食油假單胞菌聚羥基脂肪酸酯之脂質生化合成反應路徑研究

不能降解的石化塑膠廢棄物的增加是一個日益嚴重的環境問題。聚羥基脂肪酸酯（Polyhydroxyalkanoates, PHAs）稱為生物塑膠，是微生物由糖或脂質自然發酵所得的線性聚脂產物，合成來源的糖或脂肪取得容易，亦可藉由環境菌進行生物代謝、分解為無毒的基本元素返回自然界，以減少環境汙染問題，同時具再生性與永續性。而利用合成生物學的基因調控技術來控制特定結構PHA的生產，正是生物塑膠的優勢。已知微生物合成PHA所需的逆境為碳源豐富而缺乏氮、磷、鎂、鉀等營養，但如此物料逆境可能會造成將來量產PHA的限制。因此，本研究在探討環境菌處於營養條件下，如何調整環境菌合成PHA基因來增加PHA產量。食油假單胞菌已被證實能合成中長鏈PHA，本研究以即時定量聚合酶連鎖反應技術測量在使用富含碳、氮的NB培養基下，比較不加、或添加5%葡萄糖、或5%甘油三種不同生長條件下，phaC1、phaC2、phaZ、phaG以及phaJ等五個基因的相對表現量。在預設為40個循環反應下，phaZ基因的RNA表達量明顯高於其餘四個基因。當黏合溫度同為55℃時，產生16S rRNA Ct值過小，待測基因phaC1、phaC2、phaG 以及phaJ四個基因Ct值過大的問題，利用黏合溫度改變後，16S rRNA 在65℃以及待測基因在50℃或45℃下，Ct值即可陸續落於10－35的可信範圍之間。以△△Ct值經公式回推基因拷貝數，得到在合成PHA時phaC1與phaC2基因都會出現，而脂肪酸生物合成路徑代表基因phaG沒有明顯的表達、β-氧化路徑代表基因phaJ在添加甘油的生長條件下以黏合溫度45℃檢測，RNA表達量明顯。本研究得到，食油假單胞菌PHA合成反應路徑，偏向於β-氧化路徑而較不偏好脂肪酸生物合成路徑，藉此建立了PHA合成路徑監測基礎平台，未來可應用於控制PHA產量以及調控路徑的研究。

關鍵字

食油假單胞菌；聚羥基脂肪酸酯；即時定量聚合酶連鎖反應

並列摘要

The accumulation of nondegradable petroleum plastic waste has become one of the most pressing environmental problems. Polyhydroxyalkanoates (PHAs), also known as bioplastics, are renewable and sustainable natural linear polyesters fermented from readily available sugars or lipids by environmental microbes, and decomposed to non-toxic natural elements. One of the promising advantages of bioplastics is to regulate PHA related genes using synthetic biology techniques to control the structure and production of PHA. It was known that PHA production requires a stress condition of rich in carbon and lack of nitrogen, phosphorus, magnesium, potassium and other nutrients, but limited nutrient resources might hinder mass production of PHA. Therefore in this study, the possibility of increasing PHA synthesis by controlling PHA related genes in bacteria grown in rich media were discussed. Pseudomonas oleovorans has been shown capable of synthesizing MCL-PHA. In this study, quantitative realtime polymerase chain reaction technology was used to measure the transcription expression of phaC1, phaC2, phaZ, phaG and phaJ genes on carbon-rich NB medium only, with 5% dextrose, or with 5% glycerol of three different growth conditions. In the default 40 cycles set, significantly over-expression was found in phaZ as compared to the rest of the four genes. When the annealing temperature is set to be the same at 55℃, a problem was found that the too small Ct value for control 16S rRNA and too large Ct value for the tests of phaC1, phaC2, phaG and phaJ genes. The optimal reliable range of Ct values of 10-35 were obtained by changing annealing temperature in 16S rRNA gene at 65℃ and the tested genes at 50℃ or 45℃.The △Ct values from the above were subject to the transition formula to obtain the copy numbers of individual genes. The results showed that the PHA synthase phaC1 and phaC2 genes co-expressed of similar amounts in similar conditions, while phaG gene representing the de novo fatty acid biosynthesis pathway showed no significant expression, and the phaJ gene representing theβ-oxidation pathway showed stronger expression in glycerol added conditions as detected under 45℃ annealing temperature. These suggest that Pseudomonas oleovorans favors the β-oxidation reaction pathway over the novo fatty acid biosynthesis pathway. Taken together, a basic qPCR platform was established to monitor genes of PHA synthesis pathways, which might be applicable in controlling the structure and the yield of PHA production and regulating PHA biosynthetic pathways in future researches.

並列關鍵字

Pseudomonas oleovorans ； Polyhydroxyalkanoates ； PHA ； quantitative real-time PCR ； qPCR

國際替代計量

食油假單胞菌聚羥基脂肪酸酯之脂質生化合成反應路徑研究

全文下載

主題瀏覽