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研究生:林紀宏
研究生(外文):Ji-Hong Lin
論文名稱:將嗜熱性Caldimonas manganoxidans之phaCAB基因轉殖於大腸桿菌進行聚羥基丁酸酯生產
論文名稱(外文):Cloning of phaCAB genes from thermophilic Caldimonas manganoxidans in Escherichia coli for PHB production
指導教授:李思禹
指導教授(外文):Si-Yu Li
口試委員:吳宗明魏毓宏
口試委員(外文):Tzung-Ming WuYu-Hong Wei
口試日期:2015-07-28
學位類別:碩士
校院名稱:國立中興大學
系所名稱:化學工程學系所
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:2015
畢業學年度:103
語文別:中文
論文頁數:67
中文關鍵詞:嗜熱菌聚羥基丁酸酯重組大腸桿菌
外文關鍵詞:thermophilesPHBrecombinant E. coli
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Caldimonas manganoxidans JCM 10698 (BCRC 17858)是一種在日本的溫泉分離出的嗜熱菌,在1998年第一次被發現,屬於β-變形菌 (β-Proteobacteria),也是第一個以Caldimonas命名的菌種,具有生產及降解PHB的能力。Caldimonas manganoxidans的PHA生產基因確認是由β-ketothiolase (phaACm gene, 1179 bp), acetoacetyl-CoA reductase (phaBCm gene, 738 bp) and PHA synthase, (phaCCm gene, 1694 bp)所組成,PHA synthase的序列比對發現,與Class I菌株Alcaligenes latus、Cupriavidus taiwanensis、Comamonas sp. EB 172和Cupriavidus necator有至少60%以上的相似性,但是與Pseudomonas aeruginosa (class II)、Allochromatium vinosum (class III)和Bacillus megaterium (class IV)只有41、22和23%的相似性,本研究是首例成功將C. manganoxidans之PHA合成基因轉殖入重組大腸桿菌的研究,E. coli BL21 (DE3)/pT7pha1A2B/pBADphaC在Nile Blue stain assay測試結果,在UV光照射下會放出螢光,TEM圖也可以清楚看到菌體內的PHB顆粒。耗氧批次培養是使用LB含20 g/L葡萄糖進行,wild type大腸桿菌的OD600約7-8,而重組大腸桿菌則是高達39,PHB content和PHB濃度分別為52.2±2.0%和6.38±0.15 g/L。實驗結果證實PhaB的表現量,會影響PHB合成反應中單體的生成,進而影響產量。L-arabinose除了可以當作誘導劑,也可以作為碳源利用,實驗結果確實可以增加PHB產量。實驗結果也證實剃除ldh和frd往乳酸 (Lactate)和琥珀酸 (Succinate)的路徑,將碳流導回pyruvate往acetyl-CoA後,能幫助菌體生長,增加PHB產量。

Caldimonas manganoxidans, a thermophilic bacterial strains of β-Proteobacteria isolated from the hot spring in Japan, was attractive for its ability to accumulate and degrade PHB. PHA biosynthesis genes of Caldimonas manganoxidans consisting of three genes encoding β-ketothiolase (phaACm gene, 1179 bp), acetoacetyl-CoA reductase (phaBCm gene, 738 bp) and PHA synthase, (phaCCm gene, 1694 bp) were identified in this study. Sequence alignment of PHA synthases revealed that phaCCm showed at least 60% identity with those of class I strains Alcaligenes latus, Cupriavidus taiwanensis, Comamonas sp. EB 172, and Cupriavidus necator. However, the phaCCm showed low identities of 41%, 22%, and 23% with those of Pseudomonas aeruginosa (class II), Allochromatium vinosum (class III), and Bacillus megaterium (class IV). The PHA biosynthesis genes were successfully cloned in Escherichia coli. E. coli BL21 (DE3) harboring pT7pha1A2B and pBADphaC grown on LB agar plate containing Nile Blue A emitted fluorescence under UV light irradiation (312 nm). PHB granule could clearly be seen in TEM. These results revealed that recombinant E. coli could accumulate PHB. LB medium containing 20 g/L glucose was also used to confirm PHB production in this study. The result of OD600 showed that the control group of wild type E. coli BL21 (DE3) is only 7-8 whereas E. coli BL21 (DE3) harboring pT7pha1A2B and pBADphaC is as high as 39 (data not shown). The PHB content and the PHB concentration of 52.2±2.0% and 6.38±0.15 g/L can be reached in recombinant E. coli.

誌謝 i
摘要 ii
Abstract iii
目錄 v
表目錄 viii
圖目錄 ix
第一章 緒論 1
1.1 研究背景 1
1.2 研究動機 2
第二章 文獻回顧 4
2.1 P(3HB)及其他PHA介紹 4
2.1.1 物化性質 4
2.1.2 生物特性 5
2.1.3 應用範圍 6
2.2 微生物生產PHA 6
2.2.1 PHA Biosynthetic Pathway 6
2.2.2 重組大腸桿菌 7
2.3 嗜熱菌及其生產酵素之簡介 8
第三章 材料與方法 11
3.1 實驗菌株、質體和引子序列 11
3.2 實驗藥品、器材與分析設備 11
3.3 Next Generation Sequencing (NGS) 11
3.4 DNA純化 12
3.5 DNA膠體電泳 12
3.6 質體構築 12
3.6.1 PCR 12
3.6.2 Digestion 13
3.6.3 Ligation 13
3.6.4 Transformation 13
3.7 蛋白質電泳 (SDS-PAGE) 14
3.8 菌體培養 15
3.8.1 培養基配製 15
3.8.2 菌體活化 15
3.8.3 LB和M9 medium之耗氧搖瓶批次培養 16
3.8.4 M9 medium之厭氧批次培養 16
3.9 Nile Blue A stain assay 16
3.10 TEM 16
3.11 還原糖濃度測定 17
3.12 PHB content 17
第四章 結果與討論 19
4.1 C. manganoxidans之phaCAB基因和胺基酸序列比對 19
4.2 質體構築和SDS-PAGE之結果 20
4.3 Nile blue A stain assay和TEM 21
4.4 重組E. coli的菌體生長、PHB合成之結果 21
4.5 PhaA和PhaB表現量對重組E. coli合成PHB之影響 22
4.6 不同L-arabinose誘導量對重組E. coli合成PHB之影響 24
4.7 不同起始葡萄糖濃度對重組E. coli生產PHB的結果 25
4.8 M9 medium厭氧發酵結果 26
第五章 結論 28
第六章 參考文獻 56
第七章 附錄 63
附錄一 63
附錄二 65


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