Tryptophan%20production

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Tryptophan production

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• ?????? 938341 ???

2
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
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
C
B
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
Mutated (dulled)
(DAHP synthetase, tyrosine-repressible)
X
(DAHP synthetase, phenylalanine repressible)
X
(DAHP synthetase, tryptophan-repressible )

• tyrR b1323 transcriptional regulation of aroF,
  aroG, tyrA. tyrR will be inactivate.

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A
C
B
D
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Cut the branch down
tyrA5.4.99.5 1.3.1.12 bifunctional
1.chorismate mutase T (N-terminal) 2.prephenate
dehydrogenase (C-terminal) pheA5.4.99.54.2.1.
51 bifunctional 1.chorismate mutase P
(N-terminal) 2.prephenate dehydratase
(C-terminal)
1
2
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tyrA5.4.99.5 1.3.1.12 bifunctional
1.chorismate mutase T (N-terminal)2.prephenate
dehydrogenase (C-terminal)pheA5.4.99.54.2.1.
51 bifunctional 1.chorismate mutase P
(N-terminal) 2.prephenate dehydratase
(C-terminal)
Gene map gt
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Trp operon, inactive TrpR
13
A
C
B
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 tRNATrp 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
C
B
D
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Stop tryptophan metabolism
tnaA EC4.1.99.1 b3708 tryptophan deaminase
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Overview
Linkgt
How will bioinformatics influence Metabolic
Engineering? 1998, Jeremy SE.
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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)
ltback

• How will bioinformatics influence Metabolic
  Engineering? 1998, Jeremy SE.

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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
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