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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
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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.
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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.
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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.
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Amino acid biosynthesis: aromatic family
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A B C D
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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
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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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A B C D
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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
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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 >
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Trp operon, inactive TrpR
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A B C D
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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.
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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.
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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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A B C D
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Stop tryptophan metabolism tnaA [EC:4.1.99.1] b3708 tryptophan deaminase
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Overview How will bioinformatics influence Metabolic Engineering? 1998, Jeremy SE. Link>
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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.
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The end Thanks for paying attentions We are Group 10
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Flux-balance analysis (stoichiometric matrix) How will bioinformatics influence Metabolic Engineering? 1998, Jeremy SE. <back
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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
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Complete genome gene map < Back
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