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Applied and Environmental Microbiology, December 1998, p. 4897-4903, Vol. 64, No. 12
Department of Chemical
Engineering1 and
BioProcess Engineering
Research Center,
Received 23 July 1998/Accepted 30 September 1998
Polyhydroxyalkanoates (PHAs) are microbial polyesters that can be
used as completely biodegradable polymers, but the high production cost
prevents their use in a wide range of applications. Recombinant
Escherichia coli strains harboring the Ralstonia
eutropha PHA biosynthesis genes have been reported to have
several advantages as PHA producers compared with wild-type
PHA-producing bacteria. However, the PHA productivity (amount of PHA
produced per unit volume per unit time) obtained with these recombinant
E. coli strains has been lower than that obtained with the
wild-type bacterium Alcaligenes latus. To endow the
potentially superior PHA biosynthetic machinery to E. coli,
we cloned the PHA biosynthesis genes from A. latus. The
three PHA biosynthesis genes formed an operon with the order PHA
synthase, Recently, problems concerning the
global environment have created much interest in the development of
biodegradable polymers. Polyhydroxyalkanoates (PHAs) are polyesters of
hydroxyalkanoates that are synthesized and intracellularly accumulated
as an energy and/or carbon storage material by numerous microorganisms
(1, 5, 15, 28). PHAs are considered to be good candidates
for biodegradable plastics and elastomers since they possess material properties similar to those of synthetic polymers currently in use and
are completely biodegradable after disposal (9).
A major problem in the commercialization of PHAs in a wide range of
applications is their high production cost (3, 4). Much
effort has been devoted to lowering the production cost by developing
more efficient fermentation and recovery processes (15, 16,
28). Poly(3-hydroxybutyrate) (PHB) is the best known member of
the PHAs and has been studied most often as a model product in the
development of fermentation strategies.
To understand the mechanisms of PHA biosynthesis, studies on the
metabolic pathways for PHA biosynthesis and molecular analyses of PHA
biosynthesis genes in various bacteria have been conducted. In
Ralstonia eutropha (formerly known as Alcaligenes
eutrophus), acetyl coenzyme A is converted to PHB in three
enzymatic steps (1, 35). A biosynthetic One of the major factors affecting the overall production cost is
productivity, defined as the amount of PHB produced per unit volume per
unit time. R. eutropha and Alcaligenes latus have been used most often for the production of PHB, since PHB could be
produced to a high concentration with high productivity (29, 39). Recombinant Escherichia coli strains harboring
the R. eutropha PHA biosynthesis genes have also been used
for the production of PHB (19, 22). A PHB concentration of
as high as 157 g/liter could be obtained with a pH-stat fed-batch
culture (40). Recombinant E. coli has been
considered a strong candidate as a PHB producer due to several
advantages over wild-type PHB producers, such as a wide range of
utilizable carbon sources, PHB accumulation to a high content (up to
90% of cell dry weight), fragility of cells allowing easy recovery of
PHB, and no degradation of PHB during fermentation due to the lack of
intracellular depolymerases (6, 17). However, the highest
productivity obtained with recombinant E. coli was 3.4 g of
PHB/liter/h (40), considerably lower than that obtained with
A. latus (4.94 g of PHB/liter/h) (39). Since A. latus is able to produce PHB with the highest
productivity reported to date, it is reasonable to assume that this
bacterium possesses more efficient PHA biosynthesis enzymes. It was
therefore thought that recombinant E. coli harboring the
A. latus PHA biosynthesis genes might produce PHB with a
higher productivity without a loss of the advantages mentioned earlier.
In this study, we report the cloning and molecular analysis of the
A. latus PHA biosynthesis genes in E. coli. We
also report the development of several different recombinant E. coli strains harboring these genes, the characteristics of these
strains with regard to growth and PHB production, and the results
obtained from high-cell-density fed-batch cultures. There was a 36%
improvement in PHB productivity (from 3.4 to 4.63 g/liter/h) by the
newly developed recombinant E. coli strains, a result which
will make it possible to economically produce this biodegradable polymer.
Bacterial strains, plasmids, and growth conditions.
The
bacterial strains and plasmids used in this study are listed in Table
1. A. latus was cultivated in
nutrient broth medium (Difco Laboratories, Detroit, Mich.) at 30°C.
E. coli was routinely grown in Luria-Bertani (LB) medium at
37°C. LB medium supplemented with 20 g of glucose per liter was
used as a PHB accumulation medium. When required, ampicillin (50 mg/liter) was added to the medium.
DNA manipulation and library construction.
Total genomic DNA
of A. latus was isolated by the procedure described by
Marmur (25). A plasmid library of A. latus total DNA was constructed by inserting A. latus genomic DNA
fragments partially digested with Sau3AI into
BamHI-digested pUC19, followed by transformation into
E. coli XL1-Blue. Plasmid DNA isolation, agarose gel
electrophoresis, and transformation of E. coli were carried
out as described by Sambrook et al. (30). Restriction enzymes and modifying enzymes were purchased from New England Biolabs,
Beverly, Mass., and were used as recommended by the manufacturer.
DNA sequence analysis.
DNA sequencing was carried out by the
site-sequencing method with an ABI model 377 automated DNA sequencer
(The Perkin-Elmer Corp.). Computer analysis of the resulting nucleotide
sequence was performed with the DNASIS DNA and protein sequence
analysis program (Hitachi Software Engineering Co., Yokohama, Japan).
Culture conditions for the production of PHB.
For flask and
fed-batch cultures of recombinant E. coli, chemically
defined R (14) or MR (40) medium was used.
Separately sterilized glucose and thiamine were used as supplements at
final concentrations of 20 g/liter and 10 mg/liter, respectively.
Fed-batch cultures were incubated at 30°C in a 6.6-liter jar
fermentor (Bioflo 3000; New Brunswick Scientific Co., Edison, N.J.)
containing 1.2 liters of MR medium. The culture pH was kept at 6.9 by
the addition of 28% (vol/vol) ammonia water. Antifoam 289 (0.02%
[vol/vol]; Sigma Chemical Co., St. Louis, Mo.) was added at the onset
of cultivation. The feeding solution used for the fed-batch culture contained, per liter, the following: glucose, 700 g;
MgSO4 · 7H2O, 15 g; and thiamine,
250 mg. The pH-stat feeding strategy was used for 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 solution volume was calculated on-line with fermentation
software (AFS3.42; New Brunswick Scientific Co.).
Analytical procedures.
Growth was monitored by measuring the
optical density at 600 nm. Cell concentration, defined as cell dry
weight per liter of culture broth, was determined by weighing dry cells
as described previously (40). The PHB concentration was
determined by gas chromatography (HP5890; Hewlett-Packard, Wilmington,
Del.) with benzoic acid as an internal standard (2). PHB
content was defined as the ratio of PHB to cell dry weight and
expressed as a percentage.
Nucleotide sequence accession number.
The nucleotide
sequence data reported here will appear in the GenBank nucleotide
sequence database under accession no. AF078795.
Cloning and molecular analysis of the A. latus PHA
biosynthesis genes.
To clone the A. latus PHA
biosynthesis genes, we used the screening strategy of conferring the
ability to accumulate PHB to E. coli by an introduced
recombinant plasmid. The transformed E. coli cells were
replica plated on solid PHB accumulation medium. Because
PHB-accumulating cells form opaque colonies, the E. coli transformants harboring all of the A. latus PHA biosynthesis
genes will show a turbid colony phenotype if the genes are functionally expressed. The opaque colonies were isolated and separately cultivated in LB medium containing 20 g of glucose per liter. The presence of
PHB in recombinant E. coli was confirmed by microscopic
observation of intracellular inclusion bodies and by gas
chromatographic analysis. One recombinant clone accumulating a large
amount of PHB was isolated and characterized further. It was found to
harbor a 6.3-kb A. latus genomic DNA fragment, referred to
as AL63. Recombinant plasmid pUC19 containing AL63 was referred to as pJC1.
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Cloning of the Alcaligenes latus Polyhydroxyalkanoate
Biosynthesis Genes and Use of These Genes for Enhanced Production
of Poly(3-hydroxybutyrate) in Escherichia coli
ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
-ketothiolase, and reductase genes and were constitutively
expressed from the natural promoter in E. coli. Recombinant
E. coli strains harboring the A. latus PHA
biosynthesis genes accumulated poly(3-hydroxybutyrate) (PHB), a model
PHA product, more efficiently than those harboring the R. eutropha genes. With a pH-stat fed-batch culture of recombinant
E. coli harboring a stable plasmid containing the A. latus PHA biosynthesis genes, final cell and PHB concentrations
of 194.1 and 141.6 g/liter, respectively, were obtained, resulting in a
high productivity of 4.63 g of PHB/liter/h. This improvement
should allow recombinant E. coli to be used for the
production of PHB with a high level of economic competitiveness.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-ketothiolase
catalyzes the formation of a carbon---carbon bond by biological Claisen
condensation of two acetyl coenzyme A moieties. An NADPH-dependent
acetoacetyl coenzyme A (acetoacetyl-CoA) reductase catalyzes the
stereoselective reduction of acetoacetyl-CoA to
D-(
)-3-hydroxybutyryl coenzyme A. The third reaction of
this pathway is catalyzed by a PHA synthase, which links the
D-()-3-hydroxybutyryl coenzyme A to the growing chain of
PHB by an ester bond. After the first cloning of the PHA biosynthesis
genes from R. eutropha (26, 32, 34), more than 30 different PHA biosynthesis genes were cloned from various bacteria
(15). Cloning of various PHA biosynthesis genes not only has
provided detailed information on the structure and organization of the
PHA biosynthesis genes but also has allowed the creation of genetically
engineered microorganisms or even plants for more efficient production
of these biodegradable polymers and for the production of novel PHAs
(16, 28).
MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
TABLE 1.
Bacterial strains and plasmids used in this study
RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
View larger version (17K):
[in a new window]
FIG. 1.
Organization of A. latus PHA biosynthesis
genes. (A) Restriction map of the AL63 fragment. (B) Organization of
phaCAl, phaAAl,
phaBAl, and ORF4. aa, amino acids.
TABLE 2.
Partial alignment and identity of the deduced amino acid
sequence of PHA synthase from A. latus with those from
other organisms
-ketothiolases of R. eutropha (67%) (26, 32, 34), Paracoccus
denitrificans (57%) (37), and Thiocystis
violacea (60%) (23). Therefore, ORF2 was concluded to
represent a structural gene for the A. latus
-ketothiolase (referred to as phaAAl).
The putative gene product translated from ORF3 (735 bp) had high
sequence identities with acetoacetyl-CoA reductases of R. eutropha (75%) (26, 32, 34) and Chromatium
vinosum (64%) (24). Therefore, ORF3 was concluded to
represent a structural gene for the A. latus acetoacetyl-CoA
reductase (referred to as phaBAl).
As shown in Fig. 1B, these three genes form an operon in the order
phaCAl-phaAAl-phaBAl,
coding for PHA synthase, -ketothiolase, and reductase, respectively.
The putative ribosome binding sites were identified in the spaces
between phaCAl and phaAAl
and between phaAAl and
phaBAl.
A putative E. coli 
70-dependent promoter-like
sequence was found in the region upstream of the
phaCAl gene. An inverted-repeat structure, which
may serve as a transcription termination signal, was found in the
region downstream of phaBAl and had a structural free energy of 25.1 kcal/mol.
ORF4 (636 bp), which would code for a protein with a calculated
molecular mass of 24,121 Da, was located upstream of
phaCAl. The consensus sequence of the
70-dependent promoter was found in the region upstream
of ORF4. An inverted repeat was identified in the region downstream of ORF4 (nucleotides 1030 to 1053) and had a structural free energy of
23.9 kcal/mol.
Construction of recombinant plasmids harboring the A. latus PHA biosynthesis genes. Since pJC1 had a segregational instability problem during fed-batch culturing in the absence of antibiotic pressure (data not shown), a stable plasmid, pJC2, was constructed by cloning the 6.3-kb EcoRI-HindIII fragment containing the A. latus PHA biosynthesis genes into a stable high-copy-number plasmid, pSYL104 (22), digested with the same restriction enzymes; thus, the R. eutropha PHA biosynthesis genes were replaced (Fig. 2). Plasmid pJC3 was constructed by removing a fragment of about 1 kb of unnecessary DNA upstream of the promoter region of the A. latus PHA biosynthesis genes in pJC1. The fragment removed from pJC1 included the structural gene of ORF4 and its putative promoter and inverted-repeated regions. Plasmid pJC4, a stable version of pJC3, was constructed by cloning the 5.3-kb EcoRI-HindIII fragment containing the A. latus PHA biosynthesis genes into similarly digested pSYL104 (Fig. 2). Plasmids pJC3 and pJC4 were stably maintained during fed-batch culturing in the absence of antibiotic pressure (data not shown).
|
-D-thiogalactopyranoside (IPTG) at a final
concentration of 1 mM did not increase PHB production in XL1-Blue(pJC1)
and XL1-Blue(pJC3). These results suggested that the A. latus PHA biosynthesis enzymes were constitutively expressed in
E. coli from the natural promoter.
Cell growth and PHB synthesis in various recombinant E. coli strains. Recombinant E. coli strains harboring four different plasmids (pJC1, pJC2, pJC3, and pJC4) containing the A. latus PHA biosynthesis genes and recombinant E. coli strains harboring the pSYL series of plasmids (pSYL104, pSYL105, and pSYL107) containing the R. eutropha PHA biosynthesis genes were cultivated for 66 h in defined R medium supplemented with 20 g of glucose per liter and 50 mg of ampicillin per liter at 30°C (Fig. 3).
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Fed-batch cultures of recombinant E. coli harboring the A. latus PHA biosynthesis genes. pH-stat fed-batch culturing of recombinant E. coli strains harboring the A. latus PHA biosynthesis genes was carried out. For the recombinant strains harboring plasmids pJC3 and pJC4, ampicillin was not added during the entire cultivation. All recombinant E. coli strains harboring the A. latus PHA biosynthesis genes, XL1-Blue(pJC1), XL1-Blue(pJC2), XL1-Blue(pJC3), and XL1-Blue(pJC4), grew to cell concentrations higher than 177 g/liter. XL1-Blue harboring pJC3 or pJC4 accumulated more PHB (up to 73% cell dry weight) than XL1-Blue harboring pJC1 or pJC2 (50 to 60% cell dry weight). As an example, the time profiles of cell growth and PHB production during fed-batch culturing of XL1-Blue(pJC4) are shown in Fig. 4. The final cell concentration, PHB concentration, and PHB content obtained in 30.6 h were 194.1 g/liter, 141.6 g/liter, and 73%, respectively, resulting in a very high PHB productivity of 4.63 g/liter/h.
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DISCUSSION |
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Introduction
Materials & Methods
Results
Discussion
References
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The use of PHA as a substitute for nondegradable petroleum-derived plastics hinges on the ability to produce it at a cost that is competitive with that incurred in the production of conventional plastics. From studies on the design and economic evaluation of the processes used for the production of PHAs (4), it was found that PHA productivity is one of the most important factors determining overall production cost.
Even though there are more than 300 different microorganisms that are known to synthesize PHAs in nature, only a few bacteria can produce PHB to an extent that meets commercial interest (15, 16). The highest PHB productivity was achieved with the fed-batch culture of A. latus (39). It was reasoned that the efficient synthesis of PHB in A. latus was due to the greater catalytic power of the PHA biosynthesis enzymes in this bacterium. Previous studies showed that recombinant E. coli was superior to wild-type PHB producers in all aspects except for productivity (6, 12, 18, 21, 33). Therefore, economical production of PHB by recombinant E. coli was thought to be possible if PHB productivity could be increased by cloning the A. latus PHA biosynthesis genes.
The cloning strategy was based on the previous observation that recombinant E. coli harboring the R. eutropha PHA biosynthesis genes could synthesize and intracellularly accumulate PHB when grown in a suitable medium, such as LB medium containing glucose (26, 32, 34). A 6.3-kb A. latus genomic DNA fragment (AL63) coding for the PHA biosynthesis enzymes was cloned in E. coli by screening the transformants that appeared as opaque colonies. Nucleotide sequence analysis of the cloned AL63 fragment indicated that the phaCAl, phaAAl, and phaBAl genes were clustered in a single operon (phaCAB), as in R. eutropha (26, 32, 34). There was one more open reading frame (ORF4) in front of the promoter region of the PHA biosynthesis operon. Its product had high amino acid sequence identity (58%) with glutathione S-transferase, but its actual function is not known. Since ORF4 did not seem to affect PHB synthesis, two derivatives, pJC3 and pJC4, were constructed by deleting this region.
The use of antibiotics in a large-scale fermentation is not desirable. Since pJC1 and pJC3 showed instability problems during PHB production in the absence of antibiotic pressure, stable derivatives pJC2 and pJC4 were constructed by transferring the A. latus PHA biosynthesis genes into the pSYL104 backbone, which contains the parB locus of plasmid R1 for plasmid stabilization (22). The growth of recombinant E. coli strains harboring four different plasmids containing the A. latus PHA biosynthesis genes and PHB production were compared with those of recombinant E. coli strains harboring the R. eutropha PHA biosynthesis genes. PHB production was higher for recombinant E. coli XL1-Blue(pJC3) and XL1-Blue(pJC4) than for recombinant E. coli strains harboring the R. eutropha PHA biosynthesis genes. A better comparison could be made with plasmids pJC4 and pSYL104, since they share the same plasmid backbone and have the PHA biosynthesis genes cloned in the same orientation. The only difference is the source of the PHA biosynthesis genes (A. latus for pJC4 and R. eutropha for pSYL104). When XL1-Blue(pJC4) and XL1-Blue(pSYL104) were cultured under the same conditions, XL1-Blue(pJC4) had higher cell and PHB concentrations than XL1-Blue(pSYL104) (Fig. 3).
To determine if the new recombinant E. coli strains harboring the A. latus PHA biosynthesis genes could produce PHB more efficiently, fed-batch culturing was carried out. XL1-Blue(pJC1) and XL1-Blue(pJC2) grew to high concentrations (higher than 177 g of cell dry weight/liter), but the PHB contents were below 60%. On the other hand, fed-batch cultures of XL1-Blue(pJC3) and XL1-Blue(pJC4) showed high concentrations of cells and PHB (higher than 140 g of PHB/liter) with high productivity. In particular, PHB concentration and PHB productivity obtained with XL1-Blue(pJC4) were as high as 141.6 g/liter and 4.63 g of PHB/liter/h, respectively.
Recently, metabolic engineering approaches were taken to develop several recombinant bacteria and transgenic plants (16, 28) for more efficient production and recovery of PHB. Recombinant E. coli strains harboring the R. eutropha PHA biosynthesis genes have been one of the most successful examples in this aspect, except for PHB productivity. This study provided a strategy of enhancing PHB productivity by developing recombinant E. coli strains harboring the more efficient PHA biosynthesis machinery of A. latus.
The advantages of the use of recombinant E. coli plus the ability to produce PHB with high productivity should make it possible to produce PHB with a high level of economic competitiveness.
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ACKNOWLEDGMENTS |
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We thank S. H. Choo and H. S. Yoon for help in DNA sequencing.
This work was supported by the Ministry of Science and Technology and by LG Chemicals, Ltd.
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FOOTNOTES |
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* Corresponding author. Mailing address: 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. Phone: 82-42-869-3930. Fax: 82-42-869-8800. E-mail: leesy{at}sorak.kaist.ac.kr.
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REFERENCES |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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|---|
| 1. |
Anderson, A. J., and E. A. Dawes.
1990.
Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates.
Microbiol. Rev.
54:450-472 |
| 2. |
Braunegg, G.,
B. Sonnleitner, and R. M. Lafferty.
1978.
A rapid gas chromatographic method for the determination of poly--hydroxybutyric acid in microbial biomass.
Eur. J. Appl. Microbiol. Biotechnol.
6:29-37.
|
| 3. | Byrom, D. 1987. Polymer synthesis by microorganisms: technology and economics. Trends Biotechnol. 5:246-250. |
| 4. | Choi, J., and S. Y. Lee. 1997. Process analysis and economic evaluation for poly(3-hydroxybutyrate) production by fermentation. Bioprocess. Eng. 17:335-342. |
| 5. | Doi, Y. 1990. Microbial polyesters. VCH, New York, N.Y. |
| 6. | Fidler, S., and D. Dennis. 1992. Polyhydroxyalkanoate production in recombinant Escherichia coli. FEMS Microbiol. Rev. 103:231-236. |
| 7. |
Fukui, T., and Y. Doi.
1997.
Cloning and analysis of the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) biosynthesis genes of Aeromonas caviae.
J. Bacteriol.
179:4821-4830 |
| 8. | Gerngross, T. U., K. D. Snell, O. P. Peoples, A. J. Sinskey, E. Csuhai, S. Masamune, and J. Stubbe. 1994. Overexpression and purification of the soluble polyhydroxyalkanoate synthase from Alcaligenes eutrophus: evidence for a required posttranslational modification for catalytic activity. Biochemistry 33:9311-9320[Medline]. |
| 9. | Holmes, P. A. 1988. Biologically produced PHA polymers and copolymers, p. 1-65. In D. C. Bassett (ed.), Developments in crystalline polymers, vol. 2. Elsevier, London, England. |
| 10. |
Huisman, G. W.,
E. Wonink,
R. Meima,
B. Kazemier,
P. Terpstra, and B. Witholt.
1991.
Metabolism of poly(3-hydroxyalkanoates) by Pseudomonas oleovorans: identification and sequences of genes and function of the encoded proteins in the synthesis and degradation of PHA.
J. Biol. Chem.
266:2191-2198 |
| 11. | Hustede, E., A. Steinbüchel, and H. G. Schlegel. 1992. Cloning of poly(3-hydroxybutyric acid) synthase genes of Rhodobacter sphaeroides and Rhodospirillum rubrum and heterologous expression in Alcaligenes eutrophus. FEMS Microbiol. Lett. 93:73-80. |
| 12. | Kusaka, S., H. Abe, S. Y. Lee, and Y. Doi. 1997. Molecular mass of poly[(R)-3-hydroxybutyric acid] produced in a recombinant Escherichia coli. Appl. Microbiol. Biotechnol. 47:140-143[Medline]. |
| 13. | Lee, I., S. Nam, Y. Rhee, and J. Kim. 1996. Cloning and functional expression in Escherichia coli of polyhydroxyalkanoate synthase (phaC) gene from Alcaligenes sp. SH-69. J. Microbiol. Biotechnol. 6:309-314. |
| 14. | Lee, S. Y. 1994. Suppression of filamentation in recombinant Escherichia coli by amplified FtsZ activity. Biotechnol. Lett. 16:1247-1252. |
| 15. | Lee, S. Y. 1996. Bacterial polyhydroxyalkanoates. Biotechnol. Bioeng. 49:1-14. |
| 16. | Lee, S. Y. 1996. Plastic bacteria? Progress and prospects for polyhydroxyalkanoate production in bacteria. Trends Biotechnol. 14:431-438. |
| 17. | Lee, S. Y. 1997. E. coli moves into the plastic age. Nat. Biotechnol. 15:17-18[Medline]. |
| 18. | Lee, S. Y., and H. N. Chang. 1993. High cell density cultivation of Escherichia coli W using sucrose as a carbon source. Biotechnol. Lett. 15:971-974. |
| 19. | Lee, S. Y., and H. N. Chang. 1995. Production of poly(3-hydroxybutyric acid) by recombinant Escherichia coli strains: genetic and fermentation studies. Can. J. Microbiol. 41(Suppl. 1):207-215. |
| 20. | Lee, S. Y., K. M. Lee, H. N. Chang, and A. Steinbüchel. 1994. Comparison of Escherichia coli strains for synthesis and accumulation of poly-(3-hydroxybutyric acid), and morphological changes. Biotechnol. Bioeng. 44:1337-1347. |
| 21. | Lee, S. Y., A. P. J. Middelberg, and Y. K. Lee. 1997. Poly(3-hydroxybutyrate) production from whey using recombinant Escherichia coli. Biotechnol. Lett. 19:1033-1035. |
| 22. | Lee, S. Y., K. S. Yim, H. N. Chang, and Y. K. Chang. 1994. Construction of plasmids, estimation of plasmid stability, and use of stable plasmids for the production of poly(3-hydroxybutyric acid) in Escherichia coli. J. Biotechnol. 32:203-211[Medline]. |
| 23. | Liebergesell, M., F. Mayer, and A. Steinbüchel. 1993. Analysis of polyhydroxyalkanoic acid-biosynthesis genes of anoxygenic phototrophic bacteria reveals synthesis of a polyester exhibiting an unusual composition. Appl. Microbiol. Biotechnol. 40:292-300. |
| 24. | Liebergesell, M., and A. Steinbüchel. 1992. Cloning and nucleotide sequences of genes relevant for biosynthesis of poly(3-hydroxybutyric acid) in Chromatium vinosum strain D. Eur. J. Biochem. 209:135-150[Medline]. |
| 25. | Marmur, J. 1961. A procedure for the isolation of deoxyribonucleic acids from microorganisms. J. Mol. Biol. 3:208-218. |
| 26. |
Peoples, O. P., and A. J. Sinskey.
1989.
Poly--hydroxybutyrate biosynthesis in Alcaligenes eutrophus H16. Identification and characterization of the PHB polymerase gene (phbC).
J. Biol. Chem.
264:15298-15303 |
| 27. | Pieper, U., and A. Steinbüchel. 1992. Identification, cloning and molecular characterization of the poly(3-hydroxyalkanoic acid) synthase structural gene of the gram-positive Rhodococcus ruber. FEMS Microbiol. Lett. 96:73-80. |
| 28. | Poirier, Y., C. Nawrath, and C. Somerville. 1995. Production of polyhydroxyalkanoates, a family of biodegradable plastics and elastomers, in bacteria and plants. Bio/Technology 13:142-150[Medline]. |
| 29. | Ryu, H. W., S. K. Hahn, Y. K. Chang, and H. N. Chang. 1997. Production of poly(3-hydroxybutyrate) by high cell density fed-batch culture of Alcaligenes eutrophus with phosphate limitation. Biotechnol. Bioeng. 55:28-32. |
| 30. | Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. |
| 31. | Schembri, M. A., R. C. Bayly, and J. K. Davies. 1994. Cloning and analysis of the polyhydroxyalkanoic acid synthase gene from an Acinetobacter sp.: evidence that the gene is both plasmid and chromosomally located. FEMS Microbiol. Lett. 118:145-152[Medline]. |
| 32. |
Schubert, P.,
A. Steinbüchel, and H. G. Schlegel.
1988.
Cloning of the Alcaligenes eutrophus genes for synthesis of poly--hydroxybutyric acid (PHB) and synthesis of PHB in Escherichia coli.
J. Bacteriol.
170:5837-5847 |
| 33. | Sim, S. J., K. D. Snell, S. A. Hogan, J. Stubbe, C. Rha, and A. J. Sinskey. 1997. PHA synthase activity controls the molecular weight and polydispersity of polyhydroxybutyrate in vivo. Nat. Biotechnol. 15:63-67[Medline]. |
| 34. |
Slater, S. C.,
W. H. Voige, and D. E. Dennis.
1988.
Cloning and expression in Escherichia coli of the Alcaligenes eutrophus H16 poly--hydroxybutyrate biosynthetic pathway.
J. Bacteriol.
170:4431-4436 |
| 35. | Steinbüchel, A. 1991. Polyhydroxyalkanoic acids, p. 124-213. In D. Byrom (ed.), Biomaterials: novel materials from biological sources. Stockton, New York, N.Y. |
| 36. | Timm, A., and A. Steinbüchel. 1992. Cloning and molecular analysis of the polyhydroxyalkanoic acid gene locus of Pseudomonas aeruginosa PAO1. Eur. J. Biochem. 209:15-30[Medline]. |
| 37. |
Ueda, S.,
T. Yabutani,
A. Maehara, and T. Yamane.
1996.
Molecular analysis of the poly(3-hydroxyalkanoate) synthesis gene from a methylotrophic bacterium, Paracoccus denitrificans.
J. Bacteriol.
178:774-779 |
| 38. | Valentin, H. E., and A. Steinbüchel. 1993. Cloning of the Methylobacterium extorquens polyhydroxyalkanoic acid synthase structural gene. Appl. Microbiol. Biotechnol. 39:309-317[Medline]. |
| 39. | Wang, F., and S. Y. Lee. 1997. Poly(3-hydroxybutyrate) production with high polymer content by fed-batch culture of Alcaligenes latus under nitrogen limitation. Appl. Environ. Microbiol. 63:3703-3706[Abstract]. |
| 40. | Wang, F., and S. Y. Lee. 1997. Production of poly(3-hydroxybutyrate) by fed-batch culture of filamentation-suppressed recombinant Escherichia coli. Appl. Environ. Microbiol. 63:4765-4769[Abstract]. |
Applied and Environmental Microbiology, December 1998, p. 4897-4903, Vol. 64, No. 12
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