Tryptophan production 系統生物學 第十組 資工系碩士班 林柏亨 資應所碩士班 陳昱廷 資工系博士班 沈家麟 資工系博士班 鄭佳揚

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.

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.

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.

Amino acid biosynthesis: aromatic family

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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

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)

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

tyrA;[ ] [ ] bifunctional: 1.chorismate mutase T (N-terminal); 2.prephenate dehydrogenase (C-terminal) pheA;[ ][ ] bifunctional: 1.chorismate mutase P (N-terminal); 2.prephenate dehydratase (C-terminal) Gene map >

Trp operon, inactive TrpR

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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.

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.

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.

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Stop tryptophan metabolism tnaA [EC: ] b3708 tryptophan deaminase

Overview How will bioinformatics influence Metabolic Engineering? 1998, Jeremy SE. Link>

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.

The end Thanks for paying attentions We are Group 10

Flux-balance analysis (stoichiometric matrix) How will bioinformatics influence Metabolic Engineering? 1998, Jeremy SE. <back

Reference [1]. Metabolic engineering, Gregory N.S., 1997, Textbook. [2]. How will bioinformatics influence metabolic engineering?, Jeremy S.E., biotechnology and bioengineering, vol. 58,

Complete genome gene map < Back