematically dissected the effects of temperature, dissolved oxygen (DO), and dynamic control strategies on DHA biosynthesis in Aurantiochytrium sp. PKU#Mn16, linking environmental cues to intracellular metabolic states. Biomass accumulation peaked at 65.7 g/L at 32 °C, but 28 °C offered the best balance, yielding 24.5 g/L total fatty acids (TFA) and 9.4 g/L DHA at 96 h. Low temperatures (16-20 °C) enriched DHA to > 50 % of TFA, while high DO (40-50 %) boosted early biomass (68.9 g/L) but caused > 40 % DHA loss post-96 h. Metabolite profiling revealed that low temperature and DO elevated acetyl-CoA, NADPH, and ATP, priming cells for PUFA synthesis. Guided by these insights, we designed a three-stage fed-batch strategy that raised TFA and DHA to 30.3 and 14.7 g/L (1.24- and 1.56-fold improvements), with DHA comprising 48.5 % of TFA. Pilot-scale (50 L) fermentation confirmed reproducibility and scalability, establishing metabolite-informed dynamic control as a robust and industrially relevant strategy for sustainable DHA production.