菌株Cupriavidus sp. L7LΔwcaJ可利用果糖酸（levulinic acid）為單一碳源進行生長與PHA累積，於胞內累積68 dry wt% 具有良好延展性的P(3-hydroxybutyrate-co-35 mol% 3-hydroxyvalerate) PHA共聚物。為進一步提升此共聚物的延展性與抗拉強度，本研究以Cupriavidus sp. L7LΔwcaJ的聚羥基烷酸酯解聚酶（phaZ）為目標進行基因剔除試驗。由Cupriavidus sp. L7LΔwcaJ的NGS結果顯示，此菌株具有至少八個可能的細胞內聚羥基烷酸酯解聚酶。與現有文獻結果比對，本研究挑選具有潛力之phaZ基因進行剔除試驗，並評估剔除後對生合成PHA的累積量、單體組成、分子量與機械性質之影響。PHA累積試驗中，插入變異株Cupriavidus sp. L7LΔwcaJΩphaZ2及剔除株Cupriavidus sp. L7LΔwcaJΔphaZ2兩者胞內皆可累積達73% 的PHA，均略高於原始菌株。插入變異株L7LΔwcaJΩphaZ2所合成的PHA共聚物其單體組成P(70 mol% 3HB-co-30 mol% 3HV)，不同於原始菌株P(65 mol% 3HB-co-35 mol% 3HV)。拉伸測試中，Cupriavidus sp. L7LΔwcaJΩphaZ2的PHA聚合物其楊氏模量（Young’s modulus）（806 MPa）、斷裂伸長度（Elongation at break）（715%）、抗拉強度（Tensile strength）（20 MPa）。皆優於原始菌株，具有較佳的延展性與較柔軟的性質，並更適合商業化應用。這些結果表明，剔除聚羥基烷酸酯解聚酶基因能提高菌株PHA累積量，並改變其機械性質。

Bacteria Cupriavidus sp. L7LΔwcaJ can use levulinic acid for growth and PHA accumulation, and accumulate 68 dry wt% with good ductility of P(3-hydroxybutyrate-co-35 mol% 3-hydroxyvalerate) PHA copolymer. In order to improve the ductility and tensile strength of PHA copolymer, this study targeted the polyhydroxyalkanoate depolymerase (phaZ) of Cupriavidus sp. L7LΔwcaJ and performed gene knockout experiments. The NGS results of Cupriavidus sp. L7LΔwcaJ has eight possible intracellular polyhydroxyalkanoate depolymerases. In this study, the potential phaZ gene was selected for the knockout test, and evaluated the effect on the PHA accumulation, monomer composition, molecular weight and mechanical properties of the experimental strain. In the PHA accumulation test, the insertion mutant Cupriavidus sp. L7LΔwcaJΩphaZ2 and the deletion strain Cupriavidus sp. L7LΔwcaJΔphaZ2 showed that the PHA accumulation can reached 73%, which was slightly higher than the original strain. The monomer composition of the PHA copolymer synthesized by the insertion mutant L7LΔwcaJΩphaZ2 was P(70 mol% 3HB-co-30 mol% 3HV), which was different from the original strain of P(65 mol% 3HB-co-35 mol% 3HV). In the tensile test, the PHA polymer of Cupriavidus sp. L7LΔwcaJΩphaZ2 shown as Young's modulus: 806 MPa, Elongation at break: 715%, Tensile strength: 20 MPa. Compared with previous studies, the PHA produced in this study has better ductility and softer properties than the PHA produced by the original strain, and more suitable for commercial development. These results indicated that deletion of the polyhydroxyalkanoate depolymerase gene phaZ2 could increase the PHA accumulation and change its mechanical properties.

摘 要 i
Abstract iii
目錄 v
表目錄 viii
圖目錄 ix
附圖目錄 x
第一章 前言 1
一、 石化塑膠與環境危害 1
二、 聚羥基烷酸酯 (polyhydroxyalkanoates, PHA) 1
1. 來源與介紹 1
2. 化學結構及物理性質 2
3. PHA的生合成路徑 3
4. PHA的應用 5
三、 聚羥基烷酸酯解聚酶（PHA depolymerase, PhaZ） 6
1. PHA的生物可降解性 6
2. PhaZ的種類及作用機制 7
3. 剔除PHA解聚酶的代謝工程 8
四、 果糖酸（levulinic acid, LA） 10
五、 Cupriavidus sp. L7LΔwcaJ 11
六、 研究目的及策略 11
1. 研究目的 11
2. 研究策略 11
第二章 研究材料與方法 12
一、 實驗菌株及培養基 12
二、 化學藥品及儀器 12
三、 使用質體之建構 13
1. 黏合作用 13
2. 轉形作用 13
3. 基因插入性破壞載體製備 13
4. 基因剔除載體製備 13
5. 互補試驗載體製備 14
四、 建構突變菌株 14
1. 基因插入性破壞菌株 14
2. 基因剔除菌株 14
五、 建構互補試驗菌株 15
六、 PHA累積試驗 15
七、 PHA累積含量及單體組成分析 15
1. 轉酯化反應 15
2. 樣品製備及氣相層析儀分析 16
八、 饋料批式醱酵 (fed-batch fermentation) 試驗 16
九、 PHA萃取及純化 17
十、 PHA聚合物相關分析 17
1. PHA聚合物熱分析 17
2. PHA聚合物結晶度的分析 17
3. PHA聚合物化學結構分析 18
4. PHA聚合物機械性質分析 18
5. PHA聚合物分子量分析 19
第三章 結果 20
一、 Cupriavidus sp. L7LΔwcaJ菌株phaZ基因比對結果 20
二、 Cupriavidus sp. L7LΔwcaJ菌株phaZ基因的插入破壞 20
三、 Cupriavidus sp. L7LΔwcaJ phaZ基因剔除株的建構 21
四、 剔除phaZ基因對Cupriavidus sp. L7LΔwcaJ累積PHA的影響 22
五、 Cupriavidus sp. L7LΔwcaJΔphaZ2的互補試驗 22
六、 插入性破壞phaZ2基因對PHA組成單體的影響 22
七、 以饋料批式醱酵生合成大量PHA聚合物 23
八、 Cupriavidus sp.L7LΔwcaJΩphaZ2 PHA聚合物之物理性質 23
1. PHA聚合物單體組成分析 23
2. P(3HB-co-3HV-co-4HV) 三元共聚物的熱性質 24
3. P(3HB-co-3HV-co-4HV) 三元共聚物的機械性能 24
九、 Cupriavidus sp. L7LΔwcaJ插入破壞及剔除phaZ基因對PHA分子量的影響 25
第四章 討論 20
結論 31
參考文獻 32


表目錄
表一、本研究所使用之菌株。 39
表二、本研究使用之質體。 40
表三、本研究使用之引子。 41
表四、描述Cupriavidus sp.L7LΔwcaJ菌株中八個可能的phaZ基因。 42
表五、 Cupriavidus sp.L7LΔwcaJ菌株中插入破壞不同的phaZ解聚酶基因對PHA累積的影響（培養48小時）。 44
表六、 Cupriavidus sp.L7LΔwcaJ菌株中插入破壞不同的phaZ 解聚酶基因對PHA累積的影響（培養72小時）。 45
表七、 Cupriavidus sp.L7LΔwcaJ菌株中剔除不同的phaZ 解聚酶基因對PHA累積的影響（培養48小時）。 46
表八、 Cupriavidus sp.L7LΔwcaJ菌株中剔除不同的phaZ 解聚酶基因對PHA累積的影響（培養72小時）。 47
表九、 Cupriavidus sp.L7LΔwcaJΔphaZ2菌株恢復phaZ2蛋白質表現對PHA累積的影響（培養48小時）。 48
表十、 Cupriavidus sp.L7LΔwcaJΩphaZ2突變株對PHA聚合物單體組成的影響。 50
表十一、Cupriavidus sp.L7LΔwcaJΔphaZ2剔除株插入破壞AACS基因對PHA聚合物單體組成的影響。 51
表十二、P(3HB-co-3HV-co-4HV) 三元共聚物的熱性質分析。 58
表十三、P(3HB-co-3HV-co-4HV) 三元共聚物的機械性質分析。 59
表十四、PHA聚合物的分子量。 60

圖目錄
圖一、phaZ基因剔除株的確認。 43
圖二、位於Cupriavidus sp.L7LΔwcaJ菌株phaZ的上游及下游基因。 49
圖三、菌株Cupriavidus sp. L7LΔwcaJΩphaZ2饋料批式醱酵槽試驗。 52
圖四、1H-NMR 分析PHA共聚物單體組成。（搖瓶累積） 53
圖五、1H-NMR 分析PHA共聚物單體組成。（醱酵試驗） 54
圖六、13C-NMR分析Cupriavidus sp. L7LΔwcaJΩphaZ2生成的PHA聚合物D值。 55
圖七、聚合物P(3HB-co-3HV-co-4HV) DSC熱分析的第一次加熱掃描。 56
圖八、聚合物P(3HB-co-3HV-co-4HV) DSC熱分析的第二次加熱掃描。 57

 
附圖目錄
附圖 一、PHA之化學結構。 2
附圖 二、PHBV共聚物。 3
附圖 三、PHA之生合成路徑。 4
附圖 四、PHA的各種應用。 6
附圖 五、PHA顆粒的物理狀態。 9
附圖 六、PHA解聚酶的降解機制：以PHB解聚酶為例子。 9
附圖 七、PHA啞鈴型試片尺寸規格。 18
附圖 八、AACS合成酶的生化反應機制。 28
附圖 九、PHA代謝途徑。 29

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