基于重组毕赤酵母的fusaruside生物合成

目的:构建产fusaruside的毕赤酵母菌株,解决天然小分子免疫抑制剂fusaruside的来源问题。方法:从禾谷镰刀菌Fusarium graminearum PH-1中扩增获得合成fusaruside的相关基因-3位去饱和酶Δ3(E)-SD]和10位去饱和酶Δ10(E)-SD]基因;并通过2A肽策略构建两种基因的共表达载体,转化到毕赤酵母GS115中进行双酶的诱导表达;对诱导后的毕赤酵母菌体进行甲醇和二氯甲烷的处理后,经高效液相色谱质谱联用仪(HPLC-MS)检测其中产物变化。结果:3位去饱和酶和10位去饱和酶在毕赤酵母中成功共表达,SDS-PAGE显示3位去饱和酶分子量约为48kDa,10位去饱和酶分子量约为65kDa; HPLC-MS显示重组酵母可以产生fusaruside。结论:与fusaruside原产菌株镰刀菌相比,该酵母菌的发酵时间更短、产量更高,为fusaruside的进一步开发与应用奠定基础。

Biosynthesis of Fusaruside Based on Recombinant Pichia pastoris

Objective: A strain of Pichia pastoris producing fusaruside was constructed to solve the source problem of fusaruside, a selective immunosuppressive molecule.Method: Two related biosynthetic genes coding delta 3(E)-sphingolipid desaturase Δ3(E)-SD] and delta 10(E)-sphingolipid desaturase Δ10(E)-SD] were amplified from fungus Fusarium graminearum PH-1. 2A peptide based strategy was used to construct the co-expression vector. Then the 2A polyprotein construct was transformed into Pichia pastoris GS115 for induction. Finally, P. pastoris cells were extracted with methanol and dichloromethane, and the extract was detected by high performance liquid chromatography mass spectrometer (HPLC-MS).Result: The two desaturases were successfully co-expressed in P. pastoris. SDS-PAGE showed that the molecular weight of Δ3(E)-SD and Δ10(E)-SD were about 48kDa and 65kDa, respectively. HPLC-MS indicated that fusaruside could be produced by the recombinant yeast.Conclusion: Compared with fusarium which producing fusaruside, the engineered yeast had shorter fermentation time and higher yield, laying a foundation for further development and application of fusaruside.