臺灣博碩士論文加值系統

近幾年來，面臨石化燃油耗竭及污染的問題，人們致力於尋找合適的替代能源，而當可再生能源成為一項新興議題後，微藻、藍綠菌等自營生物便逐漸受到關注，因其能在僅有光照及二氧化碳的條件下生產碳水化合物，然而，因產量有限與回收成本等缺點使整體程序不具經濟效益，因此，若自營菌出口的碳水化合物能作為碳源，直接提供給具有生產增值產品的異營菌，建立一具經濟價值的共培養系統，問題將被克服。
本研究以自營菌S. elongatus PCC7942及異營菌P. putida S12為宿主細胞，透過基因工程技術，使異營菌能以自營菌出口的蔗糖為唯一碳源，轉化HMF並生產高價值產品FDCA，並從中探討培養液酸鹼值、異營菌濃度及菌株透過表面佈置相互連接對FDCA產率之影響。
首先，成功將目標蛋白cscB建立於S. elongatus PCC7942中，且培養七天所得之最大蔗糖濃度為0.54g/L，另外cscA與HMFH蛋白也成功建立於P. putida S12中，活性結果顯示菌株能成功代謝蔗糖並轉化HMF，且反應效率與其以單醣為碳源時幾乎相同。接著，成功建立表面佈置菌株，使S. elongatus PCC7942與P. putida S12額外展示融合蛋白SomA-3(G4S)-SH3 ligand及INP-YFP-SH3 domain。
最後，利用上述工程菌株建立一生產高價值FDCA的共培養系統，結果顯示，當培養液酸鹼值調整至接近中性，同時提高異營菌的起始反應濃度，能使FDCA產率從39.6%增加到52%，且發現異營菌的存在能促進光合菌生長，而當菌株進行表面佈置共培養所測得的FDCA產率可再提高至59.5%。
本研究成功建立生產高價值產品FDCA的共培養系統，且表面佈置技術在共培養系統的應用中具有其潛力。

It becomes a major challenge for people to find suitable substitutes for the fossil resources in recent years. Autotrophs such as cyanobacteria and microalgae have been discussed for the ability to fix CO2 through photosynthetic metabolism and produce carbohydrates when renewable energy becomes an emerging issue. However, due to the limited production and the cost of product recovery, it seems like that if the carbohydrate can be used directly as a carbon source to support the growth of heterotrophs with production of high-value products, the overall procedure will be more economical. In this study, sucrose was exported from engineered S. elongatus PCC7942 and was utilized by engineered P. putida S12 that can oxidize HMF to form high-value product, FDCA. To this end, the sucrose transporter cscB was successfully expressed in S. elongatus PCC7942, and the maximum concentration of sucrose that accumulated in the medium was 0.54g/L in 7 days. In addition, cscA and HMFH were also successfully expressed in P. putida S12 and showed almost the same reaction efficiency as the unmodified one. In addition, the two strains were further modified with the fusion proteins SomA-3(G4S)-SH3 ligand and INP-YFP-SH3 domain on cell surface respectively. The effect of pH, cell density and cell attachment through surface modified strains on FDCA production was discussed. Results showed that the co-culture when the pH value of the culture medium was adjusted to neutral with the increased cell density of heterotrophs, the FDCA yield can increased from 39.6. % to 52%. The presence of the heterotrophs was found to promote the growth of autotrophs. In the surface modified co-culture system, the FDCA yield could be further increased to 59.5%. This study successfully established a co-culture system for the production of high-value product FDCA, and the surface display technology has its potential in the application of co-culture systems.

第一章 緒論
1.1 研究背景
1.2 研究動機與目的
1.3 研究內容
第二章 文獻回顧
2.1 光合菌醣類生產機制
2.2 蔗糖代謝蛋白
2.3 Pseudomonas putida S12
2.4 HMF生物降解路徑
2.5 光合菌Surface display
2.6 INP蛋白
2.7 蛋白質交互作用
第三章 實驗材料與方法
3.1 菌種與質體
3.2 實驗藥品
3.3 實驗器材
3.4 實驗方法
第四章 結果與討論
4.1 質體建構Plasmid construction
4.2 戀臭假單胞菌之蛋白表達與活性測試
4.3 光合菌之蛋白表達與活性測試
4.4 共培養實驗
第五章 結論
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