APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1999, p. 4363–4368 Vol. 65, No.

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0099-2240/99/$04.00⫹0
Copyright © 1999, American Society for Microbiology. All Rights Reserved.

High-Level Production of Poly(3-Hydroxybutyrate-co-3-

Hydroxyvalerate) by Fed-Batch Culture of
Recombinant Escherichia coli
JONG-IL CHOI AND SANG YUP LEE*
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
Received 13 May 1999/Accepted 15 July 1999

Fermentation strategies for production of high concentrations of poly(3-hydroxybutyrate-co-3-hydroxyval-

erate) [P(3HB-co-3HV)] with different 3-hydroxyvalerate (3HV) fractions by recombinant Escherichia coli
harboring the Alcaligenes latus polyhydroxyalkanoate biosynthesis genes were developed. Fed-batch cultures of
recombinant E. coli with the pH-stat feeding strategy facilitated production of high concentrations and high
contents of P(3HB-co-3HV) in a chemically defined medium. When a feeding solution was added in order to
increase the glucose and propionic acid concentrations to 20 g/liter and 20 mM, respectively, after each feeding,
a cell dry weight of 120.3 g/liter and a relatively low P(3HB-co-3HV) content, 42.5 wt%, were obtained.
Accumulation of a high residual concentration of propionic acid in the medium was the reason for the low
P(3HB-co-3HV) content. An acetic acid induction strategy was used to stimulate the uptake and utilization of
propionic acid. When a fed-batch culture and this strategy were used, we obtained a cell concentration, a
P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content, and a 3HV fraction of 141.9 g/liter, 88.1 g/liter, 62.1
wt%, and 15.3 mol%, respectively. When an improved nutrient feeding strategy, acetic acid induction, and oleic
acid supplementation were used, we obtained a cell concentration, a P(3HB-co-3HV) concentration, a P(3HB-
co-3HV) content, and a 3HV fraction of 203.1 g/liter, 158.8 g/liter, 78.2 wt%, and 10.6 mol%, respectively; this
resulted in a high level of productivity, 2.88 g of P(3HB-co-3HV)/liter-h.

Polyhydroxyalkanoates (PHAs) are intracellular carbon and P(3HB) homopolymer, several processes which result in pro-
energy reserve materials that are accumulated by a variety of duction of high concentrations of P(3HB) with a high level of
microorganisms under certain unbalanced growth conditions productivity have been developed (12, 13). In particular, it has
(1, 7, 12, 19, 23). Since PHAs possess thermoplastic or elasto- been shown that recombinant E. coli harboring the heterolo-
meric properties depending on the monomer composition and gous PHA biosynthesis genes has several advantages over wild-
are completely biodegradable when they are disposed, they type PHA producers; these advantages include a wide range
have been considered good candidates for biodegradable poly- of utilizable carbon sources, accumulation of a large amount of
mers (9). Poly(3-hydroxybutyrate) [P(3HB)] is accumulated by P(3HB) with a high level of productivity, and the fragility of
numerous microorganisms and is the best-characterized PHA cells, which allows easy recovery of PHA (8, 12, 14). Recently,
(12, 23). Several bacteria, such as Ralstonia eutropha, Alcali-
we reported that an unprecedentedly high concentration of
genes latus, Azotobacter vinelandii, methylotrophs, and recom-
P(3HB) (141.6 g/liter) and a high level of productivity [4.63 g
binant Escherichia coli harboring the heterologous PHA bio-
synthesis genes, have been employed for efficient production of of P(3HB)/liter-h) could be obtained with a fed-batch culture
P(3HB) (12, 13). However, P(3HB) is a highly crystalline and of recombinant E. coli harboring the A. latus PHA biosynthesis
brittle homopolymer, which restricts its use to a limited range genes (6). Therefore, we decided to determine if P(3HB-co-
of applications (9). Because of this, it has been suggested that 3HV) copolymer could also be produced at a high level of
poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] efficiency by recombinant E. coli harboring the A. latus PHA
is better than P(3HB) because it is more flexible and stronger biosynthesis genes. There have been several reports of produc-
(7, 9). P(3HB-co-3HV) has been produced on a fairly large tion of high concentrations of P(3HB-co-3HV) by wild-type
scale by fed-batch cultures of R. eutropha from glucose and PHA producers, such as R. eutropha (11, 18, 24), A. latus (20),
propionic acid (3). A. vinelandii (17), Alcaligenes sp. (10), and Paracoccus denitri-
A major problem in commercialization of PHAs as substi- ficans (26). The highest level of copolymer P(3HB-co-3HV)
tutes for conventional petrochemical-based polymers is the productivity obtained so far was 2.55 g of PHA/liter-h by a
high production cost of these compounds (3, 4). Much effort fed-batch culture of R. eutropha (11). If P(3HB-co-3HV) can
has been devoted to lowering the production cost of PHAs by be produced by recombinant E. coli with a similar or higher
developing better bacterial strains and more efficient fermen- level of productivity, the other advantages of recombinant E.
tation and economical recovery processes. In the case of the coli described above should reduce the overall cost of produc-
tion of P(3HB-co-3HV).
In this paper, we describe a cultivation strategy for produc-
* Corresponding author. Mailing address: Department of Chemical
tion of a high concentration of P(3HB-co-3HV) with a high
Engineering and BioProcess Engineering Research Center, Korea
Advanced Institute of Science and Technology, 373-1 Kusong-dong, level of productivity. Strategies for producing P(3HB-co-3HV)
Yusong-gu, Taejon 305-701, Korea. Phone: 82-42-869-3930. Fax: 82- with different 3-hydroxyvalerate (3HV) mole fractions are also
42-869-8800. E-mail: [email protected]. described.

4363
4364 CHOI AND LEE APPL. ENVIRON. MICROBIOL.

TABLE 1. Production of P(3HB-co-3HV) by recombinant E. coli XL1-Blue(pJC4) incubated under various conditions at 30°C for 60 h
Cell dry wt PHA concn PHA content 3HV fraction
Culture conditions
(g/liter) (g/liter) (wt%) (mol%)

Glucose (20 g/liter) ⫹ 20 mM propionic acid 4.6 2.6 56.8 7.2

Glucose (20 g/liter) ⫹ 20 mM propionic acid ⫹ acetic acid induction 7.9 5.9 74.8 13.8
Glucose (20 g/liter) ⫹ 20 mM propionic acid ⫹ oleic acid supplementation 5.2 3.2 61.2 19.5
Glucose (20 g/liter) ⫹ 20 mM propionic acid ⫹ acetic acid induction ⫹ oleic 7.5 5.6 74.0 18.1
acid supplementation

MATERIALS AND METHODS PHA content, could be increased by induction with 10 mM

Bacterial strains and plasmid DNA. The E. coli strain used in this study was acetic acid. In the culture without acetic acid induction, the
XL1-Blue (supE44 hsdR17 recA1 endA1 gyrA96 thi relA1 lacF⬘[proAB⫹ lacIq 3HV fraction was increased significantly by adding oleic acid.
lacZ⌬M15 Tn10(tetr)]) (15). Plasmid pJC4 harboring the A. latus PHA biosyn-
thesis genes and the parB locus of plasmid R1 has been described previously (6). A high PHA concentration (5.6 g/liter) and a PHA content of
Culture conditions. Cells were maintained as a 20% (vol/vol) glycerol stock 74.0 wt% with a relatively high 3HV fraction (18.1 mol%)
preparation at ⫺80°C after growth in Luria-Bertani medium. Seed and fed-batch could be obtained by using acetic acid induction and oleic
cultures were grown in chemically defined MR medium (pH 6.9) (25). Separately
sterilized glucose and thiamine were added to MR medium at final concentra-
acid supplementation. Therefore, P(3HB-co-3HV) can be ef-
tions of 20 g/liter and 10 mg/liter, respectively. For production of P(3HB-co- ficiently produced by using acetic acid induction and/or oleic
3HV), propionic acid was used as a cosubstrate in order to provide the precur- acid supplementation with recombinant E. coli harboring the
sors of 3HV monomers (28). For the acetic acid induction experiments, MR A. latus PHA biosynthesis genes.
medium was supplemented with 2 g of tryptone (Difco Laboratories, Detroit,
Mich.) per liter in order to reduce the lag period (28). For the oleic acid P(3HB-co-3HV) production by fed-batch cultures. Based on
supplementation experiments, 1 g of oleic acid (Junsei Chemical Co., Tokyo, the flask culture results, fed-batch cultures of recombinant E.
Japan) per liter was added to MR medium (28). coli XL1-Blue(pJC4) were used to produce P(3HB-co-3HV)
For the fed-batch cultures, seed cultures were prepared by growing cells in a
shaking incubator overnight at 30°C and 250 rpm. For acetic acid induction
with several different nutrient feeding strategies. First, a nutri-
experiments, cells were cultivated in MR medium supplemented with 2 g of ent solution was added so that each dose increased the con-
tryptone per liter, 10 mM acetic acid, and 20 mg of thiamine per liter without centration of propionic acid in the culture by 20 mM. Figure 1
glucose. Fed-batch cultures were grown at 30°C in a 6.6-liter jar fermentor shows the time profiles of cell growth and PHA production.
(Bioflo 3000; New Brunswick Scientific Co., Edison, N.J.) initially containing 1.6
liters of MR medium. The culture pH was controlled at 6.9 except for short The cell concentration, PHA concentration, PHA content, and
periods of nutrient feeding (see below) by adding 28% (vol/vol) ammonia water. 3HV fraction obtained in 56.8 h were 120.3 g/liter, 51.1 g/liter,
The dissolved oxygen concentration was controlled (see below) by automatically 42.5 wt%, and 10 mol%, respectively. The maximum 3HV
changing the agitation speed to 1,000 rpm and adjusting the pure oxygen per-
centage. During the active PHA synthesis phase, the dissolved oxygen concen- fraction in PHA was 13.8 mol% at 32.2 h. Propionic acid
tration was maintained at 1 to 3% of air saturation (25). The feeding solution accumulated continuously in the medium to a concentration of
contained (per liter) 700 g of glucose, 15 g of MgSO4 䡠 7H2O, 250 mg of thiamine, 22.6 g/liter.
and different amounts of propionic acid. The pH-stat feeding strategy was em-
ployed for fed-batch cultures. When the pH rose to a value greater than its
Fed-batch culture with acetic acid induction or oleic acid
setpoint (pH 6.9) by 0.1 pH unit, an appropriate volume of feeding solution was supplementation. It has been shown that the mechanisms for
automatically added in order to increase the glucose concentration in the culture uptake and degradation of propionic acid in E. coli seem to be
medium to 20 g/liter (25). The propionic acid concentration in the culture the same as the mechanisms for uptake and degradation of
medium was increased depending on the ratio of glucose to propionic acid in the
feeding solution. For the fed-batch cultures supplemented with oleic acid, oleic acetic acid (16, 21, 28). Based on the previous findings and the
acid was added so that each dose increased the concentration of oleic acid in the results obtained with flask cultures, the conditions that result in
culture by 1 g/liter. more efficient uptake and utilization of acetic acid (namely,
Analytical procedures. Cell growth was monitored by measuring the absor-
bance at 600 nm with a model DU Series 600 spectrophotometer (Beckman,
induction with acetic acid or oleic acid) were used with fed-
Fullerton, Calif.). The cell concentration, defined as the dry weight of cells per batch cultures. To investigate the effect of acetic acid induc-
liter of culture broth, was determined by weighing dry cells as described previ- tion, cells were first grown on acetic acid until the absorbance
ously (15). PHA concentrations were determined with a gas chromatograph at 600 nm was 0.8. Then pH-stat nutrient feeding was started in
(model HP5890; Hewlett-Packard, Wilmington, Del.) by using n-benzoic acid as
the internal standard (2). The PHA content was defined as the percent ratio of order to increase the glucose and propionic acid concentra-
PHA concentration to cell concentration. tions to 20 g/liter and 20 mM, respectively, after each feeding.
The concentration of propionic acid in the culture medium was measured by Figure 2 shows the time profiles for cell and PHA concentra-
high-performance liquid chromatography by using a model L-3300 RI monitor,
a model L-600 pump, and a model D-2500 chromatointegrator, (all obtained tions, PHA content, and the 3HV fraction during this experi-
from Hitachi, Tokyo, Japan) equipped with an ion-exchange column (type HPX- ment. The cell concentration, PHA concentration, and PHA
87H; 300 by 7.8 mm; Aminex, Hercules, Calif.); 0.01 N H2SO4 was the mobile content obtained in 50.9 h after acetic acid induction were
phase.
141.9 g/liter, 88.1 g/liter, and 62.1 wt%, respectively. The max-
imum 3HV fraction in PHA was 17 mol% at 34.1 h. Therefore,
RESULTS the cell concentration, PHA concentration, and PHA content
Cell growth and P(3HB-co-3HV) accumulation in flask cul- could all be increased by acetic acid induction. Even with acetic
tures. The characteristics of cell growth and P(3HB-co-3HV) acid induction, propionic acid still accumulated, but to a lesser
production were first examined by growing flask cultures of extent (up to 11 g/liter).
recombinant E. coli XL1-Blue(pJC4) under various conditions. Next, a fed-batch culture of recombinant E. coli with oleic
The results are summarized in Table 1. When the recombinant acid supplementation was studied. The final cell and PHA
E. coli XL1-Blue(pJC4) was cultivated in a chemically defined concentrations and PHA content after 53.5 h were 129.6 g/liter,
medium containing 20 g of glucose per liter and 20 mM pro- 54.1 g/liter, and 41.8 wt%, respectively. The 3HV fraction in
pionic acid, the cell dry weight, PHA concentration, and PHA PHA was increased from 10 to 19.3 mol% by oleic acid sup-
content obtained were 4.6 g/liter, 2.6 g/liter, and 56.8 wt%, plementation (time profiles not shown). Again, there was ac-
respectively. The cell and PHA concentrations, as well as the cumulation of propionic acid, but the amount of propionic acid
VOL. 65, 1999 P(3HB-co-3HV) PRODUCTION BY RECOMBINANT E. COLI 4365

78.2 wt%, respectively. The maximum 3HV fraction in PHA

increased from 3.3 to 10.6 mol%.
When cells were cultivated under the same conditions except
that the propionic acid concentration was increased to 10 mM
after each feeding, the cell concentration, PHA concentration,
and PHA content obtained in 52.1 h were 189.1 g/liter, 135.1
g/liter, and 71.4 wt%, respectively (Fig. 4). The maximum 3HV
fraction in PHA was 16.1 mol%. Therefore, the 3HV fraction
could be increased by adding more propionic acid, while the
detrimental effect of a high concentration of propionic acid
could be alleviated by acetic acid induction and/or oleic acid
supplementation. The results of the fed-batch culture experi-
ments are summarized in Table 2.

DISCUSSION
P(3HB-co-3HV) has been considered a better candidate for
producing biodegradable plastic material than P(3HB), be-
cause it is more flexible, stronger, and easier to process. Slater
et al. (22) demonstrated that P(3HB-co-3HV) could be syn-
thesized by a special mutant (atoC fadR) strain of E. coli
LS5218 harboring the R. eutropha PHA biosynthesis genes
(29), because this E. coli strain allowed constitutive expression
of the enzymes involved in utilization of short-chain fatty acids.

FIG. 1. Time profiles for cell dry weight (CDW), PHA concentration, and
residual propionic acid concentration in the medium (A) and PHA content and
3HV fraction in PHA (B) during fed-batch culture of strain XL1-Blue(pJC4).
The feeding solution was added in order to increase the concentrations of
glucose and propionic acid to 20 g/liter and 20 mM, respectively, after each
feeding.

was less than the amount observed in the absence of oleic acid
supplementation or acetic acid induction.
Reduction of propionic acid accumulation. Even though
acetic acid induction and oleic acid supplementation could
enhance propionic acid utilization, propionic acid still accumu-
lated. To reduce the accumulation of propionic acid during
culture, a feeding solution containing a lower propionic acid
concentration was used without acetic acid induction or oleic
acid supplementation. This solution was designed to increase
the propionic acid concentration to 5 mM after each feeding.
The cell concentration, PHA concentration, and PHA content
obtained in 51.9 h were 179.4 g/liter, 134.7 g/liter, and 75.1
wt%, respectively. The maximum fraction of 3HV in PHA was
3.3 mol%. The final residual concentration of propionic acid
was 5.2 g/liter, which is much lower than the concentration
obtained during the three fed-batch culture experiments de-
scribed above (time profiles not shown).
Figure 3 shows the time profiles for cell concentration, PHA
concentration, and PHA content when cells were cultivated
with oleic acid supplementation after acetic acid induction. In FIG. 2. Time profiles for cell dry weight (CDW), PHA concentration, and
this experiment, the nutrient solution was added in order to residual propionic acid concentration in the medium (A) and PHA content and
3HV fraction in PHA (B) during fed-batch culture of XL1-Blue(pJC4) after
increase the propionic acid concentration to 5 mM after each induction with 10 mM acetic acid. The feeding solution was added in order to
feeding. The cell concentration, PHA concentration, and PHA increase the concentrations of glucose and propionic acid to 20 g/liter and 20
content obtained in 55.1 h were 203.1 g/liter, 158.8 g/liter, and mM, respectively, after each feeding.
4366 CHOI AND LEE APPL. ENVIRON. MICROBIOL.

tained was 120.3 g/liter, but the PHA content was rather low
(42.5 wt%). We found that propionic acid accumulated during
incubation of the fed-batch culture. The residual concentration
of propionic acid after 56.8 h was as high as 22.6 g/liter, which
seemed to be the reason for relatively low level of PHA. To
stimulate the uptake of propionic acid, acetic acid induction
experiments were carried out. The cell and PHA concentra-
tions, the PHA content, and PHA productivity all increased
when acetic acid induction was used. The residual concentra-
tion of propionic acid in the medium decreased considerably.
In the fed-batch culture with oleic acid supplementation, the
cell and PHA concentrations increased a little but were lower
than the concentrations obtained with acetic acid induction.
On the other hand, the 3HV fraction increased twofold. On the
basis of these results, we reasoned that oleic acid supplemen-
tation mainly increased the 3HV fraction in PHA. The 3HV
yield on propionic acid was also increased by acetic acid in-
duction and oleic acid supplementation.
However, the high residual concentration of propionic acid
in the medium and the low PHA content were problems that
had to be solved for efficient production of P(3HB-co-3HV) by
recombinant E. coli. In the fed-batch cultures, the feeding
solution containing propionic acid was added when the glucose
in the medium was depleted. Because the uptake rate of pro-
pionic acid seems to be different from the uptake rate of

FIG. 3. Time profiles for cell dry weight (CDW), PHA concentration, and
residual propionic acid concentration in the medium (A) and PHA content and
3HV fraction in PHA (B) during fed-batch culture of XL1-Blue(pJC4) with oleic
acid supplementation after acetic acid induction. The feeding solution was added
in order to increase the concentrations of glucose and propionic acid to 20 g/liter
and 5 mM, respectively, after each feeding.

However, E. coli LS5218 did not grow to a high cell density, nor
did it accumulate much polymer (27). We examined other E.
coli strains to determine whether they produced P(3HB-co-
3HV) more efficiently (28). Non-atoC fadR E. coli strains har-
boring the R. eutropha PHA biosynthesis genes accumulated
P(3HB-co-3HV) with 3HV fractions as high as 33 mol% from
glucose and propionic acid in flask cultures.
In this study, we investigated strategies for production of
P(3HB-co-3HV) by a high-cell-density culture of non-atoC
fadR recombinant E. coli harboring the A. latus PHA biosyn-
thesis genes with different 3HV fractions. Recombinant E. coli
harboring the A. latus PHA biosynthesis genes produced a
large amount of P(3HB) with a higher level of productivity
than recombinant E. coli harboring the R. eutropha PHA bio-
synthesis genes (6). In a flask culture used for production of
P(3HB-co-3HV), the final cell and PHA concentrations ob-
tained with recombinant E. coli XL1-Blue(pJC4) harboring the
A. latus PHA biosynthesis genes were higher than the final
concentrations obtained with recombinant E. coli XL1-Blue
(pSYL105) harboring the R. eutropha PHA biosynthesis genes.
When we used the fed-batch culture containing recombinant FIG. 4. Time profiles for cell dry weight (CDW), PHA concentration, and
E. coli XL1-Blue(pJC4) harboring the A. latus PHA biosynthe- residual propionic acid concentration in the medium (A) and PHA content and
3HV fraction in PHA (B) during fed-batch culture of XL1-Blue(pJC4) with oleic
sis genes and the feeding strategy that increased the glucose acid supplementation after acetic acid induction. The feeding solution was added
and propionic acid concentrations to 20 g/liter and 20 mM, in order to increase the concentrations of glucose and propionic acid to 20 g/liter
respectively, after each feeding, the cell concentration ob- and 10 mM, respectively, after each feeding.
VOL. 65, 1999 P(3HB-co-3HV) PRODUCTION BY RECOMBINANT E. COLI 4367

TABLE 2. Summary of P(3HB-co-3HV) production by fed-batch cultures of recombinant E. coli under various conditions
3HV yield Productivity
Fermentation Cell dry wt PHA concn PHA content 3HV fraction
Culture conditions (g of 3HV/g of (g of PHA/
time (h) (g/liter) (g/liter) (wt%) (mol%)
propionic acid) liter-h)

Glucose added to a concn of 20 g/liter and 56.8 120.3 51.1 42.5 10 0.23 0.90
propionic acid added to a concn of 20
mM after each feeding
Glucose added to a concn of 20 g/liter and 50.9 141.9 88.1 62.1 15.3 0.34 1.73
propionic acid added to a concn of 20
mM after each feeding with acetic acid
induction
Glucose added to a concn of 20 g/liter and 53.5 129.6 54.1 41.8 19.3 0.35 1.01
propionic acid added to a concn of 20
mM after each feeding with oleic acid
supplementation
Glucose added to a concn of 20 g/liter and 51.9 179.4 134.7 75.1 3.3 0.33 2.60
propionic acid added to a concn of 5
mM after each feeding
Glucose added to a concn of 20 g/liter and 55.1 203.1 158.8 78.2 10.6 0.47 2.88
propionic acid added to a concn of 5
mM after each feeding with acetic acid
induction and oleic acid
supplementation
Glucose added to a concn of 20 g/liter and 52.1 189.1 135.1 71.4 14.8 0.43 2.59
propionic acid added to a concn of 10
mM after each feeding with acetic acid
induction and oleic acid
supplementation

glucose, propionic acid accumulates if its concentration is not with a fed-batch culture of recombinant E. coli harboring the
optimized. To decrease the level of propionic acid in the me- A. latus PHA biosynthesis genes.
dium, we performed fed-batch culture experiments with differ- In order to compare the processes for production of P(3HB-
ent feeding solutions containing lower concentrations of pro- co-3HV) by recombinant E. coli with other processes in which
pionic acid. When the feeding solution was added in order to wild-type organisms are used, an economic evaluation of the
increase the propionic acid concentration to 5 mM, the con- processes was carried out by using the method described pre-
centration of PHA and the PHA content increased and pro- viously (4). In this evaluation, a P(3HB-co-3HV) production
ductivity was higher, while the residual concentration of pro- scale of 100,000 metric tons per year was used. According to
pionic acid was much lower. With acetic acid induction and the economic evaluation of the process for production of PHA
oleic acid supplementation, the PHA concentration and the having a 3HV fraction of 14.3 mol% by R. eutropha, the PHA
PHA content increased to 158.8 g/liter and 78.2 wt%, respec- production cost was as high as $9.75/kg of PHA when the
tively. The productivity was as high as 2.88 g of P(3HB-co- recovery method involved surfactant-hypochlorite digestion.
3HV)/liter-h. When the feeding solution was added in order to For the process described here in which recombinant E. coli
increase the propionic acid concentration to 10 mM with acetic was used, the PHA production cost with a 3HV fraction of 10.6
acid induction and oleic acid supplementation, the concentra- mol% was only $5.05/kg of PHA when the same recovery
tion of PHA and the PHA content decreased slightly compared method was used. Furthermore, when PHA was recovered
to the values obtained when 5 mM propionic acid feeding was from E. coli cells by the simple NaOH digestion method de-
used. However, the final 3HV fraction was higher (14.8 mol%). scribed recently (5), the PHA production cost was $3.95/kg of
On the basis of these results, we concluded that a high con- PHA.
centration of PHA and a high PHA content could be obtained In conclusion, P(3HB-co-3HV) having a 3HV fraction of 3
by using a feeding solution with a low concentration of propi- to 20 mol% could be efficiently produced by recombinant E.
onic acid, but the 3HV fraction in PHA was low. Table 2 also coli containing the A. latus PHA biosynthesis genes by simply
shows that when the feeding solution containing a low propi- varying the propionic acid concentration in the feeding solu-
onic acid concentration was added, the 3HV yield on propionic tion. Our results along with other advantages of employing
acid increased. recombinant E. coli described previously, should make recom-
Prior to this study, it was reported that the highest concen- binant E. coli a good candidate for production of PHA. Re-
tration of P(3HB-co-3HV), a PHA content, and a 3HV frac- cently, research on production of PHA by a transgenic plant
tion were 117 g/liter, 74 wt%, and 4.3 mol%, respectively, when has been carried out. However, the concentration and yield of
a fed-batch culture of R. eutropha was used and that the highest PHA should be increased in order to reduce the PHA produc-
level of PHA productivity was 2.55 g of P(3HB-co-3HV)/liter-h tion cost to a value close to the cost of starch production.
(11). However, when the 3HV fraction in the PHA increased Therefore, PHA production will rely on efficient bacterial fer-
to 14.3 mol%, the cell and PHA concentrations and the PHA mentation until the early 21st century, and recombinant E. coli
content decreased to 129 g/liter, 74 g/liter, and 57 wt%, respec- will play an important role in this production.
tively, which resulted in a level of productivity of 1.67 g of
P(3HB-co-3HV)/liter-h. The results reported in this paper ACKNOWLEDGMENTS
show that a higher concentration of PHA and a higher PHA This work was supported by the Ministry of Science and Technology
content with a relatively high 3HV fraction can be obtained and by LG Chemicals, Ltd.
4368 CHOI AND LEE APPL. ENVIRON. MICROBIOL.

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