System Biology Term ProjectTryptophan 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 )
A. 增加代謝途徑中限速步驟酶編碼基因的複製數
B. 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 )
C. 提高目標途徑活化因子的合成速率
D. 去活化目標途徑抑制因子的編碼基因
E. 阻斷與目標途徑相競爭的代謝途徑
Tryptophan synthesis pathway
Object
Strategy of enhancing the tryptophan production
1. Increase the glucose uptake without depleting the available PEP.• Recycle PEP• Reduce PEP usage in PTS.
2. Increase the E4P supply, especially the late stage of fermentation.• Non-oxidatively increase E4P.
3. Decrease the feedback-inhibition.• Feedback-insensitive isozyme• Trp operon repressor mutant.
4. Optimize the glucose feed rate.
PEP:carbohydratephosho-transferase system (PTS)
(Yi et al., 2002. Biotechnol. Prog., 18, 11411148)
PEP supply
Recycle PEP: PEP synthase (ppsA) recycles pyruvate generated by PTS back to PEP. (Yi et al., 2002. Biotechnol. Prog., 18, 11411148)
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)
PEP synthase help recycle the PEP
glucose facilitator proteinor
galactose permeasereplace
(Yi et al., 2002. Biotechnol. Prog., 18, 11411148)
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)
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: TrpR2 has a frame-shift mutation in trpR that eliminates the produc
tion of functional trp repressor.
Anthranilate synthase mutant (trpE19): trpE19 has a mutation in trpE that eliminates feedback inhibiti
on of anthranilate synthase by tryptophan.
aroFaroGaroH
trpE19
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)
Optimize the glucose feed rate- Baseline
Optimize the glucose feed rate
High Feed rate Modified Feed rate
Optimize the glucose feed rate- Glutamate formation
Low growth rate, low
[glutamate].
high growth rate, low [glutamate].
Low growth rate, high
[glutamate]
high growth rate, low [glutamate]
Results comparison
Before
After
Metabolic Flux Analysis
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
Metabolic Flux Analysis
0 cTcm
Tm vGvG
mTm
Tcc vGGv 1)(
ratesreaction measured ofVector matrix tscoefficien tricstoichiomereaction Measured
matrix tscoefficien tricstoichiomereaction measured-Non
.
.
. 14
3
2
nv
v
v
v
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 !
Reference
1. Gregory N. Stephanopoulos, et al., Metabolic Engineering - Principles and Methodology , pp.309-p351
2. 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
3. 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.
Reference
4. 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.
5. 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.
6. 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
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。
Thanks for your attention and comments!