Biology Reference
In-Depth Information
CASE STUDY
3:
PREDICTION AND VALIDATION OF THE IMPORTANCE OF THE
ENTNER
-
DOUDOROFF
(
ED
)
PATHWAY IN PHB BIOSYNTHESIS BY METABOLICALLY
ENGINEERED
E. coli
Poly(3-hydroxybutyrate), PHB, is one of the most common members
of the polyhydroxyalkanoates (PHAs) and is a biodegradable polymer
that can be used as an excellent alternative to conventional petro-
chemical-based polymers [87,88]. However, the production cost of PHB
should be considerably reduced to make it competitive with petroleum-
derived plastics. To achieve this goal, much effort has been exerted to
develop metabolically engineered strains for the efficient production
and recovery of PHB [88-90]. In this case study, we show the systems
biotechnological procedures taken to understand the metabolic charac-
teristics of an engineered
E. coli
strain producing large amounts of PHB.
Step 1: Development of metabolically engineered E. coli strain.
The PHB
biosynthesis operon of
Wautersia eutropha
and
Alcaligenes latus
encodes three enzymes: b-ketothiolase, reductase, and PHB synthase
[89,91,92]. b-Ketothiolase condenses two acetyl-CoA moieties to form
acetoacetyl-CoA, which is then reduced to (
R
)-3-hydroxybutyryl-CoA
by an NADPH-dependent acetoacetyl-CoA reductase. PHB synthase
finally links (
R
)-3-hydroxybutyryl-CoA to the growing chain of PHB
(figure 7.10). Plasmid-based expression of the
W. eutropha
or
A. latus
PHB biosynthesis genes in
E. coli
led to the accumulation of a large
amount of PHB from glucose [92,93]. In order to understand the metabolic
characteristics of the engineered
E. coli
strain, which accumulates PHB
to a surprisingly high level (up to 90% of dry cell weight) inside the cell,
systems biotechnological research was carried out as described below.
Step 2: Construction of in silico model.
The in silico metabolic model
describing recombinant
E. coli
metabolism is derived from the publicly
available information and database [75,94]. The three-step PHB biosyn-
thetic pathway, which is heterologous to
E. coli
, is also introduced in
the in silico metabolic model. For simplicity, a small metabolic model
consisting of 310 reactions and 295 metabolites was constructed and
employed [95].
Step 3: In silico experiment.
The constraints-based flux analysis was
carried out to quantify flux distribution under various conditions.
During the simulation, the previously reported fermentation data
during PHB production were imposed as additional constraints [96,97].
Metabolic fluxes under PHB-producing conditions were determined
for two different PHB contents of 49% (figure 7.11a) and 78%
(figure 7.11b) of dry cell weight, and were compared with those under
non-PHB-producing condition. Bold arrows indicate the increased
fluxes by more than 2-fold while dotted arrows indicate the decreased
fluxes to less than half under PHB-accumulating conditions.
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