Strontium Malonate

To address the critical industrial bottlenecks in Aurantiochytrium sp. fermentation, specifically the poor synergy between cell growth and lipid synthesis, as well as the low conversion rate of docosahexaenoic acid (DHA), this study established a technical system combining "malonic acid stress screening" with "ARTP mutagenesis directed breeding." By integrating key metabolic enzyme analysis with dynamic metabolic modeling, we constructed a "mutagenesis - enzyme response - metabolic flux regulation" framework to systematically elucidate the metabolic regulation mechanisms of high-yield DHA mutants. Malonic acid was selected as the screening pressure due to its specific inhibition of the tricarboxylic acid (TCA) cycle, thereby redirecting carbon flux towards lipid synthesis. Combined with atmospheric and room temperature plasma (ARTP) mutagenesis (120 W, 80 s, 98.26% lethality) and using a relative fluorescence intensity increase of over 30% as the selection threshold, the mutant strain MA-4, characterized by a high growth rate and high lipid content, was successfully isolated. Compared to the wild-type strain, the MA-4 mutant exhibited a 20.89% increase in cellular DHA content, with DHA constituting 39.20% of total lipids. Enzyme activity analysis revealed temporal shifts in key TCA cycle enzymes, isocitrate dehydrogenase (IDH) and malate dehydrogenase (MDH). These enzymes provided sequential energy support during the logarithmic growth phase and lipid accumulation phase, respectively, coordinating the energetic demands of cell growth and lipid synthesis. Metabolic flux regulation analysis indicated a 45.45% reduction in fatty acid synthase (FAS) pathway activity and a 30.14% increase in polyketide synthase (PKS) pathway activity in the mutant. This shift directed the precursor Acetyl-CoA specifically towards unsaturated fatty acid accumulation. Furthermore, a temporal regulation mechanism for NADPH supply was identified, providing precise reducing power for saturated and unsaturated fatty acid synthesis, respectively. The dynamic metabolic model further verified a 20.21% increase in glycolysis (EMP) pathway activity. This accelerated glucose decomposition provided ample carbon skeletons for downstream metabolism, ultimately driving efficient carbon flux migration towards DHA and other polyunsaturated fatty acids. This study not only elucidates the core metabolic mechanisms of high-yield DHA production in Aurantiochytrium sp. under ARTP mutagenesis and malonic acid stress but also establishes a systematic "mutagenesis screening - mechanism analysis - flux optimization" research paradigm. These findings provide critical targets and technical support for the rational design of high-yield DHA engineering strains and for overcoming bottlenecks in industrial microalgae functional oil production.