High-throughput Screening Of EPA-producing Soil Fungi And The Regulation Of Fermentation

EPA (5, 8, 11, 14, 17-Eicosapentaenoic acid), belongs to theω-3 polyunsaturated fatty acids. It plays an important role in preventing and treating cardiovascular disease, anti-cancer, anti-inflammation, and so on. Production of EPA by microbial fermentation is the hotspot in recent years because of its merits such as short cycle, free influence in raw material, climate, producing area and easy-to-scale production.In this study, a high-throughput screening method was established to screen EPA-producing fungi. The genes of key enzymes in EPA biosynthesis were used as molecular markers for targeted screening based on PCR amplification. And the lipid composition of the obtained strains was detected by GC-MS, then the EPA-producing strains were finally confirmed, in which the EPA contents were 6.95% and 9.90%, respectively, and EPA yield were 215.81 mg/L and 263.80 mg/L, respectively. Thus, the screening process was simplified, and the efficiency was greatly improved by the method.The strain RBB-5 of higher EPA yield were classified and identified from the morphological, physiological and biochemical characteristics and 18S rRNA sequence analysis and other aspects. The results showed that the strain RBB-5 has the same morphological and physiological characteristics of Pythium, and the 18S rRNA sequences of RBB-5 and Pythium splendens 117 have the gene homology of 99%. Therefore, RBB-5 was attributed to Pythium splendens and named as Pythium splendens RBB-5.The optimized regulation methods of the fermentation was established through studying the impact of EPA synthesis of the main factors, combining with the fermentation process of cell growth, lipid accumulation, EPA synthesis, and the variation of dissolved oxygen throughout fermentation. The optimized process were described as follows: the temperature was decreased to 15°C after 96 h fermentation, and the ventilation ratio was adjusted to 0.8 vvm at 0-72 h, then to 1.2 vvm at 72-168 h; and 60 g/L bean sprout juice was added into the medium at 72 h, then continue fermentation to 168 h. The biomass and EPA content were 26.20 g/L and 22.87%, respectively, the EPA yield was up to 963.50 mg/L. The genetic transformation system of the condinia of Pythium splendens RBB-5 by Agrobacetrium tumefaciens LBA4404-mediated transformation was established after optimizing some factors, such as the concentration of LBA4404, acetosyringone concentration of co-culture medium, temperature and time. It laid the foundation for construct trans-genetic strains of high EPA yield.