過表現 LRO1 基因對於Yarrowia lipolytica Po1g生長速率與脂質產量之影響

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研究生：	張雅惠 Ya-hui Zhang
論文名稱：	過表現 LRO1 基因對於Yarrowia lipolytica Po1g生長速率與脂質產量之影響 Effects of over-expressing LOR1 gene in Yarrowia lipolytica Po1g on enhancing its growth rate and lipid production
指導教授：	朱義旭 Yi-Hsu Ju 蔡伸隆 Shen-Long Tsai
口試委員:	Suryadi Ismadji Suryadi Ismadji
學位類別：	碩士 Master
系所名稱：	工程學院 - 化學工程系 Department of Chemical Engineering
論文出版年：	2014
畢業學年度：	102
語文別：	英文
論文頁數：	80
中文關鍵詞：	hp4d啟動子、TEFintron啟動子、磷脂甘油二酯醯基轉移酶、耶氏解脂酵母
外文關鍵詞：	hp4d promoter, TEFintron promoter, LRO1, Yarrowia lipolytica
相關次數：	點閱：216 下載：3
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上一筆

生質柴油被視為是一種可再生的替代能源，生產生質柴油的商業原料主要是植物和/或動物脂肪。微生物的單一細胞油是生產生質柴油一種有潛力的原料。在眾多微生物中，含油酵母菌Yarrowia lipolytica是生產單一細胞油的一種有潛力的選擇，它能將脂質儲存在脂質體（LB）內，而其脂質主要由三酸甘油酯(TAGs)和甾醇酯（SEs）組成。三酸甘油酯合成的最終步驟是藉由二醯基甘油醯基轉移酶（DGA1）或磷脂甘油二酯醯基轉移酶（LRO1）醯化二醯基甘油酯（DAG）而生產三酸甘油酯。在本研究中，選用LRO1基因以過表現在Y. lipolytica中，並探討其對生物質量及脂質含量的影響。本研究發現所有的品種偏好於在較低溫的環境下生長以及TEFintron啟動子有補救生長缺陷的能力。本研究顯示出用hp4d啟動子過表現LRO1基因的物種，脂質產率在28 oC 及 25 oC下分別是控制組的1.1倍及1.2倍。而用TEFintron啟動子過表現LRO1基因的物種，脂質產率在28 oC 及 25 oC下分別是控制組的1.17倍及1.31倍。

Biodiesel has been considered as a renewable, alternative energy. Commercial feedstock of biodiesel production mainly is vegetable and/or, animal fat. Single cell oil from microorganism is a potential feedstock for biodiesel production. Among microorganisms, the oleaginous yeast Yarrowia lipolytica is a potential candidate for single cell oil production, which store lipid in lipid body (LB) and it is mainly composed of triacylglycerols (TAGs) and steryl esters (SEs). The final step of TAG synthesis is acylation of diacylglyceride (DAG) either by diacylglycerol acyltransferase (DGA1) or by phospholipid diacylglycerol acyltransferase (LRO1) to produce TAG. In this study, LRO1 gene was choose to over-express in Y. lipolytica and its effect on biomass and lipid content was investigated. It was found that all strains showed higher preferences at lower temperature and TEFintron promoter has the ability to remedy growth defect. The results show that the strain over-expressing LRO1 gene driven by hp4d promoter exhibited 1.1-fold and 1.2-fold in lipid yield compared to control strain at 28 oC and 25 oC, respectively. The strain over-expressing LRO1 gene driven by TEFintron promoter exhibited 1.17-fold and 1.31-fold in lipid yield of that of the control strain at 28 oC and 25 oC, respectively.

中文摘要...................................................................... I
Abstract.....................................................................II
Acknowledgement.............................................................III
Contents.................................................................... IV
List of Figures............................................................VIII
List of Tables...............................................................XI
Chapter 1.....................................................................1
Introduction..................................................................1
1	Background of study...................................................1
2	Objectives of this study..............................................2
Chapter 2.....................................................................3
Literature review.............................................................3
1 Yarrowia lipolytica.......................................................3
1.1 Y. lipolytica Po1g strain...............................................3
1.2 Lipid accumulation in Y. lipolytica.....................................4
2 Genetic manipulation for Y. lipolytica....................................7
2.1 pYLEX1 vector...........................................................8
2.2 Hp4d promoter...........................................................9
2.3 TEFintron promoter......................................................10
3 LRO1 gene.................................................................10
3.1 Previous studies on LRO1 gene...........................................11
Chapter 3.....................................................................13
Methodology...................................................................13
1 Experimental materials....................................................13
1.1 Microorganism...........................................................13
1.2 Plasmid.................................................................13
1.3 Primer..................................................................13
1.4 Enzyme..................................................................14
1.5 Kits....................................................................14
1.6 DNA Marker..............................................................14
1.7 Chemicals...............................................................14
2 Culture medium and reagent................................................15
2.1 Culture medium..........................................................15
2.1.1 LB medium + ampicillin medium.........................................15
2.1.2 YPD medium............................................................15
2.1.3 YPD medium pH4........................................................15
2.1.4 YNB solid medium......................................................15
2.1.5 LB medium + ampicillin solid medium...................................16
2.1.7 YNB solid plate.......................................................16
2.2 Reagent.................................................................16
2.2.1 DNA reagent...........................................................16
2.2.2 Transformation reagent................................................16
3 Apparatus and equipments..................................................17
4 Conceptual framework of methodology.......................................17
5 Genetic manipulation......................................................24
5.1 Genomic DNA Extraction..................................................24
5.2 Polymerase Chain Reaction (PCR) ........................................25
5.3 Agarose gel electrophoresis.............................................26
5.4 Gel extraction..........................................................26
5.5 Plasmid extraction......................................................27
5.6 Restriction enzyme .....................................................28
5.7 Enzymatic DNA solution purification.....................................29
5.8 Restriction enzyme digestion............................................30
5.9 Formation of recombinant DNA molecule (ligation)........................31
5.10 Preparation of E. coli competent cell..................................32
5.11 Transformation of E. coli Top10 by heat shock..........................33
5.12 Colony PCR of E. coli..................................................33
5.13 Linearization of plasmid...............................................34
5.14 Preparation of Yarrowia lipolytica competent cell......................34
5.15 Transformation of Yarrowia lipolytica..................................35
5.16 Colony PCR of Yarrowia lipolytica......................................36
6 Fermentation..............................................................37
6.1 Inoculum................................................................37
6.2 Biomass measurement.....................................................38
7 Soxhlet extraction........................................................38
8 Total reducing sugar estimation...........................................39
Chapter 4.....................................................................40
Results and Discussion........................................................40
1 Vector construction.......................................................40
1.1 Construction of pYLRO1 vector and Y. lipolytica transformation..........40
1.2 Construction of pYTEFin vector..........................................44
1.3 Construction of pYTEFinLRO1 vector and Y. lipoylitica transformation....45
2 Effect of over expression of LRO1 driving by hp4d.........................48
2.1 Growth rate.............................................................48
2.2 Lipid analysis..........................................................52
3 Effect of over-expression of LRO1 driving by TEFintron promoter...........55
3.1 Growth rate analysis....................................................55
3.2 Lipid analysis..........................................................57
Chapter 5.....................................................................63
Conclusion and Future Prospects...............................................63
Reference.....................................................................64

                                

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