Molecular engineering of isoquinoline alkaloid biosynthesis in higher plant using Coptis O-metheyl-transferases cDNAs

研究了异喹啉生物碱生物合成的分子工程。利用花椰菜花叶病毒35 S启动子和增强子元件，构建了小檗碱生物合成的3个外膜转运蛋白（OMT）的高效表达载体，并将其导入3种不同的植物中：黄连（同源植物）、黄药子（一种产生来源于黄连素的苯并菲啶生物碱的植物，黄连素的中间体）和烟草（一种不产生异喹啉类生物碱的烟碱生物碱植物）。虽然黄连细胞的转化非常困难，但至少为所有宿主植物物种建立了具有SMT（Scoulavirus 9-0-methyltransferase）表达的转化体。转化的黄连细胞表现出增加的SMT活性和改变的生物碱组成;即更多的小檗碱和更少的黄连碱（与黄连碱不同的最终产物）。这一结果表明，分支点酶（SMT）的修饰将是一个有用的方法来改变生物碱的组成，而修饰是有限的。转化的大肠杆菌细胞也显示出明显的SMT活性和修饰的代谢产物组成;四氢氨酰苯丙氨酸的高积累，SMT反应的产物。另一方面，烟草的转化体不显示SMT活性，而检测到SMT转录本。讨论了寄主植物种类对生物碱生物合成分子工程的影响。

The molecular engineering of isoquinoline alkaloid biosynthesis in plant cells was investigated. Using cauliflower mosaic virus 35S promoter with enhancer elements, high expression vectors for 3 OMTs of berberine biosyntheis were constructed and introduced into 3 different plants ; Coptis (homologous plant), Eschscholtzia (a plant which produces benzophenanthridine alkaloid derived from scoulerine, an intermediate for berberine) and Nicotiana tabacum (nicotine alkaloid plant which does not produce isoquinoline type alkaloid). Although transformation of Coptis cells was very difficult, at least transformants with SMT (scoulerine 9-0-methyltransferase) expression were established for all host plant species. Transformed Coptis cells showed increased SMT activity and modified alkaloid composition ; I.e. more berberine and less coptisine (different end product from scoulerine). This result suggest the modification of branching point enzyme (I.e. SMT) would be a useful method to modify the composition of alkaloid, whereas modification was limited. Transformed Eschscholtzia cells also showed clear SMT activity and modified metabolite composition ; high accumulation of tetrahydrocolumbamine, a product of SMT reaction. On the other hand, the transformants of tobacco did not show SMT activity, whereas SMT transcripts were detected. Effect of host plant species on molecular engineering of alkaloid biosynthesis is discussed.