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研究生:周大中
研究生(外文):Ta-Chung Chou
論文名稱:克雷伯氏肺炎菌中影響1,3-丙二醇生產相關基因之研究
論文名稱(外文):The study of 1,3-propanediol production byengineering glycerol metabolic related genes in Klebsiella pneumoniae
指導教授:蘇伯琦
指導教授(外文):Po-Chi Soo
學位類別:碩士
校院名稱:元智大學
系所名稱:生物科技與工程研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:102
中文關鍵詞:克雷伯氏肺炎菌13-丙二醇細菌血紅素
外文關鍵詞:Klebsiella pneumoniae13-PropanediolVitreoscilla hemoglobin
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1,3- 丙二醇是一種重要的有機化工原料, 最重要的用途是作為聚酯
(polyester)、聚醚(polyether)和聚亞氨酯(polyurethanes)的單體。由它合成的聚酯有獨特的性質和優異的性能,而且可以使聚酯塑膠具有易於自然循環的可生物降解特性。目前已知有許多微生物諸如克雷伯氏菌屬之Klebsiella pneumoniae、 Klebsiella oxytoca,梭狀芽孢桿菌屬之Clostridium butyricum,檸檬酸桿菌屬之Citrobacter freundii,Enterobacter agglomerans,嗜酸乳酸桿菌屬之Lactobacillus brevis 等等皆可生合成1,3-丙二醇。本研究利用同源性染色體基因重組技術將克雷伯氏肺炎菌(Klebsiella pneumoniae)中,甘油氧化代謝途徑之重要基因dhaD、dhaK 剔除,剔除之菌株將無法產生甘油代謝後之副產物且試圖將代謝途徑轉向有效利用甘油合成1,3-丙二醇。根據實驗結果顯示dhaD、dhaK剔除菌株TC100,在氧化代謝甘油途徑中,乳酸、2,3-丁二醇與酒精等副產物被順利剔除。另外在產量上,野生型菌株與突變株TC100 之1,3-丙二醇產量分別為5.59(g/L)與1.91 (g/L);TC100 並沒有如預期般因缺少生成部分副產物而產生更多的1,3-丙二醇。
因此本研究持續以提升菌株生產1,3-丙二醇為目的,將其代謝甘油路徑中直接與1,3-丙二醇生產有關的dhaB 與dhaT 基因進行過量表現,利用基因剪接技術將兩基因接至表現質體上並將其分別轉入野生型菌株與TC100。進一步利用酵素活性分析證實過量表現的dhaB 與dhaT 活性皆較無過量表現此二酵素之對照組菌株為高。在利用液相層析法中比較各菌株間1,3-丙二醇的產量關係,研究結果顯示過量表現dhaB 之菌株TCKP01,其每單位菌量之1,3-丙二醇產量大約高出野生型菌株1.01% (5.62g/L)、過量表現dhaT 之菌株TCKP05中,每單位菌量之1,3-丙二醇產量大約高出野生型菌株1.01%;在TC100 方面,
過量表現dhaB 之菌株TC101,其每單位菌量之1,3-丙二醇產量高出TC100 約20.4%、過量表現dhaT 之菌株TC105,每單位菌量之1,3-丙二醇產量高出TC100約56.6%。因此本研究證實將dhaD、dhaK 剔除有助於減少副產物的產生以利純化1,3-丙二醇的過程與將dhaB 或dhaT 分別加以過量表現,將有益於增加單位菌量之1,3-丙二醇產量。這些重組菌株對於工業生產1,3-丙二醇有很大的助益。
1,3-propanediol (1,3-PD) is an important organic compound in chemical industry,and this compound could potentially be used for many synthesis reactions, such as a monomer to produce polyesters, polyethers and polyurethanes. The polyesters which were synthesized by 1,3-PD has unique and outstanding performance; moreover, 1,3-PD may enable the polyesters easy to have the characteristic of natural circulation and biodegradation . In recent study, there are many different microorganisms that produce 1,3-propanediol, such as Klebsiella pneumoniae, Klebsiella oxytoca, Clostridium butyricum, Citrobacter freundii, Enterobacter agglomerans and Lactobacillus brevis. In this study, in order to facilitate the conversion of glycerol to
1,3-PD in K. pneumoniae, we employed the homologous recombination to knock out two important genes, dhaD and dhaK, in glycerol oxidation pathway to eliminate side
products during glycerol metabolism. The result indicated that although the side products in glycerol oxidation pathway such as lactate, 2,3-butanediol and ethanol
were successfully eliminated in mutant strain TC100, the productivity of 1,3-propanediol of wild type (WT) and TC100 were 5.59(g/L) and 1.91(g/L), respectively.
To further promote the 1,3-PD productivity, we directly overexpressed the genes dhaB and dhaT, involved in 1,3-PD formation, by transforming expression vector into
TC100. We verified that the enzyme activities of DhaB or DhaT were higher in the recombinant strains TC101 (TC100/pBAD-dhaB) and TC105 (TC100/pBAD-dhaT)
compared with TC100. Using high performance liquid chromatography, the 1,3-PD productivity of TCKP01(WT/pBAD-dhaB) and TCKP05(WT/pBAD-dhaT) were both 1.01% higher than wild type strain respectively. Onther the other hand, the 1,3-PD productivity of TC101 and TC105 were 20.4% and 56.6% higher than TC100. In this study, we corroborated that deletion of dhaD and dhaK led to reduce side products
and overexpression of dhaB and dhaT facilitated the 1,3-PD productivity in K. pneumoniae. The recombinant strain(s) would be a great industrial application in the production of 1,3-PD.
中文摘要 ..................................................................................................... i
英文摘要 ................................................................................................... ii


目錄 ............................................................................................................. iii
圖目錄 ....................................................................................................... viii
表目錄 ..................................................................................................... x
第一章、文獻回顧 .................................................................................... 1
1.1 簡介克雷伯氏肺炎菌 (Klebsiella pneumoniae) .......................... 1
1.2 1,3-丙二醇與其生產方法簡介 ............................................................ 1
1.2.1 1,3-丙二醇簡介 ................................................................... 1
1.2.2 現今1,3-丙二醇之生產方法 ............................................. 3
1.2.2.1 化學合成法 ...................................................................... 3
1.2.2.2 生物合成法 ...................................................................... 4
1.3 克雷伯氏肺炎菌(K. pneumoniae) 中與甘油代謝之相關基因 ... 6
1.3.1 dhaB ( glycerol dehydrotase ) 與 dhaT ( 1,3-PD
oxidoreductase) .......................................................................... 6
1.3.2 dhaD ( glycerol dehydrogenase ) 與dhaK
(dihydroxyacetone kinase) ....................................................... 7
1.3.3 dha regulon .......................................................................... 7
1.4 梭狀芽孢桿菌Clostridium butyricum 中與1,3-丙二醇生產之相關基因 ............................................................................................................. 8
1.5 細菌血紅素(Bacterial Hemoglobin) .............................................. 9
1.6 研究目的 ............................................................................................ 10
第二章、材料與方法 .............................................................................. 12
2.1 實驗材料 ........................................................................................... 12
2.1.1 實驗使用之菌株與質體 .................................................... 12
2.1.2 實驗藥品 ............................................................................ 13
2.1.3 實驗儀器 ........................................................................... 14
2.1.4 引子 ................................................................................... 16
2.1.5 前培養之培養基質成分 .................................................... 16
2.1.6 主培養之培養基質成分 .................................................... 17
2.1.7 製備勝任細胞之培養基 ................................................... 18
2.1.8 酵素 ................................................................................... 18
2.1.9 質體DNA 純化試劑 ........................................................ 18
2.1.10 抗生素 ............................................................................. 19
2.1.11 DNA 電泳試劑 ................................................................ 19
2.1.12 革蘭氏陰性菌染色體純化試劑..................................... 20
2.1.13.1 分析Glycerol dehydratase(DhaB)所使用的分析溶
液 ................................................................................................. 20

2.1.13.2 分析1,3-Propanediol oxidoreductase(DhaT)所使用
的分析溶液 ................................................................................. 21
2.1.14 其他 ................................................................................. 21
2.2 實驗方法 ........................................................................................... 22
2.2.1 染色體DNA 之純化(Chromosomal DNA purification)
..................................................................................................... 22
2.2.2 質體DNA 之純化(Plasmid DNA purification) .......... 22
2.2.3 洋菜膠體萃取DNA(DNA extraction) ........................ 23
2.2.4 DNA 接合作用(Ligation) ............................................ 24
2.2.5 轉型作用(Transformation) .......................................... 24
2.2.5.1 大腸桿菌勝任細胞(Competent cell)之製備 ........... 24
2.2.5.2 DNA 轉型作用(Transformation) .............................. 25
2.2.6 重組質體之篩選 ................................................................ 25
2.2.7 細菌接合作用(Conjugation) ....................................... 26
2.2.8 同源性染色體重組作用(Homologous recombination)
..................................................................................................... 27
2.2.9 蛋白質定量分析(Protein content assay) .................... 29
2.2.10 蛋白質活性分析(Protein activity assay) .................. 30
2.2.10.1Glycerol dehydratase(DhaB)活性分析 .................... 30
2.2.10.2 1,3-Propanediol oxidoreductase(DhaT)活性分析 .. 31

2.2.11 HPLC 產物分析….……………………………………..31
2.2.12 Western blot 分析 ............................................................ 33
第三章、結果........................................................................................... 34
3.1 選殖Klebsiella pneumoniae dhaD 與dhaK 同源性基因片段 ........ 34
3.2 dhaD 與dhaK 基因剔除質體的構築 ................................................ 35
3.3 同源性染色體基因重組置換 ............................................................ 37
3.4 dhaD 與dhaK 剔除菌株之基因型確認 ............................................ 41
3.5 克雷伯氏肺炎菌野生株與基因剔除突變株之甘油代謝產物分析
................................................................................................................... 41
3.6 構築過量表現dhaB 與dhaT 之質體 ................................................ 49
3.7 在克雷伯氏肺炎菌中過量表現dhaT 之1,3-PD oxidoreductase 酵素
活性分析 .................................................................................................. 50
3.8 在克雷伯氏肺炎菌中過量表現dhaT 對生產1,3-丙二醇之影響 . 54
3.9 利用阿拉伯糖誘導過量表現dhaT 質體pTC-4 對於1,3-PD
oxidoreductase 酵素活性與生產1,3-丙二醇之影響 .............................. 56
3.10 在克雷伯氏肺炎菌中過量表現dhaB 之Glycerol dehydratase 酵素
活性分析 .................................................................................................. 57
3.11 克雷伯氏肺炎菌中過量表現dhaB 對1,3-丙二醇生產之影響 ... 59
3.12 對菌株TC100 過量表現dhaT 對1,3-丙二醇生產影響之研究 ... 61
3.13 對菌株TC100 過量表現dhaB 對1,3-丙二醇生產影響之研究 ... 64

3.14 延長培養時間對TC100 系列菌株生產1,3-丙二醇之影響 ......... 67
3.15 調整培養基之pH 值對剔除型菌株TC100 生長及產物之影響 . 69
3.16 對菌株TC100 同時過量表現dhaB 與dhaT 產生1,3-丙二醇之影
響 ............................................................................................................... 74
3.17 透明顫菌血紅素 Vitreoscilla hemoglobin(VHb)對影響1,3-丙二
醇生產之研究 .......................................................................................... 75
第四章、討論........................................................................................... 85
第五章、附錄........................................................................................... 93
附錄(一)甘油與甘油代謝副產物之標準曲線 .................................. 93
附錄(二)1,3-丙二醇濃度標準曲線 .................................................... 96
第六章、參考文獻 .................................................................................. 97

圖目錄
圖(一)、1,3-propanediol 之結構 .............................................................. 2
圖(二)、PTT 聚酯之人造纖維 .................................................................. 3
圖(三)、甘油代謝主要路徑 ...................................................................... 5
圖(四)、以葡萄糖為基質用以生產1,3-丙二醇的二階段發酵法 .......... 6
圖(五)、細菌接合作用(Bacterial conjugation)之概觀圖 ................. 28
圖(六)、利用dhaD-FF/dhaD-FR 與 dhaK-BF/dhaK-BR 兩組引子對分
別將dhaD 與dhaK 兩基因分別部分放大 ............................................. 36
圖(七)、同源性染色體互換之自殺質體pTC-1 構築過程 ................... 38
圖(八)、pTC-1 之限制酵素剪切之電泳結果 ........................................ 39
圖(九)、本研究細菌接合作用之詳細過程圖 ........................................ 43
圖(十)、同源性染色體重組 .................................................................... 44
圖 (十一)、利用電泳顯示基因 .............................................................. 45
圖(十二)、將K. pneumoniae 野生型菌株培養於主培養基24 小時並將
其產物以液相層析儀分析之分析結果 .................................................. 46
圖(十三)、剔除dhaD 與dhaK 之菌株TC100 其利用主培養基24 小時
並將其產物以液相層析儀分析之分析結果 .......................................... 46
圖(十四)、dhaB 與dhaT 利用PCR 放大於電泳膠片上所顯示之結果
................................................................................................................... 48
圖(十五)、過量表現dhaB 與dhaT 之質體構築流程 ........................... 51
vii
圖(十六)、1,3-PD oxidoreductase 蛋白質活性分析之結果 .................. 53
圖(十七)、阿拉伯糖誘導之1,3-PD oxidoreductase 活性分析結果圖 . 58
圖(十八)、Glycerol dehydratase 過量表現之酵素活性分析結果圖 .... 60
圖(十九)、在TC100 菌株中過量表現1,3-PD oxidoreductase 酵素活性
分析結果圖 ............................................................................................... 65
圖(二十)、在TC100 菌株中過量表現glycerol dehydratase 之酵素活性
分析結果圖 ............................................................................................... 70
圖(二十一)、帶有透明顫菌血紅素(VHb)之質體pTC-3 構築過程 .... 82
圖(二十二)、證實TCKP03 確實表現VHb 蛋白而進行之SDS PAGE 與
Western Blotting 的結果 .......................................................................... 83
圖(二十三)、TC101 經阿拉伯糖誘導後生成原本被剔除之產物2,3-丁
二醇與酒精 ............................................................................................... 91
圖(二十四)、TC105 經阿拉伯糖誘導後生成原本被剔除之產物2,3-丁
二醇與酒精 ............................................................................................... 92
viii
表目錄
表(一)、野生型菌株與剔除型菌株之生長與產物分析 ....................... 47
表(二)、蛋白質總量分析。各菌株蛋白質總含量之結果 ................... 53
表(三)、針對TCKP05 與TCKP06 進行液相層析之產量分析結果 ... 55
表(四)、以阿拉伯糖水溶液對TCKP05 進行誘導之液相層析分析結果
................................................................................................................... 58
表(五)、Glycerol dehydratase 酵素活性分析實驗中各菌株之蛋白質總
含量 ........................................................................................................... 60
表(六)、針對TCKP01 誘導有無與TCKP02 進行液相層析之產量分析
結果 ........................................................................................................... 62
表(七)、1,3-PD oxidoreductase 酵素活性分析實驗中各菌株之蛋白質總
含量 ........................................................................................................... 65
表(八)、針對TC105 誘導有無與TC106 進行液相層析之產量分析結
果 ............................................................................................................... 66
表(九)、glycerol dehydratase 酵素活性分析實驗中各菌株之蛋白質總
含量 ........................................................................................................... 70
表(十)、針對TC101 誘導有無與TC102 進行液相層析之產量分析結
果 ............................................................................................................... 71
表(十一)、TC101 之延長培養時間實驗結果 ....................................... 72
ix
表(十二)、TC105 之延長培養時間實驗結果 ....................................... 73
表(十三)、針對野生型菌株與TC100 進行培養基pH 值之調整對產物
與菌體生長之結果 .................................................................................. 76
表(十四)、未調整pH 值之TC100 與調整pH 值後之TC100 比較表
................................................................................................................... 76
表(十五)、TC107 之酵素活性分析與其1,3-丙二醇產量比較表 ........ 76
表(十六)、共同過量表現dhaB、dhaT 之菌株TC107 與TC102 之產物
分析比較表 ............................................................................................... 80
表(十七)、共同過量表現dhaB、dhaT 之菌株TC107 與TC104 之產物
分析比較表 ............................................................................................... 80
表(十八)、帶有VHb 菌株之TCKP03 細胞生長、產量分析之結果 . 81
表(十九)、帶有VHb 菌株之TC103 細胞生長、產量分析之結果 .... 81
表(二十)、針對TC103 延長培養時間之細胞生長與產量分析結果 .. 84
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