藍綠菌現今常被用來做為生產生質燃料或生質化學品的生物工廠。但是藍綠菌如Synechococcus elongatus PCC 7492常常在基因工程中遇到目標基因無法穩定的問題。同時CRISPR-Cas9系統是現今最新開發出來經由RNA引導的基因體編輯技術，但該系統尚未有任何文獻報告應用於藍綠菌中。在本研究中我們證明了CRISPR-Cas9系統可以有效地對於PCC 7492中的染色體進行雙股斷裂 (DSB)並且使細菌死亡。然而當我們同時共轉型CRISPR-Cas9系統含有基因組及同源臂之模板質體時，CRISPR-Cas9系統造成的雙股斷裂可以使外源基因精確的嵌入目標染色體位置。在增進同源互換效率的同時也可以減少模板DNA的劑量並減少同源臂的長度。此外CRISPR-Cas9造成的DSB也可做為篩選工具來增進外源基因組嵌入PCC7942所有染色體的機會。因此可以加速篩選出具有同質且穩定之重組染色體菌株。我們接著進一步探討利用CRISPR-Cas9系統對藍綠菌進行代謝途徑的調控。首先將glgc基因剔除同時將gltA以及ppc基因嵌入，藉此增進琥珀酸的產量達到707.0 g/L/OD730，相較野生型的琥珀酸產量提升了接近11倍。這些結果表示CRISPR-Cas9系統可以成功地修改並調控藍綠菌之的代謝途徑。接著第二部分我們進一步探討是否能利用CRISPR-Cas9系統對藍綠菌進行代謝途徑的調控促進2,3-丁二醇(2,3-BDO)在藍綠菌中之產量。在本研究中我們將分為三個階段做探討：(1)希望從許多不同來源菌株的Cas9蛋白中找到合適可替代之前研究所用的Cas9蛋白(SpCas9)，令後續使用CRISPRi系統時不會出現受到無預期干擾的抑制結果。(2)以找出之替代Cas9系統在藍綠菌中建構2,3-BDO代謝途徑生產2,3-BDO。(3)以CRISPRi library的概念進行CRISPRi抑制代謝途徑來優化2,3-BDO產量。最後結果我們發現利用SaCas9可高效將CRISPRi系統 (含SpdCas9與sgRNA兩外源基因組) 嵌入藍綠菌染色體中並利用CRISPRi library的概念篩選出fbp、sps、ppc與pdh基因對於2,3-BDO生產的影響。最後經過基因調控我們將2,3-BDO產量由818.4 mg/l提升至1583.8 mg/l (約提升93.5%)。這表示利用CRISPRi library系統可用於更有效率篩選出調控細菌代謝路徑的關鍵基因，方便我們後續更有效的調控並生產各種生質能源/生質化學品。

Cyanobacteria hold promise as a cell factory for producing biofuels and bio-derived chemicals, yet genome engineering of cyanobacteria such as Synechococcus elongatus PCC 7492 poses challenges because of their oligoploidy nature and long-term instability of the introduced gene. CRISPR-Cas9 is a newly developed RNA-guided genome editing system, yet its application for cyanobacteria engineering has yet to be reported. Here we demonstrated that CRISPR-Cas9 system can effectively trigger programmable double strand break (DSB) at the chromosome of PCC 7492 and provoke cell death. With the co-transformation of template plasmid harboring the gene cassette and flanking homology arms, CRISPR-Cas9-mediated DSB enabled precise gene integration, ameliorated the homologous recombination efficiency and allowed the use of lower amount of template DNA and shorter homology arms. The CRISPR-Cas9-induced cell death imposed selective pressure and enhanced the chance of concomitant integration of gene cassettes into all chromosomes of PCC 7942, hence accelerating the process of obtaining homogeneous and stable recombinant strains. We further explored the feasibility of engineering cyanobacteria by CRISPR-Cas9-assisted simultaneous glgc knock-out and gltA/ppc knock-in, which improved the succinate titer to 707.0g/l/OD730, a 11-fold increase when compared with that of the wild-type cells. These data altogether justify the use of CRISPR-Cas9 for genome engineering and manipulation of metabolic pathways in cyanobacteria. In second part, we try to regulate the pathway of cyanobacteria (gene fbp, sps, ppc and pdh was be regularated) to enhance the titer of 2,3-BDO by using CRISPR-Cas9 assiocited system. There are three stages: (1) Finding the altinative Cas9 system that can work correctly with dCas9 system. (2) Using alternative Cas9 system to construct 2,3-BDO metabolic pathways and produce 2,3-BDO in cyanobacteria. (3) Interference the metabolic pathway to optimize 2,3-BDO production and analysis the interaction of the pathway by CRISPRi library system. The result showed the SaCas9 can instead SpCas9 to improve the homologous recombination efficiency and integrate CRISPRi into the genome. Meanwhile, we found the regulation of these 4 genes lead to the different changing of the 2,3-BDO yield (such as enhance, decrease or no effect) and finally enhance the titer of 2,3-BDO up to 93.5% (from 818.4 mg/l to 1583.8 mg/l). This system can be used to screen the key genes that can regulate the pathways of bacterial metabolism and improve the production of various bioenergy / biochemical.

摘要
Abstract
目錄
圖目錄
表目錄
第一章 緒論------------------------------------------1
第二章 文獻回顧---------------------------------------2
2-1藍綠菌S. elongatus PCC7942簡介--------------------2
2-2 CRISPR相關技術介紹--------------------------------3
2-2-1 CRISPR基因編輯系統------------------------------3
2-2-2 CRISPRi系統之簡介------------------------------4
2-2-3 CRISPR及CRISPRi系統於藍綠菌之應用---------------5
2-2-4 CRISPR library之簡介---------------------------5
2-3琥珀酸介紹----------------------------------------6
2-4 2,3-丁二醇介紹-----------------------------------7
2-5 研究動機-----------------------------------------8
第三章 實驗材料及方法--------------------------------14
3-1建構質體方法-------------------------------------14
3-1-1模板質體與CRISPR質體建構------------------------14
3-1-2同源臂更換-------------------------------------14
3-1-3三種CRISPR系統質體之建構------------------------17
3-1-4含有2,3-BDO代謝途徑必須基因同源互換模版質體及含有dCas9蛋白基因同源互換模板質體之建構--------------------------------------18
3-2 Synechococcus elongatus PCC 7942---------------19
3-2-1 Synechococcus elongatus PCC 7942培養條件------19
3-2-2 Synechococcus elongatus PCC 7942質體轉型------20
3-3 CRISPR相關分析----------------------------------21
3-3-1 Synechococcus elongatus PCC 7942細胞死亡分析--21
3-3-2 Synechococcus elongatus PCC 7942細胞同源互換效率分析--------21
3-3-3 不同CRISPR蛋白對於Synechococcus elongatus PCC 7942細胞是否造成毒性之分析----------------------------------------------22
3-3-4以CRISPR-Cas9系統對細長聚球藻PCC7942進行代謝工程--------------22
3-3-5不同CRISPR系統間交互干擾之探討------------------22
3-3-6 CRISPRi library 設計--------------------------23
3-4其他分析方法-------------------------------------24
3-4-1以colony PCR驗證同源重組至染色體正確位置---------24
3-4-2即時偵測同步定量反轉錄聚合酶連鎖反應(qRT-PCR)----26
3-4-3胞內肝醣定量分析(Quantification of glycogen)---28
3-4-4琥珀酸產量分析(Succinate analysis)-------------29
3-4-5以GC-BID分析2,3-BDO產量------------------------29
3-4-6 F6P、蔗糖、草醯乙酸與乙醯輔酶A分析--------------30
3-4-7統計分析---------------------------------------31
第四章 實驗結果與討論(I) 利用CRISPR/Cas9技術在藍綠菌中以基因工程生產琥珀酸 ---------------------------------------------------49
4-1 CRISPR-Cas9 在細長聚球藻PCC 7942中可造成DSB導致細胞死亡--------49
4-2由CRISPR造成之DSB可促進同源互換的效率-------------50
4-3經由改變轉型質體的劑量來優化同源互換效率-----------51
4-4 CRISPR-Cas9系統可促進染色體隔離篩選效率----------53
4-5以CRISPR-Cas9系統對細長聚球藻PCC7942進行代謝工程來生產琥珀酸-----55
4-6討論--------------------------------------------56
第五章 實驗結果與討論(II) 在藍綠菌中建立CRISPRi Library平台以篩選分析多基因對於目標代謝網路的影響並優化生產----------------------71
5-1 測試各CRISPR系統在藍綠菌中造成DNA雙股斷裂效率-----71
5-2 測試各CRISPR系統在藍綠菌中同源互換促進效率--------73
5-3 利用CRISPRi library調控基因表現並優化2,3-BDO產量--------------74
5-4 經CRISPRi library調控後四基因表現與副產物產量變化對2,3-BDO產量影響 ---------------------------------------------------79
5-5 經由統計分析四基因表現量與2,3-BDO產量之關係並優化培養條件進一步提升2,3-BDO產量--------------------------------------------80
5-6 討論-------------------------------------------82
第六章 未來展望-------------------------------------97
第七章 參考文獻-------------------------------------99

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