ABSTRACT
The key aspects in the analysis of a metabolic network are
identification of the bioreaction network, quantification of
internal fluxes, and identification of the control structure
within the network.[1-4] The metabolic network of an
organism can be constructed using information available
from biochemistry and further enzyme assays provide
information about activity of different pathways.[3] Upon
identification of the metabolic network, quantification of
fluxes through different pathways could be done using
metabolic flux analysis (MFA), which has essentially two
components: (i) in silico flux analysis, also called flux
balance analysis (FBA), based on optimization algorithms
for flux estimation and (ii) estimation of internal fluxes
through metabolite balancing based on experimental mea-
surements (MFA).[1,4] An important and powerful tool for
both identification of active pathways and quantification of
fluxes through them is the use of labeled substrates in
tracer experiments and subsequent analysis of labeling
pattern of intracellular metabolites.[1-4] These experiments
(especially when using labeled carbon) help in the identi-
fication of active pathways and provide good estimates of
internal fluxes due to the high degree of redundancy
involved with carbon atom balances as opposed to meta-
bolite balances.[3,4] Another important aspect in the analy-
sis of an organism is metabolic control analysis, which
includes identification of the flux control architecture
(how the distribution of fluxes and metabolite concentra-
tions are controlled) and the enzyme regulation network in
the organism.[3,4] This kind of analysis helps in the rational
design of strategies for modification of cellular pathways
to suit engineering purposes. Also, the recent technological
advancements such as complete genome sequencing of
several organisms and elucidation of functions of a large
number of those genes has had tremendous implications in
ME by providing information about pathways present and
active in different organisms. This has facilitated compre-
hensive analysis of natural and new pathways in the con-
text of an organism, thereby aiding in the design of
strategies for modification of the metabolic network.[1,3]
Synthetic Tools in ME
The aforementioned strategies developed via analytical
tools of ME are then applied for synthesis of improved
cells using various genetic tools that allow precise modifi-
cation of the metabolic network.[3,5] Native genes can be
silenced or over expressed, foreign genes can be imported
into the genome of host cells, and natural regulation of
gene expression within a cell can also be altered by repla-
cing the promoter of that gene, thereby having different/
controlled regulation patterns.[3,5] From the ME point of
view, it is important that these genetic modifications have
certain desirable properties. Importing foreign genes into a
cell essentially involves their integration into the host
chromosome or the use of plasmids that reside indepen-
dently. It is very important for these cloning vectors to
exhibit segregational stability, ability to carry large
sequences of DNA, consistent copy numbers in cells, low
metabolic burden, tight control of expression, and little or
no interference with the native genes.[3,5] In addition to
these modifications, the enzyme(s) could also be altered to
incorporate or remove certain regulatory/control mechan-
isms by either importing genes from other organisms or via
mutagenesis of genes encoding native enzymes.[3]
There are numerous parameters in an organism that
could be manipulated so as to concoct the best possible
configuration for a particular product, and thus, ME
employs various strategies for the purpose. Application of
ME to a host of bacterial strains to produce different
chemicals, both native to the bacteria and totally novel
products, have led to efficient production of several che-
micals as illustrated in Table 1. The following sections
discuss various chemicals produced by bacteria and the
relevant ME strategies applied to them.
