高山被孢霉发酵产鸟苷酸二磷酸岩藻糖的研究

Production of GDP-L-fucose by Mortierella alpina fermentation

鸟苷酸二磷酸岩藻糖(GDP-L-Fuc)是一种重要的核糖类化合物,其作为岩藻糖基的受体具有重要的生理功能。尝试利用高山被孢霉发酵产GDP-L-Fuc。高山被孢霉经液氮研磨破壁,甲酸(含10%丁醇)溶液萃取后,利用高效液相色谱进一步分离得到了GDP-L-Fuc,并采用四极杆-静电场轨道阱高分辨质谱对结果进行定性分析。然后对高山被孢霉不同发酵时期GDP-LFuc产量进行分析,发现GDP-L-Fuc在氮源消耗之后仍然持续积累。同时,GDP-L-Fuc合成途径关键基因的转录水平在氮源耗尽时发生了上调,这表明氮源很可能是GDP-L-Fuc积累的关键因素。最后,在氮源耗尽后添加外源岩藻糖显著提高了GDP-L-Fuc的产量(50%),可达170.51μg/g。这为利用微生物发酵产GDP-L-Fuc奠定了理论基础。

Fucosylation requires the activated nucleotide form of L-fucose, GDP-L-fucose, as the donor of fucose, which is involved in a variety of biological processes. We tried to use oleaginous fungus Mortierella alpina for the production of GDP-L-fucose. After triturated under liquid nitrogen, the GDP-Lfucose content in M. alpina was extracted by formic acid（10% butanol）. Then the sample was analyzed by HPLC-MS/MS. The GDP-L-fucose content during the fermentation of M. alpina was investigated, indicating that GDP-L-fucose was still accumulated after nitrogen exhaustion. Additionally, the transcript levels of important genes involved in biosynthesis of GDP-L-fucose were up regulated by nitrogen exhaustion, which highlights the functional significance of nitrogen in the GDP-L-fucose biosynthesis in M. alpina. The addition of exogenous fucose after nitrogen exhaustion led to an increase of GDP-L-fucose production by about 50%（170.51 μg/g）. This study laid the foundation of GDP-L-fucose production by microorganism fermentation.