本論文主要分為兩大部分；第一部分為從環境中篩選可生產PHAs（polyhydroxyalkanoates）的微生物，利用設計Degenerate primer再以PCR偵測微生物中的PHAs合成酵素基因，並且用Nile red及Sudan black染色觀察菌體中脂質的堆積，最後再以比對16s rRNA基因的方式鑑定菌種，利用此方法篩得的菌株（編號KC001），以GC（gas chromatography）分析後確定可以生產PHAs。
第二部分為建構基因重組大腸桿菌，利用殖入Wautersia eutropha（BCRC13036）的phaC，phaA，phaB基因，使得原本不會生產PHB（polyhydroxybutyrate）的大腸桿菌DH5α可以生產PHB。另外也將phaC置換成環境中篩得菌種KC001之phaC1，觀察是否對於PHB的產量有不同的變化。

This thesis includes two research subjects related to the study on the polyhydroxyalkanoates (PHAs) synthesis by bacteria. The first topic involved the isolation of microorganisms that able to accumulate PHAs from different environment. Bacterial isolates were confirmed by a set of degenerate primers designed for the amplification of PHA synthase gene by PCR. Cultivation of isolates on medium containing Nile red and performing Sudan black staining were further confirm their ability to produce PHAs by phenotypic traits. Bacteria were identified by 16s rDNA analysis. Strain KC001 was isolated and characterized from this screening process and was further analyzed by gas chromatography (GC) for its PHA production.

The second part of this thesis describes the construction of recombinant PHAs-producing E. coli. We cloned the phaA, phaB and phaC genes from Wautersia eutropha (BCRC13036) and transformed them into E. coli DH5α. After introducing the PHA operon, we observed the production of PHB by recombinant E. coli by GC analysis. Recombinant E. coli with phaC1 (cloned from strain KC001) instead of phaC was also constructed and evaluated for its production of PHB.

中文摘要　……………………………………………………………....I
英文摘要　……………………………………………………………...II
目錄　……………………………………………………………….….III
表目錄　………………………………………………………......…... VI
圖目錄　……………………………….……………….………….…..VII
序列目錄　……………………………………………………………..IX

第一章 序論　………………………………………………………....1
1.1 前言　…………………………………………………………...1
1.2 文獻回顧　….…………………………………………………..3
1.2.1 PHAs的歷史　…….………….………………………..…...3
1.2.2 PHAs的性質　…………………………………..………….5
1.2.3 PHAs各種性質的鑑定方式　………….….……………….8
1.2.4 PHAs的代謝機制　………………..……………………….9
1.2.5 與PHAs合成相關的基因　…………………….…………13
1.3 實驗簡介　………………………….………….………….…..16
第二章 材料與方法　………………….………………………….…19
2.1 實驗方法　………………………….…………………….…...19
2.1.1 Primer設計　………………………...…………………….19
2.1.2 土讓樣本收集及微生物培養　…….…………….……….19
2.1.3 以PCR偵測PHAs合成酵素基因　…………..……….…20
2.1.4 Nile red及Sudan black染色法　…….……….………..….20
2.1.5 16S rRNA基因PCR　………….….…………..…..………21
2.1.6 DNA elution　 ……......…...………………..….…….……21
2.1.7 PCR產物的選殖　…….…….…….….….………...….…..22
2.1.8 勝任細胞製備方式　………….…….…….…..…..………23
2.1.9 DNA定序　…………………….…….…………….……...23
2.1.10 KC001之培養條件及培養液配方　…………….…….….25
2.1.11 PHAs萃取　………………..….….……………….….…...25
2.1.12 FT-IR及GC分析前準備　………………………………...26
2.1.13 phaC-phaA-phaB基因之cloning　…………………..…...27
2.1.14 以SDS-PAGE觀察蛋白質表現　…………………………28
2.1.15 KC001 phaC1的選殖　…….………….…….………….…29
2.1.16 建構pBC1AB質體　………………………………………30
2.1.17 重組大腸桿菌之PHAs分析及培養條件　……………….30
2.2 實驗藥品　…….…….………………………….……………..31
2.3 實驗儀器與Kit　…………….…………………….……….…34
2.3.1實驗儀器　………………………………………………….34
2.3.2實驗所用Kit　………………………………………………35
2.4 實驗所用酵素與引子（primer）　……………………………36
2.4.1實驗酵素　…………………………………………………..36
2.4.2引子合成　…………………………………………………..36
第三章 實驗結果　……………………………………………….….37
3.1 Primer設計與測試　…….….…….…….…..……...…….……37
3.2 菌種的篩選　….……….………….…………….…….………38
3.3 Recombinant E.coli　…………….………….….……………..39
第四章 討論　…………………………………………….………….57
4.1 菌種篩選　……………………….…………………………....57
4.2 Recombinant E.coli　………………………………………….59
第五章 結論　………………………………………………………..62
第六章 未來展望　…………………………………………………..63
第七章 參考文獻　……………………………………………….….64
表1-1　PHAs與常見塑膠材料比較　…………………………………6
表1-2　各項物質在自然環境中分解所需年限　.…….….…….……..7
表3-1　本論文所提到的質體　……….…….…………….………….42
表3-2　基因重組大腸桿菌所攜帶質體經由氣象層析儀分析PHAs百分比結果　……………………………………….…………….56
圖1-1　PHAs的結構示意圖　…………………………………………5
圖1-2　PHAs的代謝路徑　…………………………………………..12
圖1-3　PHAs的降解路徑　…………………………………………..13
圖3-1　利用PHAs合成酵素基因所做出之演化樹　……………….43
圖3-2　以PCR增殖PHAs合成酵素基因之測試　………………...44
圖3-3　Nile red染色圖　……………………………………………...45
圖3-4　KC001的Sudan black染色圖　……………………………..46
圖3-5　KC001以LB（Lennox）含2%葡萄糖為營養源，直接以GC分析菌體的結果圖　…………….......….….….………………49
圖3-6　KC001以KC001以LB（Lennox）含0.5%十碳酸為營養源，直接以GC分析菌體的結果圖　………………………….…..49
圖3-7　PCR增殖出Wautersia eutropha之pha operon之電泳圖　...50
圖3-8　pBCAB-WE的質體圖　………….…..…….………………...51
圖3-9　利用SDS-PAGE觀察轉殖後大腸桿菌的蛋白質表現（主要比較對象為pBCAB-WE）　…………………….……….………52
圖3-10　pBCAB-WE的GC分析圖　………….…………………….53
圖3-11　pBCAB-WE（加入IPTG誘導）的GC分析圖　………….53
圖3-12　帶pBSII質體大腸桿菌的GC分析圖　……………………54
圖3-13　pBCAB-WE的GC分析圖　………………….…..…………54
圖3-14　以SDS-PAGE觀察轉殖後大腸桿菌的蛋白質表現（主要比較對象為pBC1AB-KC1）　……………………………………55
序列3-1　KC001的部分PHAs合成酵素基因　……………….….…47
序列3-1　KC001的16S rRNA基因序列（全長）　………………..48

Anderson AJ and Dawes EA. Occurence, metabolism, metabolic rate, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Rev 1990;54(4):450-72

Brandl H, Cross RA, Lenz RW, and Fuller RC. Plastics from bacteria and for bacteria: poly(β-hydroxybutyrates) as natural, biocompatible, and biodegradable polyesters. Adv. Biotechem. Engin. Biotechnol 1990;41:77-93

Braunegg G, Sonnleitner B and Lafferty RM. A rapid gas chromatographic method for the determination of poly-β-hydroxybutyrate in microbial biomass. Eur J Appl Microbiol 1978;6:29-37

Burdon KL. Fatty material in bacteria and fungi revealed by staining dried, fixed slide preparations. J Bacteriol 1946;52:665-78

Chen JY, Liu T, Zheng Z, Chen JC and Chen GQ. Polyhydroxyalkanoate synthase PhaC1 and PhaC2 from Pseudomonas stutzeri 1317 had different substrate specificaties. FEMS Microbiology Letters 2004;234:231-237

Deng Y, Zhao K, Zhang XF, Hu P and Chen GQ. Study on the three-dimensional proliferation of rabbit articular cartilage derived chondrocytes on polyhydroxyalkanoate scaffolds. Biomaterials 2002;23:4049-4056

de Smet MJ, Eggink G, Witholt B, Kingma J and Wynberg H. Characterization of intracellular inclusions formed by Pseudomonas oleovorans during growth on octane. J Bacteriol 1983;154(2):870-8

Dewhirst FE, Chien CC, Paster BJ, Ericson RL, Schauer DB, Orcutt RP and Fox JG. Phylogeny of a group of murine defined microbiota (Altered Schaedler Flora) Appl Environ Microbiol 1999;65:3287-3292

Du1 G and Yu J. Metabolic analysis on fatty acid utilization by Pseudomonas oleovorans: mcl-poly(3-hydroxyalkanoates) synthesis versus β-oxidation. Process Biochemistry 2002;38:325-332

Forsyth WGC, Hayward AC and Roberts JB. Occurrence of poly-β- hydroxybutyric acid in aerobic Gram-negative bacteria. Nature 1958;182:800-1

Fukada E and Ando Y. Piezoelectric properties of poly-β-hydroxyvalerate. Int. J. Biol. Macromol. 1986;8:361-366


Hong K, Sun S, Tian W, Chen GQ and Huang W. A rapid method for detecting bacterial polyhydroxyalkanoates in intact cells by Fourier transform infrared spectroscopy. Applied Microbiology and Biotechnology 1999;51:523–526

Hoffimann N and Rehm BHA. Regulation of polyhydroxyalkanoate biosynthesis in Pseudomonas putida and Pseudomonas aeruginosa. FEMS Microbiology Letters 2004;237:1-7

Jacob GS, Garbow JR and Schaefer J. Direct measurement of poly(β-
hydorxybutyrate ) in a psedimonad by solid-state 13C NMR. J. Biol. Chem 1986;261:16785-16787

Jan S, Roblot C, Goethals G, Courtois J, Courtois B, Saucedo JEN, Seguin JP and Barbotin JN. Study of parameters affecting poly- (3-hydroxybutyrate) quantification by gas chromatography. Analytical Biochemistry 1995;225:258-263

Luengo JM, Garcia B, Sandoval A, Naharro G and Olivera ER. Bioplatics from microorganisms. Current Opinion in Microbiology 2003;6:251-260

Labuzek S and Radecka I. Biosynthesis of PHB tercopolymer by Bacillus cereus UW85. Journal of Applied Microbiology 2001;90:353–357


Lageveen RG, Huisman GW, Preusting H, Ketelaar P, Eggink G and Witholt B. Formation of polyesters by Pseudomonas oleovorans: effect of substrates on formation and composition of poly-(R)-3- hydroxyalkanoates and poly-(R)-3-hydroxyalkenoates. Appl Environ Microbiol 1988;54:2924-32

Madison LL and Gjalt W. Huisman Metabolic Engineering of Poly(3-Hydroxyalkanoates): From DNA to Plastic. Microbiology and Molecular Biology Review 1999;63(1):21-53

Lee SY, Lee Y and Wang F. Chiral compounds from bacterial polyesters: sugars to plastics to fine chemicals. Biotechnol. Bioeng 1999;65:363-368

Lemoigne M. Etudes sur l'autolyse microbiènne origine de l'acide β-oxybutyrique formé par autolyse. Ann Inst Pasteur 1927;41:148-65

Lemoigne M. Produit de déshydratation et de polymérisation de l'acide β-oxybutyrique. Bull SocChimBiol 1926;8:770-82

Misra AK, Thakur MS, Srinivas P and Karanth NG. Screening of poly-beta-hydroxybutyrate producing microorganisms using fourier transform infrared spectroscopy. Biotechnology letters 2000;22:1217–1219



Matsumoto K, Takase K, Aoki E, Doi Y and Taguchi S. Synergistic effects of Glu130Asp substitution in the type II polyhydroxyalkanoate (PHA) synthase: enhancement of PHA production and alteration of polymer molecular weight. Biomacromolecules. 2005;6(1):99-104

Ojumu TV, Yu J and Solomon BO. Production of Polyhydroxyalkanoates, a bacterial biodegradable polymer. Afr. J. Biotechnol 2004;3(1):18-24

Peoples OP and Sinskey AJ. Poly-β-hydroxybutyrate biosynthesis in Alcaligenes eutrophus H16. Characterization of the genes encoding β-ketothiolase and acetoacetyl-CoA reductase. J Biol Chem 1989;264:15,293-7

Peoples OP and Sinskey AJ. Poly-β-hydroxybutyrate biosynthesis in Alcaligenes eutrophus H16. Identifcation and characterization of the PHB polymerase gene (phbC). J Biol Chem 1989;264(26):15,298-303

Poirier Y, Nawrath C and Somerville C. Production of poly- hydroxyalkanoates, a family of biodegradable platics and elastomers, in bacteria and plants. Biotechnology 1995;13:142-150

Ramsay BA, Lomaliza K, Chavarie C, Dube B, Bataille P and Ramsay JA. Production of poly-(beta-hydroxybutyric-co- beta-hydroxyvaleric) acid. Applied and environmental Microbiology 1990;56:2093-2098


Rehm BHA and Steinbüchel A. Biochemical and genetic analysis of PHA synthases and other proteins required for PHA synthesis. Int J Biol Macromol 1999;25:3-19.

Robert J, Marchesini S, Delessert S and Poirier Y. Analysis of the beta-oxidation of trans-unsaturated fatty acid in recombinant Saccharomyces cerevisiae expressing a peroxisomal PHA synthase reveals the involvement of a reductase-dependent pathway. Biochim Biophys Acta. 2005;1734(2):169-77

Schubert P, Steinbüchel A and Schlegel HG. Cloning of the Alcaligenes eutrophus genes for synthesis of poly-β-hydroxybutyric acid (PHB) and synthesis of PHB in Escherichia coli. J Bacteriol 1988;170(12):5837-47

Shamala TR, Chandrashekar A, Vijayendra SVN and Kshama L. Identification of polyhydroxyalkanoate (PHA)-producing Bacillus spp. using the polymerase chain reaction (PCR). Journal of Applied Microbiology 2003;94:369-374

Sheu DS, Wang YT and Lee CY. Rapid detection of poly-hydroxyalkanoate accumulating bacteria isolated from the environment by colony PCR. Microbiology 2000;146:2019–2025

Slater SC, Voige WH and Dennis DE. Cloning and expression in Escherichia coli of the Alcaligenes eutrophus H16 poly-β- hydroxybutyrate biosynthetic pathway. J Bacteriol 1988;170(10):4431-6
Solaiman DKY, Ashby RD and Foglia TA. Rapid and specific identification of medium-chain-length polyhydroxyalkanoate synthase gene by polymerase chain reaction. Applied Microbiology and Biotechnology 2000;53:690–694

Solaiman DK and Ashby RD. Rapid genetic characterization of poly- (hydroxyalkanoate) synthase and its applications. Biomacromolecules. 2005;(2):532-7

Spiekermann P, Rehm BHA, Kalscheuer R, Baumeister D and Steinbüchel A. A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds. Arch Microbiol 1999;171:73-80

Steinbüchel A. Polyhydroxyalkanoic acids. In: Byrom D, editor. Biomaterials. Basingstoke:MacMillan 1991:125-213

Steinbüchel A and Schlegel HF. Physiology and molecular genetics of poly(beta-hydroxyalkanoic acids) synthesis in Alcaligenes eutrophus. Molecular Microbiology 1991;5:535-547

Sudesh K, Abe H and Doi Y. Synthesis, structure and properties of polyhydroxyalkanoates: biological polyesters. Prog. Polym. Sci 2000;25:1503-1555


Vaneechoutte M, Kampfer P, de Baere T, Falsen E and Verschraegen G. Wautersia gen. nov., a novel genus accommodating the phylogenetic lineage including Ralstonia eutropha and related species, and proposal of Ralstonia [Pseudomonas] syzygii (Roberts et al. 1990) comb. nov. Int J Syst Evol Microbiol. 2004;54(Pt 2):317-27

Ren Q, van Beilen JB, Sierro N, Zinn M, Kessler B and Witholt B. Expression of PHA polymerase genes of Pseudomonas putida in Escherichia coli and its effect on PHA formation. Antonie Van Leeuwenhoek. 2005;87(2):91-100

Wallen LL and Rohwedder WK. Poly-β-hydroxyalkanoate from activated sludge. Environ Sci Technol 1974;8:576-9

Ward PG, de Roo G and O'Connor KE. Accumulation of poly- hydroxyalkanoate from styrene and phenylacetic acid by Pseudomonas putida CA-3. Appl Environ Microbiol. 2005;71(4):2046-52

Ward PG and O'Connor KE. Bacterial synthesis of poly- hydroxyalkanoates containing aromatic and aliphatic monomers by Pseudomonas putida CA-3. Int J Biol Macromol. 2005;35(3-4):127-33

Wu HA, Sheu DS, and Lee CY. Rapid differentiation between short-chain-length and medium-chain-length polyhydroxy- alkanoate accumulating bacteria with spectrofluorometry. Journal of Microbiology Methods 2003;53:131– 135
Wu Q, Sun SQ, Yu P and Chen GQ. Environmental dependence of microbial synthesis of polyhydroxyalkanoates. Acta Polvm. Sin. 2000;6:751-756

Zhaolin D. and Xuenan S. A new method of recovering poly- hydroxyalkanoate from Azotobacter chroococcum. Chinese Science Bulletin, 2000;45(3):252-255

Zheng Z, Chen JC, Tian HL, Bei FF and Chen GQ. Specific identification of (R)-3-hydroxyacyl-ACP: CoA transacylase gene from Pseudomonas and Burkholderia strains by polymerase chain reaction. Sheng Wu Gong Cheng Xue Bao. 2005;21(1):19-24