APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1997, p. 4765–4769 Vol. 63, No.

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0099-2240/97/$04.0010
Copyright © 1997, American Society for Microbiology

Production of Poly(3-Hydroxybutyrate) by Fed-Batch Culture of

Filamentation-Suppressed Recombinant Escherichia coli
FULAI WANG AND SANG YUP LEE*
Department of Chemical Engineering and BioProcess Engineering Research Center,
Korea Advanced Institute of Science and Technology, Yusong-gu,
Taejon 305-701, Korea
Received 30 June 1997/Accepted 2 October 1997

Recombinant Escherichia coli XL1-Blue harboring a high-copy-number plasmid containing the Alcaligenes
eutrophus polyhydroxyalkanoate synthesis genes could efficiently synthesize poly(3-hydroxybutyrate) (PHB) in
a complex medium containing yeast extract and tryptone but not in a defined medium. One of the reasons for
the reduced PHB production in a defined medium was thought to be severe filamentation of cells in this
medium. By overexpressing an essential cell division protein, FtsZ, in recombinant E. coli producing PHB,
filamentation could be suppressed and PHB could be efficiently produced in a defined medium. A high PHB
concentration of 149 g/liter, with high productivity of 3.4 g of PHB/liter/h, could be obtained by the pH-stat
fed-batch culture of the filamentation-suppressed recombinant E. coli in a defined medium. It was also found
that insufficient oxygen supply at a dissolved oxygen concentration (DOC) of 1 to 3% of air saturation during
active PHB synthesis phase did not negatively affect PHB production. By growing cells to the concentration of
110 g/liter and then controlling the DOC in the range of 1 to 3% of air saturation, a PHB concentration of 157
g/liter and PHB productivity of 3.2 g of PHB/liter/h were obtained. For the scale-up studies, fed-batch culture
was carried out in a 50-liter stirred tank fermentor, in which the DOC decreased to zero when cell concen-
tration reached 50 g/liter. However, a relatively high PHB concentration of 101 g/liter and PHB productivity of
2.8 g of PHB/liter/h could still be obtained, which demonstrated the possibility of industrial production of PHB
in a defined medium by employing the filamentation-suppressed recombinant E. coli.

Biodegradable plastics have been drawing much attention 14). A high concentration of PHB (81 g/liter) could be pro-
due to the environmental problems caused by the accumula- duced with a high productivity of 2.1 g of PHB/liter/h by the
tion of nondegradable plastic wastes. Polyhydroxyalkanoates pH-stat fed-batch culture of recombinant E. coli XL1-Blue
(PHAs), intracellularly accumulated by various microorgan- harboring pSYL104 in a complex medium supplemented with
isms as carbon and energy storage materials, have been con- a large amount of yeast extract and tryptone (14). However, it
sidered to be strong candidates for biodegradable polymer is not desirable to use a large amount of expensive yeast extract
material (1, 6, 17). A major drawback to the commercialization and tryptone for the production of PHB, which is to be used as
of PHAs is their much higher production cost compared with an inexpensive bulk product. With the aim of lowering the
petrochemical-based synthetic plastic materials or other bio- production cost of PHB, fed-batch cultures were carried out in
degradable polymers such as poly(lactide) (7). With the aim of a chemically defined medium. Unfortunately, only less than
lowering the production cost of PHAs, various fermentation 25 g of PHB/liter could be produced by fed-batch culture of
processes employing several different bacteria have been de- XL1-Blue(pSYL104) in a defined medium (11). One of the
veloped to improve the production of PHAs (7, 13). Alcaligenes reasons for the reduced accumulation of PHB in a defined
eutrophus has been the most widely employed bacterium for medium was filamentation of cells accumulating PHB (15). It
the production of PHAs since it can accumulate a large was observed that cells became considerably elongated (up to
amount of PHAs in a simple medium (4). Poly(3-hydroxybu- 150 mm in length) in flask and fed-batch cultures (11, 15), and
tyrate) (PHB) is a homopolymer of 3-hydroxybutyrate and is the extent of filamentation was much more severe in a defined
the most widespread and best-characterized member of the medium (5). Cell filamentation was due to the inactivation of
PHAs. In A. eutrophus PHB is synthesized from acetyl coen- an essential cell division protein, FtsZ, and could be sup-
zyme A (acetyl-CoA) by three sequential enzymatic reactions pressed by amplifying the activity of FtsZ (5). More interest-
(1). Two acetyl-CoA moieties are condensed to acetoacetyl- ingly, in flask culture, recombinant E. coli with amplified FtsZ
CoA by b-ketothiolase. An NADPH-dependent reductase con- activity could more efficiently synthesize PHB in a defined
verts acetoacetyl-CoA to D-(2)-3-hydroxybutyryl-CoA, which medium compared with cells that underwent filamentation (5).
is subsequently added to the growing chain of PHB by the PHA In this study fed-batch cultures of filamentation-suppressed
synthase. The genes coding for these three enzymes were recombinant E. coli were carried out for the production of
cloned in Escherichia coli and shown to form an operon (16, 18, PHB in a defined medium. A cultivation strategy that allowed
19). We constructed the stable high-copy-number plasmid a high PHB productivity of greater than 3 g of PHB/liter/h was
pSYL104 containing the A. eutrophus PHA synthesis genes (6, developed. It was also demonstrated that PHB could be effi-
ciently produced in a pilot-scale fermentor by using this strat-
egy.
* Corresponding author. Mailing address: Department of Chemical
Engineering and BioProcess Engineering Research Center, Korea Ad-
vanced Institute of Science and Technology, 373-1 Kusong-dong, MATERIALS AND METHODS
Yusong-gu, Taejon 305-701, Korea. Fax: 42-869-8800. E-mail: leesy Microorganism and plasmid. The E. coli strain used in this study was XL1-
@sorak.kaist.ac.kr. Blue (recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac[F9 proAB, lacIqZDM15]

4765
4766 WANG AND LEE APPL. ENVIRON. MICROBIOL.

(2). Residual cell concentration was defined as cell concentration minus PHB
concentration. PHB content was defined as the percentage of the ratio of the
amount of PHB to the amount (dry weight) of cells. The PHB synthesis rate was
defined as grams of PHB synthesized per gram of residual cells per hour.

RESULTS
Fed-batch culture in a 6.6-liter fermentor. The pH-stat fed-
batch culture of XL1-Blue(pSYL107) was first carried out in
a laboratory-scale fermentor. The time profiles of cell growth,
PHB accumulation, and DOC are shown in Fig. 1. The final
cell concentration, PHB concentration, and PHB content ob-
tained in 44 h were 206 g/liter, 149.7 g/liter, and 73%, respec-
tively, which resulted in a PHB productivity of 3.4 g of PHB/
liter/h. For convenience, the fermentation process could be
divided into two phases, an active growth phase and an active
PHB synthesis phase. Before cell concentration reached 60.2
g/liter at 15 h, the PHB content was kept nearly constant at ca.
16% (active cell growth phase). Afterwards, it steadily in-
creased to 73% (active polymer synthesis phase). The DOC
could not be maintained above 12% of air saturation when the
FIG. 1. Time profiles of cell concentration, PHB concentration, residual cell cell concentration reached 97 g/liter at 18 h. From 18 h to the
concentration, PHB content, and DOC during the fed-batch culture of recom- end of the cultivation (44 h), the DOC fluctuated in the range
binant E. coli XL1-Blue(pSYL107) in a chemically defined medium. The DOC
was maintained above 7% of air saturation. of 7 to 12% of air saturation. At the end of cultivation, a total
of 4.9 liters of cell broth was harvested. A total amount of 734 g
of PHB (without considering the PHB in samples) was pro-
duced from 2,692 g of glucose (including the glucose in the
Tn10 [Tetr]). The plasmid pSYL107 containing the A. eutrophus PHA biosyn-
thesis genes and the E. coli ftsZ gene has been previously described (5). initial medium and seed culture), resulting in a yield of 0.27 g
Culture conditions. Cells were maintained as a 20% (vol/vol) glycerol stock at of PHB/g of glucose.
280°C after growing in Luria-Bertani medium. Seed and fed-batch cultures were Effect of insufficient oxygen supply on PHB synthesis. The
carried out in a chemically defined MR medium which was modified from the effect of insufficient oxygen supply on PHB production was
previously described R medium (9). The MR medium (pH 6.9) contained the
following per liter: KH2PO4, 22 g; (NH4)2HPO4, 3 g; MgSO4 z 7H2O, 0.7 g; citric investigated in fed-batch cultures. Oxygen limitation of main-
acid, 0.8 g; trace metal solution, 5 ml. The trace metal solution contained the taining the DOC at 1 to 3% of air saturation was applied
following per liter of 5 M HCl: FeSO4 z 7H2O, 10.0 g; CaCl2, 2.0 g; ZnSO4 z during two different phases of the fermentation as described
7H2O, 2.2 g; MnSO4 z 4H2O, 0.5 g; CuSO4 z 5H2O, 1.0 g; (NH4)6Mo7O24 z 4H2O, earlier. First, oxygen limitation was applied at a low cell con-
0.1 g; Na2B4O7 z 10H2O, 0.02 g. Separately sterilized glucose and thiamine
supplemented the medium to final concentrations of 20 g/liter and 10 mg/liter, centration of 8.3 g/liter (active cell growth phase). As shown in
respectively. Fig. 2, cell growth stopped after 1.5 h of oxygen limitation.
Seed cultures were prepared in flasks and incubated in a rotary shaker over- PHB concentration and PHB content increased only from 1.3
night at 30°C and 250 rpm. Laboratory-scale fed-batch culture was carried out at to 3.0 g/liter and from 15 to 28%, respectively. Therefore, it
30°C in a jar fermentor (6.6 liters) (Bioflo 3000; New Brunswick Scientific Co.,
Edison, N.J.) containing 1.2 liters of MR medium. Culture pH was controlled at was concluded that oxygen limitation during the active growth
6.9 by the addition of 28% (vol/vol) ammonia-water. The dissolved oxygen
concentration (DOC) was controlled as desired by automatic control of the
agitation speed (up to 1,000 rpm) and the pure oxygen percentage. Antifoam
(0.02% [vol/vol]) (antifoam 289; Sigma Chemical Co., St. Louis, Mo.) was added
at the onset of cultivation. The feeding solution used for the fed-batch culture
contained the following per liter: glucose, 700 g; MgSO4 z 7H2O, 15 g; thiamine,
250 mg. The pH-stat feeding strategy, which was based on the sharp pH rise upon
carbon source depletion, was employed in fed-batch cultures. When the pH rose
to a value greater than its set point (6.9) by 0.1, an appropriate volume of feeding
solution was automatically added to increase the glucose concentration in the
culture medium to 20 g/liter. The feeding volume was calculated on-line by using
fermentation software (AFS3.42; New Brunswick Scientific Co.).
For the scale-up studies in a 50-liter stirred tank fermentor (Korea Fermentor
Company, Inchon, Korea), seed culture (4 liters) was prepared in a 6.6-liter
fermentor by batch culture. When glucose was depleted, as indicated by a sharp
increase of the pH, cell broth was immediately transferred to a 50-liter fermentor
containing 12 liters of MR medium. The culture conditions were similar to those
described above except for some modifications in the feeding strategy and the
DOC control scheme to be suitable for the 50-liter fermentor. Since the 50-liter
fermentor was not interfaced to a computer, a fixed feeding volume (0.5 liters per
pulse) was added upon the increase of the pH. The DOC was maintained above
20% of air saturation during the first 15 h by varying the air flow rate at the fixed
agitation speed (500 rpm, the maximum agitation speed of this fermentor). When
the DOC could not be maintained above 20% of air saturation, oxygen-enriched
air (83% O2) generated by an oxygen generator (Korea Energy Industry Co.,
Uiwang, Korea) was provided. When the DOC could not be maintained above
20% of air saturation even after the oxygen-enriched air supply was added,
cultivation was simply continued at this maximum level of oxygen supply.
Analytical procedures. Growth was monitored by measuring the optical den- FIG. 2. Time profiles of cell concentration, PHB concentration, residual cell
sity at 600 nm. Cell concentration, defined as the amount (dry weight) of cells per concentration, PHB content, and DOC during the fed-batch culture of recom-
liter of culture broth, was determined by weighing dry cells as described previ- binant E. coli XL1-Blue(pSYL107) in a chemically defined medium. The DOC
ously (14). PHB concentration was determined by gas chromatography (HP5890; was controlled in the range of 1 to 3% of air saturation during the active cell
Hewlett-Packard, Wilmington, Del.) with n-butyric acid as an internal standard growth phase.
VOL. 63, 1997 PHB PRODUCTION BY RECOMBINANT ESCHERICHIA COLI 4767

PHB yield was 0.28 g of PHB/g of glucose (3,849 g of PHB

produced from 13,900 g of glucose).

DISCUSSION

Even though PHAs have been considered to be good can-

didates for completely biodegradable plastic or elastomer ma-
terial, the high production cost has hampered their use in a
wide range of applications. Several fermentation processes em-
ploying A. eutrophus, Alcaligenes latus, methylotrophs, and re-
combinant E. coli have been developed for more economical
production of PHB (6, 7, 13). A PHB productivity of 2 to 3 g
of PHB/liter/h has been obtained by employing A. eutrophus
and recombinant E. coli. Computer-aided process analysis and
economic evaluation for PHB production by bacterial fermen-
tation suggested that obtaining high PHB productivity and high
PHB content from inexpensive medium was important for re-
ducing the production cost of PHB (3). Among the several
bacteria that have been shown to produce PHB efficiently,
recombinant E. coli harboring the A. eutrophus PHA biosyn-
FIG. 3. Time profiles of cell concentration, PHB concentration, residual cell thesis genes has been considered to be a strong candidate as a
concentration, PHB content, and DOC during the fed-batch culture of recom- PHB producer due to several advantages such as a wide range
binant E. coli XL1-Blue(pSYL107) in a chemically defined medium. The DOC
was controlled in the range of 1 to 3% air saturation during the active PHB of utilizable carbon sources, easy recovery of PHB, and no
synthesis phase. degradation of PHB during fermentation due to the lack of
intracellular depolymerases (12). However, a high concentra-
tion of PHB (greater than 80 g/liter) with a high PHB produc-
tivity (greater than 2 g of PHB/liter/h) could be obtained only
in a complex or semi-defined medium (11, 12, 14). One of the
phase was detrimental not only to cell growth but also to PHB several reasons for the reduced synthesis of PHB in a defined
synthesis. medium was filamentation of cells accumulating PHB (5, 15).
Figure 3 shows the results obtained by applying oxygen lim- We have demonstrated in flask cultures that cell filamentation
itation during the active PHB synthesis phase. Active PHB could be suppressed by overexpressing FtsZ, an essential cell
synthesis began at 21 h when cell concentration reached 75.6 division protein (5). Furthermore, this filamentation-sup-
g/liter. The DOC was controlled in the range of 1 to 3% of air pressed recombinant E. coli XL1-Blue(pSYL107) could accu-
saturation after cell concentration reached 109.8 g/liter at 24 h. mulate more PHB in a defined medium in flask cultures (5).
Cell concentration, PHB concentration, and PHB content ob- Therefore, fed-batch cultures of XL1-Blue(pSYL107) were
tained at 49 h were 204.3 g/liter, 157.1 g/liter, and 77%, re- carried out to examine if PHB could be efficiently produced in
spectively, resulting in a PHB productivity of 3.2 g of PHB/ a defined medium. As shown in Fig. 1, an even higher concen-
liter/h. These results suggest that application of oxygen tration of PHB (149 g/liter) and a higher PHB productivity (3.4
limitation during the active PHB synthesis phase did not neg-
atively affect the production of PHB by this recombinant E. coli
in a defined medium.
PHB production in a pilot-scale fermentor. To examine if
the cultivation strategy described above could be applied to the
production of PHB in a large-scale fermentor, in which oxygen
transfer is generally poor, fed-batch culture of XL1-Blue
(pSYL107) was carried out in a 50-liter fermentor (Fig. 4).
Oxygen-enriched air generated by the pressure swing adsorp-
tion equipment, instead of pure oxygen, was used during the
fermentation. In this fermentation, the DOC was not deliber-
ately lowered but rather decreased to zero due to the oxygen
transfer limitation of the fermentor itself (maximum agitation
speed and oxygen-enriched air flow rate of 500 rpm and 5
liters/min, respectively). During the cultivation the switch from
the active growth phase to the active PHB synthesis phase
occurred at 12 h (cell concentration of 36.6 g/liter). The DOC
dropped to zero when cell concentration reached 54.8 g/liter at
15 h. However, a detrimental effect of oxygen limitation was
not observed. Cell and PHB concentrations obtained in 36 h
were 153.7 and 101.3 g/liter, respectively, resulting in a PHB
productivity of 2.8 g of PHB/liter/h. Therefore, it was con-
cluded that the filamentation-suppressed recombinant E. coli
could be employed for the efficient production of PHB in a FIG. 4. Time profiles of cell concentration, PHB concentration, residual cell
concentration, PHB content, and DOC during the fed-batch culture of recom-
defined medium in a large-scale fermentor. A total of 19.4 binant E. coli XL1-Blue(pSYL107) in a 50-liter stirred tank fermentor in a
liters of feeding solution was consumed, and 38 liters of cell chemically defined medium. The DOC dropped to zero when cell concentration
broth was harvested at the end of cultivation. Therefore, the reached 54.8 g/liter at 15 h.
4768 WANG AND LEE APPL. ENVIRON. MICROBIOL.

g of PHB/liter/h) could be obtained in a defined medium com- the time profiles of the residual cell concentrations in defined
pared with a complex or semi-defined medium. No significant (Fig. 1 and 3), complex (11), and semi-defined (10) media
cell filamentation was observed during the entire fermentation. suggests that the higher PHB concentration and productivity
Having achieved the successful production of PHB to a high obtained in a defined medium were due to the higher residual
concentration with high content and high productivity, we next cell concentration before cells entered the active PHB synthe-
examined the effects of applying oxygen limitation on cell sis phase. In a complex or semi-defined medium, the typical
growth and PHB production for two purposes. First, it may be residual cell concentration at the switch from active growth
possible to enhance the PHB synthesis rate by increasing the phase to active PHB synthesis phase was about 30 g/liter (10,
acetyl-CoA flux into the PHB biosynthetic pathway and reduc- 11), while that in a defined medium was greater than 60 g/liter
ing its flux into the tricarboxylic acid cycle. Second, it is difficult (Fig. 1 and 3). Since PHB is accumulated in the cytoplasm, the
to maintain the DOC above 10% of air saturation at high cell residual cell concentration determines how much PHA can
densities without using pure oxygen. Since oxygen transfer is potentially be accumulated. However, we cannot increase the
even worse in a large-scale fermentor and the use of a large residual cell concentration before the active PHA synthesis
amount of pure oxygen can be economically unfavorable, it will phase to too high a value because, as was shown previously,
be beneficial if PHB can be efficiently produced under oxygen- such a concentration results in incomplete PHB synthesis and,
limited conditions. subsequently, a low PHB content (7). The residual cell con-
The fed-batch process of fermentation with recombinant centration of 60 g/liter was found to be optimal for the pro-
E. coli can be conveniently divided into two phases: (i) an duction of PHB by this recombinant E. coli strain in a defined
active growth phase during which PHB content is kept rela- medium.
tively constant at a low level (15 to 20%) and (ii) an active PHB Even though recombinant E. coli has been suggested to have
synthesis phase during which PHB is actively accumulated with several advantages as a PHB producer, there were two prob-
a concomitant increase of PHB content. When oxygen limita- lems to be solved: the requirement for complex nitrogen
tion was applied during the active growth phase, cell growth sources and a high oxygen demand. In this study we demon-
stopped and no apparent enhancement of PHB accumulation strated that PHB could be efficiently produced in a defined
was observed (Fig. 2). However, cell growth and PHB produc- medium by the filamentation-suppressed recombinant E. coli
tion were not hampered when oxygen limitation was applied strain. We also solved the problem of high oxygen demand by
during the active PHB synthesis phase (the final PHB concen- demonstrating that PHB could be produced to a high concen-
tration and PHB productivity were as high as 157.1 g/liter and tration with high productivity by applying oxygen limitation
3.2 g of PHB/liter/h, respectively). A major reason for this during the active PHA synthesis phase. However, there is one
difference may have been related to acetate accumulation. new problem observed, i.e., a slightly lower PHB yield on
Oxygen limitation applied during the active growth phase re- glucose in a defined medium. The PHB yield obtained in this
sulted in accumulation of acetate to a final concentration of 14 study was 0.27 to 0.28 g of PHB/g of glucose, which is lower
g/liter, which is higher than the critical acetate concentration than that obtained in a complex medium (0.37 g of PHB/g of
that shows growth inhibition (8). The acetate concentration glucose) (12). It seems that more glucose was wasted to carbon
was less than 4 g/liter when oxygen limitation was applied dioxide in a defined medium. To investigate the possibility of
during the active PHB synthesis phase. The actual reason for improving the PHB yield, we are now carrying out fed-batch
these findings is not clear at this moment. Nevertheless, it is cultures under various conditions while monitoring the carbon
nice from a process point of view to find that oxygen limitation dioxide evolution rate by using an on-line mass spectrometer.
could enhance PHB production by filamentation-suppressed
recombinant E. coli in a defined medium. To examine if this ACKNOWLEDGMENTS
strategy could be employed for the production of PHB in a We thank the BioProcess Engineering Research Center for the
large-scale fermentor, fed-batch culture was carried out in a partial support of Fulai Wang. This work was also supported by the
50-liter pilot-scale fermentor. This fermentor is a typical Ministry of Science and Technology and by LG Chemicals, Ltd.
stirred tank bioreactor equipped with three bottom-driven
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