基于ApCPS2诱饵蛋白靶向分离穿心莲I型萜类合酶及其方法的建立

Andrographolide is the principal active component of Andrographis paniculata. It has particularly shown widespread medicinal properties, like anti-virus, antimicrobial, and anti-inflammatory activities. Characterizing their biosynthetic pathways and reconstructing them in microbe will provide an alternative for production of andrographolide. ApCPS2 which is involved in the biosynthesis of andrographolide has been identified. However, class I terpene synthase (TPS) which is involved in accumulation of andrographolide has not been identified so far. Lacking class I TPS has become the bottleneck for illuminating the biosynthetic pathway of andrographolide. Protein complex formations in plant secondary metabolism (including terpene biosynthesis) enable plants to effectively synthesize specific natural products. Tandem affinity purification (TAP), which base on two-step sequential affinity purification, is one of the most efficient methods to characterize interaction protein of a certain protein of interest (bait). In this study, ApCPS2 will be used to be a bait to co-purify class I terpene synthase (TPS) which is involved in the production of andrographolide with TAP. The function of obtained class I TPS will be further characterized in yeast. Engineered yeast which efficiently products the precursor of andrographolide will be constructed. Our study will facilitate to characterize the downstream steps of andrographolide accumulation in vivo. And TAP modified in this study will be applied to shed light on other secondary metabolism pathways.

穿心莲内酯是传统大宗中药穿心莲的主要药效成分，广泛应用于临床治疗。解析其体内合成途径，利用合成生物学进行异源高效生产，将为中药制剂生产提供大量原料。前期研究通过比较转录组学，鉴定了参与穿心莲内酯合成的II型萜类合酶ApCPS2；而后续的I型萜类合酶却始终难以分离。缺乏I型萜类合酶严重阻碍后续步骤的阐明。植物次生代谢途径的诸多催化酶类（包括萜类合酶）以蛋白复合物形式相互作用，共同催化合成下游产物。串联亲和纯化（TAP）技术以已知蛋白为诱饵，通过两步连续亲和纯化，得以高效分离体内互作蛋白。本研究拟以ApCPS2为诱饵，探索通过TAP技术，分离参与穿心莲内酯合成的I型萜类合酶；并通过基因融合构建高产穿心莲内酯二萜骨架的酵母工程菌。I型萜类合酶及底盘菌的获得将为继续解析穿心莲内酯体内合成奠定基础。本研究建立的TAP技术将可以推广应用于解析其它药用植物次生代谢途径，成为分离功能基因的重要手段。

穿心莲内酯是传统大宗中药穿心莲的主要药效成分，广泛应用于临床治疗。解析其体内合 成途径，利用合成生物学进行异源高效生产，将为中药制剂生产提供大量原料。前期研究通过比较转录组学，鉴定了参与穿心莲内酯合成的II型萜类合酶ApCPS2；而后续的I型萜类合酶却 始终难以分离。课题拟通过ApCPS为诱饵蛋白筛选及纯化与穿心莲内酯生物合成相关的I型萜类合酶。课题组通过对穿心莲再生体系激素配比进行优化及不同组织作为外植体进行筛选，成功构建穿心莲再生体系，但未能构建穿心莲稳定遗传转化体系；筛选及克隆4个穿心莲ApGGPPS作为穿心莲诱饵蛋白体系阳性对照候选基因，通过对ApGGPPS生物学功能、表达模式及亚细胞定位进行研究，结果表明ApGGPPS2可能是参与穿心莲内酯生物合成的关键基因，即筛选获得阳性对照基因；对穿心莲基因组中37个萜类合酶克隆及功能鉴定，获得6个倍半萜合酶，但未筛选获得与穿心莲内酯生物合成相关的I型萜类合酶。