基于改造P450和CPR生物合成皂皮酸及理解其底物选择机制的研究

QS-21是一种苷元为皂皮酸的天然三萜皂苷，可作为佐剂用于多种疫苗。基于皂皮酸为底物开发的非天然佐剂，具有与QS-21相似的活性，应用潜力巨大。但是，QS-21和皂皮酸均面临资源短缺的困境。生物合成有助于解决该问题，关键点是完成从β-香树脂醇到皂皮酸的6步氧化过程。目前皂树的转录组和基因组数据均未被报道，故无法通过基因挖掘手段寻找可催化该反应的P450和CPR。本项目拟利用理性和非理性策略对现有功能相似的酶进行改造：首先优化现有P450催化效果，减少β-香树脂醇到刺囊酸氧化过程中副产物的积累；再改造P450创造新反应，实现从刺囊酸到皂皮酸的氧化。通过分析P450改造前后氨基酸位点差异，阐明其在氧化刺囊酸时对底物的选择机制。以此为基础，可用于构建工程酵母菌生物合成皂皮酸，为半合成非天然佐剂和QS-21提供廉价底物；并用于糖基转移酶挖掘，阐明QS-21生物合成途径。

QS-21, a natural adjuvant with quillaic acid as backbone, has been applied to several vaccines. Recently, there are reports that semi-synthetized adjuvants based on quillaic acid have showed similar effect as QS-21. However, both QS-21 and quillaic are in shortage of supply. One solution to the shortage of QS-21 is to employ biosynthesis strategy, in which the major challenge is the compliment of biosynthetic pathway from β-amyrin to quillaic acid with six steps of oxidation. So far, neither genome nor transcriptome of Quillaja saponaria Molina has been publicized for genome mining to discover the P450 and CPR involving in the oxidation, and the most promising solution is to engineer the P450s which have the similar function. The first target is to engineer the P450 to increase the efficiency of transformation from β-amyrin to echinocystic acid and reduce the accumulation of intermediates. The second target in to engineer P450 for novel enzymatic reaction of oxidizing echinocystic acid to form quillaic acid. By analyzing the mutants which possess different activity, the mechanism of substrate specificity can also be revealed. Based on these results, yeast producing quillaic acid can be established. This strain can be used to produce quillaic for semi-synthesis of QS-21 or other unnatural adjuvants. This strain can also be employed as a platform for discovery of glucosyltransferase in order to elucidate the biosynthetic pathway of QS-21.