衣康酸為一多用途之合成前驅物，主要由生物方式生產，因其成本接近由石化生產之類似功能前驅物-丙烯酸，故期待其取代丙烯酸之市場。再者，因應全球暖化，探討轉化二氧化碳之研究備受矚目，因此本研究的第一部份主要利用藍綠菌作為轉化平台，嘗試利用代謝工程，將二氧化碳轉化為衣康酸；然而，經過13天的生產，僅得到2.3 mg/L之衣康酸，我們猜測此現象乃歸因於前驅物的不足；因此，吾人更進一步放入CRISRPi基因組合以抑制基因icd的表現，藉此累積前驅物的量。另一方面，我們懷疑AcnB蛋白的結構，會造成前驅物烏頭酸(cis-aconitate)無法累積，因此，我們嘗試利用無法轉換烏頭酸的蛋白PrpD代替AcnB。先在大腸桿菌裡測試PrpD酵素對衣康酸生產的影響，結果顯示，相較於表現原生基因acnB生產衣康酸的5.39 mg/L，表現prpD 者產量可達前者之40倍(232 mg/L)。然而，此組合並未在藍綠菌中有類似的表現。在未來工作裡，我們將利用酵素分析，測試CRISPRi的抑制效果和CAD酵素在藍綠菌中的表現。
第二部份為衣康酸生產途徑之開發，檸檬酸循環對細菌的生長扮演重要角色，因此吾人欲開發獨立於檸檬酸循環之代謝途徑，以避免影響其生長之重要因子。在計算逆分解衣康酸代謝途徑之熱力學上可行後 (∆G0 = -1.4 kJ/mol)，吾人首先設計6組用酵素菌外生產衣康酸的實驗，最高30分鐘內得到10.4 mg/L之衣康酸。並且根據過量酵素實驗的結果，將基因PaIch-YpCcl-YpIct依序設計於質體上，表現於大腸桿菌中；經過48小時的生產，分別在添加與不添加琥珀酸的培養液中，得到濃度7.7 mg/L 和 2.07 mg/L的衣康酸。另外，為了降低總反應的自由能，我們希望找到衣康酸輔酶A脫酯酶。因此，首先我們先利用最佳組合做酵素測試，然而，僅得到1.4 mg/L衣康酸。在未來工作裡，我們可以利用測試酵素Mcl2或是利用論文中的篩選平台來篩選出衣康酸輔酶A脫酯酶。
 

Itaconic acid (IA) is a promising bio-based precursor which is expected to take place of functional similar but petroleum-based precursor, acrylic acid. On the other hand, in face of global warming, many scientists put their effort to convert CO2 into valuable product. To combine those two ideas together, in the first part of this study, we engineered cyanobacteria to turn CO2 into IA. After 13-day production, we got 2.3 mg/L IA. The low titer led us to hypothesize that the native isocitrate dehydrogenase activity (Icd) is pulling away the essential precursor. Hence, we further inserted various CRISPRi icd-knockdown systems into PCC7942. Another possibility of low IA production may be the reversibility of aconitase B (AcnB), which has trouble accumulating the key intermediate cis-aconitate. As an alternative, 2-methylcitrate dehydratase (PrpD) has high specificity toward citrate and led to 232 mg/L of IA in the E. coli production test compared to the 5.4 mg/L achieved with AcnB. However, overexpressing prpD in cyano did not help increase the production efficiency. In our future work, we are going to examine the repression level of Icd in each CRISPRi strains and confirm the activity of heterologous CAD by using in vitro enzyme assay.
The second part of this study is to reverse the IA degradation pathway. Since TCA cycle is strongly related to growth, we aim to develop a pathway that is orthogonal to TCA cycle. Hence, we sought for the possibility to reverse the IA degradation pathway. After some simple calculation, we found it should be thermodynamically favorable (∆G0 =-1.4 kJ/mol). Therefore, we bioprospected various heterologous enzymes for each step and designed in-vitro IA production experiments to first demonstrate the pathway feasibility. The best enzyme combination achieved 10.4 mg/L IA within 30 minutes. Thus we further conducted excess-enzyme experiment and based on the result, we cloned the genes in the order of PaIch-YpCcl-YpIct. After 48-hour production, 7.7 mg/L and 2.07 mg/L IA was produced in the medium with and without succinate supplied individually. To make the pathway more thermodynamic favorable (∆G0 = -26 kJ/mol), we tried exploring itaconyl-CoA thioesterase with previous best combination, and got 1.4 mg/L IA after 18 hours. In our future work, we will keep working on it by trying Mcl2 or trying our designed selection method to explore the itaconyl-CoA thioesterase.

論文指導教授推薦書、學位考試委員會審定書
Abstract
摘要
Outline
I. Literature Review
1. Introduction to itaconic acid 8
2. Introduction to alternative IA production platform
2.1 Synechococcus elongatus PCC7942
2.2.1 Biological characteristics 10
2.2.2 Cyanofactories 12
2.2 Reversed IA degradation pathway 13
3. Natural IA production pathway
3.1 IA production through Cad 15
3.2 IA production through Irg1 15
3.1 IA production through Adi + Tad1 16
4. CRISPRi system 17
5. Biochemical thermodynamics calculator-eQuilibrator 18
6. Broad substrate specificity of PrpD 19
II. Motivation and Strategies
1. Cyanobacterial IA 21
2. Reversed IA degradation pathway 23
III. Material and Methods
1. Cyanobacterial IA 24
2. Reversed IA degradation pathway 32
IV. Result and Discussion Part 1- Cyanobacterial IA
1. TCA cycle intermediates feeding test 36
2. CRISPRi (NSI) + CAD (NSII) 38
3. Cad test 42
4. PrpD test (in E.coli) 45
5. PrpD test (in PCC7942) 47
IV. Result and Discussion Part 2- Reversed IA degradation pathway
1. Thermodynamic analysis 48
2. In vitro study for IA production 49
3. In vivo study for IA production 56
V. Conclusion 57
VI. Future work Part 1-Cyanobacterial IA
1. Test of SSW067, SSW068 58
2. Enzyme assay 58
3. Further research on proposed reaction 59
VI. Future work Part 1-Reversed IA degradation pathway
1. Trying Mcl2 as itaconyl-CoA thioesterase 60
2. Selection platform for itaconyl-CoA thioesterase exploration 61
VII. Reference 64

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