1. Copyright © 2004, All Rights Reserved. Tryptophan production 系統生物學 第十組 資工系碩士班 936337 林柏亨 資應所碩士班 936742 陳昱廷 資工系博士班 938347 沈家麟 資工系博士班 938341 鄭佳揚

2. Copyright © 2004, All Rights Reserved. Metabolic engineering on flux analysis Enzyme kinetics – flux bottlenecks Mutate or change key enzyme Balancing precursors and recycling cofactors Stoichiometric network, eg. block and test branch flux Regulatory network – feedback inhibition (by genetic engineering) M.E. starts with a desired target. Thus, the terminal pathway is usually the first concern.

3. Copyright © 2004, All Rights Reserved. Manipulation enzyme kinetics Rate of reaction is determined at multiple levels Enzyme & regulation protein expression, enzyme modification, enzyme degradation, enzyme activity. Protein expression Changing promoter strength level, induction level of the promoter.

4. Copyright © 2004, All Rights Reserved. Regulatory network (genetics engineering) Existing proteins (enzymes, regulators, etc.) can be blocked or removed, new proteins can be inserted. Protein activities can be changed gradually. Regulatory interactions can be altered.

5. Copyright © 2004, All Rights Reserved. Amino acid biosynthesis: aromatic family

6. Copyright © 2004, All Rights Reserved. A B C D

7. Engineering Central Metabolism, make PEP max production The yield of DAHP from glucose is still low, stoichiometric analysis shows that many enzyme compete for intracellular PEP. Over-expressed PEP synthase (pps) in the presence of glucose and increased the final concentration and the yield of DAHP by almost two fold, to a near theoretical maximum. [Ref.] Engineering of Escherichia coli Central Metabolism Engineering of Escherichia coli Central Metabolism for Aromatic Metabolite Production with Near Theoretical Yield, 1994, RANJAN PATNAIK

8. Copyright © 2004, All Rights Reserved. DAHP synthetase tyrR b1323 transcriptional regulation of aroF, aroG, tyrA. tyrR will be inactivate. (DAHP synthetase, phenylalanine repressible) (DAHP synthetase, tryptophan-repressible ) (DAHP synthetase, tyrosine-repressible) X X Mutated (dulled)

9. Copyright © 2004, All Rights Reserved. A B C D

10. Cut the branch down tyrA;[5.4.99.5] [1.3.1.12] bifunctional: 1.chorismate mutase T (N-terminal); 2.prephenate dehydrogenase (C-terminal) pheA;[5.4.99.5][4.2.1.51] bifunctional: 1.chorismate mutase P (N-terminal); 2.prephenate dehydratase (C-terminal) 1 2

11. Copyright © 2004, All Rights Reserved. tyrA;[5.4.99.5] [1.3.1.12] bifunctional: 1.chorismate mutase T (N-terminal); 2.prephenate dehydrogenase (C-terminal) pheA;[5.4.99.5][4.2.1.51] bifunctional: 1.chorismate mutase P (N-terminal); 2.prephenate dehydratase (C-terminal) Gene map >

12. Copyright © 2004, All Rights Reserved. Trp operon, inactive TrpR

13. Copyright © 2004, All Rights Reserved. A B C D

14. Making anthranilate synthetase (trpE,D) insensitive to tryptophan Overexpression of the feedback-insensitive anthranilate synthase gene in tobacco causes tryptophan accumulation, 2004, F.-Y. Tsai. AS consists of two alpha-subunits that carry the Trp binding and catalytic sites. Characterization of Rice Anthranilate Synthase – Subunit Genes OASA1 and OASA2. Tryptophan Accumulation in Transgenic Rice Expressing a Feedback- Insensitive Mutant of OASA11, 2001, Yuzuru Tozawa. Transformed and expressing a mutated OASA1 gene (D323N), that encode a protein aspartate-323 is replaced with asparagine manifested up to 35-fold increases in Trp accumulation. Increasing Tryptophan Synthesis in a Forage Legume Astragalus sinicus by Expressing the Tobacco Feedback-Insensitive Anthranilate Synthase (ASA2) Gene1, 2000, Hyeon-Je Cho.

15. Copyright © 2004, All Rights Reserved. Attenuation of inhibit tryptophan production (1). Charged tRNA-trp, (2). Trp. A. Trp over expressed, Ribosome goes too fast, let the region3, 4 fold to stem loop B. Trp low expressed, region1 has anti-codon of Trp, it move slowly, region2,3 paired and translation can go smothly.

16. Copyright © 2004, All Rights Reserved. Destruction of attenuation control by mutating trpS Regulation of Tryptophan Operon Expression by Attenuation in Cell-free Extracts of Escherichia coli, 1982, Anathbandhu Das. A tryptophanyl-tRNA synthetase mutant that reduces charging tRNA Trp in vivo. A 4- to 8-fold decrease in relative read-through transcription to wild type. trpS; tryptophanyl-tRNA synthetase.

17. Copyright © 2004, All Rights Reserved. A B C D

18. Stop tryptophan metabolism tnaA [EC:4.1.99.1] b3708 tryptophan deaminase

19. Copyright © 2004, All Rights Reserved. Overview How will bioinformatics influence Metabolic Engineering? 1998, Jeremy SE. Link>

20. Copyright © 2004, All Rights Reserved. Summary The terminal pathway is usually the most important factor in the flux. The feedback inhibition mechanism plays a major role in the regulation. Another microbial C. glutamicum is usually used on Typotophan production in industry.

21. Copyright © 2004, All Rights Reserved. The end Thanks for paying attentions We are Group 10

22. Copyright © 2004, All Rights Reserved. Flux-balance analysis (stoichiometric matrix) How will bioinformatics influence Metabolic Engineering? 1998, Jeremy SE. <back

23. Copyright © 2004, All Rights Reserved. Reference [1]. Metabolic engineering, Gregory N.S., 1997, Textbook. [2]. How will bioinformatics influence metabolic engineering?, Jeremy S.E., 1998. biotechnology and bioengineering, vol. 58, 162-169

24. Copyright © 2004, All Rights Reserved. Complete genome gene map < Back