Abstract：
Biosynthesis of Amaryllidaceae alkaloids (AA) starts with the condensation of tyramine with 3,4-dihydroxybenzaldehyde. The latter derives from the phenylpropanoid pathway that involves modifications of trans-cinnamic acid, p-coumaric acid, caffeic acid, and possibly 4-hydroxybenzaldehyde, all potentially catalyzed by hydroxylase enzymes. Leveraging bioinformatics, molecular biology techniques, and cell biology tools, this research identifies and characterizes key enzymes from the phenylpropanoid pathway in Leucojum aestivum. Notably, we focused our work on trans-cinnamate 4-hydroxylase (LaeC4H) and p-coumaroyl shikimate/quinate 3\'-hydroxylase (LaeC3\'H), two key cytochrome P450 enzymes, and on the ascorbate peroxidase/4-coumarate 3-hydroxylase (LaeAPX/C3H). Although LaeAPX/C3H consumed p-coumaric acid, it did not result in the production of caffeic acid. Yeasts expressing LaeC4H converted trans-cinnamate to p-coumaric acid, whereas LaeC3\'H catalyzed specifically the 3-hydroxylation of p-coumaroyl shikimate, rather than of free p-coumaric acid or 4-hydroxybenzaldehyde. In vivo assays conducted in planta in this study provided further evidence for the contribution of these enzymes to the phenylpropanoid pathway. Both enzymes demonstrated typical endoplasmic reticulum membrane localization in Nicotiana benthamiana adding spatial context to their functions. Tissue-specific gene expression analysis revealed roots as hotspots for phenylpropanoid-related transcripts and bulbs as hubs for AA biosynthetic genes, aligning with the highest AAs concentration. This investigation adds valuable insights into the phenylpropanoid pathway within Amaryllidaceae, laying the foundation for the development of sustainable production platforms for AAs and other bioactive compounds with diverse applications.
摘要：
石豆科生物碱（AA）的生物合成始于酪胺与3,4-二羟基苯甲醛的缩合。后者来自涉及反式肉桂酸修饰的类苯丙烷途径，对香豆酸,咖啡酸,可能还有4-羟基苯甲醛，都有可能被羟化酶催化。利用生物信息学，分子生物学技术，和细胞生物学工具，这项研究鉴定并表征了白羊座苯丙素途径中的关键酶。值得注意的是,我们的工作重点是反式肉桂酸4-羟化酶（LaeC4H）和对香豆酰基莽草酸/quinate3'-羟化酶（LaeC3'H），两种关键的细胞色素P450酶，和抗坏血酸过氧化物酶/4-香豆酸3-羟化酶(LaeAPX/C3H)。虽然LaeAPX/C3H消耗了对香豆酸，它没有导致咖啡酸的产生。表达LaeC4H的酵母将反式肉桂酸转化为对香豆酸，而LaeC3\'H特别催化了对香豆酰基莽草酸的3-羟基化，而不是游离的对香豆酸或4-羟基苯甲醛。在这项研究中在植物中进行的体内测定为这些酶对苯丙烷途径的贡献提供了进一步的证据。两种酶都证明了本氏烟草中典型的内质网膜定位，为其功能增加了空间环境。组织特异性基因表达分析显示，根是苯丙素类相关转录本的热点，球茎是AA生物合成基因的中心，与最高的AAs浓度对齐。这项研究增加了对石豆科中的苯丙烷途径的宝贵见解，为AAs和其他具有多种应用的生物活性化合物的可持续生产平台的开发奠定基础。
