E.coli K-12 MG1655 pathway

磷酸果糖激酶

烯醇酶缺少

pyruvate decarboxylase

alcohol dehydrogenase

調節

磷酸果糖激酶

烯醇酶

E.coli K-12 MG1655 pathway

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

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?

‧This method will maximize ethanol production, giving an increase of 100% relative to the reference state.

Three enzymes must beover-expressed

Two activation loops should be retained

E.coli & Yeast regulatory superstructure

‧Dashed arrows : yeast‧ Dotted arrows : E. coli‧ Dashed-dotted : yeast & E.coli

The optimal regulatory structure

This method will maximize ethanol production, giving an increase of 114% relative to the reference state.

Specific glucose uptake(B) and ethanol production rate(A) at 0.5% initial glucoseconcentration of anaerobic, resting E. coli KO20 strains with the plasmid pTrc99Aonly (control), overexpression pfkF from E. coli(PYKec), and overexpression pyk From B. stearothermohilus(PYKbs). Cells were harvested from anaerobic mid-exponential growthphase cultures. The indicated values represent the mean value of at leasttwo independent experiments, and error bars indicate the standard deviation.

11%17%

10%

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.

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 knowled

ge, must mine information from literature or obtain from experiments

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)

Linear Programming

ReactionFile

Compoundfile

Object filePerl program Manual operation

Resultfile

Execute glpk instruction

Reaction File Context

• context

R00014 C05125 + C00011 <=> C00068 + C00022R00200 C00002 + C00022 <=> C00008 + C00074R00235 C00002 + C00033 + C00010 <=> C00020 + C00013 + C00024R00658 C00631 <=> C00074 + C00001R00710 C00084 + C00003 + C00001 <=> C00033 + C00004 + C00080R00754 C00469 + C00003 <=> C00084 + C00004 + C00080R01061 C00118 + C00009 + C00003 <=> C00236 + C00004 + C00080R01070 C05378 <=> C00111 + C00118R01512 C00002 + C00197 <=> C00008 + C00236R01518 C00631 <=> C00197R02569 C00024 + C00579 <=> C00010 + C01136R02738 C04085 + C00031 <=> C00615 + C00668R02740 C00668 <=> C05345R03270 C05125 + C00248 <=> C01136 + C00068R04779 C00002 + C05345 <=> C00008 + C05378

Compound File Context

• ContextC00001: + R00658 - R00710 >= 0C00002: - R00200 - R00235 - R01512 - R04779 >= 0C00003: - R00710 - R00754 - R01061 >= 0C00004: + R00710 + R00754 + R01061 >= 0C00008: + R00200 + R01512 + R04779 >= 0C00010: - R00235 + R02569 >= 0C00022: + R00014 - R00200 >= 0C00024: + R00235 - R02569 >= 0C00033: - R00235 + R00710 >= 0C00068: + R00014 + R03270 >= 0C00074: + R00200 + R00658 >= 0C00080: + R00710 + R00754 + R01061 >= 0C00084: - R00710 + R00754 >= 0C00118: - R01061 + R01070 >= 0C00197: - R01512 + R01518 >= 0C00236: + R01061 + R01512 >= 0C00631: - R00658 - R01518 >= 0C00668: + R02738 - R02740 >= 0C01136: + R02569 + R03270 >= 0C05125: - R00014 - R03270 >= 0C05345: + R02740 - R04779 >= 0C05378: - R01070 + R04779 >= 0

C00469: - R00754 >= 0 C00000: + R02738 = 100

We must mutually addethanol compound constraint and input constraint into compound file, because It has only one reaction

Reaction & Compound Pathway

Reaction & Compound Pathway (modified)

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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