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Design of a System of
Biotransformations by means of
stoichiometric network analysis
Anne Kümmel, ETH Zürich
© ETH Zürich | Taskforce Kommunikation
Datum
Introduction
SBT-production mode
SBT-operation mode
cheap substrate
Production of all
required enzymes in a
genetically
engineered
microorganism
Production of the
product molecule
using the washed
crude extract
high-value product
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SBT design
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DHAP production path
net reaction:
ATP
glucose + P
2.7.1.2
Glucokinase
glk
DHAP + lactate
Glucose-6-phosphate
5.3.1.9
Phosphoglucose
isomerase
pgi
NADH + H+
1,3-Diphosphoglycerate
2.7.2.3
Phosphoglycerate
kinase
pgk
ADP
Fructose-6-Phosphate
Fructose-1,6-bisphosphate
2.7.1.11
Phosphofructokinase
pfkB
Glyceraldehyde-3-phosphate
1.2.1.12
Glyceraldehyde-3-phosphate
dehydrogenase
gapA
lower part:
co-subtrate
balancing
5.4.2.1
Phosphoglycerate
mutase
gpmA
2-Phosphoglycerate
4.2.1.11
Enolase
eno
Legend:
NADH balancing
Lactate
ATP balancing
NAD +
SBT design
upper part:
DHAP synthesis
Phosphoenolpyruvate
ADP
2.7.1.40
Pyruvate kinase
pyk
1.1.1.28
D-Lactate
dehydrogenase
ldhA
SBT path
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4.1.2.13
Fructosebisphosphate
aldolase
fba
P + NAD+
ATP
3-Phosphoglycerate
ADP
ATP
ADP
ATP
Pyruvate
NADH + H +
3
Dilemma
For SBT-operation, reactions that divert material into side products reduce
the yield: Isolation of the production pathway is necessary! Elimination of
side reactions can be achieved by gene deletions.
For SBT-production, a viable organism is required: Side reactions may be
essential for growth! Genes cannot be deleted as required since SBT path is
part of the central carbon metabolism.
Key question:
To which extent can the production
pathway be isolated from the rest of
the metabolic network for the
operation mode while maintaining
viability in production mode?
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Identification of elimination targets
Genome-scale metabolic reconstruction of E.coli as a compendium (iJR904):
The 11 metabolites belonging to the SBT path participate
in 90 other enzymatic reactions of E.coli. Of the 90
undesired reactions, 73 are probably not problematic
since they rely on substrates or cofactors washed out of
the SBT or proceed irreversibly in the “wrong“ direction.
17 side reactions potentially interfere with the SBT-operation
Which of these reactions can be eliminated by gene deletion?
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Methodology
Assumptions for systematic, model-driven mutant design:
ƒ Gene essentiality is predicted by stoichiometric network analysis.
ƒ Enzymes for reactions not active in the wild-type strain are not expressed,
and gene deletion is thus not required.
ƒ Mixtures of different carbohydrates are not tested in silico as they may be
not consumed simultaneously in vivo.
1. Flux balance analysis (FBA)
max (growth rate)
s.t. metabolite balances
available substrates
Enzyme expression in
wild type strain
2. Mixed-integer optimization
max (number of gene
knockouts)
s.t. viability
available substrates
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Possible knockouts and rerouting of fluxes within the
mutant strains
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Calculation results
Three different in silico media have been tested for production of a SBT path
that is as far as possible isolated.
The expression of the
genes (blue) in the wild
type
among
the
corresponding
17
undesired reactions are
shown in the table. The
expressed genes have
been classified to be
either essential (red) or
possible deletion targets
(green).
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M9 glucose
gene
KO possible?
no
gpsA
no
pgm
no
serA
no
tpiA
no
tktA, tktB
yes
zwf
no
ilvI
not active
fbp
not active
pps
not active
fsaA, fsaB
not active
mgsA
gatY, gatZ,
not active
kbaY, kbaZ
not active
dxs
not active
dgoA
not active
mtlD
not active
manA
not active
srlD
SBT design
amino acids + M9 glucose amino acids + glycerol
gene
gene
KO possible?
KO possible?
gpsA
pgm
serA
tpiA
tktA, tktB
zwf
ilvI
fbp
pps
fsaA, fsaB
mgsA
gatY, gatZ,
kbaY, kbaZ
dxs
dgoA
mtlD
manA
srlD
no
no
not active
yes
no
not active
not active
not active
not active
not active
not active
not active
not active
not active
not active
not active
not active
gpsA
pgm
serA
tpiA
tktA, tktB
zwf
ilvI
fbp
pps
fsaA, fsaB
mgsA
gatY, gatZ,
kbaY, kbaZ
dxs
dgoA
mtlD
manA
srlD
yes
no
not active
yes
no
not active
not active
no
not active
not active
not active
not active
not active
not active
not active
not active
not active
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Isolation procedure
•Glucose: deletion of zwf
gene and six remaining
side reactions
•Glucose plus amino acids:
reduction of used enzymes
•Glycerol plus amino acids:
deletion of two genes but
flux through an undesired
enzyme as well
Glucose
ATP
ADP
ATP
ADP
Glucose-6-phosphate
Fructose-6-Phosphate
NADH + H +
1,3-Diphosphoglycerate
Fructose-1,6-bisphosphate
P + NAD +
gapA
Dihydroxyacetone
phosphate
Glyceraldehyde-3-phosphate
ADP
ATP
3-Phosphoglycerate
Phosphoenolpyruvate
2-Phosphoglycerate
ADP
(a) tpiA mutant grown on
glucose/amino acids or
(b) tpiA/gpsA mutant grown on
glycerol/amino acids.
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Legend:
SBT path
Reactions eliminated
by gene deletion
Remaining side reactions
which interfere with
SBT operation
Reactions for which the
corresponding enzymes
are not expressed
SBT design
ATP
Pyruvate
Lactate
NAD +
NADH + H
8
Conclusion and outlook
ƒ One tkt gene is first target for proteomic switch.
ƒ Thermodynamic analysis can be used to rank the proteomic switch
targets.
ƒ Experimental evaluation should determine the yield of DHAP for the
SBTs produced by the two suggested strategies.
ƒ The SBT path - although mainly consisting of the core metabolism can be isolated to large extent. Only three side reactions are left.
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