Appl Microbiol Biotechnol (2006) 69: 537–542
DOI 10.1007/s00253-005-0034-6
APPLIED GENE TICS AN D MO LECULA R BIO TECH NOLOGY
Yuan-Zheng Qiu . Jing Han . Guo-Qiang Chen
Metabolic engineering of Aeromonas hydrophila for the enhanced
production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)
Received: 6 March 2005 / Revised: 26 May 2005 / Accepted: 27 May 2005 / Published online: 28 June 2005
# Springer-Verlag 2005
Abstract Wild-type Aeromonas hydrophila 4AK4 produced
35–45 wt.% poly(3-hydroxybutyrate-co-3-hydroxyhexa-
noate) (PHBHHx) consisting of 10–15 mol% 3-hydroxy-
hexanoate (3HHx). To enhance PHBHHx production, vgb
gene encoding Vitreoscilla haemoglobin or fadD gene
encoding Escherichia coli acyl-CoA synthase was co-ex-
pressed with polyhydroxyalkanoates (PHA) synthesis-re-
lated genes including phbAB from Wautersia eutropha and
phaPCJ from A. hydrophila. Expression of vgb increased
PHBHHx content from 46 to 53 wt.% without affecting
the polymer monomers composition, whereas fadD in-
creased both PHBHHx content from 46 to 64 wt.% and its
3HHx fraction from 15 to 24 mol%. Co-expression of vgb
or fadD gene with PHA-synthesis-related genes generally
increased PHBHHx content over 60 wt.%. Co-expression
of phbAB with vgb increased PHBHHx content and con-
centration up to about 70 wt.% and 4.0 g l−1, respectively.
Fermentor study also showed that in the recombinants har-
boring vgb, CDW, PHBHHx concentration and productiv-
ity were significantly elevated up to 54 g l−1, 28.5 g l−1
and 0.791 g l−1 h−1, respectively, suggesting that vgb could
promote PHA synthesis. In this strain, lac promoter could
be used to constitutively express foreign genes such as
phbA and phbB encoding β-ketothiolase and NADPH-
dependent acetoacetyl-CoA reductase of W. eutropha, re-
spectively, without use of IPTG. The results showed that
combined expression of different genes was a successful
strategy to enhance PHA production, which could be use-
ful for strain development to construct other recombinant
PHA-producing strains.
Y.-Z. Qiu . G.-Q. Chen (*)
Department of Biological Sciences and Biotechnology,
Tsinghua University,
Beijing, 100084, China
e-mail: chengq@mail.tsinghua.edu.cn
Tel.: +86-10-62783844
Fax: +86-10-62788784
J. Han
School of Life Science, Shandong Normal University,
Jinan, 250014, China
Introduction
Polyhydroxyalkanoates (PHA) are biopolyesters of (R)-
hydroxyalkanoates, which are accumulated by microorgan-
isms as carbon and energy reserves (Doi 1990; Steinbüchel
2001). These biopolymers have material properties sim-
ilar to petrochemical plastics, yet it is biodegradable and
can be produced from renewable carbon sources such as
carbohydrates and fatty acids. Therefore, PHA have at-
tracted increasing interests. Approximately 150 different
hydroxyalkanoic acids are now known to occur as con-
stituents of PHA (Doi and Steinbüchel 2002). The com-
position of PHA is mainly dependent on the substrate
specificity of PHA synthase, metabolic routes providing
precursors and cultivation conditions. Poly(3-hydroxybu-
tyrate) (PHB) is the best characterized PHA, and its pro-
duction in Wautersia eutropha has been well studied. The
PHB biosynthesis genes of W. eutropha form a single
operon known as phbCABRe, which encodes PHB syn-
thase, β-ketothiolase and NADPH-dependent acetoacetyl-
CoA reductase (Peoples and Sinskey 1989a,b; Slater et al.
1988; Schubert et al. 1988).
Copolymer poly(3-hydroxybutyrate-co-3-hydroxyhex-
anoate) (PHBHHx) consisting of 3-hydroxybutyrate (3HB)
and 3-hydroxyhexanoate (3HHx) was produced by Aero-
monas caviae and A.hydrophila 4AK4 (Doi et al. 1995; Lee
et al. 2000; Chen et al. 2001). Compared with PHB,
PHBHHx has better physical and mechanical properties,
such as enhanced flexibility and improved impact strength
(Doi et al. 1995; Feng et al. 2002). PHBHHx also has
better biocompatibility than PHB and polylactic acid
(PLA) (Yang et al. 2002; Zhao et al. 2003a). The PHBHHx
biosynthesis genes of A. caviae consisting of phaP, phaC
and phaJ encoding phasin, PHA synthase and (R)-specific
enoyl-CoA hydratase, respectively, had been cloned (Fukui
and Doi 1997). The similar operon was also found in
A. hydrophila (Zhang 2002).
A. hydrophila 4AK4 showed some advantages for
production of PHBHHx, such as robust growth property
and simple growth requirements, which make it a potential
strain for industrial production (Chen et al. 2001). Previous
538

study showed that PHBHHx production could be enhanced
by using recombinant A. hydrophila (Han et al. 2004). In
this study, PHA-synthesis-related genes were combined
with cell-growth-related gene or carbon source metabolism
gene in A. hydrophila to further increase PHBHHx pro-
duction. Genes vgb encoding Vitreoscilla haemoglobin and
fadD encoding Escherichia coli acyl-CoA synthase were
used to co-express with PHA-synthesis-related genes. Ad-
ditionally, the expression of heterogenous genes in recom-
binant A. hydrophila was also studied.
(per liter) 9.0 g Na2HPO4·12H2O, 1.5 g KH2PO4, 1.0 g
(NH4)2 SO4, 0.4 g MgSO4·7H2O and 1% (v/v) trace ele-
ment solution. The composition of trace element solution
was mentioned in previous report (Qiu et al. 2004).
Media for fermentor studies contained (in g l−1) 2.0
Na2HPO4·12H2O, 1.85 KH2PO4, 4 (NH4)2SO4, 0.5 MgSO4·
7H2O, 10 dodecanoate, 1 yeast extract and 1% (v/v) trace
element solution. A 6-l NBS computer-controlled fer-
mentor (NBS Bioflo 3000, New Jersey, USA) containing
3-l broth was used. Additional 80 g l−1 dodecanoate and
18 g l−1 (NH4)2SO4 were fed after 12-h cultivation.

Materials and methods

Bacterial strains and plasmids
A. hydrophila 4AK4 (Chen et al. 2001) and E. coli S17-1
(Simon et al. 1983) were cultivated in LB medium at 30
and 37°C, respectively. When necessary, 50 mg l−1 kana-
mycin was added. Plasmids used are listed in Table 1.
pVGAB and pTG01 were constructed previously (Qiu
et al. 2004; Zhao et al. 2003b). All plasmids were derived
from pBBR1MCS-2 (Kovach et al. 1995) by inserting a
DNA fragment of phaPC, phaCJ, phaPCJ, phbAB, vgb or
fadD genes or both two DNA fragments of those genes.
All DNA manipulations were carried out using standard
procedures (Sambrook and Russell 2001). The recom-
binant plasmids were first introduced into E. coli S17-1 by
electroporation, then conjugationally introduced into A.
hydrophila 4AK4.
Culture conditions for PHA synthesis
In culture of shake flask, overnight-cultivated A. hydro-
phila 4AK4 strains in LB medium supplemented with
50 mg l−1 kanamycin were transferred into mineral salt
(MS) medium (pH 7.2) containing 8 g l−1 dodecanoate
and 1 g l−1 yeast extract. The cells were subsequently
cultivated at 30°C for 48 h. The MS medium contained
SDS-PAGE and enzyme assay
For SDS-PAGE, 1-ml culture broth was harvested, washed
with distilled water, resuspended in 50 μl SDS-PAGE
sample buffer and boiled for 10 min. SDS-PAGE was
performed using Mini-PROTEAN II electrophoresis cell
(Bio-Rad, USA).
For enzyme assay, cells were harvested, washed with
0.1 mol l−1 Tris–HCl (pH 7.5) and resuspended in 10%
volume of the same buffer. Crude extract was prepared
from supernatant after sonication and centrifugation. Pro-
tein concentrations were determined by Bradford method
using Bio-Rad protein assay solution (Bradford 1976).
The activity of β-ketothiolase was spectrophotometri-
cally monitored at 25°C by the decrease in absorption of
acetoacetyl-CoA at 303 nm as reported by Nishimura et al.
(1978) with the following modification. The reaction
mixture contained 380 μl of 0.1 mol l−1 Tris–HCl (pH
8.1), 50 μl of 0.4 mol l−1 MgCl2, 5 μl of 7 mmol l−1
acetoacetyl-CoA, 5 μl of 10 mmol l−1 CoA and 55 μl H2O.
The reaction was initiated by the addition of 5-μl crude
extract.
Acetoacetyl-CoA reductase was spectrophotometrically
monitored at 340 nm by the oxidation of NADPH during
reduction of acetoacetyl-CoA (Senior and Dawes 1973).
The reaction was carried out at 25°C in a total volume of
500 μl containing 12.5 μl of 10 mmol l−1 NADPH and

Table 1 Plasmids used in Plasmids Relevant characteristics Source or reference

this study
pBBRvgb Containing Vitreoscilla hemoglobin gene vgb downstream This study
of its native promoter
pBBRfadD lac promoter, fadD from Escherichia coli This study
pTG01 lac promoter, phbAB from Wautersia eutropha Zhao et al. (2003b)
pVGAB lac promoter, phbAB; native promoter, vgb gene Qiu et al. (2004)
pFDAB lac promoter, phbAB and fadD This study
pAH04 lac promoter, phaPC from A. hydrophila This study
pAH04vgb lac promoter, phaPC; native promoter, vgb gene This study
pAH04fadD lac promoter, phaPC and fadD This study
pAH05 lac promoter, phaCJ from A. hydrophila This study
pAH05vgb lac promoter, phaCJ; native promoter, vgb gene This study
pAH05fadD lac promoter, phaCJ and fadD This study
pAH09 lac promoter, phaPCJ from A. hydrophila This study
pAH09vgb lac promoter, phaPCJ; native promoter, vgb gene This study
pAH09fadD lac promoter, phaPCJ and fadD This study
 539
Table 2 Shake flask culture of Strains CDW PHBHHx 3HHx fraction PHBHHx concentration
recombinant A. hydrophila
4AK4 strains (g l−1) (wt.%) (mol%) (g l−1)

4AK4 5.4±0.3 45.7±1.5 14.6±0.3 2.4±0.3

4AK4(vgb) 5.1±0.1 52.9±2.9 14.6±0.3 2.6±0.0
4AK4(fadD) 3.0±0.1 64.0±3.2 23.9±0.5 1.9±0.1
4AK4(phbAB) 5.4±0.2 59.1±1.6 10.1±0.2 3.2±0.1
4AK4(phbAB+vgb) 5.8±0.3 69.4±0.1 10.7±0.4 4.0±0.2
4AK4(phbAB+fadD) 3.0±0.2 64.3±1.4 17.4±0.3 1.9±0.2
4AK4(phaPC) 5.5±0.1 55.3±3.0 23.5±0.2 3.0±0.2
4AK4(phaPC+vgb) 5.4±0.0 63.7±2.4 25.7±0.2 3.5±0.1
4AK4(phaPC+fadD) 3.6±0.0 67.5±2.0 25.0±0.9 2.4±0.0
4AK4(phaCJ) 5.3±0.1 53.5±1.4 23.4±0.3 2.8±0.1
4AK4(phaCJ+vgb) 5.6±0.2 58.2±3.3 25.2±0.6 3.2±0.3
Cells were cultivated in MS 4AK4(phaCJ+fadD) 4.1±0.1 63.2±0.6 24.3±0.1 2.6±0.2
medium containing 8 g l−1 4AK4(phaPCJ) 5.5±0.2 55.2±4.2 20.5±0.9 3.0±0.1
dodecanoate and 1 g l−1 yeast 4AK4(phaPCJ+vgb) 2.9±0.1 63.0±0.3 23.5±0.6 1.8±0.1
extract at 30°C for 48 h 4AK4(phaPCJ+fadD) 3.1±0.1 67.8±1.8 23.8±0.2 2.0±0.2
CDW Cell dry weight

12.5 μl of 7 mmol l−1 acetoacetyl-CoA in a buffer
[120 mmol l−1 potassium phosphate (pH 5.5), 24 mmol
l−1 MgCl2, 1 mmol l−1 dithiothreitol]. The reaction was
initiated by the addition of 5-μl crude extract.
Analysis of PHBHHx
The cells were harvested by centrifugation, washed with
ethanol and distilled water and then lyophilized. The cel-
lular PHBHHx was analyzed by gas chromatography
(GC) (He et al. 1998). GC analysis was carried out using
Hewlett-Packard 6890 equipped with 30-m HP-5 capillary
column.
pression of phbAB increased PHBHHx content and low-
ered 3HHx fraction. If phbAB was co-expressed with vgb, it
significantly enhanced PHBHHx synthesis from 46 to
69%, resulting in higher PHBHHx concentration (from 2.4
to 4.0 g l−1). Expression of vgb slightly increased PHBHHx
content. When vgb was co-expressed with phaPC, phaCJ
or phaPCJ, it further increased PHBHHx content. Ampli-
fication of fadD increased PHBHHx content from 46 to
64% and increased 3HHx fraction from 15 to 24 mol%.
When fadD was co-expressed with phbAB, phaPC, phaCJ
or phaPCJ, it further increased PHBHHx content. How-
ever, for unknown reasons, amplification of fadD led to
lower cell dry weight (CDW).

Fermentor culture of recombinant A. hydrophila

Results 4AK4 strains

Shake flask culture of recombinant A. hydrophila
4AK4 strains
As shown in Table 2, amplification of phaPC, phaCJ or
phaPCJ genes increased PHBHHx content (from 46 to
55%) and 3HHx fraction (from 15 up to 24 mol%). Ex-
Recombinants harboring vgb produced higher CDW and thus
higher PHBHHx concentration and productivity (Table 3).
PHBHHx concentration and productivity in recombinant
harboring phbAB and vgb genes increased up to 28.5 g l−1
and 0.791 g l−1 h−1, respectively, compared to 22.1 g l−1
and 0.525 g l−1 h−1 in wild-type strain. Expression of

Table 3 Fermentor culture of recombinant A. hydrophila 4AK4 strains

Strains CDW PHBHHx 3HHx fraction PHBHHx concentration PHBHHx productivity
(g l−1) (wt.%) (mol%) (g l−1) (g l−1 h−1)

4AK4 40.4 54.6 14.4 22.1 0.525

4AK4(phbAB+vgb) 54.0 52.7 12.0 28.5 0.791
4AK4(phaPC+vgb) 50.2 56.3 25.1 28.3 0.674
4AK4(phaPC+fadD) 33.8 61.3 27.2 20.7 0.493
A 6-l NBS computer-controlled fermentor (NBS Bioflo 3000, New Jersey, USA) containing 3-l broth was used. Fermentations were carried
out at 30°C and pH 6.5. Dissolved oxygen concentration was maintained at 15% of air saturation in the fermentation broth. Total
fermentation time for all above studies was 36–42 h
540

phaPC in the recombinants led to higher 3HHx fraction A

in PHBHHx. In recombinant harboring phaPC and fadD 16
4AK4(pTG01)
genes, 3HHx fraction reached 27.2 mol%.
12

U/mg protein
4AK4(pTG01)+IPTG
4AK4
8
Expression of heterogenous genes in recombinant 4AK4+IPTG
A. hydrophila 4AK4 4

To study the expression characteristics of heterogenous 0

Time (h)
genes, A. hydrophila 4AK4 (pTG01) harboring phbAB 4 8 12 16 20 24 28
genes was used. The wild-type strain did not show visible B
β-ketothiolase (PhbA) and acetoacetyl-CoA reductase 4
(PhbB) bands on SDS-PAGE gel according to their mo-
3

U/mg protein
lecular mass (40.5 and 26.3 kDa). Two additional bands
(Fig. 1) were found in total protein profile of A. hydrophila 4AK4(pTG01)
4AK4 (pTG01), which represented putative PhbA and 2 4AK4(pTG01)+IPTG
4AK4
PhbB based on their calculated molecular mass. IPTG did
1 4AK4+IPTG
not affect phbA and phbB expression in the recombinant
strain since there was no significant difference of protein
0 Time (h)
profiles in the presence and absence of IPTG.
A. hydrophila 4AK4 (pTG01) significantly increased 4 8 12 16 20 24 28
PhbA and PhbB activities, which were 5- to 1,100-fold Fig. 2 Enzyme assay of β-ketothiolase (a) and acetoacetyl-CoA
higher for PhbA and 23- to 73-fold higher for PhbB com- reductase (b) in wild-type and recombinant A. hydrophila 4AK4
pared with those of wild-type strain, respectively (Fig. 2). (pTG01). Cells were cultivated in MS medium containing 8 g l−1
sodium glucoante and 1 g l−1 yeast extract at 30°C. When indicated,
IPTG significantly lowered the activities of both enzymes 0.2 g l−1 IPTG was added at 6 h. One unit of β-ketothiolase was
defined as 1 μmol of acetoacetyl-CoA depletion in 1 min. One unit
M 1 2 3 4 5 6 of acetoacetyl-CoA reductase was defined as 1 μmol of NADPH
kDa depletion in 1 min

66 in the recombinant. The recombinant strain grown with

56 IPTG showed 46, 61 and 89% PhbA activity and 10, 11 and
43 A 24% PhbB activity of that grown without IPTG at 8, 16 and
24 h, respectively. In wild-type strain, IPTG did not affect
36 the activities of the two enzymes (Fig. 2).

27 B
Discussion

A. hydrophila 4AK4 was used in industrial scale produc-

M 1 2 3 4 5 6
kDa tion of PHBHHx (Chen et al. 2001). It is crucial to increase
PHBHHx content and productivity to realize economical
66 production. It is also significant to regulate its monomer
56
composition to meet various applications (Wang et al.
2005). However, it was difficult to realize those purposes in
43 A the wild-type strain (Chen et al. 2001). Previous studies
had indicated that phaPCJ played a key role in PHBHHx
36
synthesis (Han et al. 2004), and recombinant harboring
phbAB could be used to regulate PHBHHx compositions
27 (Qiu et al. 2004). To increase PHBHHx production by
B
metabolic engineering, several pathways are crucial in-
cluding pathways from fatty acids to acyl-CoA, 3HB-CoA
Fig. 1 SDS-PAGE analysis of total cellular proteins from wild-type and 3HHx-CoA supply pathways. In addition, oxygen up-
(lanes 1, 3 and 5) and recombinant (lanes 2, 4 and 6) Aeromonas
hydrophila 4AK4 (pTG01). Cells were cultivated in MS medium take efficiency is important as this will affect β-oxidation
containing 8 g l−1 sodium glucoante and 1 g l−1 yeast extract at 30°C and cell growth. Therefore, the combination of PHA-syn-
with (A) or without (B) 0.2 g l−1 IPTG addition at 6 h. One-milliliter thesis-related genes (phaPCJ, phbAB) with cell-growth-
samples were withdrawn at 8 h (lanes 1 and 2), 16 h (lanes 3 and 4) related gene (vgb) or fatty acids metabolism gene (fadD)
and 24 h (lanes 5 and 6). Protein bands indicated by A and B
represented putative β-ketothiolase (40.5 kDa) and acetoacetyl-CoA will be a useful strategy to realize near-complete strain
reductase (26.3 kDa), respectively, based on their known molecular development so that PHBHHx production can be enhanced
mass as much as possible.

Vitreoscilla haemoglobin (VHb) encoded by vgb gene,
an oxygen-binding protein, allows the bacterium to grow
aerobically even under microaerophilic conditions (Kallio
et al. 1994). VHb was able to promote PHB synthesis in
recombinant E. coli (Yu et al. 2002). Combined expression
of phbAB, phaPC, phaCJ or phaPCJ with vgb gene further
enhanced PHBHHx production in recombinant A. hydro-
phila 4AK4. In most cases, the combined expression
resulted in higher PHBHHx content and CDW, leading to
higher PHBHHx concentration in shake flask culture.
When vgb was co-expressed with phbAB, the PHBHHx
concentration increased up to 4.0 g l−1. Since VHb affected
oxygen uptake, it will affect cell growth and β-oxidation.
By combining vgb with pha genes, the PHA accumulation
and cell growth would be promoted synergistically.
Gene fadD encoding acyl-CoA synthase (FadD) cata-
lyzes the first step of fatty acid degradation; therefore, it
would affect PHBHHx synthesis from fatty acids. It was
reported that amplification of E. coli fadD gene had sig-
nificant effect on monomer composition of medium-chain-
length PHA in recombinant E. coli (Park et al. 2003). In
recombinant A. hydrophila 4AK4, amplification of E. coli
fadD gene increased both PHBHHx content and 3HHx
fraction. Combined expression of phbAB, phaPC, phaCJ
or phaPCJ with fadD gene further increased PHBHHx
content. Amplification of fadD might accelerate the con-
version of fatty acids into acyl-CoA, resulting in increased
carbon flux in β-oxidation cycle, which sequentially pro-
moted PHBHHx synthesis. The C6 intermediate (hexa-
noyl-CoA) in the cycle is first converted to 3HHx-CoA and
presented to PHA synthase before it is degraded to shorter
fatty acids; thus, increasing carbon flux in β-oxidation in-
creased 3HHx-CoA flux more markedly compared with
that of 3HB-CoA flux, resulting in higher 3HHx fraction in
PHBHHx. However, expression of fadD gene had negative
effect on cell dry weight due to some unknown reasons.
Co-expression of fadD with phaPC, phaCJ or phaPCJ
helped to reduce the negative effect.
The enhanced PHBHHx production by combined ex-
pression of genes was further proven in fermentor culture.
When vgb was co-expressed with phbAB or phaPC, CDW,
PHBHHx concentration and productivity were signifi-
cantly elevated compared to those of wild-type strain, in-
dicating that the recombinants harboring vgb grew more
vigorously and accumulated PHBHHx faster. This is pos-
sibly due to the fact that VHb was able to promote cell
growth and β-oxidation by improving oxygen uptake. In
recombinants harboring phaPC, 3HHx fraction increased
up to 27 mol%, suggesting that the recombinants could be
used to produce PHBHHx containing high 3HHx fraction.
The phbAB in plasmid pTG01 were under the control of
lac promoter. Although IPTG was generally used as an
inducer of lac promoter in recombinant E. coli system, it
was not necessary to induce lac promoter in recombinant A.
hydrophila using IPTG. The expression of phbA and phbB
genes in recombinant A. hydrophila was constitutive. IPTG
addition did not significantly affect the expression of the
two genes since there was not a significant difference in
541
protein profiles between IPTG presence and absence. This
was possibly due to the defect of LacI protein in A.
hydrophila, the repressor of lac promoter in E. coli. That
should partially explain the fact that IPTG addition did not
affect PHBHHx biosynthesis and its monomer composition
in recombinant A. hydrophila 4AK4 (pTG01) (Qiu et al.
2004).
Enzyme assay showed that the activities of β-keto-
thiolase and acetoacetyl-CoA reductase in recombinants
significantly increased, indicating that the two genes were
successfully expressed in recombinant A. hydrophila
4AK4. It was interesting to find that IPTG addition had
negative effect on the activities of both enzymes. The
negative effect possibly occurred at the level of proteins
instead of at transcription or translation level since IPTG
did not significantly affect the expression of phbA and
phbB (Fig. 1).
In summary, the combination of PHA-synthesis-related
genes with cell-growth-related gene or carbon source me-
tabolism gene is a powerful strategy to enhance PHBHHx
production in recombinant A. hydrophila. This combi-
nation strategy may be useful for strain development to
construct other recombinant PHA-producing strains. It was
also demonstrated that lac promoter is constitutively ex-
pressed in A. hydrophila 4AK4 which makes it a good
bacterium for further genetic engineering study.
Acknowledgements We are very grateful for the kind donation of
phaA and phaB genes from Dr. A. Steinbüchel of Münster University
in Germany, vgb gene from Dr. Z.Y. Shen of Tsinghua University in
China and plasmid pBBR1MCS-2 from Dr. Philip Green of Procter
& Gamble (Cincinnati, OH, USA).
This study was financially supported by Natural Science Founda-
tion of China Grant No. 30170017 and the State Outstanding Young
Scientist Award (No. 30225001).
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