羅勒毛狀根之苯丙烷路徑代謝工程改造之研究

Abstract

鬼臼素是從鬼臼屬植物中分離出的芳基四氫化木脂素 (aryltetralin-type lignan)，其生合成路徑是從苯丙胺酸 (phenylalanine) 開始，經過多個酵素反應後可合成松柏醇 (coniferyl alcohol)，依序再經過多種酵素合成鬼臼素。鬼臼素在植物中含量不高，且複雜的化學結構使它不容易以化學方法合成。已知松柏醇為鬼臼素生合成路徑之重要中間產物，過去實驗室建構RASi質體做為RAS基因下調突變株，獲得迷迭香酸路徑上代謝物改變的結果。本研究希望在此基礎上透過代謝工程技術大量表現外源性基因酪胺酸解氨酶 (tyrosine ammonia-lyase, TAL) 使累積的迷迭香酸前驅物酪胺酸 (tyrosine) 生合成香豆酸 (coumaric acid)，隨後透過轉入外源性4-香豆酸酯3-羥化酶 (4-coumarate 3-hydroxylase, C3H)，使香豆酸轉變為咖啡酸 (caffeic acid)，期望能透過木質素生合成途徑使咖啡酸轉為松柏醇，並於羅勒中建立完整之鬼臼素代謝路徑。經由HPLC分析羅勒毛狀根中次級代謝物含量得知，當只轉形TAL基因 (TAL) 與同時轉形TAL及C3H兩個基因 (TCR) 之毛狀根與野生型毛狀根 (WT) 相比，香豆酸的含量分別提升了9.28%與30.38%；與只轉形pRASi之毛狀根 (RASi) 相比則分別增加17.19%與39.82%。而在只轉形C3H基因 (C3H) 和TCR，與WT相比其咖啡酸的含量則分別提升了83.63%與113.23.62%；與RASi比較也提升了89.74%與121.68%。此外，在羅勒毛狀根再生 (regeneration) 試驗中，嘗試以添加不同濃度比例生長激素 (auxin) 與細胞分裂素 (cytokinin) 之培養基，透過先將毛狀根組織誘導成癒傷組織，發現較適合添加之濃度比例，未來希望從癒傷組織直接進行出芽生長 (shooting) 後再生為完整植株，以用於後續的毛狀根誘導。

Podophyllotoxin is an aryltetralin-type lignan isolated from plants of the genus Podophyllum. Its biosynthetic pathway begins with phenylalanine, which is converted into coniferyl alcohol through several enzymatic reactions, followed by further enzymatic transformations to produce podophyllotoxin. Podophyllotoxin is present in low concentrations in plants, and its complex chemical structure renders chemical synthesis challenging. Coniferyl alcohol is known to be a crucial intermediate in the biosynthetic pathway of podophyllotoxin. Previous laboratory research has attempted to block the rosmarinic acid metabolic pathway using RNAi technology (RASi). Building on this, our study aims to employ metabolic engineering to overexpress the exogenous gene tyrosine ammonia-lyase (TAL) to convert the accumulated tyrosine, a precursor of rosmarinic acid, into p-coumaric acid. Subsequently, by introducing the exogenous gene 4-coumarate 3-hydroxylase (C3H), p-coumaric acid is converted into caffeic acid. The ultimate goal is to utilize the lignin biosynthesis pathway to convert caffeic acid into coniferyl alcohol, thereby establishing a complete biosynthetic pathway for podophyllotoxin in basil. HPLC analysis of secondary metabolite content in basil hairy roots revealed that the content of p-coumaric acid increased by 9.28% and 30.38% in hairy roots transformed with TAL alone (TAL) and with both TAL and C3H genes (TCR), respectively, compared to wild-type hairy roots (WT). Compared to hairy roots transformed with RASi alone (RASi), the increases were 17.19% and 39.82%, respectively. Similarly, the content of caffeic acid increased by 83.63% and 113.23% in hairy roots transformed with C3H alone (C3H) and TCR, respectively, compared to WT, and by 89.74% and 121.68% compared to RASi. Additionally, in the basil hairy root regeneration experiment, we attempted to induce callus formation from hairy root tissues using media supplemented with various concentrations of auxin and cytokinin. The optimal concentration ratio was determined, and the potential to regenerate complete plants from callus tissue was demonstrated, which could be utilized for subsequent hairy root induction experiments.