莨菪碱转运分子机理与代谢工程研究

Metabolite transportation and metabolic engineering are the hot spots of the plant science field. Biosynthesis of tropane alkaloids (TAs), such as hyoscyamine, has been intensively studied at the molecular and biochemical levels, and further metabolic engineering of TAs biosynthesis has made great progress. Unfortunately, the molecular mechnism on TAs transportation is still unknown and engineering TAs transportation is impossible without the knowkedge of TAs transporters. In the TAs biosynthetic pathway, tropinone reduction is the branch point. Tropinone reductase I (TRI) reduces tropinone to tropine that participates pharmaceutical TAs biosynthesis; tropinone reductase II reduces tropinone to pseudotropine that participates calystegin biosynthesis. These suggest that TRII might compete for the substrate tropinone with TRII. However, the effect of suppressing TRII on TAs biosynthesis is unclear. There is no report on improving TAs production by regulating TAs transportion and biosynthesis together..In this project, Atropa belladonna, the most commercially important plant for TAs production, is used as material. Hyoscyamine specific transporters will be functionally identified to understand how hyoscyamine is transported in planta and estimate their values in transport engineering. TRI and TRII of A. belladonna will be functionally identified and their roles in engineering of TAs production will be revealed. Finally, based on the genes discovered in this project, we will established a novel TAs metabolic engineering strategy through comprehensively regulating transporters-key enzymes involved in target metabolite biosynthesis-key enzymes competing for target metabolite biosynthesis. As a result of these research, the new varieties of A. belladonna with high-yield TAs will be developed.

次生代谢产物转运和代谢工程是植物科学研究热点之一。托品烷生物碱(TAs) 如莨菪碱，其生物合成途径及代谢工程研究较深入。但关于TAs转运的分子机理不清楚，基于TAs转运的代谢工程研究未见报道。托品酮还原反应是TAs合成途径中的分支点，包括生成托品(参与TAs合成)和假托品(参与打碗花精类生物碱合成)，对该分支点的代谢工程研究有待深入。综合调控代谢物转运和合成途径的TAs代谢工程研究未见报道。.本课题将从TAs商业药源植物颠茄中鉴定莨菪碱转运蛋白，解析其生物学功能，阐明莨菪碱转运分子机理；鉴定TAs合成途径的托品酮还原酶I(TRI)和竞争途径的托品酮还原酶II(TRII)，阐明其生化功能，为针对托品酮还原反应的TAs代谢工程提供理论依据。最终基于本课题鉴定的基因，采用综合调控“转运蛋白-合成途径关键酶-竞争途径关键酶”基因的方法建立新型的TAs代谢工程策略，并培育出TAs高产转基因颠茄。

药用托品烷生物碱（TAs）是茄科药用植物颠茄、曼陀罗和天仙子等生产的抗胆碱药物，包括莨菪碱及其外消旋体阿托品、山莨菪碱和东莨菪碱。莨菪碱在植物中含量极低，采用合成生物学和代谢工程技术是实现其高效生物制造最理想的手段，而这些手段都依赖于开展其代谢生物学研究。植物体中莨菪碱的代谢包括其生物合成、生物降解和转运。本项目针对莨菪碱转运的分子机制及相关代谢工程研究开展工作。.莨菪碱在植物根中特异性合成，再转运至全株各位部位，表明存在相关转运蛋白参与此过程。本研究基于颠茄等药用植物的转录组分析发现了两个根特异性表达的莨菪碱转运蛋白AbHUP1和AbPDR1。酵母转运实验证明其均具有莨菪碱转运活性，干扰AbHUP1基因和AbPDR1基因的表达均导致TAs的合成被抑制；过表达AbHUP1基因和AbPDR1基因可有效提高颠茄根和叶中莨菪碱的含量。.托品酮的还原是TAs生物合成的重要代谢节点。本研究克隆了颠茄托品酮还原酶AbTRI和AbTRII基因，通过原核表达纯化获得了AbTRI和AbTRII重组蛋白并鉴定其催化活性.AbTRI能以托品酮为底物合成托品， AbTRII能以托品酮为底物合成假托品.酶动力学常数测定结果显示AbTRII的催化效率高于AbTRII，表明颠茄中假托品的合成是托品合成的重要竞争支路。过表达AbTRI或干扰AbTRII表达均可以有效提高颠茄根中托品的含量。.为了培育出莨菪碱高产颠茄，进一步开展了“转运蛋白-合成途径关键酶-竞争途径关键酶”单基因/双基因代谢工程评价。同时过表达AbHUP1基因和AbTRI基因能最大程度提高颠茄莨菪碱的产量，根和叶中莨菪碱含量分别是对照的3.23倍和3.79倍。简而言之，莨菪碱转运蛋白AbHUP1和AbPDR1的发现对解析莨菪碱在植物中转运机制具有重要意义，也为莨菪碱高效生物制造提供了有价值的基因。