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

对异喹啉生物碱在植物细胞中生物合成的分子工程进行了研究。利用带有增强子元件的花椰菜花叶病毒35S启动子，构建了3种黄连素生物合成OMT的高效表达载体，并将其导入3种不同的植物：黄连(同源植物)、石杉(从黄连提取的苯并菲并菲生物碱的中间体)和烟草(不产生异喹啉类生物碱的尼古丁生物碱植物)。虽然黄连细胞的转化非常困难，但至少为所有寄主植物建立了表达SMT(Soulerine 9-0-甲基转移酶)的转化子。转化的黄连细胞表现出SMT活性增强和生物碱成分的修饰，即更多的黄连碱和更少的黄连碱(不同于黄连碱的最终产物)。这一结果表明，分支点酶(SMT)的修饰是一种有效的生物碱组成修饰方法，但修饰效果有限。转化细胞也表现出明显的SMT活性和修饰的代谢物组成；SMT反应产物四氢柱胺的高度积累。另一方面，烟草的转化子没有显示SMT活性，但检测到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.