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System Biology Term Project Tryptophan Production

System Biology Term Project Tryptophan Production. Group 5 Teammates: 林瑋耿 , 吳東耘 , 許博凱 , 彭嘉冠. 在現存途徑中提高目標產物代謝流 ( 五個方法 ). 增加代謝途徑中限速步驟酶編碼基因的複製數 強化啟動子為主的關鍵基因的表現系統. 在現存途徑中提高目標產物代謝流 ( 五個方法 ). 提高目標途徑活化因子的合成速率 去活化目標途徑抑制因子的編碼基因 阻斷與目標途徑相競爭的代謝途徑. Tryptophan synthesis pathway. Object.

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System Biology Term Project Tryptophan Production

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  1. System Biology Term ProjectTryptophan Production Group 5 Teammates: 林瑋耿, 吳東耘, 許博凱, 彭嘉冠

  2. 在現存途徑中提高目標產物代謝流(五個方法) • 增加代謝途徑中限速步驟酶編碼基因的複製數 • 強化啟動子為主的關鍵基因的表現系統

  3. 在現存途徑中提高目標產物代謝流(五個方法) • 提高目標途徑活化因子的合成速率 • 去活化目標途徑抑制因子的編碼基因 • 阻斷與目標途徑相競爭的代謝途徑

  4. Tryptophan synthesis pathway Object

  5. Strategy of enhancing the tryptophan production • Increase the glucose uptake without depleting the available PEP. • Recycle PEP • Reduce PEP usage in PTS. • Increase the E4P supply, especially the late stage of fermentation. • Non-oxidatively increase E4P. • Decrease the feedback-inhibition. • Feedback-insensitive isozyme • Trp operon repressor mutant. • Optimize the glucose feed rate.

  6. PEP:carbohydratephosho-transferase system (PTS) (Yi et al., 2002.Biotechnol. Prog., 18, 1141-1148)

  7. PEP supply • Recycle PEP: PEP synthase (ppsA) recycles pyruvate generated by PTS back to PEP. (Yi et al., 2002.Biotechnol. Prog., 18, 1141-1148) • Reduce PEP usage in PTS: replace PTS with glucose facilitator protein (Zymomonas mobilis’ glf) or galactose permease (E.coli gal-P ). (Yi et al., 2003.Biotechnol. Prog., 19, 1450-1459)

  8. glucose facilitator protein or galactose permease PEP synthase help recycle the PEP replace (Yi et al., 2002.Biotechnol. Prog., 18, 1141-1148)

  9. E4P supply • At the late stage of fermentation, carbon flow will shift to TCA cycle, and the available E4P is limited. • modify the pentose phosphate pathway (PPP) by overexpressing transketolase in order to change the carbon flow with a non-oxidative pathway. (Ikeda and Katsumata, Applied and Environmental Microbiol., 2497–2502)

  10. Decrease feedback inhibition • DAHP synthase isoenzymes: • Encoding by aroF– feedback inhibited by Tyr. • Encoding by aroG– feedback inhibited by Phe. • Encoding by aroH– feedback inhibited by Trp. • Trp operon repressor mutant: • TrpR2has a frame-shift mutation in trpR that eliminates the production of functional trp repressor. • Anthranilate synthase mutant (trpE19): • trpE19 has a mutation in trpE that eliminates feedback inhibition of anthranilate synthase by tryptophan.

  11. aroF aroG aroH

  12. trpE19

  13. Optimize the glucose feed rate • The glucose feed rate profile have substantial effects on both tryptophan and glutamate (by-product) production. • A feed rate profile was designed to maximize tryptophan production while minimizing the formation of the by-product glutamate. (Dodge & Gerstner, 2002, J. Chem. Technol. Biotechnol., 77: 1238-1245)

  14. Optimize the glucose feed rate- Baseline

  15. Optimize the glucose feed rate High Feed rate Modified Feed rate

  16. Optimize the glucose feed rate- Glutamate formation

  17. Low growth rate, low [glutamate]. high growth rate, low [glutamate]. high growth rate, low [glutamate] Low growth rate, high [glutamate]

  18. Before After Results comparison

  19. Metabolic Flux Analysis

  20. Metabolic Flux Analysis Degree of freedom= 總反應數目-代謝物數目 根據可以測得的反應數據，判斷系統為determined, overdetermined, or underdetermined

  21. Metabolic Flux Analysis 可以推算無法被測量到的反應其物質的通量及判斷碳源流向!

  22. Reference • Gregory N. Stephanopoulos, et al., Metabolic Engineering - Principles and Methodology , pp.309-p351 • Joachim W. Schmid, Klaus Mauch, Matthias Reuss, Ernst D. Gilles, and Andreas Kremling, 2004. Metabolic design based on a coupled gene expression-metabolic network model of tryptophan production in Escherichia coli. Metabolic Engineering 6, 364-377 • Ikeda M. and Katsumata R., 1999, Hyperproduction of Tryptophan by Corynebacterium glutamincum with the Modified Pentose Phosphate Pathway. Appl. Environ. Microbiol., 65(6):2497-2502.

  23. Reference • Yi J., Draths K. M., Li K., Frost J. W., 2002, Modulation of Phospho- enolpyruvate Synthase Expression Increases Shikimate Pathway Product Yields in E.coli. Biotechnol. Prog. 18:1141-1148. • Yi J., Draths K. M., Li K., Frost J. W., 2003, Altered Glucose Transport and Shikimate Pathway Product Yields in E.coli. Biotechnol. Prog. 19:1450-1459. • Dodge T. C. and Gerstner J. M., 2002, Optimization of the glucose feed rate profile for the production of tryptophan from recombinant E.coli. J. Chem Technol Biotechnol. 77:1238-1245

  24. Conclusion • 由文獻上的結果可以預期，我們最終的目標是得到最大的tryptophan產率且盡可能的減少副產物glutamate。 • 如何調整使E-coli維持在高產率的環境與條件而不會快速的衰退其產率，也是值得去考慮的問題。

  25. Thanks for your attention and comments!

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