基于跨界进化筛选高效糖基转移酶及其定向合成重要糖苷天然产物

Chinese herb is the largest resource of natural products. The separation and preparation of monomer natural products plays an important role in the exploitation of new drugs. However, the low content of the substances and large amount of analogs exist in corresponding plants, which make the preparation of adequately pharmaceutical pure compounds difficult in large amount. On the other hand, the complexity of structure of natural products also creates more obstacles to synthesis. Although synthetic biology provides a significant driving force to develop the synthesis of natural products by assembling biosynthetic pathways and heterologous expression, the low activity and efficiency of these enzymes in catalyzing sequential reactions still make it difficult to obtain sufficient natural products for deeply pharmacological activity research. Recently, with a large number of bioinformatics shared in the data base, the increasing massive information of enzymes involved in the synthesis of secondary metabolites makes it possible for us to mine isoenzymes bestriding plant kingdom. In this study, by analyzing key residues for substrate recognition and evolution of the distinct motif of glycosyltransferases related to biosynthesis of some important natural glycosides, several microbe isoenzymes could be rationally mined and were mainly used to solve the problems in the synthesis of crocin, salidroside and ginsenoside. Then, we will propose the mechanism of glycosyltransferase of glycosides synthesis. In virtue of higher catalytic activity and engineered genetically tractability of microbe enzymes, the highly efficient catalysis and regioselectivity regulated biosynthesis of target important natural glycosides could be achieved. Meanwhlie, the mechanism of microbe genome mining bestriding plant kingdom for synthesis of the natural glycosides would be provided an important reference for mining other functional enzymes.

药用植物是天然产物的最大资源王国，天然产物单体的分离或制备是新药开发的关键。药用植物中有效组分含量低、分子结构复杂，给天然产物单体的分离或合成带来了很大的难度。虽然合成生物学的发展给天然产物合成迎来了新突破，然而植物来源的酶在工程菌中活力低、重构的植物酶的顺序协作催化效率低等因素导致难以获得足够量的天然产物进行较深入的药理活性研究。本研究利用植物界丰富的天然产物合成酶的生物学信息，跨界进化分析获得微生物源的同工酶，重点进行藏红花、人参及红景天等植物的相关糖基转移酶序列与特征模序的进化分析，解析酶底物结合域残基，跨界理性筛选微生物来源的具有相似功能的糖基转移酶。利用微生物酶的高合成催化活性与易改造的特性，显著提高合成效率，实现多位点高选择性催化与反应高效调控。同时，提出跨界基因探矿的分子机理，为其它天然产物合成酶类的跨界基因数据挖掘提供重要参考。

红景天苷、藏红花素、人参皂苷Rh2等在冠心病治疗、抗动脉粥样硬化、抗肿瘤等方面具有重要药用价值。然而，这些天然产物在珍稀药用植物中的含量极低，单体分离困难，难以获得足够量的高纯度产物进行深入研究。本研究开展了植物源糖基转移酶全序列与特征模序进化分析，创建了跨界（植物界到微生物界）进化分析的方法发掘微生物源高效酶类，筛选获得了5个微生物源糖基转移酶；独创地提出了糖苷类天然产物合成酶与底物识别机制，解析酶底物结合域特征，提出酶分子改造策略，酶定向进化改造实现多位点高选择性催化，构建了包含>300个突变体的酶突变库，筛选获得20多个有效突变体；反应调控实现酶法制备红景天苷产物的浓度水平约2.7 g/L，总产率80%。构建了双酶催化反应体系，非水相催化反应调控实现定向合成藏红花素-3和藏红花素-5；批次补料反应技术获得藏红花酸素-5产物为4.98 mM（2.44 g/L，产率81.3%）和藏红花酸素-3为4.88 mM（3.18 g/L，产率83%）。酶YjiC突变体M315F显著提升了糖基化反应活性（80%）和区域选择性（99%），人参皂苷Rh2产物达3.7 g/L。进一步筛选获得的组合突变体Y14S/F168N/M315F合成人参皂苷Rh2的产率提升至93.51%。构建酶 YjiC突变体-蔗糖合成酶AtSuSy高效合成人参皂苷Rh2的反应体系，批次补料反应技术实现高效定向合成人参皂苷Rh2的产量水平达10.73 g/L。突变体P12A高效定向合成人参皂苷F12产物（占96.6%）达13.58 g/L；多种糖苷产物水平或产率显著高于现有的报道，突破现有的生物酶法制备天然产物产量水平低、产率低等重大瓶颈问题。糖苷类天然产物的高浓度水平或高产率生物合成为珍稀天然产物开发提供创新性思路和策略，推进具有显著临床疗效优势的中药活性成分的新药开发。同时，对于提升糖苷类天然产物的研究与应用转化水平研究具有重要借鉴意义。本课题累计获得相关发明专利10项（已获公开/授权），共发表SCI/EI论文34篇，获科研/教学奖4项，具有显著的科学价值和广阔的应用前景。