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!