系統化的方法將植物二次代謝實現於微生物中：用大腸桿菌生合成白藜蘆醇作為個案研究

Abstract

合成生物學是一個近年迅速發展的跨學門研究，結合科學與工程的技術來設計和合成一個嶄新的生物功能系統。近年來發現植物二次代謝物對人類的健康有益，大宗為酚類化合物，像是異黃酮素、兒茶素和白藜蘆醇，皆是對人類有益的酚類化合物。基於上述兩點，本研究利用合成生物學的宗旨，在微生物中合成植物二次代謝物。研究一開始先收集並整合資料庫的資料，用有系統的方法來重建代謝途徑，包括植物的二次代謝。呈現重建的代謝途徑為新穎的KEGG連接地圖：將KEGG的地圖做連接並標上經過的所有代謝物和酵素。再者，使用跨物種分析所重建的代謝途徑，可了解此代謝途徑存在哪些物種上，提供每個酵素的基因，蛋白質和後轉譯修飾之資訊讓研究者了解是否可合成感興趣的代謝物在其它物種上。白藜蘆醇是一種抗氧化劑，能抑制發炎反應和腫瘤生長，有很大的經濟和研究價值。本研究選其生合成路徑在大腸桿菌中做個案研究。首先，系統化方法有詳細且視覺化的初步結果，發現酪胺酸到白藜蘆醇這段代謝途徑必須從植物轉殖到大腸桿菌中。第二，建立代謝網路並用基本通量路徑分析，以得知最高產率的生合成路徑，進一步刪除合成苯丙胺酸和色胺酸的代謝途徑後，發現可能可以提高白藜蘆醇生合成的效率。 此系統化的分析可以快速的得到非常多合成生物學和代謝工程需要的資訊，例如酵素從哪一種物種產生？其序列為何？它轉譯的蛋白質是否有後轉譯修飾？此系統化分析方法已經建置成網路工具FMM( http://FMM.mbc.nctu.edu.tw/)。可應用在合成生物學上生產藥物和生質能源。此外，基本通量路徑分析可以評估最終產物的產率，改善傳統代謝工程隨機突變基因所消耗的金錢及時間。

Synthetic Biology, a multidisciplinary field, is growing rapidly. To understand biological systems clearly, scientists through mimicking and producing bio-orthogonal systems with new functions are two complementary pursuits. Recently, plant secondary metabolites are increasing importantly for human. The phenolic compounds, which are kinds of plant secondary metabolites, contain many important useful components, such as resveratrol, isoflavones, and catechin that are healthy for human. For the above reasons mentioned, this study for synthetic biology purposes is developed that synthesizes plant secondary metabolites in microbes. In the beginning of this study, the relative data was collected and integrated from databases. The metabolic pathway was reconstructed by systematic method containing plant secondary metabolism. Novel presentation for connecting different KEGG maps is newly provided. Furthermore, comparative analysis the reconstruction metabolic pathway will understand enzyme information about genes, proteins, and post-translational modifications (PTMs), which provide messages to find out whether a metabolite is synthesized by others species or not. Resveratrol is an antioxidant, which inhibits inflammation and tumor promotion and contains economic and scientific value. This study was chosen as a case. First, systematic method provides visualization primary results to find out the pathway synthesized from tyrosine to resveratrol having to be cloned from plant. Second, metabolic network is constructed and analyzed by elementary flux modes analysis (EFMs). The result shows that which pathway has maximum yield. Moreover, after deleting the phenylalanine and tryptophan biosynthesis pathway, the production of resveratrol may become efficient. The systematic method is an efficient analysis to obtain information rapidly for synthetic biology and metabolic engineering, such as enzyme, sequence, and PTMs. The method has been developed to web tool: http://FMM.mbc.nctu.edu.tw/, which is an effective tool for applications in synthetic biology to produce both drugs and biofuels. Besides, traditional optimization strategies of random mutagenesis and selection will eventually have limited efficacy. However, EFMs can estimate the yield of end product for improving traditional methods to spend less time and cost.