建構藍綠菌的脂質分泌系統

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Title:	建構藍綠菌的脂質分泌系統
Authors:	林思婷;Lin,Szu-ting
Contributors:	生命科學系
Keywords:	光合作用;生質柴油;脂質分泌;藍綠菌
Date:	2013-01-17
Issue Date:	2013-03-25 15:52:55 (UTC+8)
Publisher:	國立中央大學
Abstract:	由於近年石油需求量增加及開採枯竭的預期心理，造成能源物質價格不斷的上揚，加上傳統石化燃料會造成環境汙染嚴重，所引發議題引人關注。因此不斷的尋求可替代之能源，例如可再生的生物燃料，目前較注重利用微藻類的基因工程技術，尤其是單細胞原核藻類的開發，因比起其他多細胞藻類，實驗操作較單純容易，且與植物一樣能利用太陽能和將二氧化碳轉化成燃料，一方面可減少空氣中二氧化碳，另一方面，它們的生長不需要廣大的耕地等等優點，使得微藻的生質能源研究備受重視。但因目前生產的製程必須打破微藻才可從中提取細胞內脂質，不可避免地消耗大量的能源以及增加生產生質柴油的成本時間。所以我們想要跳過此耗時費能的破菌步驟，直接得到油脂，減少成本消耗。我們選擇了藍綠菌Synechocystis sp. PCC 6803作為模式生物，嘗試尋找如何在生物燃料生產中使用容易回收脂質的方法。我們的策略是建構一個有效的的脂肪酸分泌平台，在Synechocystis sp. PCC 6803。表現脂肪酸的載體蛋白，通道蛋白或轉運蛋白，促使脂肪酸被運送至細胞外，如此一來，即可以藉由培養液的萃取直接得到油脂。其不僅可以降低生物柴油的成本跳過破菌的過程，而且也有助於一貫作業式的生物製程的發展。因此，此實驗設計的重點在於分泌脂肪酸，而不是增加微藻細胞內脂質含量，以此達到細胞能不斷繼代培養及可連續由細胞外獲得原料，從而建立一個並行的生產生質柴油的製程。結果發現只有轉型Arabidopsis thaliana Temperature-induced-lipocalin (AtTIL) 及 Synechocystis sp. PCC 6803 Lipid-transfer proteins (SpLTP)的轉型株具有較明顯的脂質分泌現象。Concerns about petroleum supplies, high energy cost, and environmental pollutions are driving scientists to find renewable biofuels such as bioethanol, biodiesel, and biogas. Recently, more attention has been focused on the application of genetically engineered algae in the massive production of biodiesels. Photosynthetic microorganisms, such as microalgae and cyanobacteria, are excellent organisms for biofuel production. Their genomes are relatively small and therefore are easy to manipulate. They are efficient at converting solar energy and recycling CO2 into fuels. In particular, unlike many energy crops, they can be grown on non-arable land. One down side in using microalgae as bioenergy resources is that, an energy-consuming step is required to break down the cell wall of microalgae to extract the intracellular lipids, which would inevitably increase the cost and time of producing biodiesel. We have selected Cyanobacterium Synechocystis sp. PCC 6803 as a model organism to develop methods for easy recovery of lipids for biofuel production. Our strategy is to construct an efficient fatty acid secretion system using the cyanobacterium Synechocystis sp. PCC6803 as the platform. The targets of interest include fatty acid carrier proteins, channel proteins or transporters The secreted fatty acids can then be filtered or absorbed by filters. This strategy can not only reduce the time and cost of making biodiesel by skipping the breaking-down step, but also aid in the development of a consolidated bioprocess. Hence, this project does not aim to increase the intracellular lipid content of the microalgae, but instead to facilitate the secretion of fatty acids to the medium and consequently to establish a consolidated bioprocess for biodiesel production. FFA secretion was observed and determined by GC, our data have shown that introducing extra Arabidopsis thaliana Temperature-induced-lipocalin (AtTIL) and Synechocystis sp. PCC 6803 Lipid-transfer proteins (SpLTP) into Synechocystis sp. PCC 6803 have more secretion efficiency.
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