近來透過轉化生質原料而成的可再生材料愈來愈受到注意。衣康酸具有雙官能基以及ㄧ個不飽和雙鍵使其在高分子合成領域有相當多的應用。因為結構的相似性，衣康酸也被視為由石油衍生而來之丙烯酸之替代平台化合物。
大腸桿菌能夠有效生產衣康酸，其中檸檬酸循環的抑制被視為關鍵之代謝調控策略之一。然而，伴隨而來的是大腸桿菌會喪失內源合成穀胺酸(glutamate)之能力，導致在基礎培養基中需要額外補充穀胺酸。藉由引入Burkholderia xenovorans之非磷酸化代謝途徑於大腸桿菌中，使菌株能夠自行轉化合成alpha酮戊二酸，在不額外補充營養源的情況下，營養缺陷株能夠於36小時生長至細胞濃度達OD600 nm 0.296。透過基因組合的優化，更能將細胞生長情形優化至OD600 nm 1.62。同時利用甘油及木糖能夠提升衣康酸產量以及優化細胞生長。藉由剔除非磷酸化代謝途徑之競爭途徑，衣康酸產量可以在24小時達1.2 g/L。降低木糖於培養基中之起始濃度，能夠提升衣康酸產量在24小時達2.2 g/L IA。利用1公升生物反應器進行發酵製程放大，在基礎培養基中可以於85小時達到20.01 g/L IA、0.62 (g IA/ g glycerol) 轉化率以及0.24 g/L/hr產率。

Nowadays, increasing attention is being focused on the conversion of biomass into renewable products such as itaconic acid (IA). IA serves as a renewable alternative for petroleum-based acrylic acid, is an unsaturated dicarboxylic acid which can be utilized as a platform chemical to further synthesize various value-added chemicals and material in polymer industry.
TCA cycle disruption is essential for efficient IA production in E. coli following by the additional nutrient supply which accounting for glutamate auxotrophy. Recruiting nonphosphorylative pathway to efficiently convert xylose into α-ketoglutarate (α-KG), hence, became an alternative to circumvent additional production cost. Via applying nonphosphorylative pathway genes from Burkholderia xenovorans, we rescued the auxotroph growth to OD600 nm 0.296 (36 hrs) and further optimized it to OD600 nm 1.62 (36 hrs). Co-utilizing glycerol and xylose enhances both IA titers and cell growth. Supplement of ammonium salts prolonged IA production and achieved higher cell density. By deletion of competing pathways of nonphosphorylative metabolism, we achieved 1.2 g/L IA production (24 hrs). Lowering initial xylose concentration could increase IA production to 2.2 g/L IA (24 hrs). Scaling up IA production in a 1L fed-batch bioreactor fermentation, we achieved 20.01 g/L IA (85 hrs) with yield 0.62 (g IA/ g glycerol) and productivity 0.24 g/L/hr in minimal media .

Outline

I. Introduction 1
1. Introduction of itaconic acid 1
2. Bio-based itaconic acid production 3
3. IA production by recombinant E. coli 4
4. Solutions to glutamate auxotrophy 7
5. Lignocellulosic Biomass utilization 9
II. Motivation and strategy 13
III. Materials and methods 14
1. Chemicals and reagents 14
2. DNA manipulation 14
3. Culture medium and conditions 14
4. Chemicals analysis 16
5. IA production using 1L fed-batch bioreactor 17
6. Strains and primers table 18
IV. Results and Discussion 21
1. Proposed pathway which decoupling IA production from α-KG
biosynthesis 21
2. IA production pathway optimized in icd inactivated E. coli 23
3. Constructing nonphosphorylative pathway in Δ icd E. coli 25
4. Determination of IA production cultivation condition 28
5. Preliminary IA production in 1 L Bioreactor 31
6. Strategies for directing carbon flux into IA production 37
7. Scaling up IA production in 1 L bioreactor 40
V. Conclusion 42
VI. Reference 44

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