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研究生:范梅櫻
研究生(外文):Mei-Ying Fan
論文名稱:大腸桿菌之代謝工程研究
論文名稱(外文):Metabolic engineering of Escherichia coli
指導教授:阮俊人
指導教授(外文):Jiunn-Ren Roan
學位類別:碩士
校院名稱:國立中興大學
系所名稱:物理學系
學門:自然科學學門
學類:物理學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:90
中文關鍵詞:大腸桿菌代謝工程系統生物學
外文關鍵詞:flux balance analysismetabilismlinear optimization
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本研究以電腦模擬篩選最佳化的方法來探討大腸桿菌在不同的生長條件限制與生長速率下合成細胞所須生物合成量(biomass)與其代謝路徑流量。
我所使用的化學計量模型是由大腸桿菌代謝計量方程式與生長條件(葡萄糖、代謝產物與無機鹽)所建構,在代謝反應穩態平衡(steady-state)下,可由線性規劃(linear programming)的方法獲得最大生物合成量。此種在給定的生長條件限制之下,對代謝與生長能力作量化預測之模型稱為流量平衡分析法(flux balance analysis, FBA)。為了解何種代謝途徑能獲取最大的生物合成量,我將代謝反應產生的能量、合成蛋白質的先質、輔因子、輔基(prosthetic groups)以及合成生物量時所需的能量等生長條件併入模型中作為條件限制。除此之外,為了預測出接近實驗值的最佳化生物合成量,本研究所要求之代謝所需生物合成量與生長條件限制均來自不同生長速率下所測出之實驗數據。
我應用此模型於二種不同主題:一是大腸桿菌必須合成的12種先質與3種輔因子在能量侷限下的最大產量;另一是模擬大腸桿菌在不同生長條件限制下之最大生物合成量與流量。在第一個主題中,求得的結果與實驗符合;第二個主題以雙倍生長時間70分鐘且加入氧氣、葡萄糖與醋酸鹽以及雙倍生長時間為145分鐘且加入氧氣與醋酸鹽為條件限制,模擬所得的流量與實驗結果比較,誤差率分別是20.83%與31.45%。我也應用此模型預測基因缺失(gene deletions)對於大腸桿菌合成生物量的影響,在供給葡萄糖10 mmol/g DW ( DW = dry weight )與氧氣15 mmol/g DW時,與實驗結果比較,其預測準確率約為79%~87%之間。
As a consequence of natural selection, the metaboloic network of an organism is capable of digesting various substances and synthesizing new biomass in a wide variety of environments. This capability is examined in this work by computer simulation.
The methodology adopted in my simulations is called flux balance analysis (FBA). In FBA, a metabolic network is mathematically described by metabolic fluxes and the associated stoichiometric relations these fluxes satisfy. A steady-state condition is imposed and results in a set of linear algebraic equations. The equations usually have more than one solution. Environmental conditions such as presence or absence of glucose then serve as constraints that trim the solution space. Further constraints may come from other non-environmental conditions: a desired growth rate, a mutant that lacks certain enzyme needed in a reaction, etc. Finally, linear programming can be used to find the solution that optimizes a specific flux or a set of fluxes. To facilitate comparison
between theoretical and experimental results, experimentally determined values were used whenever possible.
Specifically, I considered two cases in E. coli. One is to optimize the fluxes for the 12 prefactors and 3 prosthetic groups under a constrainted energy. The other is to find the optimal biomass synthesized and the associated fluxes under a given growth rate. While in the first case simulations agreed with experiments, in the second case they differed, but the relative errors were all less than 30%. I also attempted in the second case to predict the effect of gene deletion on the
optimal growth rate. In about 79%~87% of the mutants examined, simulations agreed with experiments.
中文摘要----------------------------------------------------------------------------------------------------- I
Abstract------------------------------------------------------------------------------------------------------- II
總目次-------------------------------------------------------------------------------------------------------- III
表目次-------------------------------------------------------------------------------------------------------- V
圖目次--------------------------------------------------------------------------------------------------------- VII
第1章 緒言------------------------------------------------------------------------------------------------- 1
第2章 生物資訊與代謝工程背景知識---------------------------------------------------------------- 3
2.1  理論模型的資料來源------------------------------------------------------------------------ 3
2.2 生物代謝機制--------------------------------------------------------------------------------- 4
2.2.1 醣類的代謝-------------------------------------------------------------------------------- 4
2.2.2 脂質的代謝--------------------------------------------------------------------------------- 13
2.2.3 蛋白質與胺基酸的代謝------------------------------------------------------------------ 14

第3章 研究理論與方法--------------------------------------------------------------------------------- 16
3.1 理論模型定義--------------------------------------------------------------------------------- 16
3.2 理論模擬設計--------------------------------------------------------------------------------- 18
3.3 演算法------------------------------------------------------------------------------------------ 21

第4章 研究結果與分析--------------------------------------------------------------------------------- 24
4.1 大腸桿菌的生物合成先質與輔因子之合成研究分析--------------------------------- 24
4.1.1 單一代謝合成物的最大產量研究結果---------------------------------------------- 26
4.1.2 完全氧化下之耗能與碳轉換之分析------------------------------------------------- 36
4.2 討論合成生物量和能量需求的大腸桿菌化學計量代謝模型研究------------------ 39
4.2.1 生長速度與生物合成量之關係------------------------------------------------------- 39
4.2.2 生物合成量的實驗數據---------------------------------------------------------------- 42
4.2.3 最大生物合成量研究結果------------------------------------------------------------- 43
4.2.4 大腸桿菌基因缺陷之研究結果------------------------------------------------------- 50
第五章 結論與未來的工作計畫----------------------------------------------------------------------- 53
5.1 結論--------------------------------------------------------------------------------------------- 53
5.2 未來的工作計畫------------------------------------------------------------------------------ 53

References--------------------------------------------------------------------------------------------------- 55
中文參考文獻---------------------------------------------------------------------------------------------- 56
網路參考資料---------------------------------------------------------------------------------------------- 56

附錄A-1 資料庫(4.1節之代謝化學反應式)-------------------------------------------------------- 57
附錄A-2 資料庫(4.2節之代謝化學反應式)-------------------------------------------------------- 59
附錄B 資料庫(4.2節之代謝物)----------------------------- -------------------------------------- 66
附錄C C++程式碼----------------------------------------------------------------------------------- 73
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中文參考文獻:
[24] 林正宗與林榮耀, (1992). 生物化學指引,第三版 (南山堂,台北)。
網路參考資料:
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[26] http://binfo.ym.edu.tw/sb
[27] http://systemsbiology.ucsd.edu
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