pyruvate decarboxylase

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E.coli K-12 MG1655 pathway
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?? pyruvate decarboxylase alcohol
dehydrogenase
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Anaerobic fermentation pathway of non-growing
yeast, with glucose as the sole carbon source
?Solid arrows reaction steps ? Dashed arrows
activation ? Dotted arrows inhibition ?
regulatory loops (,-) 7
Vassily Hatzimanikatis accepted 30 July 1997
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How to maximize specific ethanol production?
Which of the existent regulatory loops should be
inactivated? What associated changes should be
made in enzyme expression levels?
Three enzymes must be over-expressed
Two activation loops should be retained
?This method will maximize ethanol production,
giving an increase of 100 relative to the
reference state.
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E.coli Yeast regulatory superstructure
?Dashed arrows yeast ? Dotted arrows E.
coli ? Dashed-dotted yeast E.coli
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The optimal regulatory structure
This method will maximize ethanol production,
giving an increase of 114 relative to the
reference state.
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Specific glucose uptake(B) and ethanol production
rate(A) at 0.5 initial glucose concentration of
anaerobic, resting E. coli KO20 strains with the
plasmid pTrc99A only (control), overexpression
pfkF from E. coli(PYKec), and overexpression pyk
From B. stearothermohilus(PYKbs). Cells were
harvested from anaerobic mid-exponential
growth phase cultures. The indicated values
represent the mean value of at least two
independent experiments, and error bars indicate
the standard deviation.
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Specific glucose uptake (B) and ethanol
production rate (A) at 2 initial glucose
concentration of anaerobic, resting E. coli KO20
strains with the plasmid pTrc99A only (control),
overexpressing pfkF from E. coli (PYKec), and
overexpressing pyk from B. stearothermophilus
(PYKbs).Different shading indicates different
time points after the start of the experiment 3
h (light grey), 5 h (grey), and 7 h (dark grey).
Cells were harvested from the late exponential
growth phase of aerobically grown cultures. The
indicated values represent the mean value of at
least two independent experiments, and error bars
indicate the standard deviation.
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History Records

• system biology tools we try to use
• http//sbml.org/index.psp
• Many tools for pathway analysis
• Gepsi, Copsi, and FluxAnalysis
• Advantage using kinetic parameters with
  accurate qualitative analysis.
• Disadvantage without kinetic parameters
  knowledge, must mine information from literature
  or obtain from experiments

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GNU Linear Programming Kit

• GNU Homepage
• http//www.gnu.org/home.html
• GNU Free Software Directory
• http//directory.fsf.org/GNU/
• Environment
• Unix / Win32 (4.1 version)

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Linear Programming
Reaction File
Compound file
Object file
Perl program
Manual operation
Execute glpk instruction
Result file
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Reaction File Context

• context
• R00014 C05125 C00011 ltgt C00068
  C00022
• R00200 C00002 C00022 ltgt C00008 C00074
• R00235 C00002 C00033 C00010 ltgt C00020
  C00013 C00024
• R00658 C00631 ltgt C00074 C00001
• R00710 C00084 C00003 C00001 ltgt C00033
  C00004 C00080
• R00754 C00469 C00003 ltgt C00084 C00004
  C00080
• R01061 C00118 C00009 C00003 ltgt C00236
  C00004 C00080
• R01070 C05378 ltgt C00111 C00118
• R01512 C00002 C00197 ltgt C00008 C00236
• R01518 C00631 ltgt C00197
• R02569 C00024 C00579 ltgt C00010 C01136
• R02738 C04085 C00031 ltgt C00615 C00668
• R02740 C00668 ltgt C05345
• R03270 C05125 C00248 ltgt C01136 C00068
• R04779 C00002 C05345 ltgt C00008 C05378

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Compound File Context

• Context
• C00001 R00658 - R00710 gt 0
• C00002 - R00200 - R00235 - R01512 - R04779 gt
  0
• C00003 - R00710 - R00754 - R01061 gt 0
• C00004 R00710 R00754 R01061 gt 0
• C00008 R00200 R01512 R04779 gt 0
• C00010 - R00235 R02569 gt 0
• C00022 R00014 - R00200 gt 0
• C00024 R00235 - R02569 gt 0
• C00033 - R00235 R00710 gt 0
• C00068 R00014 R03270 gt 0
• C00074 R00200 R00658 gt 0
• C00080 R00710 R00754 R01061 gt 0
• C00084 - R00710 R00754 gt 0
• C00118 - R01061 R01070 gt 0
• C00197 - R01512 R01518 gt 0
• C00236 R01061 R01512 gt 0
• C00631 - R00658 - R01518 gt 0
• C00668 R02738 - R02740 gt 0

We must mutually add ethanol compound constraint
and input constraint into compound file, because
It has only one reaction
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Reaction Compound Pathway
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Reaction Compound Pathway (modified)
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Reference

• http//biocyc.org/ECOLI/ (EcoCyc)
• http//www.genome.jp/kegg/ (KEGG)
• David R. Shonnard, Applications of Molecular
  Biotechnology Ethanol Production from Cellulosic
  Biomass,CM4710 Biochemical Processes, November
  28, 2003.

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