Production of 13C-labeled docosahexaenoic acid from heterotrophic marine microorganisms Aurantiochytrium mangrovei and Crypthecodinium cohnii enabling fluxomic applications

Docosahexaenoic acid (DHA, 22:6n-3) is the predominant polyunsaturated fatty acid in the human brain and eyes, playing a crucial role in vision and cognitive development. DHA deficiency has been associated with ocular diseases, such as macular degeneration and glaucoma, as well as neurodegenerative disorders. Since the human body has a limited ability to synthesize DHA from its precursor, alpha-linolenic acid (ALA, 18:3n-3), targeted DHA supplementation is essential for these patients. To investigate DHA metabolism and integration, researchers commonly use stable (²H,¹³C) or radioactive (³H,¹⁴C) isotopes, which are expensive and not widely accessible, restricting the scope and duration of studies. This study aimed to develop a sustainable method for biosynthesizing uniformly labeled ¹³C-DHA by culturing the heterotrophic protists <i>Aurantiochytrium mangrovei</i> and <i>Crypthecodinium cohnii</i> with ¹³C-glucose. The major fatty acids (FA) of <i>A. mangrovei</i> included 16:0, 22:5n-6 (DPA), and DHA, with DHA accounting for 50.5% ± 4.9% of the total FA. Gas Chromatography-Mass Spectrometry (GC-MS) analysis revealed a¹³C-enrichment of DHA at 96.7% ± 0.4% after the effective High Performance Liquid Chromatography (HPLC) purification. The predominant FA of <i>C. cohnii</i> were 12:0, 14:0, 16:0, and DHA, with DHA representing around 27% of the total FA and exhibiting a¹³C-enrichment of 86.3% ± 1.6%. Based on FA content, <i>A. mangrovei</i> showed a balanced distribution of neutral and polar lipids, with DHA predominantly in the polar fraction (57.8% ± 3.1%), whereas <i>C. cohnii</i> exhibited a predominance of neutral lipids (82.4% ± 0.3%), which contained the majority of its DHA (57.5% ± 1.0%).