Metabolic Flux Analysis

Outline

ƒ Introduction
ƒ Fermentation examples
ƒ Quantitative physiology for process control,
quality management and process optimizations
ƒ Overview of analytical techniques
ƒ Metabolic Flux Analysis
ƒ Stoichiometric models
ƒ Constrained based modelling
ƒ 13C tracer experiments
ƒ Lab

Bioanalytic Lecture WS05/06 LS-BT

The cellular Hierarchy

Analytical challenges

ƒGenome sequence (DNA)

ƒTranscription (RNA synthesis)

ƒTranslation (Protein synthesis)

ƒMetabolite concentrations

ƒExtracellular product/substrate
concentrations

ƒPhysical parameters (pH, T,

[O2], etc.)

Bioanalytic Lecture WS05/06 LS-BT

Metabolic Flux Glucose Reaction network
Analysis
Physiological data
(16 mmol/g/h) Biomass composition
Metabolic flux ratios

Growth Rate
(0.4 h-1)
1.6
Glucose-6-P
13.7 R-5-P

PPP

Oxaloacetate Pyruvate
0.5 Oxaloacetate
0.0 TCA
cycle

Glycerol Ethanol
(1.5 mmol/g/h) (22 mmol/g/h)
Bioanalytic Lecture WS05/06 LS-BT
Overview of cellular metabolism
 Primary & secondary metabolism

ƒ Primary metabolism
ƒ Biosynthesis and assembly of biomass components
ƒ Energy metabolism
ƒ Typically under relatively tight control
ƒ Secondary metabolism
ƒ For example, synthesis of secreted proteins and
antibiotics to modulate environment
ƒ Typically under less stringent control

Bioanalytic Lecture WS05/06 LS-BT

 Composition of biomass (E.coli)

Protein
3.9
RNA
2.5 DNA
DRY 2.5 LIPIDS
WEIGHT
3.4 LPS
30%
WATER 9.1 PG
70% Glycogen
3.1 Soluble

55

20.5

Bioanalytic Lecture WS05/06 LS-BT

 Transport
Functional
Substrate overview over
Replication
the cell
Mass
Fueling reac.
Energy
Exch
DNA Subsystems
12 precursors Material
Transcript.

Biosynthesis Energy
RNA
Information
Building blocks

Energy
Translation Flows
Polymerisation
store Mass
Protein
Macromolecule Energy

Assembly Biomass Information

Bioanalytic Lecture WS05/06 LS-BT

 Organisation

ƒ Connectivity
ƒ Shared metabolites
ƒ Shared co-factors, e.g., ATP, NADH, NADPH
ƒ Information, e.g., regulons
ƒ Structure
ƒ Chemical, reaction and control networks
ƒ Physical, e.g., cytosol, nucleus, mitochondria
ƒ Characteristic times

Bioanalytic Lecture WS05/06 LS-BT

 Relaxation times

10-6 10-4 10-2 100 102 104 106

seconds
Mass action mRNA control Mutations

Allosteric control Enzyme induction

Gradients due to mixing

Dynamics of culture transitions

Cell growth

Pseudo steady state Window of interest Constant

Bioanalytic Lecture WS05/06 LS-BT

 Transport

Type Driving force Rate

Passive transport ΔC Permeability

coefficient, P

Facilitated ΔC [Carrier]
diffusion

Active transport ATP [Carrier]

[H+] gradient

Bioanalytic Lecture WS05/06 LS-BT

 Glycolysis (1)
~P : ADP + Pi = ATP

Other sugars glucose DNA/RNA,His

~P NADPH CO2 NADPH
G6P Ribulose-5P
Carbohydrates
F6P Sedohept-7P Ribose-5P
~P

FBP
Erythrose-4P G3P Xylulose-5P

DHAP G3P

Aromatic AA (Trp,Tyr,Phe)
Lipid
Bioanalytic Lecture WS05/06 LS-BT
 Glycolysis (2)
G3P Lipid
Substrate-level phosphorylation
Pi
Energy from oxidation of aldehyde
~H2
1,3DPG captured in ~P.
~H2 : NADH = NAD+ + H+ + 2e-
~P
3PG Ser,Gly,Cys, DNA/RNA

2PG Glucose = 2 PYR + 2 ~P + 2 ~H2

PEP Aromatic AA (Trp,Tyr,Phe)

~P
PYR Ala,Val,Leu

Bioanalytic Lecture WS05/06 LS-BT

TCA cycle
Ala,Ser Thr,Lys,Leu
Cys,Gly Tyr,Phe,Trp
Lipids
CO2+~H2 ~H2

PYR Acetyl-CoA CIT

~H2 Asp,Asn OOA ICI

CO2 ~H2
CO2+~H2

MAL 2OG Glu,Gln,Pro

Arg,His
CO2+~H2
FUM S-CoA
~H2 Met,Ile,Val
SUC ~P

Bioanalytic Lecture WS05/06 LS-BT

TCA cycle
Ala,Ile,Leu
Lys,Val Leu
Lipids
CO2+~H2 ~H2

PYR Acetyl-CoA CIT

CO2+~P
OOA ICI
~H2
CO2+~H2
Asp,Asn,Ile
Lys,Met,Thr MAL 2OG Glu,Gln
Pro,Arg
CO2+~H2
FUM S-CoA
~H2
SUC ~P
PYR = 3 CO2 + ~P + 5 ~H2
Bioanalytic Lecture WS05/06 LS-BT
 Glycolysis & TCA

Glucose = 2 PYR + 2 ~P + 2 ~H2

PYR = 3 CO2 + ~P + 5 ~H2

What to do with ~H2?

Anaerobic organisms: avoid TCA & ferment
Aerobic: oxidative phosphorylation

Bioanalytic Lecture WS05/06 LS-BT

 Anaerobic metabolism (E. coli)

~H2
PYR Lactate
CO2+H2 Formate CO2+~H2
Acetyl-CoA
~H2

Acetyl-P Acetaldehyde
~H2
~P
Acetate Ethanol

Bioanalytic Lecture WS05/06 LS-BT

 Anaerobic metabolism
(Saccharomyces cerevisiae)

CO2 ~H2
PYR Acetaldehyde Ethanol
CO2+~H2
~P ~H2
Acetyl-CoA Acetate

For excess ~H2: DHAP + ~H2 = glycerol + ~P

Bioanalytic Lecture WS05/06 LS-BT

 Anaerobic (Clostridia)

CO2
PYR Acetoin
CO2+~H2
~P ~H2 ~H2
Acetate Acetyl-P Acetyl-CoA Acetaldehyde Ethanol

Acetoacetyl-CoA
~H2
~P CO2
Butyrate Butyryl-CoA Acetoacetate Acetone
~H2 ~H2

Butanol Isopropanol

Bioanalytic Lecture WS05/06 LS-BT

 Oxidative phosphorylation

Periplasm or intermembrane space

H+ H+ H+

~H2 Q Cyt C (+2) O2

QH2 Cyt C (+3) H 2O ~P

H+ AA H+

Cytosol or Mitochondrial matrix ATPase H+ symport

~H2 + 0.5 O2 = H2O + 3 ~P

Bioanalytic Lecture WS05/06 LS-BT

 Growth energetics
rATP = YxATP μ + mATP
= YxATP,growth μ + (YxATP-YxATP,growth)μ + mATP
= YxATP,growth μ + maintenance

YxATP,growth = transport, biosynthesis, polymerisation

= 35-40 mmol ATP (g DW)-1 [glucose+salts]

Maintenance include maintaining gradients (essential for

life), turnover of macromolecules, futile cycles. Some
increase in proportion to growth rate [(YxATP-YxATP,growth)μ],
While some is required even in absence of growth, mATP.

Bioanalytic Lecture WS05/06 LS-BT

 Experimental data

Species YxATP mATP Medium

Aerobactor aerogenes 71 6.8 Minimal

57 2.3 Complex

Candida parapsilosis 80 0.2 Minimal

Escherichia coli 97 18.9 Minimal
117 6.9 Minimal

Lactobacillus casei 41 1.5 Complex

Lactobacillus delbruckii 72 0 Complex

Lactococcus cremoris 73 1.4 Complex

53 Complex
15-50 7-18 Complex

Lactococcus diacetilactis 47 Complex

Saccharomyces cerevisia 71-91 <1 Minimal

Bioanalytic Lecture WS05/06 LS-BT

 Summary Biochemistry (Metabolism)

12 precursors to biomass & ~P, ~H2

Glycolysis: glucose = 2 PYR + 2 ~P + 2 ~H2
TCA: PYR = 3 CO2 + ~P + 5 ~H2
OP: ~H2 + 0.5 O2 = H2O + 3 ~P
Fermentation: removing ~H2 in absence of
ext. electron acceptor. Minimal gain of ~P
rATP = YxATP μ + mATP

Bioanalytic Lecture WS05/06 LS-BT

Industrial (Metabolic Engineering) Examples

ƒ Ethanol production
ƒ Lactate production
ƒ Amino acid production
ƒ Lysine
ƒ Threonine
ƒ Vitamins
ƒ Riboflavin (B2)
ƒ Acid (C)

Bioanalytic Lecture WS05/06 LS-BT

 Ethanol Production by Yeast

Fuel Ethanol Plant for Jilin Fuel Ethanol Co. Ltd,

Jilin, PR China: 2.5 million litre/day

Bioanalytic Lecture WS05/06 LS-BT

Lactate Production by Lactococcus lactis

2-3 mol ATP/mol glucose

Sugar NAD+
Acids, alcohols, etc

NADH
H+
Pyr
H+
ATP
Biosynthesis
ADP
α βα
β β F1 Building
α H+
γ
ε δ blocks
H+
F0
a b2 c10 H+
Lactate plant for Purac (Netherlands):
170.000 t sugar, 15,000 t sodium lactate
+ 140,000 t lactate / year

Bioanalytic Lecture WS05/06 LS-BT

Lysine (and other AA) Production by
Corynebacterium glutamicum

Threonine plant for Ajinomoto in France:

19.000 t / year

Bioanalytic Lecture WS05/06 LS-BT

B2 Production by
Bacillus subtilis

No picture found /

Riboflavin plant for DSM in Grenzach, Germany:

3.000 t / year

Bioanalytic Lecture WS05/06 LS-BT

Vitamine C Production by
Bacillus subtilis

Vitamin C etc. plant for DSM in Ayrshire, England:

3.000 t / year

Bioanalytic Lecture WS05/06 LS-BT

 Why Biocatalysts?

ƒ Faster
ƒ Bench scale optimisation
ƒ “Self-replicating” living reactors
ƒ Cheaper
ƒ Low temperature, low pressure
ƒ Water rather than organic solvents
ƒ Purer
ƒ Up to 100% stereospecificity

Bioanalytic Lecture WS05/06 LS-BT

 Why now?

ƒ Molecular biology and genomics provide the

tools for rational design
ƒ Enhance yields & productivity
ƒ Extend substrate range
ƒ Extend product spectrum & range
ƒ Improvement cellular properties

Metabolic engineering

Bioanalytic Lecture WS05/06 LS-BT

 Metabolic vs genetic engineering

ƒ $1-100/kg versus $1,000-100,000/kg

ƒ Very high yields required, often near
stoichiometric
ƒ Often integrated in central metabolism as
opposed to secondary metabolite
ƒ Need a holistic, system level approach to
redirect resources: carbon, nitrogen, energy
and reducing power

Bioanalytic Lecture WS05/06 LS-BT

Pushing the Limits of Microbial Metabolism

Engineering Cycle
Analysis

Synthesis Design

Design of methods and devices for:

=> Analysis of carbon and energy flow in cells
(productivity of catalyst)
=> Analysis of single cell metabolism
(the smallest catalytic unit)

Bioanalytic Lecture WS05/06 LS-BT

 Summary Examples

ƒ Metabolic Engineering = reactor design at

micron scale
ƒ Chemical industry is already committed to
“green factories”
ƒ We are presently at the retrofit stage and this
lecture covers one tool used for analysis
ƒ We are moving toward green field design

More in the SS2006 Systems Biotechnology lecture

Bioanalytic Lecture WS05/06 LS-BT

 Why is the
intracellular flux
distribution of
interest?

Bioanalytic Lecture WS05/06 LS-BT

Genome annotation

Metabolic
potential
(enzymes)

Bioanalytic Lecture WS05/06 LS-BT

 Enzyme
Substrate Product

Biochemistry

Genome annotation

Metabolic
network

Bioanalytic Lecture WS05/06 LS-BT

 Enzyme
Substrate Product

Biochemistry

Genome annotation

Network
composition
Transcriptome / Proteome / at a given
Metabolome condition

Bioanalytic Lecture WS05/06 LS-BT

 Enzyme
Substrate Product

Biochemistry

Genome annotation

Network
operation
Transcriptome / Proteome /
at a given
Metabolome condition
‘Fluxome‘
Bioanalytic Lecture WS05/06 LS-BT
 How can one
measure/calculate
the intracellular flux
distribution?

Bioanalytic Lecture WS05/06 LS-BT

 Constructing stoichiometric
models

Bioanalytic Lecture WS05/06 LS-BT

From black box to stoichiometric models
• extend pure black box models by including biochemical
knowledge ⇒ stoichiometric models

• describe these models by constructing stoichiometric matrices

• analyse these models with linear algebra

Bioanalytic Lecture WS05/06 LS-BT

The cell as a black box model
substrate Si
rSi
System boundary
rPi
product Pi

μ
biomass X
M N
Overall balance equation: X + ∑ Yxp ⋅ Pi − ∑ Yxs ⋅ Si = 0
i i
i =1 i =1

Bioanalytic Lecture WS05/06 LS-BT

Black box models:
Elemental balances
substrate Si
rSi

System boundary
rPi
product Pi

μ
biomass X
M N
C -balance equation: 1 + ∑ hP ,i ⋅ YxPi − ∑ hS ,i ⋅ YxSi = 0
i =1 i =1

M
⇔ N
μ + ∑ hP ,i ⋅ rP ,i − ∑ hS ,i ⋅ rS ,i = 0
Bioanalytic Lecture WS05/06 i =1 i =1 LS-BT
Modelling philosophy (a recap)

Modelling and simulation are essential:

• in well-established systems
- to predict system behavior,
in biological modelling: gene deletions, nutrient limitations
- for optimization, e.g.
in biological modelling: product formation, genetic
engineering
- process control

• in uncertain systems
- to test the current understanding (hypotheses)
- for experimental redesign, i.e. detect pivotal experiments

„Models should be as simple as possible, but not more so“,

Bioanalytic Lecture WS05/06 Albert Einstein LS-BT
Modelling philosophy (a recap)
substrate Si
rSi

System boundary
rPi

?! product Pi

μ
biomass X

⇒ rather descriptive then comprehensive

But: Biochemistry provides knowledge for more detailed

models
Bioanalytic Lecture(⇒ fill the gaps)!
WS05/06 LS-BT
Constructing a
stoichiometric C C C C C C
GLUxt
model
b1
E
GLU CC C314 C C
C CGLU
CB B
2X
E25
r1 r2 GLU
C B
6 CO2
System boundary

C C C C C C B E6
C 3C Ac b3 C
r3 6 CO2 CO2xt
r5
C C C C C C C
C C C C
r6
r4 3 Ac Acxt
b4
C C C
2X intracellular
b2 (µ) extracellular
Bioanalytic Lecture
C CWS05/06
C Xxt LS-BT
Steady state

dB
= r1 + r2 − r3
r1 r2 r 1 r2 dt

B
B
r3
r3

Bioanalytic Lecture WS05/06 LS-BT

Steady state

dB
= r1 + r2 − r3
R1 r2 r1 r2 dt

B
B
r3 r3

Bioanalytic Lecture WS05/06 LS-BT

Steady state

dB
= r1 + r2 − r3
r1 r2 r1 r2 dt

B
B
R3 r3

Bioanalytic Lecture WS05/06 LS-BT

Steady state

dB !
= r1 + r2 − r3 = 0
r1 r2 r1 r2 dt
For time scales
B
B here (>sec) we
r3 r3 assume steady
state (no
changes).

Bioanalytic Lecture WS05/06 LS-BT

Stoichiometry at steady state
GLU: b1-r1-r2 = 0
B: r1+r2-r3 = 0
C: r3-r4-r5-r6 = 0
CO2: 6·r5-b3 = 0
Ac: 3·r6-b3 = 0
X: 2·r4-b2 = 0

⎛ r11 r22 r33 r44 r5 r6 b1 b2 b3 b4 ⎞

⎜ ⎟
⎜ GLU − 1 − 1 0 0 0 0 1 0 0 0 ⎟
⎜ B 1 1 −1 0 0 0 0 0 0 0 ⎟
⎜ ⎟
S =⎜ C 0 0 1 −1 −61 −31 0 0 0 0 ⎟
⎜ CO 22 0 0 0 0 16 0 0 0 −1 0 ⎟
⎜ ⎟
⎜ Ac 0 0 0 0 0 3
1 0 0 0 − 1 ⎟
⎜ X 0 0 0 12 0 0 0 −1 0 0 ⎟⎠
⎝
Internal Exchange
fluxes fluxes

S WS05/06
Bioanalytic Lecture = (# of metabolites × # of reactions) LS-BT
Stoichiometry at steady state

⎛ r1 ⎞
⎜ ⎟
⎛ r1 r2 r3 r4 r5 r6 b1 b2 b4 ⎞
b3 ⎜ r2 ⎟
⎜ ⎟ ⎜ r3 ⎟
⎜ GLU −1 −1 0 0 0 0 1 0 0 0⎟
⎜ ⎟
⎜ B 1 1 −1 0 0 0 0 0 0 0⎟ ⎜ r4 ⎟
⎜ ⎟ ⎜ r5 ⎟
S =⎜ C 0 0 1 −1 −1 −1 0 0 0 0⎟ v=⎜ ⎟
⎜ CO 2 0 0 0 0 6 0 0 0 −1 0 ⎟ ⎜ r6 ⎟
⎜ ⎟ ⎜b ⎟
⎜ Ac 0 0 0 0 0 3 0 0 0 −1⎟ ⎜ 1⎟
⎜ X −1 0 0 ⎟⎠ ⎜ b2 ⎟
⎝ 0 0 0 2 0 0 0
⎜b ⎟
⎜ 3⎟
⎜b ⎟
⎝ 4⎠

If we assume steady state, i.e. nothing changes over time, then

S·v = 0
S·v = (6×10)· (10×1) = (6×1)

Bioanalytic Lecture WS05/06 LS-BT

Bioanalytic Lecture WS05/06 LS-BT
Covert et al., TIBS, 2001
Linear algebra of stoichiometric networks

Fluxes v are inherent variables in stoichiometric models.

Can they be calculated? Linear algebra provides the tools!
r1 r2 r3 r4 r5 r6 b1 b2 b3 b4
GLU: −1 −1 1 =0
B: 1 1 −1 =0
C: 1 −1 −1 −1 =0
CO 2 : 6 −1 =0
Ac: 3 −1 = 0
X: 2 −1 =0

Let‘s try Gaussian elimination!

Bioanalytic Lecture WS05/06 LS-BT

 Linear algebra of stoichiometric networks

Fluxes v are inherent variables in stoichiometric modelling.

Can they be calculated? Linear algebra provides the tools!

r1 r2 r3 r4 r5 r6 b1 b2 b3 b4
GLU: 1 1 −1/ 2 1/ 6 −1/ 3 = 0
B: 1 −1/ 2 1/ 6 −1/ 3 = 0
C: 1 −1 =0
CO 2 : 1 1 =0
Ac: 1 −1 = 0
X: 1 −1/ 2 1/ 6 −1/ 3 = 0

r1 ,r2, b1 ,b2, b3, b4 cannot be solved independently

⇒ set r2, b2 , b3, b4
Bioanalytic Lecture WS05/06 LS-BT
Linear algebra of stoichiometric networks
A structured approach for analysis of stoichiometric matrices:
S (n×m), i.e. S comprises n balance equations (metabolites) and m
unknown variables (fluxes).
1. Determine the number p of linearly independent equations:
p = rank(S)
2. Calculate the degree of freedom (DF):
DF(S)= m - p
There are three possibilities:
- DF(S) = 0 ⇒ well-determined: one possible solution
- DF(S) > 0 ⇒ underdetermined: more than one solution,
optimization problem (linear programming);
- DF(S) < 0 ⇒ overdetermined (e.g. experimental data):
minimize overall error (least square etc.)
Bioanalytic Lecture WS05/06 LS-BT
Linear algebra of stoichiometric networks
A structured approach for analysis of stoichiometric matrices:
⎛ r1 r2 r3 r4 r5 r6 b1 b2 b4 ⎞
b3
⎜ ⎟
⎜ GLU −1 −1 0 0 0 0 1 0 0 0⎟
⎜ B 1 1 −1 0 0 0 0 0 0 0⎟
⎜ ⎟
S =⎜ C 0 0 1 −1 −1 −1 0 0 0 0⎟ = (6×10)
⎜ CO 2 0 0 0 0 6 0 0 0 −1 0 ⎟
⎜ ⎟
⎜ Ac 0 0 0 0 0 3 0 0 0 −1⎟
⎜ X −1 0 0 ⎟⎠
⎝ 0 0 0 2 0 0 0

1. Determine the number p of linearly independent equations:

p = rank(S) = 6
2. Calculate the degree of freedom (DF):
DF(S)= m – p=10 - 6= 4
⇒ See results of our Gaussian elimination, we either have to
provide values for 4 fluxes (r2, b2, b3, b4) or optimize

Bioanalytic Lecture WS05/06 LS-BT

Calculability Analysis

⎛ v1 ⎞
v3 v7 ⎜ ⎟
v2 B E ⎛1 −1 0 −1 0 0 0⎞ ⎜ v2 ⎟
⎜ ⎟ ⎜v ⎟
⎜0 1 −1 0 0 0 0⎟
⎜ 3⎟
v1 v5 v6 M = ⎜0 1 0 1 −1 0 0 ⎟; v = ⎜ v4 ⎟
A C D ⎜ ⎟ ⎜v ⎟
⎜0 0 1 1 0 −1 0 ⎟ ⎜ 5⎟
⎜0 0 1 0 − 1⎟⎠ ⎜ v6 ⎟
v4 ⎝ 0 0
⎜ ⎟
⎝ v7 ⎠
M*v = 0

• M=(5x7);
• Rank = 5;
• Degrees of freedom: # columns – rank(M) =7-5 =2
• underdeterminded

0 = M ⋅ v = M b ⋅ vb + M n ⋅ v n ⇔ M n ⋅ v n = − M b ⋅ vb
Bioanalytic Lecture WS05/06 LS-BT
Calculability Analysis: First case is v1 = known
⎛ v1 ⎞
v3 v7 ⎜ ⎟
v2 B E ⎛1 −1 0 −1 0 0 0⎞ ⎜ v2 ⎟
⎜ ⎟ ⎜v ⎟
⎜0 1 −1 0 0 0 0⎟
⎜ 3⎟
v1 v5 v6 M = ⎜0 1 0 1 −1 0 0 ⎟; v = ⎜ v4 ⎟
A C D ⎜ ⎟ ⎜v ⎟
⎜0 0 1 1 0 −1 0 ⎟ ⎜ 5⎟
⎜0 0 1 0 − 1⎟⎠ ⎜ v6 ⎟
v4 ⎝ 0 0
⎜ ⎟
⎝ v7 ⎠
M*v = 0

1.case: v1=known
⎛ v2 ⎞
⎛ ⎞
1 ⎜ ⎟ ⎛ -1 0 -1 0 0 0⎞
⎜ ⎟ ⎜ v3 ⎟ ⎜ ⎟
⎜ 0⎟ ⎜v ⎟ ⎜ 1 −1 0 0 0 0⎟
v b = v1 ; M v = ⎜ 0 ⎟; v n = ⎜ 4 ⎟; M n = ⎜ 1 0 1 − 1 0 0⎟
⎜ ⎟ ⎜ v5 ⎟ ⎜ ⎟
⎜ 0⎟ ⎜v ⎟ ⎜ 0 1 1 0 −1 0 ⎟
⎜ 0⎟ ⎜ 6⎟ ⎜ 0 1 0 0 0 − 1⎟
⎝ ⎠ ⎜v ⎟ ⎝ ⎠
⎝ 7⎠
BUT: still 1 degree of freedom
Bioanalytic Lecture WS05/06 LS-BT
Calculability Analysis: Second case v1, v7 = known
⎛ v1 ⎞
v3 v7 ⎜ ⎟
v2 B E ⎛1 −1 0 −1 0 0 0⎞ ⎜ v2 ⎟
⎜ ⎟ ⎜v ⎟
⎜ 0 1 − 1 0 0 0 0 ⎟ ⎜ 3⎟
v1 v5 v6 M = ⎜0 1 0 1 −1 0 0 ⎟; v = ⎜ v4 ⎟
A C D ⎜ ⎟ ⎜v ⎟
⎜0 0 1 1 0 −1 0 ⎟ ⎜ 5⎟
⎜0 0 1 0 − 1⎟⎠ ⎜ v6 ⎟
v4 ⎝ 0 0
⎜ ⎟
⎝ v7 ⎠
M*v = 0

2.case: v1, v7 known

⎛1 0 ⎞ ⎛ v2 ⎞ ⎛−1 0 −1 0 0⎞
⎜ ⎟ ⎜ ⎟ ⎜ ⎟
⎜0 0 ⎟ ⎜ v3 ⎟ ⎜ 1 −1 0 0 0⎟
⎛ v1 ⎞
v b = ⎜⎜ ⎟⎟; M v = ⎜ 0 0 ⎟; v n = ⎜ v 4 ⎟; M n = ⎜ 1 0 1 −1 0 ⎟
⎝ v7 ⎠ ⎜ ⎟ ⎜ ⎟ ⎜ ⎟
⎜0 0 ⎟ ⎜ v5 ⎟ ⎜0 1 1 0 − 1⎟
⎜ 0 − 1⎟ ⎜v ⎟ ⎜0 1
⎝ ⎠ ⎝ 6⎠ ⎝ 0 0 0 ⎟⎠
well determined; 0 degree of freedom

Bioanalytic Lecture WS05/06 LS-BT

Calculability Analysis: Third case v1,v5 = known
⎛ v1 ⎞
v3 v7 ⎜ ⎟
v2 B E ⎛1 −1 0 −1 0 0 0⎞ ⎜ v2 ⎟
⎜ ⎟ ⎜v ⎟
⎜ 0 1 − 1 0 0 0 0 ⎟ ⎜ 3⎟
v1 v5 v6 M = ⎜0 1 0 1 −1 0 0 ⎟; v = ⎜ v4 ⎟
A C D ⎜ ⎟ ⎜v ⎟
⎜0 0 1 1 0 −1 0 ⎟ ⎜ 5⎟
⎜0 0 1 0 − 1⎟⎠ ⎜ v6 ⎟
v4 ⎝ 0 0
⎜ ⎟
⎝ v7 ⎠
M*v = 0

3.case: v1, v5 known

⎛1 0 ⎞ ⎛ v2 ⎞ ⎛−1 0 −1 0 0⎞
⎜ ⎟ ⎜ ⎟ ⎜ ⎟
⎜ 0 0 ⎟ ⎜ 3⎟
v ⎜ 1 − 1 0 0 0 ⎟
⎛ v1 ⎞
v b = ⎜⎜ ⎟⎟; M v = ⎜ 0 − 1⎟; v n = ⎜ v 4 ⎟; M n = ⎜ 1 0 1 0 0⎟
⎝ v5 ⎠ ⎜ ⎟ ⎜ ⎟ ⎜ ⎟
⎜0 0 ⎟ ⎜ v6 ⎟ ⎜0 1 1 −1 0 ⎟
⎜ ⎟ ⎜ ⎟ ⎜ ⎟
⎝0 0 ⎠ ⎝ v7 ⎠ ⎝0 1 0 0 − 1⎠
BUT: still 1 degree of freedom, as rank(Mn)=4

Bioanalytic Lecture WS05/06 LS-BT

Calculability Analysis: Fourth case v1, v2, v4 = known
⎛ v1 ⎞
v3 v7 ⎜ ⎟
v2 B E ⎛1 −1 0 −1 0 0 0⎞ ⎜ v2 ⎟
⎜ ⎟ ⎜v ⎟
⎜0 1 −1 0 0 0 0⎟
⎜ 3⎟
v1 v5 v6 M = ⎜0 1 0 1 −1 0 0 ⎟; v = ⎜ v4 ⎟
A C D ⎜ ⎟ ⎜v ⎟
⎜0 0 1 1 0 −1 0 ⎟ ⎜ 5⎟
⎜0 0 1 0 − 1⎟⎠ ⎜ v6 ⎟
v4 ⎝ 0 0
⎜ ⎟
⎝ v7 ⎠
M*v = 0
4.case: v1, v2,, v4 known
⎛ 1 − 1 − 1⎞ ⎛0 0 0 0⎞
⎜ ⎟ ⎛ v3 ⎞ ⎜ ⎟
⎛ v1 ⎞ ⎜0 1 0 ⎟ ⎜ ⎟ ⎜−1 0 0 0⎟
⎜ ⎟ ⎜ 5⎟
v
v b = ⎜ v 2 ⎟; M v = ⎜ 0 1 1 ⎟; v n = ⎜ ⎟; M n = ⎜ 0 − 1 0 0⎟
⎜v ⎟ ⎜ ⎟ v6 ⎜ ⎟
⎝ 4⎠ ⎜0 0 1 ⎟ ⎜ ⎟ ⎜1 0 −1 0 ⎟
⎜v ⎟
⎜ ⎟ ⎝ 7⎠ ⎜1 − ⎟
⎝ 0 0 0 ⎠ ⎝ 0 0 1 ⎠
• well determined if chosen fluxes are CONSISTENT
v1= v2,+ v4 if not: system is unsolvable
Bioanalytic Lecture WS05/06 LS-BT
A possible flux solution

Set r2 = 0, b3 = 0, b4 = 0 and b2 = 50:

⇒ we can obtain a unique solution with
r1 r2 r3 r4 r5 r6 b1 b2 b3 b4
100 0 100 100 0 0 100 200 0 0

Bioanalytic Lecture WS05/06 LS-BT

Stoichiometric models can easily be extended
GLUxt O2xt
b1 b5

GLU O2
2ATP ATP r7
r1 r2 2 ATP
NADH
B
ATP b3
2 ATP, r3 6 CO2 CO2xt
1 NADH r5
C
10 ATP r6
r4 3 Ac Acxt
2 NADH b4
2X
b2
Xxt
Bioanalytic Lecture WS05/06 Additional biochemical information
LS-BT
Gene deletions
GLUxt O2xt
b1 b5

GLU O2
2ATP ATP r7
r1 r2 2 ATP
NADH
B
0= ATP b3
2 ATP, r3 6 CO2 CO2xt
1 NADH r5
C
10 ATP r6
r4 3 Ac Acxt
2 NADH b4
2X
b2
Xxt
Bioanalytic Lecture WS05/06 Gene deletions can be considered
LS-BT
Escherichia coli:
Saccharomyces cerevisiae:
And:
Bioanalytic Lecture WS05/06
- www.ecocyc.org
- www.yeastgenome.org
- www.genome.jp/kegg/
- any biochemistry textbook LS-BT
A genome-scale model of yeast metabolism
Substrates & 74%
metabolites in 71%

essential
catabolism

duplicates
singleton
Cofactors

Vitamines
Metabolites

Lipids, cell
membrane

Nucleotides

Amino acids

Central carbon
Central carbon

catabolic rxns
biosynthesis

Energy met.
metabolism

Vitamines &
Lipids & cell
biosyntheis
Amino acid

membrane
Nucleotide

Exchange

Uptake &
cofactors
Reactions
Küpfer, Sauer, and Blank, Gen Res. 2005
Summary
Stoich. models: Advantages and disadvantages
advantages
• can easily be constructed,
„basic“ biochemistry
• allow easy analytical access,
linear algebra
• large systems (genome scale)
can be considered
Bioanalytic Lecture WS05/06
disadvantages
• linear models: units (time!)
cancel out
• model complexity is limited
• NO dynamics: steady state
LS-BT
Stoichiometric models: Recap
We learned:
• to extend pure black box models by including biochemical
knowledge ⇒ stoichiometric models
• to mathematically describe these models by constructing
stoichiometric matrices
• to analyse these models with linear algebra

Next:
• setting up a stoichiometric model (central carbon metabolism of
yeast)
• solving underdetermined systems by introducing intracellular flux
ratios from METAFoR analysis experiments

Bioanalytic Lecture WS05/06 LS-BT

 Constructing the yeast
stoichiometric model

Bioanalytic Lecture WS05/06 LS-BT

 How to get the
additional constraints
necessary to resolve
the intracellular flux
distribution?

Bioanalytic Lecture WS05/06 LS-BT

 13Ctracer based
Solution: metabolic flux analysis

ƒ Carbon is naturally occurring in a mixture of

different isotopes, i.e. with different number of
neutrons in the atomic nuclei
ƒ The natural isotope distribution is: 12C 98.89 %,
13C 1.11 %, 14C 10-10 %. 14C is not stable i.e.
radioactive
ƒ Different isotopes can be discriminated by NMR
or MS techniques
ƒ Different information from NMR or MS =>
complementary
We will use GC-MS
Bioanalytic Lecture WS05/06 LS-BT
Flux analysis based on
13C-labeling experiments

METAFoR: Metabolic flux ratio analysis

Glucose

8%
1 2 3 4 5 6 Glucose-6-P Pentose-5-P
Glucose 92%

1 2 3 1 2 3 1 2 3
Fructose-6-P
Pyruvate
(inferred from
Alanine)
Entner- Glycolysis Pentose-
Doudoroff Phosphate Trioses

PEP

Bioanalytic Lecture WS05/06 LS-BT

 How to get the flux
information?

Bioanalytic Lecture WS05/06 LS-BT

METAFoR Analysis

Mass distribution
of amino acid
PHE m57
correction for
- natural isotope abundance
- unlabeled biomass from inoculum
mit
Corrected mass PEP13 OAA24
distribution of amino
PHE19 acid fragment 82% 18%

cyt
OAA24

PEP13 PEP23 E4P14

OAA24cyt – OAA24mit
OAAcyt from PYRcyt =
PEP13 – OAA24mit

Bioanalytic Lecture WS05/06 LS-BT

METAFoR Analysis

Mitochondrion

PEP Oxalo- TCA

cycle mit
Pyruvate acetatecyt PEP13 OAA24
carboxylase
82 18
Pyruvate Oxalo-
82% 18%
acetatemit
cyt
Oxoglutarate OAA24
AcCoA

OAA24cyt – OAA24mit
OAAcyt from PYRcyt =
PEP13 – OAA24mit

Bioanalytic Lecture WS05/06 LS-BT

13C-constrained net flux analysis

Glucose
Input:
15 mmol/g/h
ƒ Reaction network 1.2 8%
mmol/g/h
ƒ Physiological data Glucose-6-P Pentose-5-P
ƒ Biomass composition
92%mmol/g/h
13.8
ƒ Metabolic flux ratios
Output: Fructose-6-P
ƒ in vivo reaction velocities
e.g. mmol/g/h

Trioses

PEP
Fischer et al., Anal. Biochem. 2004

Bioanalytic Lecture WS05/06 LS-BT

Physiology Glucose
(16 mmol/g/h)

Growth Rate
(0.4 h-1)

?
Glycerol Ethanol
(1.5 mmol/g/h) (22 mmol/g/h)
Bioanalytic Lecture WS05/06 LS-BT
METAFoR
Glucose

?
Glucose-6-P
10%
?
Growth Rate

90% R-5-P

PPP

Oxaloacetate Pyruvate
95%Oxaloacetate
5% TCAcycle

?
Glycerol
?Ethanol
Bioanalytic Lecture WS05/06 LS-BT
Net Flux Analysis Glucose Reaction network
Physiological data
(16 mmol/g/h) Biomass composition
Metabolic flux ratios

Growth Rate
(0.4 h-1)
1.6
Glucose-6-P
13.7 R-5-P

PPP

Oxaloacetate Pyruvate
0.5 Oxaloacetate
0.0 TCA
cycle

Glycerol Ethanol
(1.5 mmol/g/h) (22 mmol/g/h)
Bioanalytic Lecture WS05/06 LS-BT
Bioanalytic Lecture WS05/06 LS-BT
Summary Flux analysis

Bioanalytic Lecture WS05/06 LS-BT

 What has to be done
experimentally?

Bioanalytic Lecture WS05/06 LS-BT

Amino acid Derivatisation: Silylation

Bioanalytic Lecture WS05/06 LS-BT

GC-MS Chromatogram
Sample ID:

10.725
100
747

11.805
867
8.393 12.813
488 979

A 6.089
232
9.177
575 K
4.405
45 5.639
182
L
4.567
63
V 6.413
S 9.501
611 11.391
268 821

G I 9.969
%
T 663

6.719
302 E 13.597

P 1066

Y
10.266
5.720 696 11.859
191 12.579

M
8.906
545
10.959
773
873

12.111
953

H
9.897
4.648 901 13.380
7.736 655 13.921
72 5.126 1042
415 8.114 1102
125 457

0
50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100

Bioanalytic Lecture WS05/06 LS-BT

MS Massdistribution of the amino acid glycine

LB_10 45 (4.405) Scan EI+

73 1.76e6
100

158

147

232

260
160

59

75
103 234

133
45 57 102 263
115 132
86 161 189
56 60 94 145 235
76 190 258 274 302
162 174 216 276 305 316 448
409 454 456
0 m/z
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600

Bioanalytic Lecture WS05/06 LS-BT

Steps in Data processing

ƒ Data file from GC-MS software

ƒ Convert to CDF file (by e.g. DataBridge)
ƒ Convert to FF file by FiatFlux
ƒ FiatFlux:
ƒ removes dramatization
ƒ takes non-labeled inoculum into account (User)
ƒ takes natural isotope abundance into account
ƒ Redundant information is fitted (least square
minimization of the errors)
ƒ User intervention of experimental doubtful amino
acid fragments

Bioanalytic Lecture WS05/06 LS-BT

 Software: Fiat Flux

GC-MS data analysis

Bioanalytic Lecture WS05/06 LS-BT

Yeast growth experiment: quantitative
physiology to distinguish a respiratory
and a respiro-fermentative yeast

Pichia stipitis Saccharomyces cerevisiae

ƒ Isolate from insect larvae
ƒ Respiratory yeast
ƒ Can grow on 5 carbon sugars
like xylose, potential for
ethanol production from
hemicellulose
ƒ Baker‘s yeast
ƒ Respiro-fermentative yeast
ƒ Ethanol production from
hexoses, protein production,
food ingredient

Bioanalytic Lecture WS05/06 LS-BT

 Which data do we
need to calculate an
intracellular carbon
flux distribution?

Bioanalytic Lecture WS05/06 LS-BT

 Which analytical
techniques do you
suggest for the
different molecules to
be analyzed?

Bioanalytic Lecture WS05/06 LS-BT

 Analytics

ƒ Glucose ƒ Enzyme kit or RI-HPLC

ƒ Ethanol ƒ Enzyme kit or RI-HPLC or GC
ƒ Glycerol ƒ Enzyme kit or RI-HPLC
ƒ Acetate, Succinate ƒ Enzyme kit or UV-HPLC (or GC)
ƒ Labeling pattern of ƒ NMR or GC-MS or LC-MS
proteinogenic amino acids
ƒ Biomass ƒ Vis-spectrophotometer

Bioanalytic Lecture WS05/06 LS-BT

 Work plan

ƒ Perform growth experiment with both yeasts

ƒ Harvest biomass (how?)
ƒ Hydrolyze proteins by addition of 6 M HCl at 105°C in an oven
ƒ Dry sample
ƒ Resuspend solids in DMF
ƒ Derivatize amino acids with a silane at 85°C for 1 hour
ƒ Perform GC-MS analysis
ƒ Reformat data into CDF format
ƒ Calculate flux ratios
ƒ Measure extracellular metabolites by HPLC (and enzyme kits)
ƒ Combine data sets for intracellular net flux calculations

Bioanalytic Lecture WS05/06 LS-BT

Summary Experimental Procedures

Bioanalytic Lecture WS05/06 LS-BT

Summary Metabolic Flux Analysis

ƒ Allows the quantification of the

intracellular carbon flux distribution
ƒ Based on 13C tracer experiments
ƒ Steady state assumption
ƒ GC-MS analysis

Essential for tracing the impact of

Metabolic Engineering on microbial
metabolism. Can be used for cellular
catalyst optimization.

Have fun in the practical class!!! See you there.

Bioanalytic Lecture WS05/06 LS-BT

 Acknowledgments

Lars Küpfer, Bayer Technologies, Leverkusen

Lars K. Nielsen, University of Queensland, Australia

Recommended Literature:
Stephanopoulos, Aristidou, Nielsen. Metabolic engineering: principles and
methodologies. San Diego: Academic Press, c1998.

Bioanalytic Lecture WS05/06 LS-BT