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249
8
Metabolic Flux Analysis
on the Production of
Poly(3-hydroxybutyrate)
Prof. Dr. Sang Yup Lee1, M. Eng. Soon Ho Hong2, M. Eng. Si Jae Park3,
Dr. Richard van Wegen4, Dr. Anton P. J. Middelberg5
1
Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical Engineering and BioProcess Engineering Research Center, Korea
Advanced Institute of Science and Technology 373-1 Kusong-dong, Yusong-gu,
Taejon 305-701, Korea; Tel.: ‡ 82-42-869-3930; Fax: ‡ 82-42-869-3910;
E-mail: [email protected]
2
Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical Engineering and BioProcess Engineering Research Center, Korea
Advanced Institute of Science and Technology 373-1 Kusong-dong, Yusong-gu,
Taejon 305-701, Korea; Tel.: ‡ 82-42-869-5970; Fax: ‡ 82-42-869-3910;
E-mail: [email protected]
3
Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical Engineering and BioProcess Engineering Research Center, Korea
Advanced Institute of Science and Technology 373-1 Kusong-dong, Yusong-gu,
Taejon 305-701, Korea; Tel.: ‡ 82-42-869-5970; Fax: ‡ 82-42-869-3910;
E-mail: [email protected]
4
Department of Chemical Engineering, University of Adelaide, SA 5005, Australia;
Tel.: ‡ 43(01) 796-6362-311; Fax: ‡ 43(01) 796-6362-333;
E-mail: [email protected]
5
Department of Chemical Engineering, University of Cambridge, Pembroke Street,
Cambridge, CB2 3RA, UK; Tel.: ‡ 44-1223-335245; Fax: ‡ 44-1223-334796;
E-mail: [email protected]
1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
250
2
Historical Outline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
251
3
Production of Poly(3HB ) by Ralstonia eutropha . . . . . . . . . . . . . . . . . .
252
4
Production of Poly(3HB ) by Recombinant Escherichia coli . . . . . . . . . . . .
255
5
Conclusions and Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
258
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8 Metabolic Flux Analysis on the Production of Poly(3-hydroxybutyrate)
6
Patents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
259
7
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
260
3HB
CoA
DOC
MCA
MFA
NADH
NADPH
PHA
SCL
TCA
3-hydroxybutyrate
coenzyme A
dissolved oxygen concentration
metabolic control analysis
metabolic flux analysis
nicotinamide adenine dinucleotide (reduced form)
nicotinamide adenine dinucleotide phosphate (reduced form)
polyhydroxyalkanoate
short-chain-length
tricarboxylic acid cycle
1
Introduction
Polyhydroxyalkanoates (PHAs) have been
considered to be good candidates as alternatives to synthetic nondegradable polymers
due to their similar mechanical properties to
petroleum-derived polymers and their complete biodegradability (Steinb¸chel, 1992;
Brandl et al., 1995; Lee, 1996a). A number of
microorganisms have been found to accumulate PHAs under unfavorable growth
conditions and in the presence of excess
carbon source (Anderson and Dawes, 1990;
Doi, 1990; Steinb¸chel, 1991; Lee, 1996b;
Steinb¸chel and F¸chtenbusch, 1998; Madison and Huisman, 1999). Among the
various members of PHAs, poly(3-hydroxybutyrate), poly(3HB ), a member of shortchain-length (SCL ) PHAs, is best characterized and has been produced on a semicommercial scale. One of the major drawbacks in the commercialization of PHAs is
the much higher production cost of PHAs
compared with petrochemical-based polymers. Therefore, much effort has been
devoted to reduce the production costs of
PHAs by developing better bacterial strains
and downstream processes such as more
efficient fermentation and more economical
recovery processes (Lee, 1996a,b; Choi et al.,
1998; Choi and Lee, 1999a,b). Process
design and economic analysis of SCL-PHA
production by various bacteria have been
reported, which provided the guidelines for
designing an efficient means of PHA production (Choi and Lee, 1997, 1999c, 2000;
Lee and Choi, 1998). Several factors affecting
the production cost of PHA, including PHA
productivity, PHA content and yield on
carbon substrate, the cost of carbon substrate, and the recovery yield of PHA have
been examined in detail. Based on the results
of economic evaluation, Ralstonia eutropha,
Alcaligenes latus, and recombinant Escherichia coli have been suggested as good
candidates for the efficient production of
SCL-PHAs (Lee, 1996a,b).
By using a metabolic flux analysis (MFA )
technique, the intracellular metabolic fluxes
can be quantified by the measurement of
extracellular metabolite concentrations in
2 Historical Outline
combination with the stoichiometry of intracellular reactions (Nielsen and Villadsen,
1994; Edwards et al., 1999). MFA is based on
the pseudo-steady-state assumption, which
means that there is no net accumulation of
intermediates (Stephanopoulos, 1999). To
analyze the system using the MFA technique, the system should be determined or
over-determined, which means that the
number of constraints is equal to or greater
than that of the reactions. To analyze the
under-determined system, more constraints
are required. This is often replaced by setting
up an objective function, such as maximum
growth or maximum metabolite production,
and solving by linear programing ( Varma
and Palsson, 1994). MFA has been applied to
calculate the maximum theoretical yield of a
desired metabolite to be produced. Another
application is to identify the rigidity of
branch points in metabolic pathways. The
rigidity of a branch point is important, as a
rigid branch point resists changes in flux
split ratios, while a flexible branch point
tends to be more accommodating (Stephanopoulos and Vallino, 1991). The third
possible application is the identification of
alternative metabolic pathways. The detailed
theories and applications of the MFA can be
found in recent reviews (Edwards et al.,
1999; Stephanopoulos, 1999).
Metabolic control analysis (MCA ) is a
statistical modeling technique that can be
used to understand the control of metabolic
pathways and pathway regulations. MCA
allows us to understand how metabolic
fluxes are controlled by certain enzyme
activities and metabolite concentrations
(Kacser and Burns, 1973; Heinrich and
Rapoport, 1974). The responses of small
changes in enzyme activities and metabolite
concentrations to metabolic flux distribution
can be predicted by MCA (Nielsen and
Villadsen, 1994). If we consider a linear
chain of N enzymatic reactions, there are
N 1 intermediates Xj, j ˆ 1, º, N 1. The
response coefficient of a certain metabolite is
defined as the ratio of the relative change in
the reaction rate brought about by the
change in the metabolite concentration, viz:
eji ˆ
Xj @ri
;
ri @Xj
i ˆ 1; ::; N and j ˆ 1; ::; N
1
where ri is the net rate of the i-th enzymatic
reaction and Xj is the size of the j-th
metabolite pool. The flux control coefficient
of a certain enzyme is the relative change in
the steady-state flux resulting from the
change in the activity of an enzyme of the
pathway, viz:
Cir ˆ
Xei @r
;
r @Xei
i ˆ 1; :::; N
where Xei is the activity of i-th enzyme and r
is the overall steady-state flux. By analyzing
the flux control coefficients and response
coefficients, one can propose which enzymatic reaction step is rate controlling (e.g., a
reaction step with high flux control coefficient is the rate-controlling step), and also
predict the results of deviations in certain
enzymatic reactions. Readers are encouraged to refer to an excellent monograph on
MCA by Fell (1997).
In this chapter, we review the applications
of MFA and MCA on the production of
poly(3HB ) by various bacterial strains. The
effects of various environmental conditions
on poly(3HB ) production are evaluated, and
the important factors such as intracellular
metabolite concentrations and enzyme activities on poly(3HB ) biosynthesis are examined by MFA and MCA.
2
Historical Outline
A brief historical outline of the commercial
production of poly(3HB ) is shown below:
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252
8 Metabolic Flux Analysis on the Production of Poly(3-hydroxybutyrate)
Year History
Strain
1925 Production of poly(3HB) Bacillus
first discovered
megaterium
1973 MCA first proposed
1988 The first phb full operon Ralstonia
cloned
eutropha
1988 Poly(3HB) first proEscherichia
duced in E. coli
coli
1991 Metabolic engineering
first proposed
1993 Effect of nitrogen limRalstonia
itation analyzed
eutropha
1997 Effect of carbon sources Ralstonia
analyzed
eutropha
1998 Poly(3HB)-producing
mechanism identified
1999 Effect of oxygen limita- Escherichia
tion analyzed
coli
2001 Control factors of poEscherichia
ly(3HB) production
coli
identified
3
Production of Poly(3HB) by Ralstonia
eutropha
Ralstonia eutropha (formerly Alcaligenes eutrophus), has been intensively examined for
the efficient production of SCL-PHAs. The
PHA biosynthetic pathway in R. eutropha has
been well characterized: two acetyl-CoA
moieties are condensed to form acetoacetyl-CoA by b-ketothiolase. AcetoacetylCoA is then reduced to (R )-3-hydroxybutyryl-CoA by an NADPH-dependent reductase. PHA synthase finally links (R )-3hydroxybutyryl-CoA to the growing chain
of poly(3HB ). The genes coding for the three
enzymes were found to form a phb operon in
the order of PHA synthase, b-ketothiolase,
and reductase (Schubert et al., 1988; Slater
et al., 1988; Peoples and Sinskey, 1989). The
three-step poly(3HB ) biosynthesis pathway
from acetyl-CoA is shown in Figure 1. Fedbatch culture strategies to achieve high
productivity of PHAs have also been developed (Kim et al., 1994a,b; Ryu et al., 1997).
An optimal fed-batch culture strategy resulted in both high poly(3HB ) concentration
and productivity of 232 g L 1 and 3.14 g
poly(3HB ) L 1 h 1, respectively (Ryu et al.,
1997).
The cost of raw materials (especially of the
carbon source) is a very important factor
affecting the overall production cost of poly(3HB ). Therefore, the effect of different
carbon sources on the production of poly(3HB ) was extensively analyzed using
MFA (Shi et al., 1997). R. eutropha can
utilize various organic acids as carbon
sources which, from a practical stand-point,
is important for the production of poly(3HB )
from organic wastes, for example food waste.
Consequently, butyrate, lactate, and acetate
were examined as carbon sources. When R.
eutropha was cultivated on a mixture of three
carbon sources, lactate was consumed first
as a large amount of ATP is needed for the
transport of acetate and butyrate. The central
metabolic pathways for the utilization of
three carbon sources are very similar, except
for the anaplerotic pathway which replenishes carbon intermediates to the tricarboxylic acid (TCA ) cycle. As shown in Figure 2,
carbon dioxide is wasted during the conversion of pyruvate to acetyl-CoA when
lactate is used as a carbon source. To decide
which carbon source is better for the production of poly(3HB ), simulations were
carried out at different specific growth rates
with three carbon sources. The maximum
poly(3HB ) yields (g g 1) without cell growth
obtainable with acetate, lactate, and butyrate
were 0.33, 0.33, and 0.67, respectively. The
poly(3HB ) yield was limited by NADPH
regeneration, as it is required for the
conversion of acetoacetyl-CoA to (R )-3-
3 Production of Poly(3HB) by Ralstonia eutropha
Fig. 1
Simplified central metabolic pathway of E. coli and PHB biosynthesis pathway.
hydroxybutyryl-CoA. NADPH is regenerated
solely by isocitrate dehydrogenase in the
TCA cycle. Butyrate was found to be more
efficient for the production of poly(3HB ),
because about 67% of butyrate was directed
to the TCA cycle, in which the required
NADPH was regenerated, while only 33% of
acetate and lactate were metabolized through
the TCA cycle (Figure 2B, C, and D ).
Poly(3HB ) production by R. eutropha H16
(ATCC 17699) was also examined by fedbatch cultivation using butyrate as a carbon
source (Shimizu et al., 1993). The maximum
poly(3HB ) yield of 0.85 (gg 1) was obtained
at a butyrate concentration of 3 g L 1 and at
pH 8.0. The final poly(3HB) content was
75 wt.% dry cell weight. The changing profiles of the intracellular metabolic flux distribution during the fermentation were evaluated by MFA (Shi et al., 1997). The accumulation of poly(3HB) was induced by depletion
of the nitrogen source. When the strain was
shifted from the growth phase to the poly(3HB ) production phase, flux into the poly(3HB) biosynthetic pathway increased, whilst
flux into the glyoxylate bypass decreased
(Figure 2A, B ). There was no significant
change in flux to the TCA cycle through
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254
8 Metabolic Flux Analysis on the Production of Poly(3-hydroxybutyrate)
Fig. 2 Flux distribution during fed-batch culture on (A ) butyrate at cell growth phase and (B ) at poly(3HB)
production phase, and on (C ) acetate and (D ) lactate at poly(3HB) production phase. Fluxes were
normalized by substrates uptake rates. [ Redrawn from Shi et al. (1997) with permission.]
isocitrate dehydrogenase. It can be concluded that glyoxylate bypass is required for the
growth on butyrate, and this competes with
the poly(3HB ) biosynthetic pathway. Since
flux into the TCA cycle was almost constant
throughout the cultivation, the amount of
NADPH produced during cultivation was
not significantly changed. The NADPH
consumption rate, however, was decreased
because limitation of the nitrogen source
blocked the amino acid synthesis pathways,
especially the reaction from a-ketoglutarate
to glutamate, which assimilates ammonium
ions into the cell. Therefore, the residual
NADPH was accumulated, which in turn
enhanced poly(3HB ) biosynthesis.
As mentioned earlier, the cost of raw
materials is one of the most important
factors affecting the economics of PHA
production. Since the cost of glucose is
lower than that of butyrate, glucose is better
for the industrial production of PHAs. To
4 Production of Poly(3HB) by Recombinant Escherichia coli
use glucose directly as a carbon source, a
mixed culture system was examined, in
which glucose was converted to lactate by
Lactobacillus delbrueckii and the lactate was
converted to poly(3HB ) by R. eutropha
(Katoh et al., 1999). To evaluate the effects
of environmental conditions on cell growth
and poly(3HB ) production, MFA was carried
out under various conditions. It was found
that NH3 plays an important role in the
production of poly(3HB ). When NH3 was
sufficient, most of the NADPH was utilized
for amino acids biosynthesis, and cell growth
was activated. NADPH was used as a
coenzyme of acetoacetyl-CoA reductase for
the conversion of acetoacetyl-CoA to (R )-3hydroxybutyryl-CoA under nitrogen-limiting
conditions. From these results, it can be said
that poly(3HB ) production can be enhanced
by providing a condition in which NADPH is
in excess.
4
Production of Poly(3HB ) by Recombinant
Escherichia coli
Since the first demonstration that a large
amount of poly(3HB ) could be synthesized
in recombinant E. coli harboring the PHA
biosynthesis genes of R. eutropha (Schubert
et al., 1988), recombinant E. coli has been
intensively examined for the production of
poly(3HB ). Recombinant E. coli has been
considered to be a good candidate as a
producer for SCL-PHAs as it has several
advantages over wild-type PHA producers
such as R. eutropha and A. latus (Lee, 1997).
Various metabolically engineered E. coli
strains have been developed for the efficient
production of poly(3HB ), with high productivity of up to 4.63 g L 1 h 1 ( Wang and
Lee, 1997, 1998; Choi et al., 1998; Ahn et al.,
2000). Recombinant E. coli equipped with a
heterologous PHA biosynthetic pathway
produces PHA in a growth-associated manner. Since poly(3HB ) is a new intracellular
metabolite of E. coli, the effect of poly(3HB )
biosynthesis on the alteration of metabolic
flux distribution should be evaluated in
order to understand the physiological consequences of poly(3HB ) accumulation in the
cells. The dissolved oxygen concentration
(DOC ) is one of the important factors in
poly(3HB ) production by recombinant
E. coli, since oxygen often becomes limited
during high cell density cultivation ( Wang
and Lee, 1997; Wong et al., 1999). It was
reported recently that poly(3HB ) production
by recombinant E. coli could be enhanced
under oxygen-limiting conditions ( Wang
and Lee, 1997). To understand this phenomenon, the effects of DOC on metabolic flux
distribution were examined using the MFA
technique. The result of simulation using
this approach suggested that 100 mol and
67 mol of poly(3HB ) were produced from
100 mol of glucose under oxygen-sufficient
and oxygen-limiting conditions, respectively.
It was also found that the flux through
pyruvate formate-lyase increased without
any change in pyruvate dehydrogenase flux
under oxygen-limiting conditions, causing
the accumulation of intracellular acetyl-CoA
from 350 mg g 1 RCM to 600 mg g 1 RCM
(van Wegen et al., 2001). This accumulated
acetyl-CoA was efficiently channeled to the
poly(3HB ) synthetic pathway, and more
poly(3HB ) could be accumulated under the
oxygen-limited condition.
The amount of acetyl-CoA and NADPH
available have also been found to be important factors in poly(3HB ) production by
recombinant E. coli, and this agrees well with
the results of MFA that acetyl-CoA and
NADPH play important roles in poly(3HB )
biosynthesis by R. eutropha (Lee et al., 1996).
The regulatory effects of NADPH and enzyme activities on poly(3HB ) biosynthesis
were examined in recombinant E. coli XL1-
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8 Metabolic Flux Analysis on the Production of Poly(3-hydroxybutyrate)
Blue harboring the R. eutropha PHA biosynthesis genes. Cells were grown in various
culture media including complex LuriaBertani (LB ) medium, LB supplemented
with glucose, and chemically defined medium. Among these, poly(3HB ) was most
favorably accumulated in LB ‡ glucose medium, which supported the highest
NADPH/NADH ratio. The activity of citrate
synthase, which competes with b-ketothiolase for acetyl-CoA, was also much lower
when cells were cultured in LB ‡ glucose
medium (Lee S. Y. et al., 1995; Lee I. Y. et al.,
r
rmax
availability of two substrates for poly(3HB )
synthesis, NADPH and acetyl-CoA.
MCA was also carried out in order to
evaluate thoroughly the effects of NADPH
and acetyl-CoA on poly(3HB ) biosynthesis
(van Wegen et al., 2001). The kinetics data of
related enzymes are required for the calculation of flux control and elasticity coefficients. The b-ketothiolase catalyzes the conversion of acetyl-CoA to acetoacetyl-CoA, and
it follows a ping-pong, Bi-Bi mechanism
(Leaf and Srienc, 1998). The rate equation is:
ˆ
AB PQ Keq
Kb A‡Ka B‡AB‡Kq V1 P V2 Keq ‡Kp V1 Q V2 Keq ‡V1 PQ V2 Keq ‡Kq V1 AP V2 Kia Keq ‡Ka BQ Kiq
1996) also indicated the importance of the
availability of NADPH on PHA biosynthesis
in recombinant E. coli. Supplementation of
complex nitrogen sources, oleic acid, or
amino acids to the chemically defined
medium significantly enhanced poly(3HB )
production. Because the biosynthesis of
amino acids and oleic acid requires large
amounts of reducing equivalents, poly(3HB )
production by recombinant E. coli in chemically defined media was inefficient compared with poly(3HB ) production in complex media (Lee et al., 1995). These experimental findings were supported by studies
on the effects of acetyl-CoA and NADPH on
the intracellular metabolic flux distribution
of recombinant E. coli (Shi et al., 1999). The
results of MFA suggested that in order to
achieve the maximum poly(3HB ) yield,
about one-half of the carbon flux should be
directed to the pentose phosphate (PP )
pathway, and flux to the TCA cycle should
be shut down. These two pathways affect the
where
r
ˆ forward reaction rate (mM substrate min 1)
rmax ˆ maximum forward reaction rate
(mM substrate min 1)
A
ˆ first substrate (acetyl-CoA ) concentration (mM )
B
ˆ second substrate (also acetyl-CoA )
concentration (mM )
P
ˆ first product (CoA ) concentration
(mM )
Q
ˆ second product (acetoacetyl-CoA )
concentration (mM )
V1/V2 ˆ ratio of maximum forward reaction
rate to maximum reverse reaction
rate
Keq ˆ equilibrium constant
Ka, Kb, Kp, Kq, Kia, Kib, Kip, Kiq,
ˆ various kinetic constants (mM )
The kinetics of acetoacetyl-CoA reductase
has been studied to a lesser extent, but most
likely follows a rapid equilibrium, random,
Bi-Bi mechanism. The rate equation is:
AB PQ Keq
r
ˆ
rmax Kia Kb ‡ Kb A ‡ Ka B ‡ AB ‡ Kq V1 P V2 Keq ‡ Kp V1 Q V2 Keq ‡ V1 PQ V2 Keq
4 Production of Poly(3HB) by Recombinant Escherichia coli
where
A
B
P
Q
ˆ first substrate (acetoacetyl-CoA )
concentration (mM )
ˆ second substrate (NADPH ) concentration (mM )
ˆ first product ((R )-3-hydroxybutyrylCoA ) concentration (mM )
ˆ second product (NADP ) concentration (mM )
The last enzyme, PHA synthase, catalyzes
the polymerization of (R )-3-hydroxybutyrylCoA, and the reaction is considered to be
diffusion-limited.
r
‰…R†-3-hydroxybutyryl-CoAŠ
ˆ
rmax Km ‡ ‰…R†-3-3-hydroxybutyryl-CoAŠ
The kinetic constants for three enzymes are
summarized in Table 1 (Leaf and Srienc,
1998).
The results of MCA suggested that the
poly(3HB ) biosynthesis flux was highly
sensitive to the acetyl-CoA/CoA ratio (response coefficient 0.8) and total acetyl-CoA
‡ CoA concentration (response coefficient
0.7), while it is less sensitive to the NADPH/
NADP ratio (response coefficient 0.25) (TaTab. 1
ble 2). This means that to increase the flux to
the same extent, the NADPH/NADP ratio
needs to be increased more than the acetylCoA/CoA ratio. Finally, it was proposed that
the overexpression of acetoacetyl-CoA reductase seems to be the most efficient way to
enhance poly(3HB ) productivity, as the flux
control coefficients were 0.6, 0.25, and 0.15
for acetoacetyl-CoA reductase, PHA synthase, and b-ketothiolase, respectively (see
Table 2).
From the results of MFA and MCA, it was
found that acetyl-CoA and NADPH are the
most important factors affecting poly(3HB )
production. To identify whether these two
actually affect poly(3HB ) biosynthesis, two
different mutant E. coli strains were examined as host strains for the production of
poly(3HB ). The first strain was TA3516
harboring pJM9131 and containing the R.
eutropha phb operon. In this strain, acetylCoA was expected to be overproduced because of the inactivation of phosphotransacetylase and acetate kinase (Shi et al., 1999).
The production rate of lactic acid decreased
significantly, whilst those of pyruvate and
acetic acid decreased only slightly. However,
Kinetic constants for b-ketothiolase, acetoacetyl-CoA reductase and PHA synthasea
b-Ketothiolase
Keq
V1/V2
Ka
Kb
Kp
4 î 10 5
2.5 î 10 4
3.78 î 10 3 mM
840 mM
31.4 mM
Kq
Kia
Kib
Kip
Kiq
64.6 mM
5.96 mM
841 mM
12.4 mM
1.62 î 10 2 mM
500
5 mM
19 mM
Kp, Kip
Kq, Kiq
16.5 mM
31 mM
Acetoacetyl-CoA reductase
Keq
Ka, Kia
Kb, Kib
PHA synthase
Km
a
720 mM
Data taken from Leaf and Srienc (1998).
257