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研究生:葛麗絲
研究生(外文):Grace-Marlina Winata
論文名稱:Expression of 5-Aminolevulinic Acid Synthase (ALAS) for 5-Aminolevulinic Acid (ALA) Production in Recombinant Escherichia coli
論文名稱(外文):Expression of 5-Aminolevulinic Acid Synthase (ALAS) for 5-Aminoelvulinic Acid (ALA) Production in Recombinant Escherichia coli
指導教授:李振綱李振綱引用關係
指導教授(外文):Cheng-Kang Lee
口試委員:李振綱
口試委員(外文):Cheng-Kang Lee
口試日期:2006-07-20
學位類別:碩士
校院名稱:國立臺灣科技大學
系所名稱:化學工程系
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:英文
論文頁數:120
中文關鍵詞:5-Aminolevulinic acid synthase
外文關鍵詞:5-Aminolevulinic acid synthase
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5-Aminolevulinic acid (ALA) is the precursor of protoporphyrin IX (PP IX) in the heme biosynthesis pathway and has been suggested for photodiagnosis and photodynamic therapy of epithelial cancer. The Rhodobacter sphaeroides hemA gene encoding 5-aminolevulinic acid synthase (ALAS) was cloned into the expression vector pWHmA and transformed into Escherichia coli BL21(DE3) for its heterologous expression under the control of Bacillus xylA promoter. By introducing sopABC gene into pWHmA to make the expression plasmid become more stable, pGHmA plasmid was generated so that higher ALAS expression level and higher amount of ALA production was achieved. With the expression of ALAS and addition of glycine and succinate as substrates, the recombinant E. coli could produce ALA extracellularly. In shaker-flask cultures at 37oC for 36 h with addition of D(+)-xylose as inducer enhanced the ALA concentration produced.
The expression level of ALAS under the control of xylA promoter was also compared with that under the control of T7 promoter. It was found that the basal expression of ALAS driven by xylA promoter is higher than that of T7 promoter and with no inclusion body formation based on Western Blot analysis.
It was observed that recombinant E. coli BL21(DE3) harboring pGHmA cultivated under dark condition, with the repeated addition of ALA precursors, succinate (90 mM) and glycine (30 mM), and LA (inhibititor of ALA dehydratase (ALAD) in heme biosynthesis), can increase the ALA accumulation to 15.5 mM. On the other hand, in the culture under light illumination, the more ALA produced was converted into prophyrins and resulted in a lower ALA concentration. Besides, the cell concentration obtained was also lower in the light culture.
5-Aminolevulinic acid (ALA) is the precursor of protoporphyrin IX (PP IX) in the heme biosynthesis pathway and has been suggested for photodiagnosis and photodynamic therapy of epithelial cancer. The Rhodobacter sphaeroides hemA gene encoding 5-aminolevulinic acid synthase (ALAS) was cloned into the expression vector pWHmA and transformed into Escherichia coli BL21(DE3) for its heterologous expression under the control of Bacillus xylA promoter. By introducing sopABC gene into pWHmA to make the expression plasmid become more stable, pGHmA plasmid was generated so that higher ALAS expression level and higher amount of ALA production was achieved. With the expression of ALAS and addition of glycine and succinate as substrates, the recombinant E. coli could produce ALA extracellularly. In shaker-flask cultures at 37oC for 36 h with addition of D(+)-xylose as inducer enhanced the ALA concentration produced.
The expression level of ALAS under the control of xylA promoter was also compared with that under the control of T7 promoter. It was found that the basal expression of ALAS driven by xylA promoter is higher than that of T7 promoter and with no inclusion body formation based on Western Blot analysis.
It was observed that recombinant E. coli BL21(DE3) harboring pGHmA cultivated under dark condition, with the repeated addition of ALA precursors, succinate (90 mM) and glycine (30 mM), and LA (inhibititor of ALA dehydratase (ALAD) in heme biosynthesis), can increase the ALA accumulation to 15.5 mM. On the other hand, in the culture under light illumination, the more ALA produced was converted into prophyrins and resulted in a lower ALA concentration. Besides, the cell concentration obtained was also lower in the light culture.
TABLE OF CONTENTS



ABSTRACT i
ACKNOWLEDGEMENT ii
TABLE OF CONTENTS iii
LIST OF FIGURES vi
LIST OF TABLES ix
CHAPTER I INTRODUCTION 1
CHAPTER II LITERATURE STUDY 4
2.1 5-Aminolevulinic acid (ALA) structure and chemical stability 4
2.2 Metabolic pathway and regulation of ALA biosynthesis 4
2.2.1 ALA production by photosynthetic bacteria 7
2.2.2 Genetic manipulation and ALA production 9
2.2.3 Chemical synthesis and biotechnological synthesis 10
2.3 Applications of ALA 11
2.3.1 Agricultural applications 12
2.3.1.1Biodegradable herbicide 12
2.3.1.2 Growth promoting factor for plants 12
2.3.1.3 Increased salt and cold temperature tolerance 13
2.3.2 Medical applications of ALA 14
2.3.2.1 Diagnosis of heavy-metal poisoning 14
2.3.2.2 Photodynamic therapy for cancer treatment 14
2.3.2.3 Photodynamic diagnosis 15
2.3.2.4 Other medical applications 15
2.4 Quantitative method to determine the amount of ALA 16
2.5 Qualitative method to determine porphyrin 21
2.6 F plasmid and sopABC partitioning system function 20
CHAPTER III EXPERIMENT 25
3.1 Experiment procedures 25 3.1.1 Construction of pWHmA vector for expression of
R. sphaeroides hemA in recombinant Escherichia coli 25
3.1.2 Construction of pGHmA vector for expression of
R. sphaeroides hemA in recombinant Escherichia coli 28
3.1.3 5-Aminolevulinic acid synthase (ALAS)
protein expression 30
3.1.4 5-Aminolevulinic acid (ALA) production 30
3.2 EXPERIMENT MATERIAL 31
3.2.1 Bacterial strain 31
3.2.2 Plasmid vector 31
3.2.3 Primer design 31
3.2.4 Enzyme 31
3.2.5 DNA test kit 32
3.2.6 Molecular weight standard solution 32
3.2.7 Antibody 32 3.3 Other chemicals 33
3.4 Experiment culture medium and reagent 33
3.4.1 Culture medium 33
3.4.2 Reagent 34
3.5 Experiment apparatus 36
3.6 Experiment step 37
3.6.1 Polymerase Chain Reaction (PCR) primer design 37
3.6.2 Genetic manipulation of hemA gene 37
3.6.2.1 Construction of pWHmA plasmid 37
3.6.2.2 Construction of pGHmA plasmid 48
3.7 Batch fermentation for ALA production 55 3.8 Jar-fermenter preparation 55
3.9 Competent cell preparation 57
3.10SDS-PAGE gel analysis 58
3.11Immuno/Western blot analysis 60
3.12Colorimetric quantification of ALA 61
3.13Fluorescence measurement of porphyrin 63
CHAPTER IV RESULT AND DISCUSSION 64
4.1 Expression of Rhodobacter sphaeroides hemA gene
in Escherichia coli 64
4.1.1 pWHmA plasmid construction 64
4.1.2 pGHmA plasmid construction 66
4.2 BL21(DE3)/pWHmA for ALAS expression and
ALA production 68
4.3 Reuse of BL21(DE3)/pWHmA to produce extracellular ALA 73
4.4 BL21(DE3)/pGHmA for ALAS expression and
ALA production 75
4.5 Xylose as catabolite repressor in E. coli BL21(DE3)
harboring pPhmA and pWHmA 79
4.6 Xylose as inducer in BL21(DE3)/pGHmA strain for
ALAS expression and ALA production 83
4.7 Aeration effect on ALA production 86
4.8 Batch fermentation of E. coli BL21(DE3)/pGHmA
cultivation for ALA production 89
4.9 Effect of dark and light cultivation conditions on
ALA production 91 4.10 Effect of levulinic acid (LA) on ALA production 95
4.11 Repeated addition of succinate, glycine, and levulinic acid
for the mass production of extracellular ALA 99
CHAPTER V CONCLUSION 101
REFERENCES x
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