基于重要糖苷天然产物合成的酶分子改造研究

Natural glycoside products have received much attention for their specifical targeting ability, clear therapeutic target, and exact efficacy. Due to the lack of nature resources, effective components such as salidroside, gastrodine, and cinnamoside from endmic and rare plants, are very difficult to prepare in large-scale to meet the needs of the clinical medication, even further exploitation, whereas chemical synthesis of glycosides has the key drawbacks in regio- and stereoselectivity. Under such circumstances, we explored the novel glycosidase in the first time from bacterium that catalyzed efficiently synthesis of salidroside. We investigate the mechanism of substrate recognition of the glycosidase by bioinformatic approaches, analyze the proximity and orientation between the different substrates with the key amino acid residues nearby the enzyme active center, furthermore put forward a proposal for the rational design of molecular alteration of glycosidase, and then prepare the mutants of glycosidase by means of protein engineering. We set up a high-throughput method to screen the effecient mutants of glycosidase, and project non-aqueous systems to synthetize high value natural glycosides, as well as desired novel analogues. This may give a new way to explore natural glycoside products, also provide instances of molecular modification of the glycosidase, and reliable biotechnique for exploitation of efficient mutants of glycosidase directing at synthesis of natural glycoside products.

糖苷天然产物具有特异的寻靶能力，靶点明确，药效确切。一些珍稀物种的活性成份如红景天苷、天麻苷、肉桂苷等由于资源稀有缺乏，以及糖苷的化学合成法又存在选择性问题，难以大量制备限制了其开发利用，本项目以首次发现的细菌来源的具有高效催化红景天苷合成的新型糖苷酶为研究对象，利用生物信息学等手段探讨糖苷酶与天然产物苷元的识别机制，分析糖苷酶与底物/糖苷产物复合物结合状态下的活性中心微环境变化，针对重要糖苷天然产物（如红景天苷、天麻苷、肉桂苷等）分析酶活性中心与不同底物结合关键氨基酸残基的邻近与定向作用，提出酶分子的理性设计与改造策略，利用蛋白质工程手段实现酶分子改造，并利用组合生物催化高效筛选糖苷酶突变体，进一步构建非水相体系合成糖苷天然产物及其类似物的催化体系，为重要糖苷天然产物的开发提供酶分子改造技术和高效糖苷酶的深入挖掘提供借鉴。

红景天苷、天麻素、虎杖苷等重要天然产物在冠心病、眩晕症、抗休克等疾病治疗方面具有疗效显著且副作用小的优势。红景天苷等活性成分在天然珍稀植物中含量较低、提取繁琐且成本高昂，限制了其开发利用。本课题在红景天苷类天然产物合成的微生物菌株筛选基础上，表达策略优化实现了多个功能酶的高效可溶性表达。我们首次提出了跨界（植物界到微生物界）进化方法发掘微生物源高效酶类，基于红景天苷产物合成的酶基因序列进化分析，首次成功地筛选获得了红景天苷类产物合成的原核微生物源高效酶类，其催化红景天苷产物的合成活性是现有报道同类酶的50倍，取得了创新性的突破。首次筛选并表达获得新红景天苷产物合成的耐有机溶剂糖苷酶。分子对接独创地提出红景天苷等重要天然产物合成的酶与底物识别机制和酶分子设计与改造策略，构建了包含100个突变体的酶突变库；酶突变体的表达与组合生物催化筛选获得了有效的酶突变体6个，有效地拓展了红景天苷类天然产物合成的酶源。利用非水相生物催化调控技术建立了多个高效催化体系，酶及其突变体能高效地催化合成十几种重要糖苷天然产物，其中催化合成红景天苷产物的转化率达93%；天麻素产物的转化率达84%；非水相定向合成虎杖苷的转化率达90%；新红景天苷（酪醇半乳糖苷）类产物水平可达30 g/L；显著优于现有报道水平，突破了现有生物法制备红景天苷类天然产物的产量水平低、产率低等重大瓶颈问题。红景天苷类等多种重要天然产物的高浓度水平或高产率生物合成为高效制取糖苷类天然产物提供创新性思路和策略；将助推生物酶工程技术等领域的发展，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，对于提升红景天苷类天然产物的研究与应用转化水平研究具有重要的借鉴意义。本课题累计申请获得相关的国家发明专利8项（已获公开/授权），共发表标注本项目资助的SCI/EI论文17篇和中文核心期刊论文4篇，具有显著的科学价值和广阔的应用前景。