生質柴油是一種新興的替代能源，能夠取代石化柴油的燃料，其原料可來自含有高含量三酸甘油脂的植物油或廢棄生物質，藉由轉酯化反應產生單烷基酯長鏈脂肪酸與短鏈醇，如脂肪酸甲基酯。在最近幾年應用基因工程微藻生產生質柴油受到許多注目。脂肪酸合成是先將acetyl-CoA轉變成malonyl-CoA，這個反應由acetyl-CoA carboxylase (ACCase)的催化。因此ACCase往往被視為脂肪酸合成途徑中的關鍵酵素。根據這論點，我們希望增加ACCase的表現以達到增加細胞內脂質的含量，進一部增加生質柴油的量產。藍綠菌 (Synechocystis sp. PCC 6803)屬於藍綠菌的一種，因為其易於培養的生長特性，成為生質能源的首選。然而，現今沒有適用於藍綠藻的穿梭載體可用；因此本論文主要包括兩大方向：(1) 構築ACCase至表現載體中。(2) 設計和建構大腸桿菌與藍綠菌適合的穿梭載體，以利將來用於基因選殖和功能分析。研究結果顯示在大量表現ACCase時，細胞的脂質組成有所改變，改變的差異可以當作是否採用增加細胞ACCase來增加細胞內的脂質作為生質柴油研發的策略。而穿梭載體部分，我們將構築好的穿梭載體送入藍綠菌中，觀察穿梭載體是否能保留在菌體內，同時我們也測試出藍綠菌對ampicillin耐受性。 Biodiesel is an alternative energy source and a substitute for petrochemical diesel fuels. Transferification of triacylglycerols from plant oil or waste biomass yields monoalky esters of long-chain fatty acids with short chain alcohol such as fatty acid methyl esters. More attention has recently focused on the application of genetically engineered microalgae in the production of biodiesel. The reaction of acetyl-CoA to malonly-CoA is catalyzed by acetyl-CoA carboxylase (ACCase). ACCase is often regarded as the first committed step of the fatty acid synthetic pathway. My aim is to increase the lipid content of the cell through enhancing the expression of ACCase. Cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis) is one of the best-characterized microalgae species and, given its robust growth characteristic, becomes the organism of choice for applications. Detailed studies on techniques for genetic manipulation of this species are available. Unfortunately, no suitable vector for cyanobacteria is courrently available. Therefore, the thesis has two main aims: (1) cloning ACCase on expression vector. (2) constructing a E.coli / Synechocystis suitable shuttle vector for efficient gene cloning and functional assays. My results show that overexpression of ACCase increases the lipid content of the cells. In addition, the shuttle vector constructed was transformed into Synechocystis for testing. As it turned out, it conferred antibiotic resistance to the cell and could be recovered as a plasmid form.
