衣康酸是一具有多種用途之五碳二羧酸，而生物合成化學品中面臨到的主要問題是其高成本之碳源。而在細胞代謝過程中，醋酸是一個常見的副產品，醋酸不僅在細胞代謝過程中能得到，也可以由厭氧消化工業廢水中大量取得，因此希望能以醋酸做為較便宜之替代碳源。
本研究主要是調控檸檬酸合成酶之轉譯表現量，為了最適化由醋酸轉化至衣康酸之生產。由結果來看，在中等強度表現量下可得到最高產量之衣康酸0.17 g/L，而當表現量在極端條件時，衣康酸產量反而降低或保持不變，因此證明控制關鍵酵素之表現量可以優化生產效率及產量

Itaconic acid is a five carbon dicarboxylic acid with versatile uses, one of the major setbacks that most metabolic engineered biochemicals is faced with is the high production costs contributed mostly by the carbon source. Acetate, is a chemical that is usually a byproduct of metabolic processes when there is an imbalance in diabolic activities of cells, it is also abundantly available in anaerobically digested industrial waste water, making it an appealing and cheap alternative carbon source for the production of bio-based chemicals.
This study focuses on tuning the expression levels of citrate synthase on a translation level in order to obtain a translation expression that is most optimal for producing Itaconic acid from Acetate. From results obtained, expression levels that were on a medium level range showed improved productivity titers with the highest titer measured at 0.17g/L itaconic acid and when expression levels are at the extreme ends of the spectrum titer levels decreased or remained relatively unchanged, thus proving the fact that is indeed a need to control expression levels of key enzymes in order to optimize production titer and yield.

Table of Contents
1. Introduction 6
1.1 Itaconic acid 6
1.1.1.Potential as a core chemical building block 6
1.1.3 Itaconic acid production by Aspergillus Terreus 7
1.1.4 Itaconic Acid production in Escherichia Coli 9
1.2 Alternative carbon feedstocks 10
1.2.1 Waste management 10
1.3 Acetate as an alterative carbon resource 12
1.3.1 Native Acetate assimilating pathways in E.coli 12
1.3.2 Biochemicals produced from Acetate 13
1.3.3 Antimicrobial effects of Acetate 14
1.4 Altering Ribosome binding sites to enhance production 15
2. Experiment purpose 17
3. Experiment Method 20
3.1 Chemical and reagents 20
3.2 Bacterial strains 20
3.3 Plasmids 21
3.4 Synthetic RBS design 26
3.5 Production media and cultivation condition in flask 26
3.5 Screening for Acetate assimilating pathways 27
3.6 Enzyme assay 27
4. Results and Discussion 28
4.1 Screening for acetate assimilating pathways 28
4.2 Identifying suitable strain for production 30
4.3 RBS calculations 32
4.3.1 Prediction of plasmid pPC59 RBS expression level 32
4.3.2 Synthetic RBS design 33
4.4 varied expression lead to varied productivty 34
4.5 Determing enzyme activty 36
5. Conclusion 37
Reference 39

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