E.coli K-12 MG1655 pathway 調節 磷酸果糖激酶 烯醇酶 缺少 pyruvate decarboxylase alcohol dehydrogenase

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? 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.

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.

17% 11% 10% 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.

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 knowledge, 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 Reaction File Compound file Object file Perl program Manual operation Execute glpk instruction Result file

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

Compound File Context Context We must mutually add C00001: + R00658 - R00710 >= 0 C00002: - R00200 - R00235 - R01512 - R04779 >= 0 C00003: - R00710 - R00754 - R01061 >= 0 C00004: + R00710 + R00754 + R01061 >= 0 C00008: + R00200 + R01512 + R04779 >= 0 C00010: - R00235 + R02569 >= 0 C00022: + R00014 - R00200 >= 0 C00024: + R00235 - R02569 >= 0 C00033: - R00235 + R00710 >= 0 C00068: + R00014 + R03270 >= 0 C00074: + R00200 + R00658 >= 0 C00080: + R00710 + R00754 + R01061 >= 0 C00084: - R00710 + R00754 >= 0 C00118: - R01061 + R01070 >= 0 C00197: - R01512 + R01518 >= 0 C00236: + R01061 + R01512 >= 0 C00631: - R00658 - R01518 >= 0 C00668: + R02738 - R02740 >= 0 C01136: + R02569 + R03270 >= 0 C05125: - R00014 - R03270 >= 0 C05345: + R02740 - R04779 >= 0 C05378: - R01070 + R04779 >= 0 C00469: - R00754 >= 0 C00000: + R02738 = 100 We must mutually add ethanol 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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