由於大量使用化石燃料，造成大氣中的CO2含量大幅增加，導致全球暖化與天然資源的短缺，為全球永續發展帶來巨大的挑戰(Intergovernmental Panel on Climate Change, 2007)。藻類中的微藻CO2固定效率及生產力遠大於高等植物, 且可直接利用煙道氣之CO2，進行固碳及生產有用物質及生物燃料 (biofuel)，例如醣類、油脂等，大規模戶外養殖微藻以固定CO2被認為是減少大氣中CO2濃度的可行方法之一 (Murakami and Ikenouchi, 1997)。因此篩選出高光合固碳效率微藻藻株是有效固定CO2的開發關鍵。目前以天然篩選的藻株進行CO2固定的效率方線之研究仍然有限，且其CO2固定效率遠小於化學方法，因此利用基因工程手段提供另一種方法，除了可以提高微藻光合固碳效率外，也可藉由代謝路徑的修飾，使微藻生產大量高價值的副產物，包括糖、纖維素、酒精、氫氣、異丁醇等 (Ducat et. al., 2012)，因此藉由基因工程方式開發高效能的光合作用固碳藻株，以供後續生產利用，被認為是目前相當有潛力的方法之一。
本研究目的以基因轉殖方式建構高效能藍綠菌(Synechococcus elongatus PCC7942)之carbohydrate生產藻株，藉由提高ribulose 1,5-bisphosphate carboxylase/ oxygenase (RuBisCO)週邊CO2濃度、提高耐高溫能力，使成為適合在戶外光反應管以CO2氣體進行carbohydrate生產。研究策略為：1. 建構高光合作用效率的S. elongatus PCC7942: 以逐步轉殖CO2濃縮機轉 (CO2-concentration mechanism) 之HCO3- transporter (ictB及 BicA)、carbonic anhydrase (icfA及ecaA)，獲得具[高HCO3- 吸收能力並可將HCO3-轉換為CO2]之高光合作用效率轉殖藻株。 2. 建構耐逆境之轉殖藻株: 過量表現osmotin與hspA於高光合作用效率轉殖藻株及確認osmotin與hspA轉殖藻株逆境耐受性。 3. 共轉殖HCO3- transporter (ictB)、carbonic anhydrase (ecaA)之相關基因於纖維素合成轉殖藻株以生產carbohydrate。
結果發現與野生型相比，培養於添加NaHCO3培養基和通入5% CO2時，HCO3- transporter (ictB、BicA) 與carbonic anhydrase (icfA、ecaA)轉殖細胞光合速率及生物量明顯增加。藍綠菌S. elongatus PCC7942表現逆境耐受性相關基因（osmotin、 hspA）後，也提高了藻株的耐逆境能力，使其可在戶外利用海水培養，而產生的碳水化合物可做為生物精煉之原料。在藍綠菌S. elongatus PCC7942中表現細菌纖維素合成之acsAB基因,可有效提高藍綠菌之碳水化合物, 同時在細胞中表現HCO3- transporter與carbonic anhydrase的相關基因ictB、ecaA後，可以促進光合作用和細胞生長的速率，並提高其生物量和碳水化合物產率。
本研究預期效益為創造可以在戶外高溫環境生長快速及生產carbohydrate之高光合作用效率轉殖藍綠菌，降低養殖微藻固定CO2及生產生物燃料之成本，克服微藻生質能工業化生產之關鍵。

An increase in atmospheric CO2 and fuel shortage resulted from the overuse of fossil fuel pose tremendous challenges to global sustainability (Intergovernmental Panel on Climate Change, 2007). Microalgae have much higher CO2 fixation rates and productivity than higher plants. They can directly use CO2 from flue gas, and convert it into biomass, which then be used to produce valuable products and biofuels, such as sugars, lipids, etc. Therefore, CO2 fixation by outdoor cultivation of microalgae has been considered as one of the feasible approaches for CO2 mitigation and biofuel production (Murakami and Ikenouchi, 1997). While the CO2 fixation efficiency of microalgae isolated from natural environment is still low compared to that of the chemical fixation methods, using genetic engineering of microalgae to achieve high photosynthetic efficiency and greater ability to convert CO2 to high value products, such as sugar, cellulose, ethanol, H2, and isobutunol (Ducat et. al., 2012) will be a promising approach for CO2 bio-fixation. Therefore, developing microalgae with high photosynthetic efficiency is a viable approach to mitigate biological CO2 and achieve sustainable biofuels production.
The objective of this study is to construct efficient carbohydrate producing cyanobacteria (Synechococcus elongatus PCC7942) with the ability to grow under outdoor conditions in high CO2 concentration and heat, by genetic engineering. This objective will be accomplished through the following approaches: 1. Developing S.elongatus PCC7942 with high photosynthetic efficiency: CO2 Concentrating Mechanism (CCM) genetically engineered cyanobacteria are constructed by co-expression of bicarbonate transporter (ictB, BicA), and carbonic anhydrase (ecaA, icfA) in S. elongatus PCC7942. 2. Construction of stresses tolerance transgenic strains by expressing osmotin and hspA in S. elongatus PCC7942. 3. Construction of high CO2 fixing transgenic strains with production of carbohydrate by co-expressing HCO3- transporter (ictB)、carbonic anhydrase (ecaA) in cellulose synthesis (acsAB) S. elongatus strains.
Compared with the wild type, overexpressing HCO3- transporter (ictB, BicA), carbonic anhydrase (icfA、ecaA) significantly increased cell growth and photosynthesis rates, when the cells grown in 5% CO2, in medium supplemented with NaHCO3. The expression of the partial cellulose synthase genes, acsAB, improved carbohydrate productivity of S. elongatus PCC7942. The expression of hspA and osmotin conferred tolerances to multiple stresses encountered during outdoor cultivation and both maximum biomass and glucose productivities by S. elongatus PCC7942 transformed cells in closed outdoor photobioreactor with sea water were improved, the carbohydrates can be used as bio-refinery feedstock. Co-expressing HCO3- transporter (ictB) , carbonic anhydrase (ecaA), and bacterial cellulose (acsAB) significantly improved its biomass productivity and total carbohydrate productivity.
The expected result of this study is to obtain high CO2 fixing cyanobacteria that can tolerate the high temperature of outdoor cultivation for carbohydrate production. It will certainly reduce the cost of microalgae cultivation and biofuel production.

Verification letter from the Oral Examination Committee i
Acknowledgements ii
Abstract in Chinese iii
Abstract in English v
Contents vii
List of Figures xii
List of Tables xv
Chapter 1. Introduction 1
1.1. General background 1
1.2. Aim of this study 6
1.3. References 9
Chapter 2. Enhancement of photosynthetic efficiency and biomass by overexpressing bicarbonate transporters and carbonic anhydrase in synechococcus elongatus PCC7942 13
2.1 Abstract 13
2.2 Introduction 14
2.3 Materials and methods 17
2.3.1 Bacterial strains 17
2.3.2 Growth conditions 17
2.3.3 Vector construction 17
2.3.4 Transformation of cyanobacteria cells and selection of positive clones 18
2.3.5 RNA isolation and RT-PCR 19
2.3.6 Protein extraction 19
2.3.7 Electrophoresis and Immunoblotting 20
2.3.8 Growth of cyanobacteria under 5% CO2 in air 20
2.3.9 Biomass Quantification 21
2.3.10 Determination of growth biomass productivity 21
2.3.11 Determination of CO2 fixation rate 21
2.3.12 Measurements of photosynthetic oxygen evolution 22
2.3.13 Determination of chlorophyll a contents 22
2.3.14 Determination of carbon content 23
2.3.15 Statistical analysis 23
2.4 Results 24
2.4.1 Genetic engineering of cyanobacteria S. elongatus PCC7942 with over-expression of GFP, ictB, BicA, ecaA, and icfA 24
2.4.2 Investigation of rbcL promoter activity in S. elongatus PCC7942 grown under different levels of light intensities and CO2 concentrations 24
2.4.3 Physiological and biochemical characterization of bicarbonate transporter ictB and BicA transgenic strains of S. elongatus PCC7942 26
2.4.3.1 Construction and analysis of bicarbonate transporter ictB and BicA transgenic strains 26
2.4.3.2 Evaluation of the effects of overexpresson of ictB and BicA on photosynthesis efficiency, growth performance and biomass production in 5% CO2 26
2.4.4 Physiological and biochemical characterization of carbonic anhydrase ecaA and icfA transgenic strains of S. elongatus PCC7942 28
2.4.4.1 Construction and analysis of carbonic anhydrase ecaA and icfA transgenic strains 28
2.4.4.2 Evaluation of the effects of overexpresson of ecaA and icfA on photosynthesis efficiency, growth performance and biomass production in 5% CO2 28
2.5 Discussion 30
2.6 Conclusions 32
2.7 References 33
Chapter 3. Enhancement of outdoor seawater culture efficiency in a freshwater cyanobacterium by heterologous expression of hspA and osmotin genes 57
3.1. Abstract 57
3.2. Introduction 58
3.3. Materials and Methods 61
3.3.1 Bacterial strains 61
3.3.2 Plasmids and strains construction 61
3.3.3 Growth conditions 62
3.3.4 RNA isolation and RT-PCR 62
3.3.5 Growth of transgenic strains under high temperature conditions 63
3.3.6 Growth of transgenic strains under high light conditions 63
3.3.7 Growth of transgenic strains under different salt concentration 63
3.3.8 Growth of transgenic strains in closed photobioreactor with seawater under outdoor cultures conditions 64
3.3.9 Determination of chlorophyll a contents 64
3.3.10 Measurements of photosynthetic oxygen evolution 65
3.3.11 Determination of glucose content of S. elongatus PCC7942 transgenic strains 65
3.3.12 Statistical analysis 66
3.4. Results 67
3.4.1 Construction and analysis of osmotin and hspA expression strain of S. elongatus PCC7942 67
3.4.2 Effects of overexpression hspA and osmotin genes on cell growth, photosynthesis rates of S. elongatus PCC7942 68
3.4.3 Effect of high temperature on cell growth, photosynthesis rates of S. elongatus PCC7942 transgenic strains 69
3.4.4 Effect of high light on cell growth, photosynthesis rates of S. elongatus PCC7942 transgenic strains 69
3.4.5 Effects of salt stress on cell growth and photosynthesis rates of S. elongatus PCC7942 transgenic strains 70
3.4.6 Increased outdoor photobioreactor culture efficiency in seawater by overexpression of hspA and osmotin genes 71
3.5. Discussion 74
3.6. Conclusions 78
3.7. References 79
Chapter 4. Co-expressing ictB, ecaA, and acsAB and stress tolerance genes in Synechococcus elongatus, to increase production of carbohydrate and improved outdoor culture efficiency 106
4.1. Abstract 106
4.2. Introduction 107
4.3. Materials and Methods 110
4.3.1 Bacterial strains 110
4.3.2 Growth conditions 110
4.3.3 Plasmids and strains construction 110
4.3.4 RNA isolation and RT-PCR 112
4.3.5 Measurements of photosynthetic oxygen evolution 112
4.3.6 Determination of carbohydrate content 112
4.3.7 Growth of transgenic strains in closed photobioreactor under outdoor cultures conditions 113
4.3.8 Statistical analysis 113
4.4. Results 114
4.4.1 Analysis of acsAB over-expression strain of S. elongatus PCC7942 114
4.4.2 Construction of S. elongatus PCC7942 strains co-expressing ictB, ecaA, and acsAB 115
4.4.3 Effect of co-expression of ictB, ecaA, and acsAB on cell growth and photosynthesis rates of S. elongatus PCC7942 116
4.4.4 Effect of co-expression of ictB, ecaA, and acsAB on carbohydrate productivity of S. elongatus PCC7942 117
4.4.5 Effect of co-expression of hspA or osmotin on outdoor photobioreactor culture efficiency in the ictB, ecaA, and acsAB co-expressing strain of S. elongatus PCC7942 118
4.5. Discussion 120
4.6. Conclusions 122
4.7. References 123
Chapter 5. Conclusions 143

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