Flux Balance Analysis

The Central Dogma

Figure 1. The central dogma
Metabolic Pathways and fluxes
Metabolism - life-
sustaining chemical
transformations within the
cells of living organisms

Metabolites
intermediate products of
metabolism

Metabolic Pathways Way
to represent metabolism

Metabolic Flux - Rate of
turnover of molecules
through a metabolic
pathway



Figure 2 Metabolic pathway
Genome Scale Metabolic Networks
Genes -> Enzymes
(Central Dogma)
Enzymes ->
Metabolic Networks
(Catalysts)

Genes -> Metabolic
Networks

Integration of
genomic and
metabolomic data

Cell Modeling
Figure 3. The reconstruction process
What is FBA?

Mathematical approach to analyse flow of metabolites in
metabolic networks
Figure 4. A simplified core carbon metabolic network
Steps in FBA
(a) System Definition
(A,B,C metabolites
v
i
internal flux
b
i
exchange flux)
(b) Mass Balance
(c) Constraints
(Thermodynamic,
experimental)
(d) Optimization

Figure 5. Steps in FBA
Applications
Analysis of Genome Scale Metabolic Networks
Drug Target Identification
Metabolic Engineering
Refinement of metabolic networks
Challenges
Solution as good as constraints
Focuses only on enzyme part of genome
Reconstruction of GSMNs a challenge
Relevance of objective functions
Regulatory Networks not considered
rFBA
Modification of FBA
Includes boolean regulatory information

Figure 6. Feedback network
Steps in rFBA
Figure 7 Solution space in rFBA
(a) Introduce adjustable constraints using boolean operations
(regulatory events dependant on time)
(b) Quasi steady state assumption
(c) Reduction in solution space
Advantages over FBA
Quantitative dynamic simulation of substrate
uptake, cell growth and by-product secretion

Qualitative simulation of gene transcription
events and the presence of proteins in the cell

Investigation of the systemic effects of
imposing temporary regulatory constraints on
the solution space
Methods like rFBA

iFBA Integrated ordinary differential
equations model with rFBA

SR-FBA identifies a metabolicregulatory
steady state
Disadvantages
Manual curation a tedious process

Modelling restricted to smaller and
extensively studied models

On/Off approach gives only qualitative
analysis
PROM (probabilistic
regulation of metabolism)
Automatically quantifies the interactions from
high-throughput data, no need of manually
curation

Uses conditional probabilities for modeling
transcriptional regulation rather than boolean

Greatly increases the capacity to generate
genome-scale integrated models
PROM inputs
Reconstructed genome scale metabolic
networks

Regulatory network structure consisting of
transcription factors (TFs) and their targets

Gene expression data under various
environmental and genetic perturbations
Analysis Tools

Flux Balance Analysis (FBA)

Flux Variability Analysis (FVA)

Kolmogorov Smirnov Test
Flux Variability Analysis
Determine the variability of fluxes in the
network
Original objective value fixed
Each reaction maximized and minimized to get
feasible range of fluxes
Kolmogorov Smirnov Test
Nonparametric test for the equality of
continuous, one-dimensional probability
distributions
Compare two separate samples or a sample
with a reference probability distribution
Makes no assumption about distribution
Used to select those pairs of TFs and targets for
which the targets expression changes
significantly wrt the TF state

Steps in PROM
Kolmogorov-Smirnov statistic used to get TF
and target interactions
Probabilities used to represent gene states
and interactions between a gene and TF
Eg - the probability of gene A being active
when the regulating transcription factor B is
not active is represented by P(A=1|B=0)

Microarray data used to assign values to the
relationship between transcription factor and target
gene
Using following formula:
P(A=1|B=0)=N(A=1|B=0)N(B=0) where N is the
number of times the event is observed
For example, if in 80% of the samples we find the
gene to be on when the transcription factor is off,
then the probability P(A=1|B=0)=0.8
Therefore, the flux through the reaction regulated
by gene A when its corresponding regulator B is
turned off would be PvLAvAPvUA

Estimates for reaction bounds obtained by FVA
algorithm or by utilizing literature or other
kinds of prior knowledge

Regulatory constraints to be soft constraints to
compensate for different noises

Able to exceed regulatory constraints to
maximize growth but with penalty
Final Equation
Maximize :
Z=cjvj+j(jj+jj)
subject to:
jSijvj=0
PvLjjvj < vij <PvUj+j
j,j > 0
where PvLj and PvUj are the
transcriptional regulation
bounds, j and j are positive
variables that allow deviation
from those bounds, and j is
the cost for such deviations.

Figure 8 PROM

Image Sources
1. https://dnasu.org/DNASU/Resources/Plasmid.jsp

2.http://en.wikipedia.org/wiki/Citric_acid_cycle#mediaviewer/File:Citric_acid_cycle_with_aconitate_2.svg

3. Markus W. Coverta, Christophe H. Schillinga, Iman Familia, Jeremy S. Edwardsb, Igor I. Goryaninc, Evgeni
Selkovd, Bernhard O. Palssona, Metabolic modeling of microbial strains in silico, Trends in Biochemical
Sciences(2001), Volume 26, Issue 3, Pages 179186

4. Markus W. Covert, Christophe H. Schilling and Bernhard Palsson, Regulation of Gene Expression in Flux
Balance Models of Metabolism, J. theor. Biol. (2001) 213, 73-88

5. Kauffman KJ1, Prakash P, Edwards JS. Advances in flux balance analysis, Curr Opin Biotechnol.
(2003),5:491-6

6. Markus W. Covert, Christophe H. Schilling and Bernhard Palsson, Regulation of Gene Expression in Flux
Balance Models of Metabolism, J. theor. Biol. (2001) 213, 73-88

7. Markus W. Covert and Bernhard . Palsson Transcriptional Regulation in Constraints-based Metabolic
Models of Escherichia coli , J. Biol. Chem. (2002), 277:28058-28064.

8. Chandrasekaran S, Price ND ,Probabilistic integrative modeling of genome-scale metabolic and
regulatory networks in Escherichia coli andMycobacterium tuberculosis, Proc Natl Acad Sci U S A (2010)
107(41):1784517850


References
Orth, J.D., Thiele, I., and Palsson, B.. What is flux balance
analysis?, Nature Biotechnology (2010), 28: 245-248
Kauffman KJ1, Prakash P, Edwards JS. Advances in flux balance
analysis, Curr Opin Biotechnol. (2003),5:491-6
Karthik Raman and Nagasuma Chandra, Flux balance analysis of
biological systems: applications and challenges, Briefings in
Bioinformatics,(2009) Volume 10, No 4 435-449
Markus W. Covert, Christophe H. Schilling and Bernhard Palsson,
Regulation of Gene Expression in Flux Balance Models of
Metabolism, J. theor. Biol. (2001) 213, 73-88
Simeonidis E, Chandrasekaran S, Price ND., A Guide to Integrating
Transcriptional Regulatory and Metabolic Networks Using PROM
(Probabilistic Regulation of Metabolism) Methods Mol Biol. (2013)
985:103-12.

Thank You