臺灣博碩士論文加值系統

生質柴油發展到現在已經是一個重要的替代能源。從油脂微生物獲取油脂被視為是一個非常具有潛力的生質柴油來源。Yarrowia lipolytica是一種相當好氧的油脂酵母。為解決發酵時缺氧的缺陷，藉由Vitreoscilla hemoglobin (VHb)提升氧氣的傳遞來增加微生物的呼吸與能量代謝。在本研究中，我們使用hp4d啟動子表現VHb在Yarrowia lipolytica中以探討Vitreoscilla hemoglobin (VHb)對Yarrowia lipolytica生長與脂質含量的影響。結果指出藉由利用hp4d啟動子，蛋白質在靜止期初期被生產出來。雖然VHb被表現在Yarrowia lipolytica中，但細胞在低養分的情況下無法繼續增長。因此在低溶氧與高溶氧下，表現VHb並未提升Yarrowia lipolytica的生長速率。油脂產率在控制組為 8.667 g/l，而在VHb表現下產率為 9.410 g/l。表現VHb也許會幫助油脂的累積。然而，這些許的增加在統計上不重要。

Biodiesel has become an important alternative energy source. Recently, microbial oil from oleaginous microorganism is considered as a potential candidate for biodiesel production. Yarrowia lipolytica is a strictly aerobic oleaginous yeast. To solve the defects of hypoxic conditions, Vitreoscilla hemoglobin (VHb) has been used to enhance respiration and energy metabolism by promoting oxygen delivery. In this study, hp4d promoter was used for expressing VHb protein in Y. lipolytica. The effect of VHb protein on growth rate and lipid production of Y. lipolytica was investigated. The result indicates that protein was produced at the beginning of stationary stage by using hp4d promoter. Although VHb protein was produced at stationary stage, cells were not able to grow at low nutrients. Therefore, growth rate of Y. lipolytica was not enhanced by expressing VHb protein under low-oxygen and high-oxygen level. Lipid yield was 8.667 g/l for VHb- strain and 9.410 g/l for VHb+ strain. The expression of VHb protein may help lipid accumulation. However, the increase is not statistically important.

中文摘要............................................................................................................................ I
Abstract .............................................................................................................................II
Acknowledgement .......................................................................................................... III
Contents .......................................................................................................................... IV
List of Figures .............................................................................................................. VIII
List of Tables .................................................................................................................. IX
Chapter 1........................................................................................................................... 1
Introduction....................................................................................................................... 1
1.1 Background................................................................................................................. 1
1.2 Objects of this study.................................................................................................... 2
Chapter 2........................................................................................................................... 3
Literature review............................................................................................................... 3
2.1 Oleaginous microorganisms........................................................................................ 3
2.1.1 Introduction .......................................................................................................... 3
2.1.2 Oil content of some oleaginous microorganisms ................................................. 3
2.1.3 Lipids from oleaginous yeasts.............................................................................. 4
2.1.4 Yarrowia lipolytica............................................................................................... 5
2.1.4.1 Introduction ................................................................................................... 5
2.1.4.2 Yarrowia lipolytica Po1g strain ................................................................... 6
2.2 Genetic manipulation for Yarrowia lipolytica ............................................................ 6
2.2.1 Introduction ........................................................................................................ 6
2.2.2 Hp4d promoter ................................................................................................... 9
2.2.3 pYLEX1 vector ................................................................................................ 10
2.3 Vitreoscilla hemoglobin............................................................................................ 11
2.3.1 Introduction ...................................................................................................... 11
2.3.2 Function of VHb ............................................................................................... 12
2.3.3 Application of VHb .......................................................................................... 15
Chapter 3......................................................................................................................... 17
Methodology................................................................................................................... 17
3.1 Experimental materials ............................................................................................. 17
3.1.1 Chemicals ......................................................................................................... 17
3.1.2 Kits ................................................................................................................... 17
3.1.3 Enzyme ............................................................................................................. 17
3.1.4 Microorganism ................................................................................................. 17
3.1.5 Plasmid ............................................................................................................. 18
3.1.6 Primer ............................................................................................................... 18
3.2 Culture medium and reagent..................................................................................... 18
3.2.1 Culture medium ................................................................................................ 18
3.2.1.1 LB medium + ampicillin ........................................................................... 18
3.2.1.2 YPD medium ............................................................................................. 18
3.2.1.3 YPD medium pH4 ....................................................................................... 19
3.2.1.4 YNB medium ............................................................................................. 19
3.2.2 Reagent ............................................................................................................. 19
3.2.2.1 DNA reagent .............................................................................................. 19
3.2.2.2 Transformation of bacterial cell reagent .................................................... 19
3.2.2.3 SDS-PAGE ................................................................................................ 19
3.2.2.4 Western blot reagent .................................................................................. 19
3.3 Apparatus and equipments........................................................................................ 20
3.4 Conceptual framework of methodology ................................................................... 20
3.5 Genetic manipulation................................................................................................ 24
3.5.1 Plasmid extraction ............................................................................................ 24
3.5.2 Polymerase Chain Reaction (PCR) ................................................................ 25
3.5.3 PCR product purification ................................................................................. 25
3.5.4 Agarose gel electrophoresis .............................................................................. 26
3.5.5 Restriction enzyme digestion ........................................................................... 27
3.5.6 Gel extraction ................................................................................................... 28
3.5.7 Formation of recombinant DNA molecule (ligation)......................................... 29
3.5.8 Preparation of E. coli competent cell ............................................................... 29
3.5.9 Transformation of E. coli Top10 by heat shock ............................................... 30
3.5.10 Colony PCR of E. coli .................................................................................... 30
3.5.11 Colony PCR confirmation using agarose gel electrophoresis ........................ 31
3.5.12 Linearization of plasmid ................................................................................. 31
3.5.13 Preparation of Yarrowia lipolytica competent cell ......................................... 32
3.5.14 Transformation of Yarrowia lipolytica by heat shock .................................... 32
3.5.15 Colony PCR of Yarrowia lipolytica ............................................................... 33
3.6 VHb expression confirmation................................................................................... 34
3.6.1 Release of VHb protein .................................................................................... 34
3.6.2 SDS-PAGE ....................................................................................................... 34
3.6.3 Western Blot ..................................................................................................... 36
3.7 Fermentation ............................................................................................................. 37
3.7.1 Inoculum ........................................................................................................... 37
3.7.2 Microbial fermentation ..................................................................................... 37
3.8 Lipid analysis ............................................................................................................ 38
3.8.1 Soxhlet extraction ............................................................................................. 38
3.9 Total reducing sugar ................................................................................................. 38
Chapter 4......................................................................................................................... 39
Results and Discussion ................................................................................................... 39
4.1 Construction of pYVHb vector................................................................................. 39
4.2 Y. lipoylitica transformation...................................................................................... 40
4.3 VHb protein expression in Y. lipoylitica................................................................... 41
4.3.1 SDS-PAGE ....................................................................................................... 41
4.3.2 Western Blot ..................................................................................................... 42
4.4 Effect of VHb protein ............................................................................................... 44
4.4.1 Growth rate......................................................................................................... 44
4.4.2 Lipid content ...................................................................................................... 47
Chapter 5......................................................................................................................... 48
Conclusion and Future projects ...................................................................................... 48
References....................................................................................................................... 49

List of Figures
Figure 2.1 The gene map of pYLEX1 ............................................................................ 10
Figure 2.2 Structure of VHb homodimer........................................................................ 11
Figure 2.3 Proposed mechanism of the interaction between and subunit I of
cytochrome bo ubiquinol oxidases.................................................................................. 14
Figure 2.4 Applications of VHb protein ......................................................................... 16
Figure 3.1 Conceptual framework of this study.............................................................. 21
Figure 3.2 Conceptual framework of genetic manipulation ........................................... 22
Figure 3.3 pYVHB construction........................................................................................ 23
Figure 4.1 The DNA electrophoresis image of gel purification ..................................... 39
Figure 4.2 Transformation of E. coli Top10. (a) Control (pYLEX1+dd-H2O), (b)
Recombinant DNA(pYLEX1+vgb gene) ....................................................................... 40
Figure 4.3 The DNA electrophoresis image of E. coli colony PCR. .............................. 41
Figure 4.4 DNA electrophoresis image of yeast colony PCR ........................................ 42
Figure 4.5 (a) SDS-PAGE, (b) Western Blot.................................................................. 43
Figure 4.6 Comparison of growth profiles between VHb+ and VHb- strain under
low-oxygen level in rich YPD medium. ......................................................................... 45
Figure 4.7 Comparison of growth profiles between VHb+ and VHb- strain under
high-oxygen level in rich YPD medium. ........................................................................ 46
Figure 4.8 Western Blot analyses of VHb protein sample at different glucose
concentration. The same amounts of cells were used to release VHb protein................ 46
Figure 4.9 Dry cell weight and lipid content in different strains. Number 1: pYLEX1
(VHb-). Number 2: pYVHb (VHb+) .............................................................................. 47

List of Tables
Table 2.1:Oil content of some microorgansms ................................................................. 4
Table 2.2: Components for Y. lipolytica expression/secretion vectors............................. 7
Table 2.3: Heterologous protein expression/secretion in Y. lipolytica ............................. 9
Table 2.4: Kinetic constants for reactions of various hemoglobins with oxygen........... 12
Table 4.1: Comparison between this study and literature............................................... 45
Table 4.2: The fraction of solvent extraction.................................................................. 47

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