Violacein 和deoxyviolacein為微生物的二級代謝產物，具有抗氧化、抗菌及抗腫瘤等特性，作為天然染料也具有良好的色調及穩定性，這些特點使人們對於生產紫色桿菌素越來越感興趣。然而以往透過微生物生產紫色桿菌素時，都會透過添加色胺酸作為前驅物以提高產量，不過此方式是比較不經濟實惠的，因此本實驗建構了增強色胺酸途徑之質體及菌株，經過測試能在不額外添加色胺酸的條件下增加最終產物紫色桿菌素的產量。接著我們以RBS Library同時調控上下游的基因，再透過呈色法篩選出具有生產潛力之菌株，而這些菌株能達到不同的violacein/deoxyviolacein比例及產量，另外我們也透過再現性測試證實產量及比例改變之主因來自我們所設計之不同RBS序列組合，而非菌株或是基因序列發生突變所致。在經過生產環境的優化之後，violacein及deoxyviolacein最佳生產菌株能在搖瓶中於72小時內分別達到1.6 g/L的violacein及4 g/L deoxyviolacein。此外，我們也將菌株定序確認其RBS序列組合以分析各酵素之初始轉譯速率間的關係，從中發現violacein及deoxyviolacein之優良生產菌株，他們於high copy number及low copy number質體之轉譯速率強度關係具有相似的趨勢。
黃烷酮類化合物為另一種來自植物的二級代謝產物，具有抗氧化、抗發炎及抗癌特性，並且被證實具有治療糖尿病及肥胖等代謝綜合症的藥理活性，在醫藥產業具有一定的發展潛力，同時他們也是其他類似物的重要前驅物及基本骨架，代表可以藉由酵素或化學催化的方法從基本骨架合成其他的衍伸物，這加重了黃烷酮類化合物的重要地位，因此我們希望藉由RBS Library優化其生產，不過這類化合物並沒有像紫色桿菌素具有在顏色方面的優勢以利於篩選，因此我們尋找文獻並選擇生物傳感器做為篩選工具，利用來自H. seropedicae的fde操縱子，fdeR酵素在與柚皮素結合後能夠活化螢光蛋白，使菌落在藍光下具有綠色螢光。而在經過測試及優化後，我們透過生物傳感器系統進行RBS Library之菌落篩選，最後成功挑選到產量提升12倍之菌株。

Violacein and deoxyviolacein which possess anti-fungal, anti-cancer and antibiotic properties are the derivatives of tryptophan. Their colorful appearances also raise interests among dyeing industry. To efficiently produce violacein and its related compounds from simple carbon source, here we aim to fine-tune enzyme expression of both upstream and downstream pathway using RBS library while lowering metabolic burdens inside the cell. We first designed and constructed the RBS library to combine it with tryptophan and violacein producing genes, then screening the potential producing strains by coloring method. In this screening stage, we picked out hundreds of strains that possess different ratio and titer of violacein and deoxyviolacein. After the optimization of production parameters, 1.6 g/L violacein or 4 g/L deoxyviolacein was achieved by best-producer respectively. In addition, we also sequenced to confirm their RBS sequence combination while estimated translation rate using RBS calculator to analysis the regulation and correlation between these enzymes.
Flavanones are another secondary metabolite from plants, which also have multiple biological properties and have been proven to have pharmacological activity in the treatment of metabolic syndromes such as diabetes and obesity. They are also the important precursor and basic skeleton of other analogs, which more accentuate the importance of flavanones. That’s why we want to improve their production, but flavanones does not have the advantage of color like violacein to facilitate screening, so we searched for the screening methods and finally chose biosensor as ours tools. By applying the fde operon from H. seropedicae as our biosensor system, we can see the colonies on the plate have green fluorescence under blue light. After testing and optimization of this biosensor system, we applied RBS Library to the pathway and screened for the better producers, and finally successfully picked up a strain with 12-fold improvement in flavanones titer.

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