管花肉苁蓉中苯乙醇苷类活性成分生物合成关键糖基转移酶基因的功能鉴定及酶法合成应用

Phenylethanoid glycosides (PheGs) which have been demonstrated diverse pharmacological activities such as anti-aging, protecting neurons are the major active components in many medicinal plants such as Cistanche Herba and Rhodiola L. However, the further studies of PheGs were limited because of the low natural abundance and difficult chemical synthesis. Structure-activity relationship investigations revealed that the glycosylation of phenylethanol derivatives not only contributes significantly to the structural variation of these secondary metabolites, but also enhances their bioactivities and bioavailabilities. Thus, the manipulation of glycosylation reactions of phenylethanol derivatives by glycosyltransferases might represent an advantageous shortcut for the one-step synthesis of PheGs. In Preliminary studies, series of novel glycosyltransferase genes have been successfully cloned from Cistanches tubulosa which produce the most abundant amounts of PheGs in natural. Based on previous research, the glycosyltransferase genes involved in the biosynthesis route of PheGs were proposed to be screened out and characterized following by the engineering strain construction as well as the reaction conditions optimization to achieve chemo-enzymatic synthesis of PheGs. Furthermore, under the guidance of enzyme active sites analysis coupled with protein structural information, site-directed mutagenesis would be processed and the mutant strains with broad substrate selectivity would be obtained and used for searching leading compounds. This investigation would provide not only a new approach of PheGs synthesis and leading compounds discovery, but also useful hints for the structure and function research of glycosyltransferase of plants, with both theoretical and practical significance.

苯乙醇苷类化合物具有抗衰老、神经保护等广泛的药理活性，是许多中药如肉苁蓉、红景天的主要活性成分。但该类化合物天然来源有限，化学合成困难，限制了其作为药物先导物的进一步研究。构效关系研究显示，糖链的存在是其结构多样性及药理活性的关键，也是化学合成的瓶颈所在，而以价廉易得的苯乙醇衍生物为原料，通过生物酶法糖基化反应，可实现该类化合物的“一步合成”。管花肉苁蓉为至今发现的苯乙醇苷最丰富的药用植物，本项目前期从其中克隆得到多条新颖的糖基转移酶序列，在此基础上拟对所获基因进行功能鉴定，揭示苯乙醇苷生物合成关键糖基转移酶基因；优化酶促催化条件，建立酶法生物合成苯乙醇苷类化合物的新方法；分析酶催化醇羟基糖基化的活性位点，并在蛋白结构指导下构建突变体，拓宽底物谱，制备“苯乙醇苷类化合物库”，结合活性筛选获得药物先导物；同时为植物糖基转移酶的结构及功能研究提供科学依据，具有重要的理论及实际应用意义。

苯乙醇苷类化合物结构多样性丰富，药理活性广泛。糖链的存在是其结构多样性及药理活性的关键。本项目以富含苯乙醇苷类化合物的管花肉苁蓉为研究材料，按原有计划顺利开展，通过分子生物学手段从管花肉苁蓉基因组中克隆获得了11个糖基转移酶基因，并对其中5个糖基转移酶完成了功能鉴定，结果如下——CtGT-A：其能够特异催化红景天苷糖苷键水解生成苷元酪醇，同时也能够催化黄酮类化合物染料木素葡萄糖苷化生成染料木苷；CtGT-B & CtGT-E：两者功能相似，催化能力略有差别，均能高效、特异的催化酪醇侧链醇羟基发生β-葡萄糖苷化生成红景天苷，为苯乙醇苷类化合物生物合成途径中的首步关键反应。通过同源模建和分子对接对CtGT-B进行蛋白三维结构分析，并在结构指导下进行了突变体构建，获得了酶催化活性改善的突变体，进一步通过放大的体外酶促催化反应，制备获得了30余个不同结构类型的非天然苯乙醇苷类衍生物；CtGT-D & CtGT-I：两者功能相似，催化能力方面I蛋白明显优于D蛋白，其均能够催化不同结构类型的芳香类和芳香糖苷类化合物发生单/多糖基化反应，产物多样性丰富。通过制备获得了天然界中少有的四糖苷苯乙醇苷类化合物及二苯乙基色酮苷类化合物。酰基转移酶：酰基转移酶为苯乙醇苷生物合成途径中的另一关键酶，从管花肉苁蓉基因组中克隆获得6个酰基转移酶基因CtAT1~6，功能鉴定结果显示CtAT5为BAHD家族丙二酰基转移酶。其能够接受不同结构类型糖苷类化合物为底物，催化葡萄糖6-OH位置特异性丙二酰基化反应。鼠李糖基供体合成：项目建立了酶法合成鼠李糖基供体的方法，为后续进行葡萄糖3-OH位置特异性鼠李糖基转移酶的研究打下了基础。组织培养体系研究：项目还成功建立了管花肉苁蓉愈伤及悬浮培养体系，并对其最适培养条件、最佳碳源、氨基酸前体饲喂、生物诱导剂诱导对苯乙醇苷类化合物生物合成的影响进行了探讨。项目发表SCI论文2篇，中文期刊论文1篇，国内学术会议论文2篇，申请专利2项，培养硕士研究生2名。