海洋真菌SD116在产物合成中的辅因子谱分析

The analysis of Cofactor changes in product synthesis of marine fungus Aurantiochytrium sp.SD116

目的通过对海洋真菌Aurantiochytriumsp.SD116发酵过程中9种辅因子的定性定量分析,阐述胞内辅因子平衡及能荷水平与其生长、油脂积累及二十二碳六烯酸(DHA)合成等表型的内在联系。方法对发酵过程进行间断点采样,淬灭,采用超高效液相色谱-串联质谱技术及同位素稀释质谱分析策略进行辅因子的定性定量分析。结果在细胞快速分裂生长时,细胞内的NADH/NAD+、NADPH/NADP+的比率及能荷水平较低,细胞停止分裂快速积累油脂及DHA时,还原力保持平衡但能荷水平处于高位。而细胞生长及油脂积累均停滞时,胞内各种辅因子出现剧烈的动荡,辅因子的总量呈先升后降直至衰亡趋势。结论菌体的辅因子浓度及能荷水平与菌体的生长、油脂及DHA的积累及衰亡密切相关,高能荷水平是胞内油脂积累的必要条件,而胞内辅因子循环和氧化-还原态势的失衡则与细胞产物合成停滞及衰亡呈正相关关系。

Objective To expound the relationship of cofactor content and energy charge with the cell growth, lipid synthesis and docosahexaenoic acid（DHA） accunmlation, the in vivo analysis of nine co-factors of a marine fungus Aurantioctlytrium sp. SD116 was qualitatively and quantitatively performed during fermentation. Methods Through the fermentation process, intermittent sampling and quenching were conducted, and the ultra performance liquid chromatography-isotope dilution tandem mass spec trometry was used for the qualitative and quantitative analysis. Results In the exponential phase, the cell division and growth showed an exuberant trend, and the intracellular ratios of NADH/NAD and NADPH/NADP and the energy charge remained at very low level. In the stationary phase, the cell growth was slow, the accumulation of lipid and DHA became intensive, the redox balance and the ener gy charge level remained high. However, when both cell growth and lipid accumulation kept at low lev el, the contem of many cofactors changed vigorously following the trend of first increasing and then de-creasing until the decline phase. Conclusion According to the analysis, the cofactor concentration and energy charge level were closely related to the cell growth, lipid and I）HA accumulation, and cell apop tosis. The high-energy charge level was a primary condition for intracellular lipid accumulation. Be sides, we observed that the cofactor recycling and the oxidation-reduction state showed a positive corre lation with the cell synthesis and apoptosis.