Tryptophan production

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Transcript Tryptophan production

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
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Enzyme kinetics – flux bottlenecks
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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
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Rate of reaction is determined at
multiple levels
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Enzyme & regulation protein expression,
enzyme modification, enzyme degradation,
enzyme activity.
Protein expression
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Changing promoter strength level,
induction level of the promoter.
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Regulatory network (genetics
engineering)
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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
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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
(DAHP synthetase, tyrosine-repressible)
Mutated
(dulled)
X
(DAHP synthetase, phenylalanine repressible)
X
(DAHP synthetase, tryptophan-repressible )
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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
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)
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Gene map >
Trp operon, inactive TrpR
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A
C
B
D
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Making anthranilate synthetase
(trpE,D) insensitive to tryptophan
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Overexpression of the feedback-insensitive anthranilate synthase gene in
tobacco causes tryptophan accumulation, 2004, F.-Y. Tsai.
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Characterization of Rice Anthranilate Synthase –Subunit Genes OASA1 and
OASA2. Tryptophan Accumulation in Transgenic Rice Expressing a FeedbackInsensitive Mutant of OASA11, 2001, Yuzuru Tozawa.
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AS consists of two alpha-subunits that carry the Trp binding and catalytic sites.
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
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(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
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Regulation of Tryptophan
Operon Expression by
Attenuation in Cell-free Extracts
of Escherichia coli , 1982,
Anathbandhu Das.
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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 [EC:4.1.99.1] b3708
tryptophan deaminase
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Overview
Link>
How will bioinformatics influence Metabolic Engineering? 1998, Jeremy SE.
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Summary
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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)
<back
How will bioinformatics influence Metabolic Engineering? 1998, Jeremy SE.
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Reference
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[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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