臺灣博碩士論文加值系統

Comamonas sp. L8U為台灣環境土壤中分離獲得的菌株。在pH7.2 ~ 8.0條件下均能生長與生合成polyhydroxyalkanoates (PHA)，PHA累積的最適溫度為30oC (PHA yield有0.95 g/L)。果糖酸為可再生性碳源 (renewable feedstocks)，可由纖維素於加熱、酸性條件下轉換獲得。纖維素可以由稻稈、蔗渣、麥桿…等農業廢棄物中獲得，沒有佔據農業耕作地的問題。果糖酸對一般細菌而言具有毒性，Comamonas sp. L8U可利用果糖酸為唯一碳源生長並累積高含量的聚羥基烷酸酯 (polyhydroxyalkanoates, PHA)。分析此聚合物，其單體組成為96 mol% 3-hydroxyvalerate (3HV) 以及4 mol% 3-hydroxybutyrate (3HB)。此外，Comamonas sp. L8U可於高達1.2% (以 0.8% 為最佳) 的果糖酸鈉濃度下生長。以gel permeation chromatography (GPC) 分析此聚合物的分子量約為2.88 × 106 Da，是一超高分子量 (ultra-high-molecular-mass) 的聚合物。13C-NMR光譜分析，證明此聚合物的單體組成為poly(3HB-co-3HV)。礦物鹽培養基缺亞鐵離子條件下，此菌株能產生高達乾重78% 的PHA，且幾乎無3HB單體存在。利用批式醱酵進行PHA累積實驗，發現可以產生乾重 89% 的PHA。以果糖酸為唯一碳源進行饋料 (fed-batch) 醱酵培養，經十天的培養後菌體濃度更可高達到每公升102克乾重。Comamonas sp. L8U中具有兩個pha operons (分別為 phaC1 operon、phaC2 operon)。基因剔除實驗結果顯示，剔除PHA合成酶基因phaC1後，Comamonas sp. L8U即完全失去累積PHA的能力；剔除phaC2基因則對Comamonas sp. L8U累積PHA無明顯影響。表示PhaC1為Comamonas sp. L8U合成PHA的主要PHA合成酶。剔除phaC1 operon中的phaB基因，所累積的PHA聚合物中則無3HB單體。由此可知，phaB為Comamonas sp. L8U細胞中提供3HBCoA的關鍵基因，剔除PHA operon其他基因則對PHA累積無明顯影響。為了尋找獨特的代謝路徑，利用miniTn5跳躍子基因進行隨機插入突變。目前獲得PHA累積量下降的突變株M23、M30、M52、M66，其中M23完全失去PHA的累積能力。突變株M23的miniTn5插入位置為phaC1、M30為molecular chaperone dnaK、M52為MFS transporter、M66為modulator of DNA gyrase。

Comamonas sp. L8U, a wild type bacterium isolated from soil in Taiwan. It grow and biosynthesized PHA under pH from 7.2 to 8.0. The optimal growth temperature is at 30oC (0.95 g/L of PHA yield). Levulinic acid is a renewable carbon source and can be transformed from cellulose catalyzed by acid. Cellulose is the component of agricultural wastes such as straws and bangasse. Levulinic acid is toxic for many bacteria generally. Comamonas sp. L8U accumulate high content of PHA from levulinic acid as sole carbon source. The polymer consisted of 96 mol% 3-hydroxyvalerate (3HV) and 4 mol% 3-hydroxybutyrate (3HB). In addition, Comamonas sp. L8U is able to grow from concentration 0.2% to 1.2% of levulinate (peaked at 0.8%). The molecular weight of thepolymer is about 2.88 × 106 Da analyzed by gel permeation chromatography (GPC). NMR spectrometry result exhibited the PHA structure consisted of 3HB and 3HV monomer. Comamonas sp. L8U biosyntheisized a high PHA content (78 dr wt%) and a homopolymer polyhydroxyvalerate in a ferrous ion free condition. Comamonas sp. L8U accumulated a 89 dry wt% PHA in a batch fermentation. The biomass reached 102 g/L from levulinate in a fed-batch fermentation. Comamonas sp. L8U possessed two PHA operons consisted of phaC1-phaA-phaB and phaC2-phaJ-ORF-pta-ack genes. The PhaC1 was the main PHA synthase of Comamonas sp. L8U for PHA polymerization. Deletion of phaC1 gene resulted in Comamonas sp. L8U accumulated no PHA and no influence while phaC2 gene was deleted. In addition, the phaB gene corresponds with 3-hydroxybutyryl-CoA providing. Deletion of phaB gene, Comamonas sp. L8U accumulated poly(3-hydroxyvalerate) polymer without 3HB monomer. Deletion of other genes in phaC2 operon did not impact the PHA accumulation. Insertion mutation with miniTn5 found 4 mutants accumulated respectively lower PHA content than wild type (M23, M30, M52, M66). The insertion site of the mutants M23, M30, M52, M66 was phaC1, molecular chaperone gene dnaK, MFS transporter gene and modulator of DNA gyrase, respectively.

致謝………………………………………………………………………....... 2
摘要………………………………………………………………………....... 3
目錄………………………………………………………………………....... 7
表目錄……………………………………………………………………....... 10
圖目錄……………………………………………………………………....... 11
附圖目錄……………………………………………………………………... 12
附表目錄…………………………………………………………………....... 13
壹、 前言………………………………………………………………...... 14
一、 環境汙染…………………………………………………………...... 14
二、 聚羥基烷酸酯 (polyhydroxyalkanoates，PHA)…………………… 14
1. 來源與結構……………………………………………………...…….. 14
2. PHA合成酶 (PHA synthase operon) 的分類………………............... 16
3. 生合成途徑………………………………………………………….… 17
4. 物理特性………………………………………………………………. 18
5. 特點與應用……………………………………………………………. 19
6. PHA顆粒生合成相關蛋白 (PHA granule-associated proteins)……... 21
三、 果糖酸 (levulinic acid) …………………………………………….. 26
四、 實驗目的與策略…………………………………………………….. 27
1. 實驗菌株………………………………………………………………. 27
2. 研究目的………………………………………………………………. 28
3. 研究策略………………………………………………………………. 28
貳、 材料與方法………………………………………………………….. 29
一、 菌株………………………………………………………………….. 29
二、 Comamonas sp. L8U 最適生長條件……………………………….. 29
1. 最適生長溫度………………………………………………………….. 29
2. 不同pH值的礦物鹽培養基之PHA累積試驗..………………........... 29
3. 礦物鹽培養基中不同金屬離子之PHA累積試驗……………………. 29
4. 生合成PHA培養基之碳氮比 (C/N ratio) 試驗…………………….. 30
5. 不同碳源PHA累積試驗..……………………………………………. 30
6. 果糖酸鈉濃度耐受性試驗…………………………………………….. 30
三、 批式醱酵PHA累積試驗……………………………………………. 30
四、 PHA 含量與單體組成之分析……………………………………… 31
1. PHA的轉酯化………………………………………...……………….. 31
2. 氣相層基儀之分析…………………………………………………….. 31
五、 基因剔除試驗……………………………………………………….. 32
1. 基因剔除A、B片段之建構…………………………………………….. 32
2. DNA接合作用與轉形作用……………………………………………. 33
3. 質體DNA之萃取………………………………………………………. 33
4. 接合生殖 (conjugation) ……………………………………………….. 34
5. 基因剔除 (double crossover) …………………………………………. 34
6. PHA累積試驗…………………………………………………………. 35
六、 基因互補試驗……………………………………………………….. 35
1. 建構互補菌株………………………………………………………….. 35
2. PHA累積………………………………………………………………. 35
七、 miniTn5隨意插入突變株之建構與篩選…………………………… 35
1. 突變株的建構………………………………………………………….. 35
2. PHA累積試驗…………………………………………………………. 36
3. miniTn5插入位置分析………………………………………………… 36
八、 PHA聚合物化學結構分析…………………………………………. 37
九、 PHA的萃取與純化…………………………………………………. 37
十、 PHA分子量分析……………………………………………………. 37
十一、 PHA醱酵試驗……………………………………………………... 37
參、 結果………………………………………………………………… 39
一、 不同碳源對Comamonas sp. L8U PHA累積的影響……………….. 39
二、 不同培養溫度對Comamonas sp. L8U生長與PHA累積的影響….. 39
三、 培養基pH對PHA累積的影響……………………………………... 39
四、 調整礦物鹽培養基組成對PHA累積的影響………………………. 40
五、 生合成PHA之最佳碳氮比 (C/N ratio) …………………………… 41
六、 Comamonas sp. L8U對果糖酸之耐受性…………………………... 41
七、 PHA化學結構分析…………………………………………………. 41
八、 PHA分子量分析……………..……………………………………... 42
九、 批式醱酵進行PHA累積……………………………………………. 42
十、 批式饋料醱酵進行PHA累積………………………………………. 42
十一、 果糖酸代謝路徑的探討………………………………………....... 44
十二、 以miniTn5隨機插入突變試驗搜尋果糖酸代謝關鍵基因……… 45
肆、 討論…………………………………………………………………... 47
伍、 結論…………………………………………………………………... 50
陸、 未來展望……………………………………………………………... 51
柒、 參考文獻……………………………………………………………... 52

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