Studies On Preliminary Optimization And Mutagenesis Of Pythium Irregulare To Produce Eicosapentaenoic Acid By Fermentation

Eicosapentaenoic acid(EPA) is one of the important omega-3 polyunsaturated fatty acid(PUFA), possessing biological activities in the prevention and treatment of inflammatory areas, cancers and the heart or circulatory system. Consequently, EPA has important application values in pharmaceutical, food, animal feed. However, the method of large-scale industrial production of EPA has still not established. Therefore, the fermentation production of EPA by Pythium irregulare can provide a foundation for future commercial production of EPA.Using single-factor experiment and response surface design, the medium components for EPA production by P. irregulare was optimized. The optimum fermentation medium consisted: glucose 40.2 g·L-1, cottonseed meal(100 mesh) 16.1 g·L-1, Mg SO4·7H2O 1.5 g·L-1, KH2PO4 2 g·L-1, soybean oil 1%(v·v-1). Then the EPA production reached 403.8 mg·L-1. The nitrogen source played a vital role in the content of total faty acid(TFA) and its composition. And the variation in the fatty acid content of TFA indicates that EPA was synthesized through the n-6 route in P. irregulaere and Δ12 desaturase was the key enzyme for EPA biosynthesis. In addition, the effect of free gossypol(FG) on P. irregulare growth was studied, the result showed that: when the concentration of cottonseed meal in medium didn’t exceed 43.5 g·L-1, the growth of P. irregulare would not be inhibited. After fungal biomass from each flask had been harvested from Na2CO3-supplemented medium, 1 %(w·v-1) Na2CO3 solution was used to wash the mycelia three times and then the FG was not detected(detection limit 0.0018 %).Secondly, the initial p H, inoculum and cultured temperature of EPA production by P. irregulare were optimized, and results were as follows: The optimum initial p H was 6; The best inoculation amount was about 8% to 10%(v·v-1); Temperature taken stepwise shift: 25 ℃ for the first 72 h, 20 ℃ from 72 h to 120 h, and 15 ℃ for the last 48 h. Besides, the temperature was the most important factor of fermentation conditions. When the temperature decreased, the ratio of ARA and EPA decreased, and the EPA content and yield increased, but the fermentation time was too long results in a low EPA productivity; Taking temperature shift strategy, both EPA yield and productivity was higher.Finally, using ARTP mutagenesis technique, a mutant strain JN10-3 was obtained successfully on the establishment iodoacetic acid and TTC plates. The EPA yield of the strain reached up to 905.3 mg·L-1, which increased by 67.6% compared with the original strain. Besides, after passage for 6 times, the EPA yield was still stable, which indicated that the mutant strain JN10-3 possessed good hereditary stability.